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Integrin mediated mechanotransduction in renal vascular smooth muscle cells

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Title:
Integrin mediated mechanotransduction in renal vascular smooth muscle cells
Physical Description:
x, 214 leaves : ill. (col.) ; 29 cm.
Language:
English
Creator:
Balasubramanian, Lavanya
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla.
Publication Date:

Subjects

Subjects / Keywords:
Muscle Smooth, Vascular   ( mesh )
Mechanotransduction, Cellular -- physiology   ( mesh )
Calcium signaling -- physiology   ( mesh )
Calcium -- metabolism   ( mesh )
Integrins -- metabolism   ( mesh )
Kidney -- blood supply   ( mesh )
Rats   ( mesh )
Rates, Sprague-Dawley   ( mesh )
Calcium sparks
Paramagnetic beads
Myogenic response
Fluorescence confocal microscopy
Impedance sensing
Dissertations, Academic -- Molecular Pharmacology and Physiology -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in non-muscle cells. Synthetic integrin-binding peptide triggers Ca2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) from rat afferent arteriole, indicating that interactions between ECM and integrins modulate vascular tone. RGD, an integrin binding peptide, triggered contraction in cultured VSMCs as observed by Electric Cell-Substrate Impedance Sensing technique. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (FN, natural ligand of alpha5beta1 integrin in VSMCs). Pulling of fibronectin-coated beads with electromagnet triggered Ca2+ sparks, followed by global Ca2+ mobilization.Paramagnetic beads coated with low-density lipoprotein (LDL), whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca2+ sparks and global Ca2+ mobilization. Pre-incubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha5 and beta1 integrins triggered Ca2+ sparks simulating the effects of fibronectin-coated beads. Anti-beta2- integrin antibody served as the negative control. Traction force microscopy studies showed that only the force transduced via integrins could potentially trigger cytoskeletal remodeling in cultured VSMCs. Atomic force microscopy revealed a significant increase in surface roughness in VSMCs when treated with RGD peptide though there was no difference in the maximum deflection of the force curves.Pre-incubation of microperfused afferent arterioles with ryanodine or integrin specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. Thus, integrins can potentially act as sensors for myogenic response phenomenon and affect the autoregulatory mechanism in the vasculature.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references (leaves 180-204).
Additional Physical Form:
Also available online.
Statement of Responsibility:
by Lavanya Balasubramanian.
General Note:
Includes vita.

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aleph - 001968230
oclc - 269285303
usfldc doi - E14-SFE0002227
usfldc handle - e14.2227
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SFS0026545:00001


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ABSTRACT: Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in non-muscle cells. Synthetic integrin-binding peptide triggers Ca2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) from rat afferent arteriole, indicating that interactions between ECM and integrins modulate vascular tone. RGD, an integrin binding peptide, triggered contraction in cultured VSMCs as observed by Electric Cell-Substrate Impedance Sensing technique. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (FN, natural ligand of alpha5beta1 integrin in VSMCs). Pulling of fibronectin-coated beads with electromagnet triggered Ca2+ sparks, followed by global Ca2+ mobilization.Paramagnetic beads coated with low-density lipoprotein (LDL), whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca2+ sparks and global Ca2+ mobilization. Pre-incubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha5 and beta1 integrins triggered Ca2+ sparks simulating the effects of fibronectin-coated beads. Anti-beta2- integrin antibody served as the negative control. Traction force microscopy studies showed that only the force transduced via integrins could potentially trigger cytoskeletal remodeling in cultured VSMCs. Atomic force microscopy revealed a significant increase in surface roughness in VSMCs when treated with RGD peptide though there was no difference in the maximum deflection of the force curves.Pre-incubation of microperfused afferent arterioles with ryanodine or integrin specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. Thus, integrins can potentially act as sensors for myogenic response phenomenon and affect the autoregulatory mechanism in the vasculature.
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Integrin Mediated Mechanotrans duction in Renal Vascular Smooth Muscle Cells by Lavanya Balasubramanian A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Pharmacology and Physiology College of Medicine University of South Florida Major Professor: Kay-Pong Yip, Ph.D. Eric S.Bennett, Ph.D. John R.Dietz, Ph.D. Craig A. Doupnik, Ph.D. Chun-Min Lo, Ph.D. Date of Approval: October 30, 2007 Keywords: calcium sparks, paramagnetic beads, myogenic response, fluorescence confocal microscopy, impedance sensing Copyright 2007 Lavanya Balasubramanian

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First they ignore you, then they laug h at you, then they fight you, then you win. Mohandas Karamchand Gandhi

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DEDICATION I dedicate this thesis to my lo ving husband, Kannan, without whom this expedition would have been impo ssible. I also dedicate this to my parents Drs. Jayanthi and Balasu bramanian and my lovely sister Divya. Last but not the least to my beloved grandmother Mrs. Valliammal without whom this life would have no meaning.

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ACKNOWLEDGEMENTS I am grateful to my mentor, Dr. Kay-Pong Yip, for his relentless effort in teaching me and helping me become a self-reliant scientific professional. His guidance earned me the prestigious AHAs Predoctoral Fellowship and recognition in various national and international conferences. Thank you, boss, for instilling in me the humble confidence of a true scientist. I thank the members of my commi ttee, Drs. Eric Bennett, John Dietz, Craig Doupnik, Chun-Min Lo for their continued support and patience and for letting me grow and blossom in my own time and space. I have had the opportunity to interact closely with each one of you. This experience has not only taught me to be a better scientist but also a better person. Our chairm an, Dr. Bruce Lindsey, has always provided his unconditional support, leading the students by example. His kind and encouraging words ha ve taught me to persevere. I would like to extend my heartf elt gratitude to our collaborators Dr. Chun-Min Lo (for the designin g the electromagnet and providing the expertise on traction force mi croscopy), Dr. James S.K. Sham

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(who provided the IDL analysis soft ware), and Drs. Jay B. Dean and Dominic D Agostino (for sharing th e valuable technology of atomic force microscopy). Our collaborato rs have graciously accommodated my scientific work amidst their busy schedule. My sincere thanks to Ms. Caro l Landon who sh owed me the ropes and provided technical support in the lab. Needless to say that I appreciate all the help our departme ntal office staff (Ms. Barbara, Joyce, Bridget and Judy) have pr ovided. I deeply appreciate the guidance of Ms. Kathy Zahn, Ms. Susan Chapman and the office of graduate and post doctoral affairs through all the bureaucracies. I am grateful to Ms. Carol Landon and Ms Barbara Nicholson, my American mothers, who are always there fo r me and who have made my life more interesting in this country. Im indebted to my beloved Kanna n for standing by me during both the high and the low times in my graduate and personal life. He has never failed to extend his support beyond the call of duty. He has made me a more self-assured person by loving me for who I am and not for whom I could be. Many a times has he sat by me and burnt the midnight oil while trying to accomplis h my goals. Although he does not specialize in health science he is ev er eager to learn the science behind

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my work. I am glad that he is alwa ys there for me even before I knew I need him. He has been patiently lending his ears to all my hurdles and has boosted my confidence to help me plough through the rough times. Of course, I would not be here without my parents and their unconditional love and support. I thank them for their many prayers and the long conversations which have helped me find the light. They have silently stood by me and never lost faith in me even when I was not so sure. Appreciations are ex tended to my young and vibrant sister, Divya, for providing the emotio nal support that only sisters can provide. She always had a story to keep me smiling. I also take this opportunity to thank my dear uncle and aunt Mr. and Mrs. Badrinath for their unfaltering support in all my endeavors. My sincere thanks to everyone who has been a part of my life however small the duration may be, for all of you have enriched my life and helped make the person who I am today. I am who I am today because of every one of yo u thank you.

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i TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v ABSTRACT ix CHAPTER ONE INTRODUCTION AND BACKGROUND 1 Evolution 3 Renal Blood Flow 4 Urine Formation 7 Autoregulation 9 Integrins 15 Calcium and Contraction 24 Intracellular Stores of Ca 2+ 29 Calcium Channels in Plasma Membrane 32 CHAPTER TWO MATERIALS AND METHODS a. Isolation of Renal VSMCs and Monitoring of Ca 2+ Sparks 38 b. Pulling renal VSMCs with paramagnetic beads 39 c. Confocal Fluorescence Mi croscopy for the Detection of Ca 2+ Sparks 41 d. Immunofluorescence Histochemistry 45 e. Perfusion of Afferent Arteriole and Measurement of Luminal Diameter to Moni tor Myogenic Response 45

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ii f. Electric Cell-substrate Impendence Sensing (ECIS) Technique i) Cell Attachment Studies via Impedance Measurements 49 ii) Measurement of Changes in Cell Morphology 50 g. Traction Force Microsco py to Detect Remodeling by Integrin Mediated Mechanotransduction 52 h. Atomic Force Microscopy Imaging for Characterizing the Morphology of the Renal VSMCs 55 i. Atomic Force Microscopy Force Plot to Measure the Changes in the Stiffness of the Cultured VSMCs 56 j. Data Analysis for Ca 2+ Sparks 60 CHAPTER THREE THE EFFECT OF INTEGRIN BINDING PEPTIDES ON CULTURED RENAL VSMCs Introduction 62 Results 63 Discussion 88 CHAPTER FOUR THE ROLE OF INTEGRINS IN MECHANOTRANSDUCTION IN FRESHLY ISOLATED RENAL VSMCs Introduction 93 Results 95 Discussion 109 CHAPTER FIVE THE EFFECT OF INTEGRIN SPECIFIC ANTIBODIES ON FRESHLY ISOLATED RENAL VSMCs Introduction 115

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iii Results 116 Discussion 124 CHAPTER SIX THE ROLE OF MECHANOTRANSDUCTION VIA INTEGRINS ON CYTOSKELETAL STIFFNESS Introduction 126 Results 131 Discussion 159 CHAPTER SEVEN ROLE OF INTEGRINS IN INTACT RENAL AFFERENT ARTERIOLAR MYOGENIC RESPONSE Introduction 166 Results 167 Discussion 171 CHAPTER EIGHT SUMMARY AND CONCLUSIONS 174 CHAPTER NINE PERSPECTIVES 178 REFERENCES CITED 180 BIBLIOGRAPHY 204 APPENDICES 205 Appendix A Abbreviations Used 206 Appendix B Source Code for IDL Program Used to Analyze Ca 2+ Sparks 209 ABOUT THE AUTHOR End Page

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iv LIST OF TABLES Table 1 Properties of Ca 2+ sparks induced by pulling with coated paramagnetic beads 108 Table 2 Properties of Ca 2+ sparks induced by treatment with integrin specific antibodies 123 Table 3 Morphological characterization of renal VSMCs using traction force microscopy 150 Table 4 Morphological characterizati on of renal VSMCs using AFM 158

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v LIST OF FIGURES Figure 1 Nephron and its functions 2 Figure 2 Renal vasculature tree 6 Figure 3 Glomerulus and JG apparatus 14 Figure 4 Model for integrins as sensors for myogenic response 23 Figure 5 Calcium regulation of VSMC contraction 28 Figure 6 PKC activation pathway 30 Figure 7 Role of calcium releas e from ryanodine stores during myogenic response in renal VSMCs 36 Figure 8 Biorad MRC-1000 system 43 Figure 9 Leica TCS SP5 system 44 Figure 10 Perfusion setup used in the Biorad MRC-1000 system 48 Figure 11 ECIS system 51 Figure 12 Magnified view of a single well 51 Figure 13 Cell culture chamber used for traction force studies 54 Figure 14 Atomic force microscope 59 Figure 15 Silicon nitride AFM probe tips 59

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vi Figure 16 Normalized resistance showing the attachment and spreading of renal VSMCs on different ECM proteins 65 Figure 17 Changes in impedance with and without renal VSMCs 67 Figure 18 Impedance ratio of a typical electrode with and without VSMCs 70 Figure 19 Impedance variations measured using ECIS in the presence and absence of VSMCs 73 Figure 20 The effect of RGD and RGE on VSMCs grown on different ECM coatings 76 Figure 21 Time course of integrin effects at various RGDpeptide concentrations 78 Figure 22 Image of VSMCs on the ECIS electrode 79 Figure 23 Time course response for RGD peptide 81 Figure 24 Changes in impedance in the presence of coated beads 84 Figure 25 Effect of integrin sp ecific antibodies on cultured VSMCs as measured by ECIS 87 Figure 26 Transmitted light imag e of a freshly isolated renal VSMC with a fibronectin-coated paramagnetic bead attached 96 Figure 27 Linescan images of Ca 2+ sparks in renal VSMCs 98

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vii Figure 28 Disruption of cytoskel eton in freshly isolated renal VSMCs 102 Figure 29 Frequency of Ca 2+ sparks and global Ca 2+ response in VSMCs 104 Figure 30 Frequency distributions of the spatiotemporal parameters of Ca 2+ sparks 105 Figure 31 Frequency distributions of the spatiotemporal parameters of spontaneously occurring Ca 2+ sparks 106 Figure 32 Percentage of renal VSMCs in which Ca 2+ sparks were detected at different experimental conditions 107 Figure 33 Ca 2+ sparks triggered by 5 integrin antibody 118 Figure 34 Ca 2+ spark triggered by 1 integrin antibody 119 Figure 35 Percentage of renal VSMCs in which Ca 2+ sparks were detected when exposed to different integrin antibodies 120 Figure 36 Frequency distributions of the spatiotemporal parameters of Ca 2+ sparks 121 Figure 37 Frequency distributions of the spatiotemporal parameters of Ca 2+ sparks triggered by 2 integrin antibody 122 Figure 38 Transmitted light image of beads moving through dimethylpolysiloxane 133

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viii Figure 39 Force-distance relati onship for the electromagnet and the paramagnetic microbeads 134 Figure 40 Image of a renal VSM C used in traction force microscopy study 135 Figure 41 Force maps of a VSMC generated during magnetic pulling of FN-coated beads 137 Figure 42 Force maps of a VSMC generated during magnetic pulling of LDL-coated beads 138 Figure 43 Traction force per unit area of renal VSMC when pulled using FN-coated paramagnetic beads 140 Figure 44 Traction force per unit area of renal VSMC when pulled using LDL-coated paramagnetic beads 142 Figure 45 Traction force measurements in a VSMC treated with 1 integrin antibody 144 Figure 46 Force patterns of a VSMCs subjected to different conditions 147 Figure 47 Images of VSMCs acquired using the AFM 153 Figure 48 Force curves of VSMCs treated under different experimental conditions 157 Figure 49 Time course of changes in lumen diameter of renal afferent arterioles 169

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ix INTEGRIN MEDIATED MECHANOTRANSDUCTION IN RENAL VASCULAR SMOOTH MUSCLE CELLS Lavanya Balasubramanian ABSTRACT Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cyto skeleton and have been shown to function as mechanotransducers in non-muscle cells. Synthetic integrin-binding peptide triggers Ca 2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) from rat afferent arteriole, indicating that interactions between ECM and integrins modulate vascular tone. RGD, an integrin bind ing peptide, triggered contraction in cultured VSMCs as observed by Electric Cell-Substrate Impedance Sensing technique. To examin e whether integrins transduce extracellular mechanical stress into intracellular Ca 2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (FN, natural ligand of 5 1 integrin in VSMCs). Pulling of fibronectin-coated beads with electromagnet triggered Ca 2+ sparks,

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x followed by global Ca 2+ mobilization. Paramagnetic beads coated with low-density lipoprotein (LDL), whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca 2+ sparks and global Ca 2+ mobilization. Pre-incubation with ryanodine, cytochalasinD, or colchicine substantially reduced the occurrence of Ca 2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of 5 and 1 integrins triggered Ca 2+ sparks simulating the effects of fibronectin-coated beads. Anti2 integrin antibody served as the negative control. Traction force microscopy studie s showed that only the force transduced via integrins could po tentially trigger cytoskeletal remodeling in cultured VSMCs. Atom ic force microscopy revealed a significant increase in surface ro ughness in VSMCs when treated with RGD peptide though there was no difference in the maximum deflection of the force curves. Pre-incubation of microperfused afferent arterioles with ryanodine or integrin specific binding peptide inhibited pressure-induced myogenic constr iction. In conclusion, integrins transduce mechanical force into intracellular Ca 2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. Thus, integrins can potentially act as sensors for myogenic response phenomenon and affect the autoregulatory mechanism in the vasculature.

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1 CHAPTER ONE INTRODUCTION AND BACKGROUND Kidneys are one of the most im portant organs involved in homeostasis. Nephrons, the functional units of the kidneys, consists of glomerular network surrounded by Bowmans capsule (components of the renal corpuscle), a proximal convoluted tubule followed by a proximal straight tubule, a loop of Henle (comprising of thin descending limb, thin and thick asce nding limb), and a collecting duct (which includes cortical collecti ng duct, outer and inner medullary collecting duct). About 1 million neph rons are present in each human kidney. Besides excreting metabolic waste products and chemicals, kidneys also help in the maintenance of water and electrolyte balance, regulation of arterial pressure, an d regulation of acid-base balance. The two major regions in a kidney are the outer cortex and the inner medulla. There are two types of nephron namely the superficial nephron and the juxtamedullary neph ron. The former, as the name suggests, is found in the outer parts of the cortex and has a short loop of Henle which reaches into the ou ter medulla. But, the juxtamedullary nephrons arise much deeper in the cortex close to the medulla with its loop of Henle reaching into the inner medulla.

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Figure 1. Nephron and its functions Glomerulus Proximal convoluted tubule Proximal straight tubule Thin descending Loop of Henle Thick ascending Loop of Henle Thin ascending Loop of Henle Inner medullary collecting duct Cortical collecting duct Distal convoluted tubule Vasa recta Afferent arteriole Efferent arteriole Glomerular filtr a ti o n T ubular reabsorption Tubular secretion (adapted from Valtin and Schaffer, 1995) 2

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3 EVOLUTION Over 550 million years ago in the Paleozoic era, the prochordate ancestors of the vertebrates had to survive in the salty Cambrian Sea. Since their tissues were bathed in what could be very similar in composition to the extracellular flui d, they must have had a simple conduit to expel their metabolic re sidue. Later came the fresh water organisms and with them came the need to conserve salts as they were constantly submerged in hypo -osmotic environments. At this point the organisms developed glomerul us for ultrafiltration of solutes, proximal tubules to help with the reabsorption and distal tubules to expel water. Then the vertebrates mi grated to the land and the need to conserve water arose. Reptiles took a step back in evolution as they had more degenerate glomerulus an d expelled uric acid with minimal water. Mammals retained their glom erulus and evolved long loops of Henle to conserve water through counter current system (which includes the counter current ex change and the counter current multiplication). There are the birds which are in the middle because they expel uric acid and also ha ve loops of Henle, though much shorter, to conserve water.

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4 RENAL BLOOD FLOW Approximately 21 percent of the ca rdiac output is redirected to supply the two kidneys though, toge ther, they constitute less than 0.5% of the total body weight. Although the amount of blood flow to the kidneys remains constant within the species, the amount of blood that is filtered varies between the species based on the level of glomerular development and within the species based on the physiological and environmental conditions. In most mammals including the human each kidney is supplied by a single renal artery though there could be one or more accessory renal arteries. The renal artery, a major component of splanchnic blood supply, enters the kidneys through the hilum and divides into an anterior and a posterior branch. The anterior branch furthe r divides into three segmental or lobar arteries and one branch to su pply the apex of the kidney. The posterior division supplies more th an half of the posterior renal surface. The lobar arteries divide fu rther to form interlobar arteries, which extend all the way towards the renal cortex. These give rise to arcuate arteries that are seen in the border between the cortex and the outer medulla. In turn the arcuate arteries br anch to form cortical radial arteries which extend to the surface of the kidney. The smaller branches of these cortical radial arteri es give rise to afferent arterioles. Afferent arteries are lined conti nuously with endothelium and its

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5 basement membrane. They also contain a monolayer of smooth muscle cells with the walls thinning out as they approach the hilus and form the glomerular network. The glomerular capillary network is connected to the peritubular circulat ion by the efferent arterioles.

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Figure 2. Renal vasculature tree Arcuate artery Arcuate vein Cortical radial artery Cortical radial vein Afferent arteriole Efferent arteriole Ascending vasa recta Interlobar vein Interlobar arter y (adapted from Pflugers Archive. 1988) 6

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7 URINE FORMATION The afferent arteries enter the glomerular capillary network where filtration of the solutes takes place. Due to the large size the blood cells and other macromolecules cannot pass through the fenestrations in the glomerular ca pillary. The filtered fluid then traverses the tubules where solutes are reabsorbed. For many of the substances the reabsorption is prim arily in proximal tubule, though other substances like water and sodi um are also reabsorbed in more distal sites in the nephron. There ar e specific carriers involved in the transport of some of these substances. The final step in urine formation is tubular secretion. Tu bular secretion not only helps to eliminate drugs, toxins, and meta bolic byproducts, but also to maintain the acid-base balance in the body. The rate of urine formation is thus the sum of glomerul ar filtration, tubular reabsorption and tubular secretion. Only 15%-20% of the plasma that enters the glomerulus is actually filtered wh ile the remaining passes on to the efferent arterioles to be finally returned to the systemic circulation via the renal vein. In the 17 th century Marcello Malphigi pioneered to discover the glomerular corpuscle. He discove red the presence of worm-like vessels (renal tubules) on the surface of the kidneys. He also detected the presence of small glands (Malphigian corpuscle or

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8 glomeruli) and that they were conne cted to branches of arteries and veins. Giovanni Alfonso Borelli piqued by Malphigis discoveries went on to propose the theory of filtration in mid 1600s. By 1662 Lorenzo Bellini, an Italian physician and anat omist, a pupil of Borelli, described the papillary ducts or Bellinis du cts. Nearly two centuries after Malphigis discovery, Frederik Ru ysch identified the glomerular capillaries in 1729. Sir William Bowman, a British surgeon, histologist and anatomist, identified what ca me to be known as the Bowmans capsule in 1842. He proposed that glomerulus secreted water to eliminate the solutes secr eted from the renal tubules. Until then the theories on urine formation were not clear. He identified the structure and function of the basement membrane. In 1844, Carl Ludwig hypothesized on glomerular filtration and tubular reabsorption emphasizing the role of renal he modynamics. In 1874, Rudolph Peter Heinrich Heidenhain cha llenged the Ludwigs hypothesis and concluded that the urine formation is enti rely by secretion where some substances were excreted by the gl omerulus while some others by the tubular epithelium. He considered the volume of blood perfusing through the kidneys to be an import ant determinant of the quantity of substances excreted. Arthur Robert son Cushny, a Scottish physician, in 1917 claimed that the glomeruli fi ltered out harmful bodily waste products while the tubules reabsorb ed the useful nutrients into the

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9 body and denied the possibility of selective tubular secretion. Later in 1923, Eli Kennerly Marshall Jr., demons trated the presence of tubular secretion in renal tubules. Now we kn ow that it includes all the three processes glomerular filtration, selective tubular reabsorption and selective tubular secretion. AUTOREGULATION Contrary to the popular belief mammalian blood pressure has been shown to fluctuate spontaneou sly to approximately 40% of the mean arterial blood pressure (MABP). Marsh and co-workers showed that the logarithm of blood pressu re power spectral density varied inversely with the logarithm of the frequency (Marsh et al., 1990). Signals from MABP record that gene rates 1/f power spectra are fractal curves (Voss, 1988), which means that blood pressure dynamics remain invariant under transformations of scale (Marsh et al., 1990). These fluctuations in blood pressure can cause fluctuations in blood flow to the organs unless the vasculature compensates for it by varying its resistance. If these fluctu ations in pressure are translated to fluctuations in renal blood fl ow then it will render the GFR inappropriate by overwhelming th e transport/carrier proteins. But these spontaneous fluctuations in blood pressure do not affect the

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10 renal blood flow or the glomerular filtration rate (GFR) over a range of blood pressure because of the pr esence of renal autoregulation. Autoregulation is the homeostatic tendency of the organ system to maintain a constant blood flow under varying pressure conditions. This mechanism is seen in many organs including the kidneys. The primary purpose of autoregulation in most of the tissues is to maintain an optimal oxygen supply and to eliminate the metabolic waste products. However, in the kidn eys autoregulation maintains a relatively constant GFR whereby it controls the excretion of water and solutes. In 1960, Guyton an d his colleagues believed that autoregulation is absent in normal kidneys and that its presence will only impair the feedback regulation of blood volume. They also showed that the kidneys have varying degr ees of autoregulation only after renal injury (Langston et al., 1960). In 1961, they further proved that the absence of renal autoregulation in normal kidneys was not due to extrarenal blood flow (Langston et al., 1961). After a few years Guyton and co-workers developed a mathem atical model to show that the osmolality of the tubular fluid at th e macula densa regulates the renal blood flow in the afferent arteri oles (Guyton et al., 1964). Although they did not have the evidence, they suspected that spontaneous depolarizations due to changes in the smooth muscle cell-extracellular

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11 matrix (SMC-ECM) environment can trigger contraction. A mathematical model to describe th e role of kidneys in long-term pressure regulation was formul ated in 1967 (Guyton and Coleman 1967). The feedback gain of the ki dneys in maintaining the fluid volume based on variations in pr essure is infinite. The kidneys eventually return the pressure to the equilibrium value. Even after almost five decades of research th e mechanism of renal autoregulation is still not completely understood. We have come a long way from discovering the very existence of re nal autoregulation to potentially identifying and elucidating the in tercellular components of this phenomenon (or solving the se nsors of TGF mechanism). Renal autoregulation is thought to be a dynamic intrarenal reaction to variations in blood pressure occurring at different frequencies (Akselrod et al., 1985; Benton et al., 1990; Blinowska and Marsh, 1985; Broten and Zehr, 1989; Friberg et al., 1989; Hayano et al., 1990; Iberall, 1984; Kanabrocki et al., 1988; Livnat et al., 1984; Marsh et al., 1990; Messerli et al., 1982; Munakata et al., 1990; Nayha, 1985; Portaluppi et al., 1989). This autoregulation of renal blood flow (RBF) is unique in that it comprises both the rapid local myogenic response along the pr eglomerular vasculature and the slower tubuloglomerular feedba ck (TGF) mechanism regulating

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12 vascular resistance in the terminal juxta-glomerular segment of the afferent arteriole (Kiil, 2002; Loutzenhiser et al., 2002; Walker et al., 2000). The sensor for tubuloglomerular feedback is the macula densa in early distal tubule, which dete cts the flow-dependent changes in luminal [NaCl], and accordingly, adjusts the afferent arteriolar resistance (Briggs and Schnermann, 1987). Macula densa is a specialized group of epithelial cells in the distal tubule close to the afferent arteriole. Granular cells called juxtaglomerular (JG) cells present near the afferent arterioles act as intra-renal pressure sensors to secrete renin when the arterial pressure is lowered. The JG apparatus consists of the macula densa, the JG cells and the extraglomerular mesangial cells whic h contracts when stimulated with renal sympathetic nerves. However, the mechanisms of me chanotransduction in myogenic response are not well defined (Davis and Hill, 1999). Myogenic response, first described by W.M. Bayliss in 1902, is the intrinsic mechanism of the smooth muscle ce lls by which the vasculature constricts on elevation of perfusio n pressure and dilates on reduction of the pressure. It is an extrem ely quick response which happens within a matter of few seconds. This mechanism enables organ systems to receive an optimal amo unt of blood flow under varying

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13 perfusion pressure conditions. Myogenic response in skeletal myocytes was thought to indicate enhanced ex citation-contraction coupling of myofilaments through increased Ca 2+ entry into the cell (Uchida and Bohr, 1969a) from extracellular so urces. This suggested possible relationship between Ca 2+ permeability and myogenicity of skeletal myocytes. Myogenic response is ne cessary to maintain constant blood flow and capillary hydrostatic pressu re (Davis and Hill, 1999). In order to respond to changes in blood pr essure, tension-sensing mechanisms should be present (Davis and Hill, 1999; Johnson, 1980).

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Figure 3. Glomerulus and JG apparatus The site of action of myogenic response and TGF Thickness of the arrows indicates the degree of action in that region. (adapted from Guyton and Hall 1996) 14

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15 The general consensus is that the stretch-sensitive non-selective cation channels act as the sens ors in mechanotransduction. An increase in transmural pressure stretches the vascular wall and opens the stretch-sensitive channels, which initiates contraction by depolarizing vascular smooth muscle cells (VSMCs) leading to the activation of voltage-gated Ca 2+ channels (VGCC) and hence an increase in the intracellular calcium concentration ([Ca 2+ ] i ) (Davis et al., 1992; Kirber et al., 1988; Me ininger and Davis, 1992). Stretchactivated whole cells currents were found (Wellner and Isenberg, 1994; Wellner and Isenberg, 1995) in smooth muscle cells (SMCs). The stretch-induced depolarization co uld be explained by the activation of mechanosensitive ion channels promoting Na + or Ca 2+ influx, Cl efflux, or inhibiting K + efflux (Davis and Hill, 1999). This concept of stretch-sensitive channels as sensor s has a major limitation. It does not explain the continued vasoconstr iction, after the initial myogenic constriction, in the absence of er ror signal from stretch-activated channels. INTEGRINS Integrins are now being thought of as a stress sensing and transducing element. They are hete rodimeric transmembrane proteins composed of and subunits, which provide the structural link

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16 between the ECM and the internal cytoskeleton (CSK), and function as signaling receptors. It has been shown in non-muscle cells that extracellular mechanical force is transmitted across the plasma membrane via integrins to initiate intracellular signaling (Ingber, 1990; Katsumi et al., 2004; Martinez-L emus et al., 2003; Wang et al., 1993). It is plausible that increase of perfusion pressure can result in transient detachment between nati ve ECM and VSMC integrin during passive dilation and that myogenic constriction requires re-attachment between native ECM and integrins ba sed on Ingbers tensegrity model (Ingber, 1997; Ingber, 2000; Ingbe r, 2003a; Ingber, 2003b; Wang et al., 2001). There is considerable evid ence that integrins can transduce mechanical force across plasma memb rane and initiate intracellular signaling (Boudreau and Jones, 1999; Davis et al., 2001; Ingber, 1990; Sadoshima and Izumo, 1993; Vu ori, 1998; Wang et al., 1993). It is known that the ECM inte grin cytoskeleton axis is essential in mechanosensing & mechanotransduction by which the vasculature detects and responds to the changes in luminal pressure. In 1993, Ingber described that ce lls are prestressed tensegrity structures (Ingber, 1993). The term Tensegrity was coined by the architect R. Buckminster Fuller (F uller, 1961) to describe structures that stabilize their shape by providing continuous tension rather than

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17 compression (Ingber, 2003a; Ingber, 2003b). This tensional integrity is conveniently called tensegrity. Th is model describes CSK as a highly organized, three-dimensional sync ytium of compression-resistant struts (microtubules) suspended among various elastic elements (intermediate and actin filaments) (Ingber, 1993). Stress will restructure the microtubules in the CSK to offset the tension. This transduced force and the microtub ule depolymerization will then initiate secondary messengers and signaling cascades including an increase in the [Ca 2+ ] i causing contraction. The microfilaments and microtubul es provide the struts for the internal cell environment. The tr ansmembrane proteins (integrins) linking the ECM and the cytoskeleton provide an outlet to transmit external mechanical stress while other receptors dissipate the mechanical stress and fail to transmit it into the cell. Wang and coworkers showed that disruption of cytoskeleton will inhibit integrindependent cell stiffening in endothe lial cells (Wang et al., 1993). Intact cytoskeleton is essential to sense cues from the ECM which results in appropriate cellular response like migr ation, division, contraction, etc. Butler and coworkers suggested that ligation of integrins triggered cytoskeletal reorganization which in turn leads to cell adhesion (Butler et al., 2003). Pelham and Wang show ed that physical communication

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18 in a cell both within itself as well as with its external milieu is important to respond accordingly to adhesion forces (Pelham and Wang, 1997). Moreover, the disrupti on of actin cytoskeleton with cytochalasin D decreased FAK (Focal Adhesion Kinase) tyrosine phosphorylation and formation of fo cal adhesions (Defilippi et al., 1995). Studies in pressurized cerebr al arteries show that actin polymerization helps in mediating my ogenic activity (Cipolla et al., 2002). Another study showed the ab rogation of pressure-induced myogenic constriction when actin po lymerization is disrupted in tail arteries from mice (Flavahan et al., 2005). While other studies in skeletal muscles arterioles showed that disrupting microtubules enhanced the myogenic tone of the vasculature (Platts et al., 1999). Both microfilaments and microtubules are thought to be essential in mechanotransduction and developmen t of force in VSMCs. Paul and colleagues suggested that disruption of microtubules can impact signal transduction thus altering the va scular tone in porcine coronary arteries (Paul et al., 2000). Ligation of integrins with ECM molecules like fibronectin (FN) has been shown to induce clustering of actin thus regulating cytoskeletal organization in mouse embryo cells (Hocking et al., 2000). Another study showed that ligation of inte grins with RGD peptide in mouse

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19 oocytes regulated reorganization of cytoskeletal protein (Yue et al., 2004). Some techniques like traction force microscopy and atomic force microscopy (AFM) can be empl oyed to study integrin mediated remodeling of cytoskeleton with minimum damage to the cells. FNdependent increase in prestress was observed using traction force microscopy perhaps due to the altering of integrin receptor clustering in pulmonary VSMCs (Polte et al., 2004). The ability of the cells to exert force on the substr atum and react to the latters flexibility allows us to study the cells using tracti on force microscopy. Studies in fibroblasts have shown that the moveme nt of the cells is influenced by the rigidity of the substrate (Lo et al., 2000; Pelham and Wang, 1997). Atomic force microscopy can also be employed to investigate mechanotransduction in the presence of intact cytoskeleton. In this method the tip of the probe pushes down on the ce ll and records its deflection depending on the rigidity of the cytoskeleton beneath the cell membrane. Studies showed that the amount of resistive forces exerted by the cells on the AFM prob e is lower when the cytoskeleton is broken down (Girasole et al., 2007). These techniques are comparatively less invasive than cytoskeletal toxins and help us monitor the real-time changes in the rigidity of the cells in response various ligands.

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20 Recent works also show that components of the ECM and integrins can be a contributing fact or to altering the structure and function of the vasculature (Intengan and Schiffrin, 2000). Addition of RGD containing peptide to VSMC integrins can initiate Ca 2+ influx (Arnaout et al., 2002; Bhattacharya et al., 2000; D'Angelo et al., 1997; Davis et al., 2001; Shankar et al., 1995; Xie et al., 1998). Previous studies showed that applic ation of RGD to integrins resulted in increased cytoskeletal stiffening in endothelial cells (Wang et al., 1993) and also activated second me ssenger formation in fibroblasts (McNamee et al., 1993). Integrin linked kinase (ILK) provides an alternative pathway through which activation of integrins might regulate vascular resistance independent of Ca 2+ (Deng et al., 2001). Synthetic integrin-binding peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), when applied exogenously to perfused rat renal afferent artery induces vasoconstriction, which is associated with a pronounced increase of [Ca 2+ ] i in VSMCs (Yip and Marsh, 1997) Elevation of intravascular pressure in cerebral arterioles led to an increase in Ca 2+ spark, Ca 2+ wave frequency, global [Ca 2+ ] i and myogenic constriction (Jaggar, 2001). This suggests the possible existence of a sensor which acts to interface mechanical force and Ca 2+ sparks.

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21 [Ca 2+ ] i involved in various cellular functions, is a major determinant of contractility in myocytes. Interestingly, the addition of RGD-containing peptide initiated ryanodine-sensitive recurrent Ca 2+ waves in renal VSMC (Chan et al., 2001). Calcium Induced Calcium Release (CICR) is the basis for pr opagation of self-regenerating Ca 2+ waves. When the Ca 2+ waves repeat themselves they form global Ca 2+ oscillations. CICR can be preceded by calcium sparks, which indicate the opening of ryanodine stores. Ca sparks are caused by the coordinated opening of a cluster of ryanodine receptors. They were first identified in Ca 2+ activated K + currents in neurons from frog (Brown et al., 1983; Mathers and Barker, 1981). These sparks are very local events and do not di rectly increase the global Ca 2+ levels. As these sparks are seen as discret e packets, the increase in Ca 2+ levels in these microdomains can be very high. This implies that subsarcolemmal [Ca 2+ ] is a different pool from the cytoplasmic [Ca 2+ ]. The further away from such loci the lesser the increase in Ca 2+ Proximity of the next signaling molecu le in the pathway is essential for the sparks to mediate an effect. In cerebral arteries sparks can cause the Ca 2+ levels to go up 10 100 M (Jaggar et al., 1998a; Nelson et al., 1995). This high Ca 2+ concentration can influence any Ca 2+ dependent process in the nearby

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22 region. This local subcellular Ca 2+ transient (Ca 2+ sparks) can directly activate Ca 2+ activated K + channels (K Ca ) to negatively feedback on myogenic response via membrane hyperpolarization as shown in cerebral artery (Jaggar et al., 2000; Nelson and Quayle, 1995). Ca 2+ sparks can also trigger myogenic response by activating Ca 2+ activated Cl channels which can depolarize cell membrane (Gordienko et al., 1994). This depolarization can stimulat e VGCC resulting in an increase [Ca 2+ ] i The longitudinal stretching of urinary bladder smooth muscle cell induces Ca 2+ sparks, Ca 2+ waves, and inward Cl current (Ji et al., 2002). But, the effect of Ca 2+ sparks on myogenic response in renal arterioles is not known because both K Ca (Fallet et al., 2001; Gebremedhin et al., 1996) and Ca 2+ activated Cl channels are present in afferent arteriolar VSMC (Car mines, 1995; Jensen et al., 1997; Jensen and Skott, 1996). Thus sparks can act as a brake or initiate the myogenic response mechanism.

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Figure 4. Model for integrins as sensors for myogenic response 23 RGD RGD RGD RGD Vascular smooth muscle Extracellular Matrix Increase of transmural pressure integrin SS SSC = stretch-sensitive cytoskeleton RGD =Arg-Gly-Asp, (adapted from Yip)

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24 CALCIUM AND CONTRACTION Calcium, an intracellular ion, is unevenly distributed within cells and various organelles. Intracellular Ca 2+ is essential for a wide array of responses to environmental stim uli including but not limited to contractility, proliferation, apop tosis and gene expression. SMCs, especially those in the vasculature are arranged perpendicular to the long axis of the vessel so when th e SMCs contract, it will reduce the diameter of blood vessels. The SMCs are mechanically coupled to each other so they act as a single unit during contraction and communicate through gap junctions. It is known that Ca 2+ can pass through gap junctions (Dhein, 1998). This Ca 2+ can play a role in the spread of myogenic response along a single vessel or allow for a coordinated response in the whole arteriolar network. Uchida and Bohr demonstrated the importance of Ca 2+ in arteriolar tone about 40 years ago (Uchida and Bohr, 1969b) Previous work by Meininger and coworkers (Meininger et al., 1991) observed that during myogenic response increasing the pressure induced an increase in [Ca 2+ ] i in rat cremaster myocytes. Another study done in rat afferent arterioles also showed that myogenic constriction caused by increasing perfusion pressures resulted in an increase in [Ca 2+ ] i (Yip and Marsh, 1996).

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25 The basic contractile unit in a myocyte consists of a series of repeating sarcomeres. Each sarcomere consists of a thick and thin filament. The latter is connected via dense bodies which serve to couple the SMCs. In SMCs the acti n and myosin filaments are not well organized unlike in striated muscle. Sarcolemma is the membrane surrounding the myofilaments that in vaginates the myofibrils forming the membranous transverse tubules which store Ca 2+ Sarcoplasmic reticulum (SR) is a specialized fo rm of smooth endoplasmic reticulum which lies on either side of the tr ansverse tubular network. The SR Ca 2+ stores are organized into small, distinct and independent compartments. The sacrolemma posse ss microdomains called caveolae which help in cell signaling (Tagga rt et al., 2000). These invaginations contain a myriad of receptors, seco nd messengers and ion channels in close proximity. The caveolae themselves are found close to the SR thus serving to continue the signaling process to release Ca 2+ The contraction itself is effected by the sliding of the actin and myosin filaments over each other. This cytosolic Ca 2+ binds to calmodulin (CaM) to activate a kinase, the myos in light chain kinase (MLCK). The activated MLCK phosphorylates myos in light chain with an inorganic phosphate from ATP. The phosphorylated crossbridge interacts with actin and causes the cell to contract. The crossbridge cycling ceases with the decrease in the intracellular Ca 2+ levels and the crossbridges

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26 are dephosphorylated by myosin phosphatase. Intracellular Ca 2+ levels decrease thereafter, due to pumping of Ca 2+ either to stores such as sarcoplasmic reticulum, endoplasmic reticulum (via sarcoplasmic/endoplasmic reticular calcium ATPase SERCA) and mitochondria or to the extracellular fluid via Na-Ca exchanger or Ca 2+ ATPase. In SMCs the endoplasmic reticulum serves as the major Ca 2+ store. The increase in Ca 2+ can have an early transient effect as well as a late prolonged effect. In SMCs an increase in the [Ca 2+ ] i causes contraction, which affects the resist ance of the vascular bed in turn affecting the property of autore gulation of VSMCs. At rest, [Ca 2+ ] i is less than 100 nM, and goes up to about 300 nM during endothelin or angiotensin II induced contraction in renal afferent arterioles (Fellner and Arendshorst, 2004; Fellner and Arendshorst, 2005). The involvement and relative roles of specific Ca 2+ sources in the myogenic response still remain uncertain. The two major sources are calcium channel(s) mediated calcium releas e from intracellular stores and calcium influx from extracellular p ool. The increase in intracellular levels of Ca 2+ is possible by: 1) Release from intracellular stores via inositol 1,4,5trisphosphate (IP 3 ) sensitive and ryanodine receptors, and/or

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27 2) Entry from extracellular environment via nonselective cation channels and VGCC.

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Figure 5. Calcium regulation of VSMC contraction Ca2+Calmodulin ( CaM ) Myosin Myosin-P MLC-Kinase Ca2+Ca2+ / CaM-MLCK MLC-Phosphatase Cross bridge cycling Actin-activated myosin complex Actin Contraction ATP (adapted from Davis and Hill, 1999) 28

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29 Intracellular Ca 2+ stores The two major Ca 2+ stores are IP 3 -sensitive and ryanodinesensitive stores. Their receptors have a four-fold symmetry. IP 3 is an important second messenger. Agonists activate phospholipase C (PLC) which in turn cleaves phosphatid ylinositol 4,5-bisphosphate (PIP 2 ) to form diacyl glycerol (DAG) and IP 3 (figure 6). IP 3 binds to IP 3 receptor (IP 3 R are mostly found on the endoplasmic reticulum) to stimulate the release of Ca 2+ from intracellular stores DAG activates PKC, which facilitates phosphorylation of va rious proteins which may affect contractility. Agonists can also in duce phospholipase D to produce DAG. Some studies showed that PKC modulates the contractility of the vasculature by regulating the K v channels. Narayanan J. and coworkers 1994 (Narayanan et al., 1994) showed that PLC cleavage of PIP 2 increased when the renal arterial transmural pressure was elevated. There are three types of IP 3 R. Type 1 and 3 are found in VSMCs (De Smedt et al., 1994); of this type 3 is the predominant one and is distributed throughout the SR Type 3 is also known to have lower affinity for IP 3 (Tasker et al., 1999). The three known types of IP 3 R exhibit different cellular and subcellular distribution suggesting that they may have distinct functions.

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Figure 6. PKC activation pathway Phosphatidyl choline Lysolecithin DAG Ca2+ from IP3 stores via IP3R Sustained PKC activation Phospholipase D Phospholipase C Phospholipase A2 Free fatty acids Cell membrane Ca2+ Vasoconstrictors Early cellular responses Late cellular responses Inositol triphosphate Transient PKC activation Phosphatidyl inositolbisphosphate (adapted from Berne and Levy, 1998) 30

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31 Ryanodine receptors (RyR) are present in many cell types including all types of myocytes. Ther e are three distinct types of RyR RyrR1, RyR2, and RyR3. RyR1 is primarily found in skeletal muscle, while RyR2 is seen in cardiac tissue and RyR3 in brain. The major subtype involved in smooth muscle cells is thought to be RyR2 though others may also be present. The ph armacological properties of the RyR in the SMCs are similar to those in cardiomyocytes though the density in the former is considerably lowe r. Caffeine sensitizes and activates the RyR channels in SMCs. Calcium induced calcium release (CICR) by ryanodine and IP 3 sensitive stores cause amplification and regenerative propagation of the Ca 2+ signal. Ca 2+ compartments are refilled by SERCA, in the presence of ATP. The Ca 2+ sparks have either a positive or a negative feedback on the membrane potential depending on whether the increase activates the Ca 2+ activated chloride or the Ca 2+ activated potassium channel, respectively. Correspondingly th e cell membrane will either be depolarized or hyperpolarized. Knot and co-workers suggested that local Ca 2+ release originating from RyR channels (Ca 2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of K Ca channels, which regulate membrane potential through tonic hy perpolarization, thus limiting Ca 2+

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32 entry through L-type voltage-dependent Ca 2+ channels (Knot et al., 1998). Phosphorylation of phosphol amban by protein kinase G or protein kinase A increases the freq uency of sparks due to increased Ca 2+ pumping and SR Ca 2+ load in mouse ventricular myocytes (Santana et al., 1997). PKC increases vasoconstriction by decreasing the frequency of sparks, depolarizati on of membrane and activation of VGCC in rat cerebral arteries (Bonev et al., 1997). Calcium channels in plasma membrane: Vascular smooth muscle cells exhibit characteristics of both Ltype (Bean et al., 1986; Benham et al., 1987; Ganitkevich and Isenberg, 1990; Matsuda et al., 1990; Nelson and Worley, 1989; Worley et al., 1986) and T-type (Bean et al., 1986; Ganitkevich and Isenberg, 1990; Loirand et al., 1986) Ca 2+ channels. The L-type is the long lasting or slow inactivating channel, activated by high voltage while the T-type is the transient or fast inactivating channel, activated by low voltage. The L-type channel (also re ferred to as the VGCC) is thought to be more important in arterial smooth muscle (Nelson et al., 1990). L-type channels may be directly gated by membrane distension, or by intracellular seco nd messengers released due to cell volume changes which can then modu late the channel activity (e.g., cAMP as shown by Liu H. et al. (Liu et al., 1997)). Membrane

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33 depolarization opens the VGCC increasing [Ca 2+ ] i which can cause contraction of SMCs. Opening of VG CC can also increase the frequency and amplitude of Ca 2+ sparks resulting in contraction or relaxation of vessels depending on the vascular bed. Furthermore, studies using dihydropyridines (L-type Ca 2+ channel blocker) eliminated or dramatically attenuated myogenic response (Asano et al., 1993; Harder, 1984; Hill and Meininger, 1994; Hynes and Duling, 1991; Laher and Bevan, 1989; Wesselman et al., 1996) Diltiazem, another VGCC blocker, also inhibited myogenic response (Takenaka et al., 1998). On the other hand, BAYK 8644 (activator of VGCC) enhanced myogenic response (Hill and Meininge r, 1994; Kirton and Loutzenhiser, 1998; Wesselman et al., 1996). K + currents can counteract the myogenic tone. Of the various potassium channels identified in vascular smooth muscle (VSM), voltage-dependent K + (K v ) channels and large-conductance Ca 2+ activated K + (BK Ca or K Ca ) channels can provide potentially powerful repolarizing mechanisms to counteract stimuli resulting from VSM stretch. BK Ca channels are activated by Ca 2+ influx and by Ca 2+ sparks [bursts of Ca 2+ release from SR], produc ing spontaneous transient outward currents (STOCs) that substantially hyperpolarize the cell. The presence of STOCs suggest that the subsarcolemmal [Ca 2+ ] is different

PAGE 50

from global cytoplasmic [Ca 2+ ] and can be even higher than the latter under certain circumstances (Jaggar et al., 2000). Ryanodine-sensitive Ca 2+ channels cause Ca 2+ sparks which activate BK Ca channels and induce relaxation of SMCs (Nelson et al., 1995). Because myogenic tone is associated with both Ca 2+ influx (Asano et al., 1993; Harder, 1984; Hill and Meininger, 1994; Hynes and Duling, 1991; Laher and Bevan, 1989; Wesselman et al., 1996) and Ca 2+ release (Davis et al., 1992; Nakayama, 1982) BK Ca current should be tonically activated when a blood vessel is at its normal pressure. Earlier studies by Nelson and co-workers (Bonev et al., 1997; Gollasch et al., 1998; Jaggar et al., 1998a; Jaggar et al., 1998b; Knot et al., 1998; Nelson et al., 1995; Perez et al., 1999; Porter et al., 1998) showed that Ca 2+ sparks regulate BK Ca channel activity in VSMCs an d, in turn, the vascular tone by a negative feedback mechanism. Besides BK Ca channels, VSMCs also have Ca 2+ activated chloride channels (Cl Ca ), though not necessarily in the same cells. Cl Ca currents were found in a few SMC preparations inclusive of rat rena l artery (Gordienko et al., 1994) and pulmonary artery (Helliwell et al., 1994). Activation of Cl Ca channels by Ca 2+ sparks cause depolarization. Nelson (Nelson, 1998) proposed that activation of Cl channels could explain stretch-induced depolarization of VSM. Also, studies have found the presence of spontaneous transient inward currents (STICs) in SMCs isolated from pulmonary 34

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35 arteries (Remillard et al., 2002; Yuan, 1997) and trachea (ZhuGe et al., 1998). Further, ZhuGe et al.(ZhuG e et al., 1998), showed that the biphasic nature of the Ca 2+ activated current was due to contributions by both STOCs and STICs. They showed that both BK Ca and Cl Ca channels colocalized adjacent to Ca 2+ spark sites. Although this is true, STOCs and STICs still have very different characteristics. Unitary conductance of STOCs are known to be greater but STICs have slower decay times than STOCs (ZhuGe et al., 1998). When the sarcoplasmic or endoplasmic Ca 2+ store deplete, channels on plasma membrane know n as the store operated channels (SOCs) open to allow Ca 2+ flow into the cell. Ca 2+ ionophores, which increase the permeability of cell membrane, allows the influx of extracellular Ca 2+ and also cause release of stored Ca 2+ and SERCA inhibitors, which cause depletion of Ca 2+ stores, Ca 2+ chelators like BAPTA lead to the activation of capacitative Ca 2+ entry. Thus, SOCs open to increase intracellular levels of Ca 2+ thereby replenishing sarcoplasmic or endoplasmic stor es. The mechanism of coupling between SOCs and Ca 2+ stores is not current ly known due to its complex nature, as this may invo lve more than one channel. The relationships between the different Ca 2+ activated ion channels are shown in figure 7.

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Figure 7. Role of calcium releas e from ryanodine stores during myogenic response in renal VSMCs Depolarization RyR Ca2+ ClCa KCa Voltage-Gated Ca2+ Channel Hyperpolarization Myogenic Response Global Ca2+Response Positive feedback Negative feedback (adapted from Jaggar et al. 2000) 36

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37 These observations raise the intriguing hypothesis that integrins on the plasma membrane of renal VSMCs may serve as mechanotransducers to regulate vascular tone by generating Ca 2+ sparks and triggering contraction (ZhuGe et al., 1998). I undertook this study in an attempt to provide the evidence to substantiate this hypothesis. My study will provide no vel and vital information on the integrin-mediated constriction pathwa y. It will directly test whether integrins serve as mechanotransduce r and signal to the cells by an elevation of [Ca 2+ ] i This study is also designed to test the role of integrin-mediated constriction in the initiation (via Ca 2+ sparks) and maintenance of in myogenic response of afferent arterioles. As myogenic response is important in various physiological and pathophysiological conditions, it may open up new avenues for therapeutic manipulation.

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38 CHAPTER TWO MATERIALS AND METHODS a. Isolation of renal VS MCs and monitoring of Ca 2+ sparks : All experiments were performe d under protocols approved by the University of South Florida's An imal Care and Use Committee. Male Sprague-Dawley rats from Harlan Farms (120 g) were killed by anesthetic overdose (5 % halothan e in a chamber through a Fluotec Mark-3 vaporizer). The kidneys were quickly removed through a midline abdominal incision, cut longitudinally into half and placed in ice-cold dissection buffer. The enti re preglomerular vascular tree is capable of responding to changes in perfusion pressure (Carmines, 1995). Renal vascular smooth muscle cells were isolated with enzyme digestion from dissected arcuate arte ries and cortical radial arteries because the yield of isolated cells from these arterioles was much higher than using afferent arteri oles (Gordienko et al., 1994). The arteries were first digested with papain, and then digested with 2% collagenase type 4, 1% trypsin inhibitor and 0.5% elastase. All digestions were performed in low ca lcium dissociation solution at 37C. Low calcium dissociation solution co nsisted of (in mM): NaCl, 119; 4.7

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39 KCl, 4.7; 1.2 MgSO 4 1.2; KH 2 PO 4 1.18; NaHCO 3 24; Glucose, 5.5; Hepes, 10; and 50 M CaCl 2 pH 7.4. The vessels were then triturated and VSMCs were seeded into glassbottomed Petri dishes coated with Matrigel (BD Bioscience). VSMCs were then loaded with calcium indicator dye fluo-4/AM (10 M, Molecular Probes) in Hanks Balanced Salt Solution (HBSS, Mediatech, containing 1.3 mM Ca 2+ ) for 30 minutes, followed by 20 min fo r de-esterification. All Ca 2+ spark experiments were conducted at room temperature in HBSS or calciumfree HBSS (Mediatech) with 4 mM EGTA. b. Pulling renal VSMCs with paramagnetic beads: To transduce controlled external mechanical force into the VSMCs via integrins, paramagnetic microbeads coated with integrin binding ligands were used to tug on the VSMCs with an electromagnet. FN is the natural ligand for 5 1 integrins in VSMCs (Mori and Cowley, 2004; Umesh et al., 2006; Yip and Marsh, 1997). Tosyl-activated paramagnetic microbeads (4.5 m diameter, M-450, Dynal Inc) were coated with FN or low-density lip oprotein (LDL) using the protocol provided by the manufacturer. LDL is the ligand for the metabolic LDL receptor on VSMCs. Pulling of LDL-co ated bead was used as control for non-integrin-mediated mechanical sign al transduction. To apply force on renal VSMC using paramagnetic beads, coated beads were first

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40 incubated with VSMCs adhered on glass-bottomed cell Petri dish (WPI, FL) for 20 min. Only cells with indi vidual paramagnetic beads attached were used. Pulling was initiated by applying a local magnetic field directed to the cell of interest with a custom-made miniaturized electromagnet mounted on a mi cromanipulator. The tip of electromagnet has a diameter of 420 m and was positioned within 100-150 m from the cell of interest at an angle of 30 to 45. The dragging force imposed by the electromagnet on individual paramagnetic bead was calibrated by pulling the paramagnetic beads through dimethylpolysiloxane (visco sity 100 centistokes, Sigma) based on Stokes Law. (Alenghat et al., 2000; Lammerding et al., 2003; Matthews et al., 2004). The velocity of the beads migrating to the tip of electromagnet was quantified ba sed on the time lapsed images collected at 2 Hz. The dragging force was then calculated using Stokes Law (force = 6 R, where is the viscosity of the fluid, R is the radius of the bead, and is the velocity of the bead). The pulling force per se is controlled by ad justing the distance between the magnet and the bead (50-200 m), or by increasing the current to the electromagnet.

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41 c. Confocal fluorescence micros copy for the detection of Ca 2+ sparks : Changes in subcellular [Ca 2+ ] i induced by magnetic pulling were monitored in linescan mode (512 pixe ls/line, 2 ms/scan line) with a Bio-Rad MRC-1000 confocal microscope or Leica TCS SP5 confocal microscope. Linescan images were collected immediately under the plasma membrane of VSMCs. Scanni ng line was positioned in close proximity to at least one of the a ttached paramagnetic beads. Images were collected for 4-6 s as baseline, and then for another 20-30 s after the magnetic pulling was initiated. The operating system in the Bio-rad confocal microscope (COMOS Version 7.0) can collect 512 scan lines continuously scanning at 0.078 m/pixel. Some line scan images were acquired with Leica TCS SP5 system which collects data continuously for 16.385 s scanning at 0.075 m/pix el. All the images were collected with a Zeiss 40x plan-apochroma t objective (N.A. 1.2, water immersion). Ca 2+ sparks were visualized using the membrane permeable Ca 2+ -sensitive fluorescent dye (fluo-4 acetoxymethyl ester, 10 M) in freshly isolated renal VSMCs. Fluo-4 was excited at 488 nm, and its emission was collected with a bandpass filter 522/35 nm. To test whether ligating 5 1 integrins with antibodies that recognize their extracellular domain s could trigger sparks, the VSMCs

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42 were monitored in the presence of anti-integrin 5 antibody (25 g/ml, HM 5-1, Pharmingen) or anti-integrin 1 antibody (50 g/ml Ha2/5, Pharmingen). This soluble anti5 1 integrin antibody has been shown to activate 5 1 integrin in tsA-201 and HEK-293 cells (Gui et al., 2006). 2 antibody (50 g/ml, Wt.3, Pharmingen) was used as the control. To test whether intact structural linkage of integrin-cytoskeleton is required for integrins to mediate mechanotransduction and generation of Ca 2+ sparks, isolated renal arterioles were incubated with cytochalasin-D (4g/ml) or co lchicine (1mg/ml) in HBSS for 2 hours before VSMCs were harvest ed by enzyme digestion. The occurrence of Ca 2+ sparks induced by pulling of FN-beads was then monitored in these smooth muscle cells.

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Figure 8. Biorad MRC-1000 system A.C. power supply for the electromagnet Micromanipulator Electromagnet Petri dish with isolated renal VSMCs 43

PAGE 60

Figure 9. Leica TCS SP5 system 44

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45 d. Immunofluorescence histochemistry: To detect the changes in the orga nization of actin microfilaments induced by cytochalasin-D, renal VSMCs were first fixed with 2% paraformaldehyde in PBS for 20 mi n, and then permeabilized with 0.5% Triton-X in PBS for 30 min. To detect the changes in the organization of microtubule induce d by colchicine, renal VSMCs were first fixed with 0.5% glutaraldehy de for 10 min an d then quenched with 0.1% NaBH 4 (Zhang et al., 2000). Cells were then incubated with either FITC-conjugated phalloidin (4 g/ml, Sigma), or Cy3-conjugated anti--tubulin antibody (20 g/ml, goat IgG, Sigma). Confocal fluorescence images were collected with a Zeiss 63x plan-apochromat objective (N.A. 1.4, oil immersion). e. Perfusion of afferent arteri ole and measurement of luminal diameter to monitor myogenic re sponse in afferent arterioles: Experiments were conducted in afferent arterioles isolated from rat juxtamedullary nephrons as repo rted previously (Yip and Marsh, 1996; Yip and Marsh, 1997). Male Sprague-Dawley rats from Harlan Farms (120 200 g) were killed by anesthetic overdose. The kidneys were quickly removed and placed in ice-cold dissection buffer. A segment of afferent arteriole (300 m) just proximal to a glomerulus was then dissected, cannulated with two concentric glass

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46 pipettes on one end and perfused in a temperature controlled perfusion chamber (Vestavia, AL) at 37C. The perfusion chamber was mounted on a Zeiss Axiovert 100TV inverted microscope which was coupled to a Bio-Rad MRC-1000 confocal scanning unit equipped with transmitted light detector. Imag es were collected with a Zeiss 20x plan-apochromat (N.A. 0.75) or 40x plan-apochromat objective (N.A. 1.2, water immersion). An exchange pipette is positioned inside the inner pipette for rapid fluid exchan ge. The other end of the cannulated arteriole is positioned inside a holding pipette to seal the flow. The intraluminal pressure of the vesse l was initially set at 80 mmHg. Vessels were discarded if there was fluid leakage. Transmitted light images were collected at 0.25 or 0. 5 Hz throughout the experimental period. After vascular tone was established in the perfused vessel, perfusion pressure was increased from 80 to 120 mm Hg in a single step. The time course of changes in luminal diameter was monitored from the stored images with an edge detecting algorithm based on covariance (Marsh et al., 1985). Th e algorithm was implemented with a Matrox IP-8 imaging board (Wag ner et al., 1997; Yip and Marsh, 1996). The composition of the dissecting solution consisted of (in mM): NaCl, 115; NaHCO 3 25; K 2 HPO 4 2.5; MgSO 4 1.2; CaCl 2 1.8; glucose, 5.5; pyruvic acid, 2.0; and 1g/dL di alyzed bovine serum albumin (BSA, fraction V, Calbiochem). The lumi nal perfusate and bathing solution

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47 were identical to the dissecting solution except that no BSA was added to the bathing solution. BSA was excl uded in the bathing solution in order to prevent bacterial growth in the perfusion chamber. All solutions were gassed with 5% CO 2 before use, and pH was adjusted to 7.4.

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Figure 10. Perfusion setup used in the Biorad MRC-1000 system Perfusion chamber Perfusion pipette in its holder 48

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49 f. Electric Cell-substrate Impedance Sensing (ECIS) technique f. i) Cell attachment studie s via impedance measurements: Electrode arrays, relay bank, lock-i n amplifier and the software for the ECIS measurements were obtain ed from Applied BioPhysics, Inc. (Troy, N.Y.). Each electrode array consists of eight wells which are 1 cm in height and 0.9 cm 2 in area; each well contains a 250 m diameter gold electrode and a much larger gold counter electrode. The large electrode and one of the sma ll electrodes are connected via the relay bank to a phase-sensitive lock-i n amplifier, and an AC voltage is applied through a 1 M resistor. Experimental setup and circuit connection were same as we previously described (Lo et al., 1995). For impedance measurements, Ham s F12 media supplemented with 10% Fetal Bovine Serum (0.4 ml) was added over the electrode in each well. Cells were allowed to a ttach and spread for at least 24 hr before impedance was measured. After 24 hours in culture, the confluence and viability of the cell monolayer was conf irmed by light microscopy and electrically by the resistance values. Attached cells on the electrode act as insulating pa rticles and the main current must therefore flow around the cells. The data presented as inand out-ofphase voltage with the applied signal were converted as resistance and capacitance connected in series. The changes in cell dimensions manifest as changes in impedance as the cell-covered area and/or the

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50 cell-substrate separation change. RG D, RGE, or integrin specific antibodies in HBSS or complete medi um alone were added to each well and the electrical impedance of each well was measured every two minutes for cell attachment studies. In all the attachment measurements shown here, 1 V at 4 KHz was applied to the cells through a 1 M resistor. It takes 2 min per sweep to measure 16 electrodes. f. ii) Measurement of changes in cell morphology: Frequency scan is another main method in ECIS with which we can measure the impedance of the cell-el ectrode system as a function of frequency. It takes approximately 2.5 minutes to measure each electrode. We can obtain Frequency Scan before and after cells attach on the corresponding electrodes. By comparing the experimental data of confluent cell layers with the ca lculated values obtained from cellelectrode model, frequency scan measurements can provide us morphological information such as ce ll-cell and cell-substrate contacts and membrane capacitance of the adherent cells.

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Figure 11. ECIS system Figure 12. Magnified view of a single well 51 (Images courtesy of Applied BioPhysics, Inc.)

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52 g. Traction force microscopy to detect remodeling by integrinmediated mechanotransduction: VSMCs were grown on flexible substratum impregnated with fluorescent beads, and traction fo rce exerted on the substratum (by individual VSMC) was calculated ba sed on the displacement of beads and the Youngs modulus of the fl exible substratum (Dembo and Wang, 1999; Lo et al., 2000). In brief, polyacrylamide gel (5% Acrylamide, 0.1% Bis-acrylamide, 75 m thickness, Youngs modulus = 28 kN/m 2 ) will be prepared on glass co verslip with fluorescent bead (0.2 m diameter) impregnated under th e top surface. The surface is then coated with collagen-1 (0.2 mg/ml) after photoactivation with Sulfosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (SulfoSANPAH; Pierce chemical) (Beningo et al., 2002). One end of the Sulfo-SANPAH reacts non-specifically to the polyacrylamide gel on photoactivation and the other end re acts with primary amines thus crosslinking the ECM protein (co llagen) to the gel. Recently hydrazinolysis has also been shown to be an effective tool to make gels with uniform coatings and prot ein conjugation (Damljanovic et al., 2005). Primary culture of renal VSMC is then seeded and cultured overnight. Fluorescent images of the impregnated beads right under the cell of interest will be collected with a cooled CCD camera (MicroMax:512BFT; Princeto n Instruments, Trenton, NJ)

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53 attached to an inverted microsco pe (Zeiss Axiovert 200M) equipped with a 10x achromat (N.A. 0.25), 20x achroplan (N.A. 0.40) or 20x plan-apochromat (N.A. 0.75) objective lens with an incubation system surrounding the microscope. The basic sequence of image colle ction is (1) to take a control image of impregnated fluorescent beads right under the cell of interest, (2) to collect a series of image during and after the cell exposed to experimental maneuver, and (3) to collect another image after removing the cell from the fl exible substratum. Deconvolution algorithm will then be applied to these images to tract the movement of individual beads. The traction force exerted by the cell to the flexible substratum will be computed based on the displacement of the beads and the Youngs modulus of the flexible substratum. The custom-written image deconvolutio n program and algorithms for computing traction force are prov ided by Wang and Dembo (Dembo and Wang, 1999; Lo et al., 2004). Calc ulation of traction stress was carried out on a supercomputer, us ing the displacement vectors, the cell boundary, the Youngs modulus, an d the Poisson ratio as the input. The flexibility of polyacrylamide sheets was determined with an improved method based on the Hertz theory, similar to that used in

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atomic force microscopy (Radmacher et al., 1992). In short, a steel ball (0.64-mm diameter, 7.2 g/cm 3 ; Microball Company, Peterborough, NH) was placed on a stiff or a so ft polyacrylamide sheet embedded with fluorescent beads. The indentation caused by the steel ball was measured by following with the micr oscope focusing on the vertical position of the fluorescent beads un der the center of the ball. Youngs modulus was calculated as Y = 3(1-v 2 ) f /4 d 3/2 r 1/2 where f is the force exerted on the sheet, d is the indentation, r is the radius of the steel ball, and is the Poisson ratio assumed to be (0.3 in our calculation; (Li, 1993)), (Lo et al., 2000). Figure 13. Cell culture chamber us ed for traction force studies Gel Chamber dish Activated glass coverslip Vacuum grease 54

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55 h. Atomic force microscopy imaging for characterizing the morphology of the renal VSMCs: AFM, another non-invasive imaging technique, was done using a Veeco Bioscope SZ, an integrated imaging system consisting of an inverted biological microscope (Nik on TE2000) mated with an atomic force microscope (Digital Instrume nts, Santa Barbara, CA). Silicon nitride AFM probe tips (Digital In struments, #DNP-20) were used for all of the imaging. AFM cantilevers were 200 m in length with a spring constant of 0.28 N/m to 0. 32 N/m. The scanning probe tip had a nominal tip radius with a curva ture of 20-60 nm. Laser alignment was performed on the cantilever an d the photodetector before each scan for maximum sensitivity. Scanni ng was done using Contact Mode in fluid. All the imaging was pe rformed on renal VSMCs grown on 60 mm plastic cell culture dishes. The experiments were conducted in Hanks Balanced Salt Solution (HBSS, Mediatech) in the absence or presence of GRGDSP as the agonist or GRGESP for control studies. All measurements were made at room temperature and a 30 to 60 min equilibration period was allowe d before attempting to scan. The AFM probe tip was positioned using the inverted optics of the Nikon TE2000 just prior to enga ging the sample. Integral gain, proportional gain, deflection set-poin t, scan rate and scan size were all

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56 optimized to maximize sensitivity and minimize lateral friction and vertical force to cells. The scanning frequency was 1 Hz. The 512 x 512 data image point fields were taken from scan sizes approximately 100 m. Images took approximately 2 to 6 minutes to acquire. Both height and deflection data (positio n of the probe) were acquired for each horizontal scan line by applying a constant force of 0.7 nN to 1.3 nN to the cell. Analysis of membrane roughness was used to characterize changes in membrane topography with and without treatment. Membrane surface topogr aphy was characterized by Mean Roughness (R a ). R a is defined as the arithmet ic average of the absolute values of the surface height devi ations measured from the mean plane. Analysis was done using NanoScope (version 6.0 and 7.1, Veeco, CA). Statistical comparisons of the average roughness between treatment groups were made using an unpaired students t-test and P < 0.05 was considered significant. Al l roughness values were reported as the mean SEM. i. Atomic force microscopy force plot to measure the changes in stiffness of the cultured VSMCs: This technique is complementary to traction force microscopy generating information about the chan ges in cytoskeletal stiffness and hence cytoskeletal remodeling in integrin mediated

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57 mechanotransduction. Force-distance curves were generated using the AFM as described previously by Tr ache and coworkers (Trache et al., 2005). The piezotransducer drives the cantilever to approach the cell, touch it and then retract within a predefined distance in the z-axis. Typically the probe comes down from a point where its not in contact with the cell. After contact, the cant ilever pushes down on the cell to cause an indentation. Depending on the stiffness of the cells, further extension of the probe causes an increasing force in the opposing direction and this bends the cantileve r resulting in a defection signal. During the retraction phase, the ca ntilever is slowly raised until it comes to the original position. Prio r to force-distance measurements the spring constant of the cantilever was calibrated using the thermal noise method. Calibrating the spring constant gives the deflection sensitivity which helps to convert a change in deflection shown in voltage to a change in nanometers. In orde r to obtain the deflection sensitivity, we performed force-distance curves on a hard surface (plastic). The slope of the contact re gion is the deflection sensitivity (e.g., in nm/V). By drawing a line parallel to the curve we can measure the slope and then using the software we can compute a "deflection sensitivity" (in nm/V) based on the spring constant.

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58 Renal VSMCs grown on 60 mm plastic cell culture dishes incubated with in Hanks Balanced Salt Solution (HBSS, Mediatech) at room temperature were used fo r the force plot measurements. Scanning frequency was 1 to 1.5 Hz. Ramp size ranged from 2 m to 4 m. The z-scan start was adjusted manually until the AFM cantilever deflection on the cell was optima l. Adjusted parameters were maintained for a particular cell unde r control and treatment conditions. Approximately ten force distance cu rves were acquired on each cell in the presence and absence of RGD or RGE containing peptides. Forcedistance plots of stiffness meas urements were analyzed using NanoScope software (version 7.1, Di gital Instruments, Santa Barbara, CA).

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Figure 14. Atomic Force Microscope Scan head Figure 15. Silicon nitride AFM probe tips (Images courtesy of Veeco Instruments Inc, Dr.Deans lab) 59

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60 j. Data analysis for Ca 2+ sparks: Results of measurement were shown as mean SE. Only one arteriole was used from each animal for perfusion study. Ca 2+ sparks were detected in confocal images with a custom made algorithm written in Interactive Data Language software (IDL, Research Systems, Boulder, CO). The program identified Ca 2+ sparks on the basis of their statistical deviation fr om the background noise (Cheng et al., 1993; Zhang et al., 2003). Fluo rescence signals (F) of each confocal image were first normalized in terms of F/F 0 where F 0 is the baseline of fluorescence in a region of the image without Ca 2+ sparks. A denoising algorithm based on wave let-transformation was applied to remove the Gaussian noise from th e image (v Wegner et al., 2006). The mean and variance ( 2 ) of the normalized image were then determined. Ca 2+ sparks were identified based on local fluorescence intensity greater than mean + 2.5 2 The amplitude of sparks selected will be reported as the peak value of F/F 0 (fluorescence intensity normalized by background fluorescence). The duration and width of Ca 2+ sparks were quantified as the full-duration halfmaximum (FDHM) and full-width half -maximum (FWHM) respectively (Remillard et al., 2002). FDHM is th e duration (ms) in which the fluorescence intensity of a Ca 2+ sparks is greater than half of its peak fluorescence intensity. FWHM is the width (m) in which the

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61 fluorescence intensity of a Ca 2+ sparks is greater than half of its peak fluorescence intensity. The spark frequency of each cell was defined as the number of sparks detected per second in a scan line of 25 m. Statistical significance (p < 0.05) was assessed by paired or unpaired Student's t -test whenever applicable.

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62 CHAPTER THREE THE EFFECT OF INTEGRIN BINDING PEPTIDES ON CULTURED RENAL VSMCs INTRODUCTION Microelectrode arrays provide a simple interface for monitoring impedance characteristics of popu lations of cultured cells over extended periods. Renal vascular smooth muscle cells (VSMCs) are allowed to attach to the gold electr odes and, since the cell membranes exhibit dielectric properties the changes in the effective electrode impedance can be measured usin g this technique. Impedance measurements using alternate current (AC) techniques are based on the fact that intact living cells are ex cellent electrical insulators at low signal frequencies, hence a noni nvasive assay of morphological properties of cultured cells. Giaever and Keese developed the ECIS system to quantify the cell behavi or in tissue culture (Giaever and Keese, 1984; Giaever and Keese, 1991). Using this system, one can quantify changes in the capacitance of the cell membrane, cellsubstrate separation, and cell-cell separation with exquisite sensitivity and in a non-invasive manner (Giaever and Keese, 1991; Lo, 1998; Lo et al., 1998; Lo et al., 1995). ECIS was employed to contemplate the

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63 effects of integrin binding peptid es on cultured VSMCs from rat kidneys. This technique is useful in observing contractility in cultured VSMCs as it is otherwise difficult to record this aspect since the cells become very thin and spread out in cu lture. Methods used to test this hypothesis are described in the method section under 2a and 2f. RESULTS Rates of cell attachment and spreading are known to be dependent on the type of ECM protei n coated on the substratum. In order to examine the preference of the cultured VSMCs to various ECM proteins we coated ECIS electrodes with fibronectin, vitronectin, laminin, bovine serum albumin (BSA), collagen, and uncoated electrodes were used as the nega tive control where the adsorbed protein layer was simply a collection of those proteins found in the FBS used to supplement the growth me dium. Figure 16 shows a typical result obtained using the ECIS attachment assay and data are presented as the measured resistance normalized to its value at the start of each run. Here the cells were inoculated on the electrodes at time zero and the impedances were monitored for 20 hours. As shown in figure 16, there was a large diffe rence in the response of VSMCs to the different protein layers. Followi ng the inoculation, the resistance

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64 of the fibronectin-coated electrode increased more rapidly with time than those of the other fi ve electrodes. This initial quick rise in the curve was because the suspended VS MCs continuously dropped to the bottom of the ECIS wells, attached to the electrode, and effectively blocked the area available for current flow. Approximately by the end of 2 hours the resistance peaked and started to fall as the attached VSMCs started to develop focal adhe sions, spread, push each other, and form a monolayer. By 6 hour s the cells attached, spread, and reached equilibrium. Smaller change s in the cell-electrode interaction due to cell motions caused the impedance to fluctuate with time. Resistance changes of collagen and vitronectin electrodes lagged somewhat behind the fibronectin electrode, and there was hardly any increase in the resistance for lami nin, BSA, or uncoated electrode. These results agree with observation s from other laboratories that smooth muscle cells prefer fibronectin, vitronectin, and type I collagen for attachment, spreading and the formation of stable focal adhesions (Hedin et al., 1989; Naito et al., 1992; Yamamoto and Yamamoto, 1994).

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Figure 16. Normalized resistan ce showing the attachment and spreading of renal VSMCs on different ECM proteins 65 04812162Time, hrs 0 0.5 1.0 1.5 2.0 2.5 3.0Normalized resistance The different coatings used are as fibronectin (black), collagen I (red), vitronectin (blue), laminin (green) and, bovine serum albumin (pink), or uncoated electrodes (yellow).

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66 An intriguing feature of this ECIS system is the frequencydependent measured impedance, whic h is always associated with cellcell and cell-substrate interactions. In the next experiment the impedances of the electrode wells were measured under different frequencies. It records one electrod e at a time and requires about 2.5 minutes for each electrode. Figu res 17 A and B show the measured resistance and capacitance of an electrode without and with VSMCs respectively. As evidently seen fr om figure 17, both resistance and capacitance values of the cell-free electrode drop with increasing frequency; however, they differ as cells attach and spread on the electrodes. At high frequency, wh en the cells cover up some of the electrode area, the resistance incr eases and the capacitance decreases because the cells impair the moveme nt of ions, resulting in lesser current flow from the electrode. At low frequency, both resistance and capacitance do not change much ev en when there are cells on the electrode because the impedance from the electrode-electrolyte interface dominates the measured im pedance. As the resistance and capacitance are frequency dependent, the changes in the interactions between the cell membrane and the electrode at different frequencies can be monitored using this method.

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Figure 17. Changes in impedanc e with and without renal VSMCs Figure 17 (A). Frequency scan showin g the changes in resistance of electrodes with (red) and without (blue) VSMCs 1.02.03.04.05.0Log frequency, Hz 3.0 3.5 4.0 4.5 5.0 5.5Log r esistance, ohm 67

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Figure 17 (B). Frequency scan showing the changes in capacitance of electrodes with (red) and without (blue) VSMCs 1.02.03.04.05.0Log frequency, Hz 0.0 0.2 0.4 0.6 0.8Log capacitance, nF 68

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69 Figures 18 shows the plot of normalized resistance (A) and capacitance (B) as a function of log 10 (frequency) for a typical electrode with a confluent layer of VSMCs. The curves were obtained by dividing the measured values for cell covered electrodes by its corresponding value for the cell-fr ee electrodes. This measurement was taken at different frequencies. The junctional resistance between the cells influences the height of the peak. Also greater the cell substrate separation the more is the curve moved towards the right side of the plot. From this we ca n observe that approximately 10,000 Hz is the optimal frequency to measure the impedance of these electrodes when monitoring the VSM Cs for interactions with various compounds or ligands.

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Figure 18. Impedance ratio of a typical electrode with and without VSMCs Figure 18 (A). Ratio between the resi stance of a typical electrode with and without cells 1.02.03.04.05.0Log frequency, Hz 1.0 1.5 2.0 2.5 3.0 3.5No r malized r esistance 70

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Figure 18 (B). Ratio between the capacitance of a typical electrode with and without cells 1.02.03.04.05.0Log frequency, Hz 0.2 0.4 0.6 0.8 1.0 1.2Normalized capacitance 71

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72 The figures 19 A and B represent the changes in resistance and capacitance as a function of log 10 (frequency) when cells are grown over multiple electrodes. These gr aphs demonstrate that both the resistance and the capacitance valu es of the cell-free electrode drop with increasing frequency. The high er the frequency the lesser is the current flow due to the VSMCs im pairing the movement of ions. Moreover it also illustrates that th e optimal resistance of 10,000 Hz is a population behavior applicable to multiple electrode configurations. Both the resistance and capacitanc e drop after this optimal value.

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Figure 19. Impedance variations measured using ECIS in the presence and absence of VSMCs Figure (A). Logarithm of resistance of the electrodes with (red) and without (blue) cultured renal VSMCs 73 1.02.03.04.05.0Log frequency, Hz 3.0 3.5 4.0 4.5 5.0 5.5Log r esistance, ohm

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Figure 19 (B). Logarithm of capacita nce of the electrodes with (red) and without (blue) cultured renal VSMCs 1.02.03.04.05.0Log frequency, Hz 0.0 0.2 0.4 0.6 0.8Log capacitance, nF 74

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75 Previous studies showed that RGD peptide caused an increase in intracellular Ca 2+ fluorescence intensity in cultured renal VSMCs (Chan et al., 2001) and caused constriction in intact afferent arterioles (Yip and Marsh, 1997). Here ECIS was used to examine the overall cellular response resulting from integrin lig ation using soluble RGD containing peptide. Figure 20 shows typical tr acings of ECIS attachment data obtained from VSMC monolayers. In the figure the solid lines correspond to treatment with 0. 5 mM RGD while the dotted lines correspond to treatment with 0.05 mM RGE. The different color lines indicate different coatings on the el ectrode as explained in the legend. The black arrow indicates the time po int at which the different ligands were added into the well. This is marked by a transient but steep increase in resistance in response to a small but sudden decrease in temperature due to the addition of RGD or RGE containing peptide. Regardless of different ECM coatings the addition of RGD lowered the resistance after the initial spike, in dicating that lesser electrode area was covered by the VSMCs. A visual examination of the electrodes confirms that cells have a tendency to contract and round up when treated with RGD. While addition of RGE peptide did not inhibit cell spreading and it acted as the inactive control.

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Figure 20. The effect of RG D and RGE on VSMCs grown on different ECM coatings 012345Time, hr 0.5 0.7 0.9 1.1Normalized resistance The different coatings used were fi bronectin (black), collagen (red), bovine serum albumin (blue), vitron ectin (green), laminin (pink), or uncoated electrodes (yellow). The same colors signify the same coatings on different electrodes. Dotted lines indicate addition of RGE (0.05 mM) as control, and solid line s indicate addition of RGD (0.5 mM) at the point shown by the arrow. 76

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77 Previously dose dependent curve s for constriction in perfused afferent arterioles induced by RGD peptide showed a significant degree of constriction over a range of concentrations varying from 10 -7 to 10 -3 M (Yip and Marsh, 1997). Now the effe ct of varying RGD concentration on the impedance of VSMC-covered electrode was monitored by ECIS for 5 hours. In these experiments, VSMCs were cultured on collagen I (0.2 mg/mL) coated electrodes wh ich became a confluent monolayer 20 hours after inoculation. Comple te media was used as negative control. RGE containing peptide served as the inactive control. The cells treated with the highest concen tration of RGD, 1 mM, showed an initial spike following ligand additi on. This was followed by a drastic drop in resistance for a few hours im plying that most of the cells came loose at the end of the measuremen t. With 0.1 mM RGD the initial spike in resistance was similar but followed by a slower decline. A dose dependent relationship was generally observed, with negligible effects for the 0.01 mM RGD containing peptide. Images in figure 22 gives an idea of the electrode co verage when the VSMCs are treated with 0.5 mM RGD or RGE (top and bo ttom panels respectively). It can be seen that fewer VSMCs remain attached when treated with RGDthan when treated with RGEcontaining peptide.

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Figure 21. Time course of integrin effects at various RGDpeptide concentrations 012345Time, hr 0.6 0.8 1.0 1.2No r malized r esistance Complete media (red), RGD having a final concentration of 0.01 nM (brown), 0.1 mM (dark blue), 1 mM (pale blue) and 0.1 mM RGE (green). 78

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Figure 22. Images of VSMCs on the ECIS electrode The gold electrode is seen as th e circular area. Scale bar = 100 m. VSMCs after addition of 0.5 mM RGD (top panel) and 0.5 mM RGE (bottom panel) 79

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80 We also conducted time course experiments to study the effects of RGD with regard to the attachment time of the VSMCs to the substratum coated with collagen I. Figures 23 A and B show that the measured resistance did not peak when 0.5 mM RGD was added to the medium simultaneously as the cells were inoculated into the wells which indicates that there was ve ry little cell attachment to the electrodes. The red line shows the changes in impedance when RGD was added 1 hour after seeding the cells. Here the peak in resistance was observed but was less significant than that seen when RGD peptide was added 3 hours (green line) after seeding. The dotted line indicates the trend in the impedance changes on addition of cell suspension in the presen ce of 0.5 mM RGE (control peptide) at time point 0. When RGD was added over 3 hours after seeding the cells (data not shown), normalized resistance curves were similar to the result in control condition.

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Figure 23. Time course response for RGD peptide Figure 23 (A). Variations in resi stance during the time course experiment 012345Time, hr 0.6 1.0 1.4 1.8No r malized r esistance Solid lines represent addition of 0.5 mM RGD peptide at 0 (blue), 1 (red) and 3 hours (green) and broken line indicates addition of 0.5 mM RGE peptide (black) at time zero. 81

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Figure 23 (B). Variations in capacitance during the time course experiment 82 01234Time, hr 5 0.5 0.6 0.7 0.8 0.9 1.0 1.1No r malized capacitance Solid lines represent addition of 0.5 mM RGD peptide at 0 (blue), 1 (red) and 3 hours (green) and broken line indicates addition of 0.5 mM RGE peptide (black) at time zero.

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83 Fibronectin (FN) is known to predominantly bind to 5 1 integrin heterodimer. Fibronectin, a common ECM protein, was used to coat paramagnetic beads. These beads were in turn used to transduce force into VSMCs in the presence of a magnet ic field. So we tested the effect of the beads themselves falling on the cells and attaching to them. After the initial spike with the addition of the beads, FN-coated microbeads showed a trend to cause contraction of VSMCs (figure 24 (A) and (B)). Addition of uncoated beads did not show any appreciable change in impedance and served as a control for non-specific binding.

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Figure 24. Changes in impedanc e in the presen ce of coated beads Figure 24 (A). Effect after addition of FNcoated (pink) and uncoated (blue) microbeads to renal VSMCs 012345Time, hr 0.9 1.0 1.1 1.2No r malized r esistance 84

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Figure 24 (B). Image of an elec trode covered with VSMC to which paramagnetic microbeads have been added (Scale bar = 100 m) 85

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86 Finally, the effect of this integrin specific 1 antibody was studied using ECIS. Since VSMCs do not express 2 integrins, antibody to that integrin was used as the control. From figure 25 we gather that addition of 1 integrin antibody caused an immediate drop in the resistance unlike the addition of 2 integrin antibody or RGD containing peptide which was shown previous ly. This immediate decline in resistance can be interpreted as the potent contractile capability of the antibody by releasing a significant amount of Ca 2+ which is discussed in chapter 4. By the end of one hour the resistance sl owly creeps back to the pretreatment levels only to start their gradual irreversible decline soon after that. This is perhaps due to the cells trying to resist and overcome the effect of antibody but due to its more potent and irreversible nature the VSMCs finally contract and have a tendency to come loose easily. 2 integrin antibody does not show any appreciable change in resistance and allows the cells to spread on the surface of the electrode.

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Figure 25. Effect of integrin sp ecific antibodies on cultured VSMCs as measured by ECIS 0.0 0.5 1.0 1.5Time, hr 0.8 0.9 1.0 1.1No r malized r esistance 1 integrin antibody is shown in black; 2 antibody (red) was used as control. 87

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88 DISCUSSION We studied the preferential adhe rence and spreading of the cells on different ECM coatings. Although in vivo VSMCs are found in a milieu of ECM proteins, they seem to prefer collagen and fibronectin over other coatings in vitro Generally studies use collagen or fibronectin to coat the electrodes to get a quick and reliable cellular attachment (Salas et al., 1987; Wegener et al., 2002). We showed that within two hours the attachment of cells had peaked and the cells started to spread. We assumed that the cells have a rectangular shape. We tried to identify the individual capacitance values of basal and apical membranes. Generally, in a simple rectangular model the current is assumed to flow intercellularly and transcellularly without considering the different current distribution th rough basal and apical membranes. In the newer more comprehensive mode l the electrical potential inside the cells, V i is assumed to be a constant and that the transcellular current flows in through the basa l membrane and out through the apical membrane. From our study we found that the capacitance of the apical membrane is slightly hi gher than the basolateral membrane. Since the apical cell surface of an adherent cell usually has more folds than the basal membrane and henc e displays a higher capacitance

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89 value, this new consideration is im portant to make model calculation fit the experimental data, particularly in the high frequency region. The impedance of the electrodes is measured by ECIS under varying frequencies. The resistance and capacitance decreases with increase in frequency when cells cove r part of the electrode. This is because at higher frequencies there is less time for the current to come out of the electrode and he nce less current passes between the cells. But at lower frequencies there is sufficient time for the current to pass between the cells resulting in al most no change in the resistance and capacitance when cells cover the electrodes. We showed resistance and capacitance as a function of log 10 (frequency) obtained from electrodes with and without a monolayer of VSMCs. Different cell types exhibit different peak impedanc e as a function of frequency. The impedance of MDCK cells peak at 700 Hz while fibroblasts peak at 4000 Hz (Lo et al., 1995). This is be cause the shift in the peak is governed by both junctional resistance and cell-substrate separation. Frequencies close to 10,000 Hz were determined to be the optimal range for measuring the changes in impedance in the renal VSMCs. In culture, VSMCs respond to inte grin ligation by triggering an increase in [Ca 2+ ] i when treated with exogenous RGD peptide (Chan et

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90 al., 2001). RGD peptide caused constric tion in intact arterioles (Yip and Marsh, 1997). But it is a challenge to measure contractility of cultured cells as they are spread ou t in thin monolayer. So we used ECIS to study the response of cult ured VSMCs in terms of changes in cell-cell and cell-substrate interact ions. Our results illustrate that irrespective of the ECM protein co ating used on the electrode, the resistance drops drastically with the addition of RGD peptide. A drop in resistance indicates that lesser area on the electrodes is covered by the cells, which could be due to co ntraction and rounding up of the cells. Dose dependent curves for cons triction in perfused afferent arterioles induced by RGD peptid e showed a significant degree of constriction over a range of concentrations varying from 10 -7 to 10 -3 M (Yip and Marsh, 1997). From ECIS experiments we found that the VSMCs responded effectively to 0. 1 mM RGD but 1 mM RGD appeared to be at the upper limit of concentrat ion as most cells we re lifting off of the electrodes. With the help of time course experiments we showed that the resistance of the electrod es during attachment of cells did not peak in the presence of 0.5 mM RGD containing peptide. This signifies that this peptide hinders normal formation of focal adhesion complexes by ligating integrins. On the contrary 0.5 mM RGE peptide served as the

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91 control and did not markedly affect th e attachment of VSMCs. It is also noted that the effect of RGD peptide is lesser when added after the initial cell spreading. This peptide slightly hinders the formation of a monolayer of cells when added 1 ho ur after the inoculation of cells. But RGD peptide does not show any e ffect in inhibiting cell attachment when treated 3 hours later. From th ese data we can speculate that the soluble ligand (RGD peptide) bind s to integrins all over the VSMCs when they are in suspension. But, on ce the cells have established focal adhesion, RGD peptide binds more re adily to the free integrins than compete with the integrins that have formed focal adhesion complex. This is seen by the lesser drop in resistance in the firmly attached VSMCs. The response to RGE peptide at 0 hour is similar to the addition of RGD after 3 hours of attachment signifying that both these ligands at the given time frame do not hinder the formation of focal adhesion and hence the resistance of the electrodes is higher. The response to the addition of FN-coated and uncoated microbeads was also monitored. Th e uncoated beads showed a pattern similar to control peptides which is attributed to the non-specific binding to the VSMCs. FN predominantly binds to 5 1 integrin heterodimer. ECIS study showed th at addition of FN-coated beads caused a drop in resistance indicati ng contraction with a more specific

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92 integrin binding. The effect of integrin specific 1 antibody was also tested. Antibodies to integrins have generally been used as functional blockers. In our study we found that ligation of integrins using antibody also caused a drop in resi stance attributed to contraction of cell. Besides this, the immediate response to 1 antibody is a sharp decline in the resistance which could indicate that the cells lost contact due to the replacement of foca l adhesion complexes with the irreversible binding of the anti bodies. The cells may still try to establish contact and resist the co ntraction which by the third hour seemed to be in vain.

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93 CHAPTER FOUR THE ROLE OF INTEGRINS IN MECHANOTRANSDUCTION IN FRESHLY ISOLATED RENAL VSMCs INTRODUCTION The primary mechanism for renal blood flow autoregulation is the regulation of afferent arte riolar vascular resistance by tubuloglomerular feedback and myog enic response (Holstein-Rathlou and Marsh, 1994). The dynamics of the myogenic response in afferent arteriole is modulated by tubuloglom erular feedback in terms of both the amplitude and frequency (Cho n et al., 2005; Marsh et al., 2005a; Marsh et al., 2005b; Yip et al ., 1993). The sensor for the tubuloglomerular feedback is the macula densa in the early distal tubule, which detects the flow-dependent changes in luminal [NaCl] and accordingly adjusts th e afferent arteriolar resistance (Briggs and Schnermann, 1987). However, the mechanotransduction mechanisms in the myogenic response are not well defined (Davis and Hill, 1999). Integrins, the heterodimeric transmembrane proteins composed of and subunits, provide the structural linkage between ECM and CSK, and function as signaling receptors. Studies showed that extracellular mechanical force is transmitted across the plasma membrane via

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94 integrins to initiate intracellular signaling in non-muscle cells (Ingber, 1990; Katsumi et al., 2004; Martinez-L emus et al., 2003; Wang et al., 1993). Synthetic integrin-binding pe ptide GRGDSP (Gly-Arg-Gly-AspSer-Pro), induces vasoconstriction in rat afferent arterioles, which is associated with a pronounced increase of [Ca 2+ ] i in VSMCs (Yip and Marsh, 1997). Longitudinal stretch of urinary bladder smooth muscle cell induces Ca 2+ sparks (coordinated op ening of a cluster of ryanodine receptors), Ca 2+ waves, and inward Cl current (Ji et al., 2002). Interestingly the Ca 2+ mobilization in renal VSMCs induced by integrin-binding peptide is ryanodine -sensitive and is associated with recurrent Ca 2+ waves (Chan et al., 2001). These observations raise the intriguing hypothesis that integrin s on the plasma membrane of renal VSMCs may serve as mechanotransduce rs to regulate vascular tone by generating Ca 2+ sparks and triggering contraction (ZhuGe et al., 1998). The present study was undertake n in an attempt to provide the evidence to substantiate this hypothesis. To investigate whether integrins transduce mechanical force into Ca 2+ sparks, we monitored the occurrence of Ca 2+ sparks when magnetic pulling of fibronectin-coat ed paramagnetic beads (FN-beads) was applied to freshly isolated rena l VSMCs. FN predominantly binds to 5 1 integrin heterodimer in SMCs (Hor witz et al., 1985; Pytela et al.,

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95 1985; Takada et al., 1987). Our result s demonstrate for the first time that integrin-mediated mechanotrans duction is coupled to localized subcellular Ca 2+ release in form of Ca 2+ sparks. RESULTS We coated paramagnetic beads with FN, a common ECM protein that binds to integrin. Figure 26 shows a typical VSMC used for magnetic pulling study, in which there was one FN-bead attached. There was no discernable membrane deformation or cell dislocation when magnetic field was applied to FN-bead. There was only a transient re-orientation of the FN-bead towards the electromagnet. Some of the cells used for imaging had multiple beads attached (up to 5 beads/cell).

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Figure 26. Transmitted light imag e of a freshly isolated renal VSMC with a fibronectin-coated paramagnetic bead attached 96

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97 We first tested whether mech anical force applied through 5 1 integrins triggers Ca 2+ signal cascade in renal VSMCs. Before the magnetic field was applied to the FN-beads, no spontaneous Ca 2+ spark was detected in the baseline. Pulling of FN-beads triggered Ca 2+ sparks, Ca 2+ wave and global Ca 2+ increase in 85% of cells with a variable time delay of 0.5 3 s. Global increase of [Ca 2+ ] i followed the occurrence of multiple Ca 2+ sparks (Figure 27 A). The fluorescence intensity profiles of individual Ca 2+ sparks are shown in Figure 27 B. The time course of increase in Ca 2+ spark frequency (number of sparks in every 512 scan lines) and spatially averaged global [Ca 2+ ] i (average fluorescence intensity of every 512 scan lines) are shown in Figure 29 A and B. We characterized the distribution of the spatiotemporal parameters of Ca 2+ sparks in terms of FDHM (FullDuration Half-Maximum) and FWHM (Full-Width Half-Maximum) as shown in Figure 30 A and B. The me dian of FDHM and FWHM were 24 ms and 1.0 m respectively. Other parameters of the Ca 2+ sparks were tabulated in Table 1.

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Figure 27. Linescan images of Ca 2+ sparks in renal VSMCs Figure 27 (A, B). Ca 2+ sparks triggered by FN -bead pull in renal VSMCs A B 200 ms 10 m 1000 ms 10 m (A) A record of 16 s showing Ca 2+ sparks, Ca 2+ waves, and global increase of [Ca 2+ ] i induced by pulling of FN -bead. (B) High temporal resolution image of two Ca 2+ spark induced by FN-bead in 1 s and their fluorescence profiles. Dotted line indicates the initiation of magnetic pulling. White arrowheads point to examples of Ca 2+ sparks. Images were collected with a Leica TCS SP5 confocal system. 98

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Figure 27 (C, D). Spontaneous Ca 2+ sparks in renal VSMCs C D 200 m s 10 m 1000 m s 10 m (C) A record of 16 s showing spontaneous Ca 2+ sparks. (D) High temporal resolution im age of a spontaneous Ca 2+ spark and its fluorescence profile. Dotted line indi cates the initiation of magnetic pulling. White arrowheads point to examples of Ca 2+ sparks. Images were collected with a Leica TCS SP5 confocal system. 99

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100 Ca 2+ sparks are the consequence of coordinated opening of ryanodine receptors in clusters (Nelson et al., 1995). In renal VSMCs pretreated with 50 M ryanodine for 30 min, pulling of FN-beads triggered Ca 2+ sparks in only 4% of the cells tested (figure 32). Removal of extracellular Ca 2+ (Ca 2+ -free HBSS + 4 mM EGTA in the bathing solution) did not block the occurrence of Ca 2+ sparks induced by FN-beads. These observations indicate that Ca 2+ sparks induced by FN-beads does not depend on the influx of extracellular Ca 2+ but on the gating properties of ryanodine receptors. Next, we tested whether the st ructural linkage of integrincytoskeleton is required for ma gnetic pulling to generate Ca 2+ sparks. Incubation of renal VSMCs with cy tochalasin-D induced fragmentation of actin microfilament (figure 28 A and B). 4% of cytochalasin-D treated cells still responded to ma gnetic pulling by generating Ca 2+ sparks (figure 32). Incubation of renal VSMCs with colchicine induced disruption of microtubule netwo rk (figure 28 C and D). 16% of colchicine-treated cells responded to magnetic pulling by generating Ca 2+ sparks (figure 32). These observa tions are consistent with the notion that structural linkage of in tegrin-cytoskeleton is required for the induction of Ca 2+ sparks. Disruption of internal cytoskeleton inhibited the transmission of extracellular mechanical force into

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101 intracellular organelles (Wang et al., 2001). The force imposed by magnetic pulling was then confined to the plasma membrane, which might still activate other inte grin-independent mechanisms.

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Figure 28. Disruption of cytoskel eton in freshly isolated renal VSMCs Fluorescence of FITC-conjuga ted phalloidin (A, B) and immunofluorescence of -tubulin (C, D). Cells were treated with cytochalasin-D (B) and colchicine (D ) before fixation. The images are the projections of five optical sectio ns with a step size 0.4 m. Scale bar is 10 m. 102

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103 To test whether transmembrane receptors that are not linked to the cytoskeleton can also trigger Ca 2+ sparks, LDL-beads were used instead of FN-beads. Magnetic pulling of LDL-beads triggered Ca 2+ sparks in 14% of cells tested (figure 32). The mean Ca 2+ sparks frequency was significantly less compar ed to that induced by FN-beads (Table 1). There was no global [Ca 2+ ] i increase after the occurrence of Ca 2+ sparks (figure 29 C and D). The dist ribution of the spatiotemporal parameters of Ca 2+ sparks in terms of FullDuration Half-Maximum and Full-Width Half-Maximum were show n in figure 30 C and D. Other parameters of the Ca 2+ sparks were tabulated in Table 1. These data suggest that pulling of non-integrin receptors has minimal effects in triggering Ca 2+ sparks and global [Ca 2+ ] i response when compared to pulling integrins. Uncoated parama gnetic beads were used in the pulling study as a control for nonspecific adhesion between VSMCs and the beads. Pulling of uncoated beads attached on VSMCs did not trigger Ca 2+ sparks.

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Figure 29. Frequency of Ca 2+ sparks and global Ca 2+ response in VSMCs -4048121620Time, s 0.0 0.4 0.8 1.2 1.6Spark frequency, s-1 -4048121620Time, s 0.8 1.0 1.2 1.4 1.6 1.8 2.0F/F0 -404812162Time, s 0 0.0 0.4 0.8 1.2 1.6Spark frequncy, s-1 -4048121620Time, s 0.8 1.0 1.2 1.4 1.6 1.8 2.0F/F0 A B C D Mean occurrence frequency of Ca 2+ sparks (number of Ca 2+ sparks in every 512 scan lines) and spatially averaged Ca 2+ transient (average fluorescence intensity in every 512 scan lines) before and during pulling of (A,B) FN-beads (n=26), and (C,D) LDLbeads (n=10). F, fluorescence; F 0 baseline fluorescence. Pulling was initiated at t=0. Scan lines were collected at 500 Hz. 104

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Figure 30. Frequency distribu tions of the spatiotemporal parameters of Ca 2+ sparks 105 0102030405060708090FDHM, ms 0 5 10 15 20 25Counts 0.0 1.0 2.0 3.0FWHM, m 0 10 20 30 40Counts 010203040506070809FDHM, ms 0 0 5 10 15 20 25Counts 0.0 1.0 2.0 3.0FWHM, m 0 10 20 30 40Counts A C B Dmedian = 24 ms median = 25 ms median = 1.0 m median = 0.9 m Ca 2+ sparks were triggered by pul ling with (A,B) FN-beads (263 sparks, 52 cells) and (C,D) LDL-bead s (24 sparks, 8 cells). Numbers of events are expressed as dura tion (FDHM, Full-Duration HalfMaximum) and spatial spread (F WHM, Full-Width Half-Maximum).

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Figure 31. Frequency distribu tions of the spatiotemporal parameters of spontaneously occurring Ca 2+ sparks A Bmedian = 12 ms median = 0.6 m 010203040506070809FDHM, ms 0 0 10 20 30 40Counts 0.0 1.0 2.0 3.0FWHM, m 0 10 20 30 40Counts Data shown here represents 137 Ca 2+ sparks from 12 cells. Numbers of events are expressed as dura tion (FDHM, Full-Duration HalfMaximum) and spatial spread (F WHM, Full-Width Half-Maximum). 106

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Figure 32. Percentage of renal VSMCs in which Ca 2+ sparks were detected at different experimental conditions 0 20 40 60 80 100FN bead s FN beads + Ryanodine FN beads + Cy to D F N beads + Colchicin e L D L beads Spo nt aneous sp ark sPercentage1/22 10/69 4/25 1/24 84/99 12/117 FN-coated paramagnetic beads (n = 99), FN-coated paramagnetic beads + ryanodine (n = 22), FN-coated paramagnetic beads + cytochalasin-D (n = 24), FN-coated paramagnetic beads + colchicine (n = 25), LDL-coated paramagnetic beads (n = 69), spontaneous Ca 2+ sparks (n=117). No Ca 2+ spark was detected in cells pulled with uncoated paramagnetic beads (n= 27). 107

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108 Table 1. Properties of Ca 2+ sparks induced by pulling with coated paramagnetic beads Spark Frequency (sparks/s) Spark Amplitude (F/F 0 ) FDHM (ms) FWHM (m) Fibronectincoated beads 0.75 0.05 (n = 99) 1.61 0.03 (n = 263) 27.0 0.8 (n = 263) 1.1 0.03 (n = 263) LDL-coated beads 0.12 0.02* (n = 69) 1.61 0.04 (n = 24) 25.9 2.3 (n = 24) 1.1 0.1 (n = 24) Spontaneous sparks** 0.36 0.07 (n = 12) 1.5 0.02 (n = 137) 13.3 0.3 (n = 137) 0.7 0.03 (n = 137) F, peak fluorescence; F 0 baseline fluorescence; FDHM, Full-Duration Half-Maximum; FWHM, Full-Width Half-Maximum. Spark amplitude was measured at the peak of each Ca 2+ spark. Asterisk (*) indicates that the difference is significant when compared to the same parameter of sparks induced FN-beads (p<0.03). All data were collected with a Bio-Rad MCR-1000 confocal system unless specified. ** Data were collected with a Leic a TCS SP5 confocal system for 32 s continuously from each cell. Only cells with spontaneous Ca 2+ sparks were used for calculation the spark frequency.

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109 DISCUSSION Integrins have been implicated as the mechanosensor in the myogenic response to transduce mechan ical force into an intracellular Ca 2+ signal (Davis et al., 2001; Martinez-Lemus et al., 2003; Yip and Marsh, 1997). There is no direct evidence that integrins function as mechanotransducer in renal VSMCs to initiate intracellular Ca 2+ signals. An earlier st udy showed that addition of RGD in cultured VSMCs triggered Ca 2+ release from receptor s (Chan et al., 2001). Our studies using the ECIS technique re vealed that RGD peptide caused contraction in cultured VSMCs which is consistent with the observation in cultured VSMCs where RGD pept ide triggered an increase in intracellular Ca 2+ (Chan et al., 2001). Microbeads coated with RGD peptide have been successfully used in magnetic twisting experiments to transduce mechanical force into endothelial cells (Wang et al., 1993). In studies where the force wa s applied through collagen-coated magnetic beads to fibroblasts, an increase in actin assembly and cytoskeletal stiffening was seen. This was dependent on both [Ca 2+ ]i and tyrosine-phosphorylation (Gloga uer et al., 1997). Thus employing magnetic beads allows us to ma nipulate external force while simultaneously measuring dynamic changes in intracellular calcium.

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110 In the present study, we demonstr ated that integrins transduced external mechanical force into intracellular Ca 2+ sparks by modulating the gating property of ryanod ine receptors. Spontaneous Ca 2+ sparks are usually detected in VSMCs isolated from other vascular beds (Nelson et al., 1995; Pucovsky an d Bolton, 2006; Remillard et al., 2002; Umesh et al., 2006; Zhang et al., 2003). However, spontaneous Ca 2+ sparks were not detected in pre-pull baseline when the Bio-rad confocal system was used. It is most likely because the sampling interval is not long enough plus th e image collection is not continuous (1 s time lapse between each 512 scan lines). We tested this possibility by using a Leica TCS SP5 confocal system to overcome the limitation of the Bio-rad system. By using a continuous sampling period of 32 s, we de tected spontaneous Ca 2+ sparks in 12 out of 117 cells in 7 preparations (figure 27 C and D; figure 31 A and B). The properties of these Ca 2+ sparks were tabulated at Table 1. In the cells that displayed spontaneous Ca 2+ sparks, their frequency is 50% less than that in pulmonary VSMCs (Remillard et al., 2002). Since only 10% of cells displayed spontaneous Ca 2+ sparks in a period of 32 s, this does not abrogate our obse rvations that integrin-mediated mechanical force triggers Ca 2+ sparks.

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111 The force and distance relati onship between the electromagnet and a single paramagnetic bead was established based on Stokes Law. Since the magnetic pulling force is very sensitive to the distance between the magnet and the bead, the bead that is the closest to the magnet exerts most pulling force. Assuming that there are 5 beads attached to a VSMC and all beads are 100 m away from the magnet, the total force exerted is only 0.5 nN (5 X 100 pN). This magnitude of the pulling force is comparable to the force used to study the phenomenon of integrin-mediated cell adhesion (Alenghat et al., 2000; Lammerding et al., 2003; Matthews et al., 2004). The maximal pulling force exerted by the paramagnetic beads (0.5 nN) is an order of magnitude less than that of the in crease of wall tension, which is approximately calculated using the law of Laplace. Therefore, the Ca 2+ transient triggered by pulling of FNbeads is unlikely due to excessive pulling force. Wang and coworkers demonstrated that disruption of microfilaments or microtubules did not completely inhibit the stiffening response when RGD-coated beads we re used in magnetic twisting experiments in endothelial cells. But disrupting all three CSK filaments completely suppressed this response (Wang et al., 1993). It is suggested that molecular connections between integrins, cytoskeleton

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112 filaments, and nuclear scaffolds may conduct mechanical signal transfer throughout the cells, and provide a mechanism for producing integrated changes in cells in resp onse to even local changes in ECM mechanics (Maniotis et al., 1997). Th e observations that cytoskeleton disruption agents, cytochalasin-D and colchicine, attenuated the induction of Ca 2+ sparks by FN-beads are consistent with this hypothesis. Similar observations we re also reported from colonic smooth muscle cells, in which Ca 2+ release from internal Ca 2+ stores induced by mechanical stimulation requires intact actin filament (Young et al., 1997). The observations from LDL-beads substantiated the notion that structural linkage with internal cytoskeleton is required for transmembrane mechanical sign al transduction. Although LDLbeads are commonly used as the co ntrol for non-integrin mediated mechanical stress triggered by magnetic field (Ingber, 1997; Wang et al., 1993; Wang et al., 2001), there is a remote possibility that the different effects of FN-beads and LDL-beads on Ca 2+ sparks are simply due to the difference in mechanical stress (force/unit area) imposed by the beads to the cells. Even though the amount of force imposed by a FN-bead or LDL-bead to a cell ca n be identical (same intensity of magnetic field imposed on the same size of paramagnetic bead), local mechanical stress measured under th e bead can be varied depending on the contact area.

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113 Ca 2+ sparks triggered by FN-bead might be the consequence of enhanced activity of Ca 2+ channels. However, opening of ryanodine receptors is only loosely couple d to the gating of L-type Ca 2+ channels in smooth muscle cells. Opening of L-type Ca 2+ channels is not necessary to trigger Ca 2+ sparks (Collier et al., 2000). Linear stretch of urinary bladder smooth muscle cell triggers Ca 2+ sparks in the absence of extracellular Ca 2+ (Ji et al., 2002). In the present study, Ca 2+ sparks could be triggered by FN-beads in the absence of extracellular Ca 2+ Therefore, these Ca 2+ sparks are most likely mediated by mechanical/chemical signals conducting along the ECMintegrin-cytoskeleton axis, and is not dependent on L-type Ca 2+ channel activity. The time delays in the occurrence of Ca 2+ sparks were probably overestimated becaus e of the time gap between every 512 scan lines. Ca 2+ sparks might induce relaxation or contraction in VSMCs, depending on which types of Ca 2+ -dependent channels are activated on the plasma membrane. Ca 2+ sparks stimulate Ca 2+ -activated K + channels in cerebral VSMCs and lead to membrane hyperpolarization and relaxation (Jaggar et al., 1998; Nelson et al., 1995). Ca 2+ sparks stimulate Ca 2+ -activated Cl channels in pulmonary VSMCs and airway

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114 smooth muscle cells and lead to membrane depolarization and contraction (Remillard et al., 2002; Zhang et al., 2003; ZhuGe et al., 1998). Stretch induced Ca 2+ sparks evokes Ca 2+ -activated Cl currents in mouse urinary bladder myocytes (Ji et al., 2002). There are both Ca 2+ -activated Cl and Ca 2+ -activated K + channels in renal VSMCs (Carmines, 1995; Gebremedhin et al., 1996; Gordienko et al., 1994). When intracellular Ca 2+ was mobilized using en dothelin-1 in renal VSMCs, membrane depolarization and Ca 2+ -activated Cl inward current were observed (Gordien ko et al., 1994). Blocking of Cl channels with DIDS inhibits contra ction of afferent arterioles (Jensen and Skott, 1996), while blocking Ca 2+ -activated K + channels does not exaggerate agonist induced constriction in afferent arterioles (Fallet et al., 2001). Collectively these observation s suggest that the activity of Ca 2+ -activated Cl channels is predominant over Ca 2+ -activated K + channels in determining the contra ctile state of renal VSMCs. Future studies are required to test whether Ca 2+ sparks induce membrane depolarization in renal VSMCs.

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115 CHAPTER FIVE THE EFFECT OF INTEGRIN SPEC IFIC ANTIBODIES ON FRESHLY ISOLATED RENAL VSMCs INTRODUCTION Conventionally antibodies are used to specifically bind to antigens thus inhibiting agonist-antigen binding and preventing downstream signaling. An earlier work suggested that anti5antibody blocked the Ca 2+ current triggered FN-coated beads in SMCs from rat cremaster arterioles (Wu et al., 1998). However, beads coated with 5 antibody caused a marked increase in Ca 2+ current while anti3antibody inhibited the increase. They postulated that v 3 and 5 1 work differently to modulate Ca 2+ currents in cremaster VSMCs. Works by Martinez-Lemus an d co-workers showed that anti5or anti1antibodies inhibited myogenic constriction as seen by the response to pressure increments in rat skeletal muscle arterioles (Martinez-Lemus et al., 2005). Further, they showed that anti3 antibody and RGD also inhibited myogenic constriction. Maybe RGD peptides bound to integrins hind er mechanotransduction via ECMintegrin-CSK axis. On the contrary a study in endothelial cells showed that V integrin antibody induced an increase in [Ca 2+ ] i unlike 5

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116 integrin antibody which did not elicit Ca 2+ response, though all the three integrins participated in the cells binding capability to FN (Schwartz and Denninghoff, 1994). Fu rthermore, we showed that mechanotransduction via integrins using FN-coated beads (ligating to 5 1 integrins) trigger Ca 2+ sparks (Chapter 4). All these data motivated us to determine whet her integrin antibodies to 5 1 integrins trigger Ca 2+ sparks. RESULTS In our research we made the no vel observation that both antiintegrin 5 and anti-integrin 1 antibodies induced recurrent Ca 2+ sparks as observed in pulling wi th FN-beads. This observation challenges the concept of using anti bodies as blockers. In a sampling period of 20 s immediately after the treatment of antibodies, Ca 2+ sparks were detected in > 85% of cells being tested (figure 35). Preincubation with ryanodine inhibited the occurrence of Ca 2+ sparks induced by anti-integrin antibodi es. Figures 33 and 34 show Ca2+ sparks triggered by integrin specific antibodies. While in a similar sampling period, Ca 2+ sparks were detected in < 14% of cells treated with anti-integrin 2 antibodies. The 2 integrins

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117 are not expressed in VSMCs, theref ore they were used as timed control for laser illumination. Local Ca 2+ release could be induced by prolonged laser scanning. For the first time we characterized the sparks triggered by integrin antibo dies in renal VSMCs (figures 36 and 37). The spatial and temporal parameters of Ca 2+ sparks triggered by anti-integrin antibodies are tabula ted in Table 2. The frequency and the amplitude of the sparks triggered by 5 integrin antibody and the amplitude of spark triggered by 1 antibody are significantly different from those triggered by pulling with FN-coated beads.

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Figure 33. Ca 2+ sparks triggered by 5 integrin antibody 118 200 ms 5 m The white arrows indicate Ca 2+ sparks.

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Figure 34. Ca 2+ spark triggered by 1 integrin antibody 200 ms 5 m The white arrows indicate Ca 2+ sparks. 119

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Figure 35. Percentage of renal VSMCs in which Ca 2+ sparks were detected when exposed to different integrin antibodies 0 20 40 60 80 100A n t i -i n t e grin a lph a 5 a nti b od y An t ii n t e g r i n b e t a 1 a ntibo d y An t ii n t e g r i n b e t a 2 a ntibo d yPercentage7/50 26/29 28/33 Anti-integrin 5 antibody (n = 33), anti-integrin 1 antibody (n = 29), anti-integrin 2 antibody (n = 50). Pre-incubation with ryanodine abolished Ca 2+ sparks induced by anti-integrin 5 antibody (n = 11), anti-integrin 1 antibody (n = 13). 120

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Figure 36. Frequency distribu tions of the spatiotemporal parameters of Ca 2+ sparks 121 A C B Dmedian = 22 ms median = 22 ms median = 0.8 m median = 1.0 m 0.0 1.0 2.0 3.0FWHM, m 0 10 20 30 40Counts 0102030405060708090FDHM, ms 0 5 10 15 20 25Counts 010203040506070809FDHM, ms 0 0 5 10 15 20 25Counts 0.0 1.0 2.0 3.0FWHM, m 0 10 20 30 40Counts Ca 2+ sparks were triggered by (A,B) 5 integrin antibody (118 sparks, 23 cells) and (C,D) 1 integrin antibody (78 sparks, 17 cells). Numbers of events are expressed as dura tion (FDHM, Full-Duration HalfMaximum) and spatial spread (F WHM, Full-Width Half-Maximum).

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Figure 37. Frequency distribu tions of the spatiotemporal parameters of Ca 2+ sparks triggered by 2 integrin antibody A Bmedian = 18 ms median = 0.8 m 0.0 1.0 2.0 3.0FWHM, m 0 10 20 30 40Counts 102030405060708090FDHM, ms 0 5 10 15 20 25Counts Data shown here represent 33 sparks from 4 cells. Numbers of events are expressed as duration (FDHM, Full-Duration Half-Maximum) and spatial spread (FWHM, Full-Width Half-Maximum). 122

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123 Table 2. Properties of Ca 2+ sparks induced by treatment with integrin specific antibodies Spark Frequency (sparks/s) Spark Amplitude (F/F 0 ) FDHM (ms) FWHM (m) Anti-integrin 5 Antibody 0.57 0.08* (n = 33) 1.78 0.06* (n = 118) 24.1 1.1 (n = 118) 0.9 0.1 (n = 118) Anti-integrin 1 Antibody 0.70 0.08 (n = 29) 1.77 0.07* (n = 78) 25.1 1.4 (n = 78) 1.0 0.1 (n = 78) F, peak fluorescence; F 0 baseline fluorescence; FDHM, Full-Duration Half-Maximum; FWHM, Full-Width Half-Maximum. Spark amplitude was measured at the peak of each Ca 2+ spark. Asterisk (*) indicates that the difference is significant when compared to the same parameter of sparks induced FN-beads (p<0.03). All data were collected with a Bio-Rad MCR-1000 conf ocal system unless specified.

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124 DISCUSSION The common integrin binding motif, RGD, is found in many ECM proteins including FN. FN is the natural ligand of for 5 1 integrin heterodimer. 5 1 integrin regulates L-type voltage-gated Ca 2+ channels (Cav1.2) activity via phosphorylation of 1C C-terminal residues Ser 1901 and Tyr 2122 (Gui et al., 2006). Currents through L-type voltage-gated Ca 2+ channels are acutely potentiated following 5 1 integrin activation by fibronectin and anti5 1 integrin antibodies in VSMCs of rat cremaster skeletal arterioles (Gui et al., 2006; Wu et al., 1998). It has been shown that Ca 2+ sparks can be induced by membrane depolarization in SMCs via current through L-type Ca 2+ channel (Collier et al., 2000; Morimura et al., 2006). Our studies demonstrate that the mere addition of 5 and 1 integrin specific an tibodies triggered Ca 2+ sparks which were inhibited by ryanodine. The occupancy of 5 and 1 integrins with monoclonal antibodies have been shown to trigger phosphorylation and accumulation of focal adhesion ki nase (Miyamoto et al., 1995). This iterates that ligation of integrins can cause clustering of the receptors thus triggering downstream response. The spatiotemporal characteristics of the sparks obse rved in my study triggered by integrin antibodies are comparable to those triggered by pulling with

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125 FN-coated beads. Not only is this a novel finding but this also puts forth the idea that certain integrin s on ligation can cause changes in the conformational coupling whic h may lead to downstream Ca 2+ response. This fits the notion that integrins are constantly changing their linkages to the ECM with chan ges blood pressure thus providing continuous signaling mechanism to alter the diameter of the vasculature. These may in turn phosphorylate other proteins including ion channels which can help in the refilling of Ca 2+ stores. Monoclonal antibody to 7 integrin stimulated IP 3 hydrolysis to trigger Ca 2+ release from SR/ER which activates calreticulin to promote Ca 2+ influx and enhance integrin-mediated cell adhesion in rat skeletal myoblasts (Kwon et al., 2000). Observations from my experiments also revealed that anti5 and 1 integrin antibodies do, in fact, ligate integrins to operate as functional agonists to trigger downstream Ca 2+ response.

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126 CHAPTER SIX THE ROLE OF MECHANOTRANSDUCTION VIA INTEGRINS ON CYTOSKELETAL STIFFNESS INTRODUCTION Integrins help the ECM and CSK form a continuous network. Mechanical alterations in the extr acellular environment can modulate the conformation of the cytoskeleton via integrins. Cells always exert force through the cytoskeleton on the ECM and this force varies during different cellular responses includin g migration and contraction. Cells are known to have bidirectional signaling via integrins. Ligand occupancy and clustering can trigger outside-in signaling via integrins (Giancotti and Ruoslahti, 1999), while a change in the conformation of the subunits on the cytoplasmic side can trigger activation of the receptor through inside-out signa ling (Lu et al., 2001; Takagi et al., 2001). The mechanical stress from th e ECM is transmitted to the cells via focal adhesions. This is counter-b alanced by the cell traction forces generated by the cytoskeleton and exerted on the focal adhesions via integrins. Past studies have showed that the stiffness of the ECM structure can influence tumorogenici ty of cells (Ingber et al., 1981). Normal mammary epithelial cells conv erted themselves to a malignant

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127 phenotype when cultured on stiff ECM gels (Paszek et al., 2005). The stiffer the gel the more it resist ed the force exerted by the cell. Increased stiffness may promote integr in clustering and activation of Erk and Rho-mediated contraction. An increase in cellular tension promotes a stiffer ECM thus feeding into a vicious positive feedback cycle (Huang and Ingber, 2005). Now that we have established that mechanical force can be transduced into the VSMCs via integrins, we explored the possibility of cytoskeletal remodeling due to integrinmediated mechanotransduction. In order to test our hypothesis that integrin mediated force triggers cy toskeletal remodeling we used two approaches 1) Traction Force Microscopy, and 2) Atomic Force Microscopy. The former technique allows us to measure the force exerted by the whole cell on its su bstratum. While the AFM works at the nanoscale looking at interact ions within a very small area. Traction force microscopy is a powerful technique to study the interaction between cultured VSMCs /cells and their environment. A unique feature in this technique is the deformable polyacrylamide substrate. Collagen coating is applie d to this substrate to provide the VSMCs a more physiological environm ent. We study the force exerted by the cells on the substrate using this technique. We calculate the changes in the force exerted by the cells by tracking the displacement

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128 of the fluorescent microbeads which are impregnated in this substrate. The deformable substrate is made by controlling the percentage of polyacrylamide. This approach prov ides reproducible control of the flexibility (stiffness) of the subs trate. The transparency and the thickness of the gel perm it the observation of beads and cells through both fluorescence and phase contra st microscopy. Besides, it is relatively easy to characterize the mechanical properties of the substrate as described in the me thods section in Chapter 2 (g) (Pelham and Wang, 1997). The basic principle of this techni que is quantifying the direction and the magnitude of the interactiv e forces between the cell and its substrate in order to correlate it wi th cytoskeletal and focal adhesion dynamics. This technique has been used in previously to study the migration of fibroblasts (Lo et al., 2004; Lo et al., 2000). Different types of cells have been studie d using this technique including fibroblasts, epithelial cells, endothelial cells, macrophages, neutrophils, neurons, smooth muscle cells, and cardiomyocytes. Atomic force microscopy is another modern tool to measure biological samples at a high reso lution using a minimally invasive approach. This technique, invented in the late 1900s by Binnig and co-

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129 workers (Binnig et al., 1986), images the topography of the cells by scanning with a tip mounted to a cant ilever spring. A feedback loop is used to maintain a constant force between the sample and the tip of the probe. The AFM is a type of scanning probe microscope designed to measure the physical and chemical properties of samples, including but not limited to surf ace topography, height, friction and viscoelasticity. The AFM raster scans the probe over a small area of the sample simultaneously measuring the local property. Two kinds of data are obtained from this type of sca nning deflection and height. The former, is obtained by plotting the deflection of the cantilever against its position on the cell. The latter is acquired from the height of the cantilever on the sample. AFM can be used only on cells that form good adhesion and are able to wi thstand the scanning movement of the probe. Since freshly isolated renal VSMCs have a short time frame for survival and can easily come off of the dish, we used cultured renal VSMCs for these experiments. Sun and co-workers characterized the interactions between fibronectin and 5 1 integrins in VSMCs from rat cremaster (skeletal) muscle arteriol es in terms of force curves. The interactions between FN and 5 1 integrin on VSMCs was tested by using coated cantilevers and beads ei ther fused or covalently attached to the AFM probe (Sun et al., 2005).

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130 AFM is also used to measure the mechanical properties of cells with the help of a nano probe which can be used to scan or push down on the cells. The spatial and tempor al alterations in the mechanical properties of cells are a consequence of the complex physiological processes taking place below the cell surface. Studies have looked at mechanical properties of the cells by poking the cells (Petersen et al., 1982), twisting them with magnetic tweezers (Amblard et al., 1996; Bausch et al., 1998) and using micropipettes to hold the cells by aspiration (Evans and Yeung, 1989; Hochmuth and Evans, 1982; Sato et al., 1987). In 1993 Kasas and co-workers (Kasas et al., 1993) studied the elastic properties of l ung cancer cells and paved the way for single cell imaging with AFM. An other lab investigated the surface morphology and mechanical properties of MDCK cells by AFM (Hoh and Schoenenberger, 1994). This techniqu e facilitates measuring single molecular events such as single inte grin molecule adhesion forces in osteoclasts and osteoblasts (Leh enkari and Horton, 1999) while simultaneously providing high-resolut ion structural imaging. Also due to the quality of spatial resolution AFM can be used to measure the adhesion of a single leukocyte to endothelial monolayer (Goligorsky et al., 1993). When AFM is used on live cells it helps to monitor the dynamic changes which manifest as variations in the mechanical properties of the cells. This relatively new technique was used to

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131 explore the VSMCs and investigate the cytoskeletal changes that may be associated with integrin me diated mechanotransduction. The methodology is given in detail in Chapter 2 (h,i). RESULTS Traction force microscopy: In order to calibrate the dr agging force imposed by the electromagnet on the paramagnetic beads, the beads were pulled in dimethylpolysiloxane with a viscosi ty of 100 centistokes. The force experienced by the paramagnetic microbeads was calculated using Stokes Law (Force = 6 R ) as shown previously (Alenghat et al., 2000; Matthews et al., 2004). The magn etic force applied is directly dependent on the distance between the tip of the magnet and the microbead. As expected for the sa me amount of current (0.7 amp), the velocity with which the beads move towards the magnet increases as the distance decreases. Figure 38 shows the trajectory of the magnetic beads as they move towa rds the electromagnet. Figure 39 shows the force distance relation ship between the magnet and the microbeads. Measurement was made from 34 beads at multiple positions from the tip of the magnet The distance from the tip of the electromagnet to the bead was kept between 50 and 200 m in our

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132 magnetic bead pull experiments. Fi gure 40 shows an image of the cell grown on top of the deformable substrate impregnated with fluorescent beads. The paramagnetic microbeads were added on top of the gel after selecting the cell to run the experiment. This image is a combined fluorescence image with the light microscopy image.

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Figure 38. Transmitted light im age of beads moving through dimethylpolysiloxane Magnet Paramagnetic microbead Bead trajectory 133

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Figure 39. Force-distance relationship for the electromagnet and the paramagnetic microbeads 0100200300400500Distance, m 0 100 200 300 400 500Fo r ce, pN 134

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Figure 40. Image of a renal VS MC used in traction force microscopy study This figure depicts a fluorescenc e image of the polyacrylamide substrate impregnated with microbea ds combined with phase contrast image of renal VSMC grown on the surface of th e gel. Scale bar = 100 m. 135

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136 From the force maps generated for VSMCs pulled with FNor LDLcoated beads we can study the amount and the distribution of the forces exerted by these cells. Figures 41 A and 42 A show the distribution of force on the substr atum as exerted by a resting cell. This force pattern is altered after the additions of the microbeads as seen in figures 41 B and 42 B. When the beads are pulled the cell tries to hold onto the substrate more fi rmly and as a consequence the force patterns are realigned and increase in activity (figures 41 C and 42 C). In figure 41 D, we can see that only the cells pulled with the FNbeads have a lingering higher traction fo rce even after the pull was stopped. While in figure 42 D the post pull tr action force goes back to the prepull amount.

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Figure 41. Force maps of a VS MC generated during magnetic pulling of FN-coated beads D C A B Force maps of (A) of (A) a renal VS MC, (B) after addition of FN-coated magnetic beads, (C) during magnetic pulling, and (D) after termination of pull. 137

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Figure 42. Force maps of a VS MC generated during magnetic pulling of LDL-coated beads A B C D Force maps of (A) a renal VSMC, (B) after addition of LDL-coated magnetic beads, (C) during magnetic pulling, and (D) after termination of pull. 138

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139 The next figure (figure 43) show s the stress as calculated by traction force microscopy, in terms of dynes/cm 2 When this stress is normalized for the pre-pull condition, we can see that magnetic pulling increases the stress exerted by the cells. When the pull is stopped the average force per unit area falls bu t it still remains higher than the pre-pull baseline.

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Figure 43. Traction force per un it area of renal VSMC when pulled using FN-coated paramagnetic beads Pre-pull Pull After pull 0.0x1001032.0x1033.0x1034.0x1035.0x1036.0x103St r ess, dyne/cm2 140

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141 LDL-coated beads were used as control for non-integrin mediated mechanical signal transduction. Stress experienced by the cells increased when the LDL-coated beads pulled. But after stopping the pull, the force decreased back to the pre-pull level unlike when pulled using FN-coated beads. Although the value shown is zero, in reality the cell exerts some amount of force on the substratum; its just too low to be estimated by the current algorithm (figure 44).

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Figure 44. Traction force per un it area of renal VSMC when pulled using LDL-coated paramagnetic beads Pre-pull Pull After pull 0.0x1001032.0x1033.0x1034.0x1035.0x1036.0x103St r ess, dyne/cm2 142

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143 Next, we tested the effect of in tegrin antibody on the traction force. The predominant integrin subunit is 1 so we applied antibody to 1 integrin, in solution. Conventiona lly traction force microscopy is used to measure the amount of force exerted by the cells on the flexible substratum when the forc e experienced by the cell due to external factors (magnetic pulling in our case) is varied. But when we add antibody in the solution, we are trying to study the possible loss of traction on the substratum due to the contraction of the VSMCs. The 1 integrin antibody was added at t= 0 (figure 45 A). As seen from the representative graph, the cells lost traction and started contracting when the antibody was added. The 1 integrin predominantly dimerizes with 1 through 6 and V subunit. Figure 45 B shows that the ratio of force along the long axis versus the short axis decreased which indicates that the VSMC is less po larized and shows a tendency to round-up.

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Figure 45. Traction force measur ements in a VSMC treated with 1 integrin antibody Figure 45 (A) Normalized total force output -5051015202530Time, min 0.2 0.4 0.6 0.8 1.0No r malized ave r age f o r ce output o f the cell Arrow indicates the time at which with 1 integrin antibody was added to the chamber. 144

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Figure 45 (B). Ratio of the force ex erted by a VSMC along the long and short axis after treatment with 1 integrin antibody -5051015202530Time, min 0.0 1.0 2.0 3.0 4.0 5.0 6.0Ratio of fo r ce along the long Vs sho r t axis of the VSMC Arrow indicates the time at which with 1 integrin antibody was added to the chamber. 145

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146 The angular distribution of traction force under different experimental conditions can be seen in figures 46 A, B and C. In most cases the magnitude of the tracti on forces exhibited a sinusoidal dependence on the angle, i.e. the peaks and valleys are separated by 90. Experiments using FN-coated beads to transduce mechanical force into the VSMCs showed consiste nt lining up of peaks and valleys. The peaks represent the maximal forc e along the long axis while the valleys denote the maximal force al ong the short axis of the cell. Although the force itself varied du ring the magnetic pull experiments the force along the long and short axes were aligned through the different conditions. The clear dema rcation of the crests and valleys seem to be ablated on treatment with 1 integrin antibody. Also the maximal force decreased after the addition of the antibody. Addition of RGD peptide caused intermittent vari ations in the maximal force along the various axes of the cells. The maximal force seems to oscillate though every cell tested had its own pattern.

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Figure 46. Force patterns of VSMCs subjected to different conditions Figure 46 (A). Force pattern of a VSMC pulled with FN-coated beads 0306090120150Angle 0.000 0.020 0.040 0.060Force, dyne The maximal force exerted by an untreated VSMC is represented by the black line, force changes on ad dition of FN-coated beads (red), during magnetic pulling (blue) and after the pull (yellow) are also shown. 147

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Figure 46 (B). Force pattern of a VSMC treated with 1 integrin antibody 0306090120150Angle 0.000 0.002 0.004 0.006 0.008 0.010Force, dyne The time course distribution of the force pattern on treatment with 1 integrin antibody with black line representing untreated VSMC is shown here. The subsequent line s were taken at 1 minute intervals for 6 minutes. The following readings were taken after 5 minutes each and finally after 10 minutes. 148

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Figure 46 (C). Force pattern of a VSMC treated with RGD containing peptide 0306090120150Angle 0.000 0.005 0.010 0.015 0.020 0.025Force, dyne The time course distribution of the force pattern on treatment with RGD containing peptide with black line representing untreated VSMC is shown here. The subsequent lines were taken at 2 minute intervals for 6 minutes. The following readings were taken after 10 minutes each. 149

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150 Besides these studies, we also used traction force microscopy to characterize some of the features of the cultured renal VSMCs. The cells grown on top of the flexible substratum exerted force on its substratum and was applied to the deconvolution algorithm to calculate the average traction forc e from the displacement of the fluorescent beads (Dembo and Wang, 1999; Lo et al., 2004). Total traction force exerted by the cells is the product of average traction force (dynes/cm 2 ) and the cell area; it was found to be 0.20 0.04 dynes. The other parameters are tabulated in table 3. Table 3. Morphological characte rization of renal VSMCs using traction force microscopy Cell area (cm 2 ) Ratio of force along long axis Vs short axis Average traction force (dynes/cm 2 ) Total traction force (Dynes) Renal VSMC 2.37E-05 1.61E-06 (n=39) 2.51 0.26 (n=50) 8260.64 1749.27 (n=39) 0.20 0.04 (n=39)

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151 AFM: Prior studies done on corona ry venular endothelial cells employed AFM to monitor morphological changes on histamine treatment. The cytoskeleton had visible rearrangement and force curves showed changes in adhesion forces on the surface (Trache et al., 2005). In order to exemplify the surface topology of renal VSMCs cells for the first time, we scanne d them in contac t mode in the presence HBSS. Monolayer of cultur ed VSMCs was resilient to scanning with AFM probes. On scanning we observed that the cells were flat and spread out with a prominent central nucleus. The cytoskeleton beneath the cell membrane was seen as long fibers mostly parallel to the cell boundary figures 47 A, B and C. From these experiments we found the average height of the VSMCs to be 2230.37 96.7 nm. We also estimated the surface roughness to be 44.52 2.3 nm (table 4). Studies measuring Ca 2+ have shown an increase in [Ca 2+ ] i when 1M RGD containing peptide was added to the cultured VSMCs (Chan et al., 2001). Moreover, in Chapter 3, we have shown using ECIS that ligation of integrins using GRGDSP pe ptide causes the cultured cells to contract. This motivated us to exam ine the effects of RGD peptide on the topography of the cell. Addition of 1M RGD in solution significantly

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152 increased the height of the cells. It also significantly increased the surface roughness of the cultured VSMCs. RGE containing peptide served as the control. The height and roughness of the cells were not diffe rent with or without the presence of 1M RGE peptide. Although there was no significant difference in the height of the cell when treated with either peptide, the surface roughness was significantly higher when treated with RGD than with RGE peptide (table 4).

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Figure 47. Images of VSMCs acquired using the AFM (A) Height data, (B) Deflection data in the presence of Hanks BSS. (A) (B) 20 m 20 m 153

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Figure 47 (C). 3-dimensional image of the cell in the presence of Hanks BSS 154

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155 As a next step we studied the force curves generated on RGD treatment. The vertical position of the tip and the deflection of the cantilever are recorded and converted to force-versus-distance curves, known as force curves. Force curves typically show the deflection of the cantilever as the probe is br ought vertically towards and then away from the sample surface us ing the vertical motion of the scanner. Originally force curves were used to analyze the mechanical properties of solid surfaces in te rms of nanometers scale. While using live cells, the probe pushes down on the cell, which exerts some resistive force that is dependant on how fluid the cell is. The fluidity of the cells is in turn dependent on the intracellular milieu which follows the shape and rigidity of the cytosk eleton beneath this membrane. The maximum deflection is a measure of the position on the graph where the approach and the retract curve s are separated maximally. This gives us an idea of how elastic or rigid the cells are. This is tabulated in table 4. Studies have showed that deform ability of the endothelial cells increased after contact with monocy tes which played a role in early stage of atherosclerosis (Kataoka et al., 2002). While treatment of E.coli with an antimicrobial peptide le d to the loss of cell stiffness and caused cells to rupture (da Silva and Teschke, 2003). Experiments

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156 have also been shown the changes in cell stiffness due to aging in rat cardiomyocytes (Lieber et al., 2004; Shroff et al., 1995). Another study has also shown an increase in cell stiffness when contractility is increased in fibroblasts (Nagayama et al., 2004). Furthermore, from the previous ch apters we know that integrin ligation triggers downstream signalin g which can lead to contraction in VSMCs. Ligation of integrins recruits kinases which are linked to integrins via cytoskeleton. Wev e also shown that an intact cytoskeleton is essential to propagate integrin mediated responses. Thus with the help of force curves we studied the possible effect of integrin binding peptide on the cyto skeleton. From such force curves, we found that the cell is softer near the nucleus and stiffer towards the periphery which agrees with studies on atrial myocytes (Shroff et al., 1995). The maximum deflection is the point at which the approach and retract curves are separated maxi mally. Although there is no significant difference between the cells treated with Hanks and RGD or RGE peptide, the cells tended to swell up and contract more when treated with RGD. Figure 48 shows a typical force curve generated from one such experiment.

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Figure 48. Force curves of VS MCs was treated under different experimental conditions 3.02.52.01.51.00.50.0Z, m -0.4 -0.2 0.0 0.2 0.4Deflection, m D C 2.01.51.00.50.0Z, m -0.4 0.0 0.4 0.8Deflection, m A 2.01.51.00.50.0Z, m -0.4 0.0 0.4 0.8 1.2Deflection, m 3.02.52.01.51.00.50.0Z, m -0.6 -0.4 -0.2 0.0 0.2 0.4Deflection, m B Force curves generated from VSMC that was treated with A) RGD, B) RGE, C) and D) Hanks BSS. 157

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158 Table 4. Morphological characte rization of renal VSMCs using AFM Hanks BSS RGD (1 M) RGE (1 M) Cell Height (nm) 2230.37 96.7 (n = 47) 2527.73 120.2 (n=22) 2449.46 749.4 (n=4) Roughness (nm) 44.52 2.3 (n=49) 74.23 5.3 (n=21) 40.64 11.7 ** (n=4) Maximum deflection (microns) 0.31 0.1 (n=17) 0.33 0.1 (n=17) -Maximum deflection (microns) 0.22 0.03 (n=10) -0.35 0.1 (n=10) Single asterisk (*) indicates the difference is significant when the same parameter is compared to that in the presence of HBSS (p<0.03). Double asterisk (**) indicates the difference is significant when the same parameter is compared to that in the presence of RGD (p<0.03).

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159 DISCUSSION From our studies using traction force microscopy we could characterize the amount of force ex erted by VSMCs an d how this force was modulated in integrin mediated mechanical signal transduction. From the force maps we see that the VSMCs exert unequal amount of force on the substratum. This force is redistributed on addition of the microbeads. The cells are trying to compensate for the binding of the microbeads on the cell surface. There was a large increase in traction force as soon as the pull was initiate d. This implies that the external force is transduced into the cells. Thus, confirming that the magnetic bead pull study was an effective meth od to transduce mechanical force into the cells. 5 1 integrin is the natural ligand of fibronectin. An intriguing finding is that even on arresting the pull the traction force continues to remain higher than in th e pre-pull condition. This post pull feature is not seen when LDL-coated beads were used. This may signify some cytoskeletal rearrangement due to the integrin mediated mechanotransduction. The persistenc e of higher force even after the pull is stopped only when FN-coa ted beads denotes some type of memory in signaling when the forc e is transduced via integrins. 1 integrin antibody added in solution to the cells caused the cells to progressively lose the tracti on on its substrate. One can easily

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160 speculate that at the beginning this antibody forms new linkages with integrin on the surface of the cell. This in turn can trigger downstream signaling which can cause the cells to contract. In the process of contraction the cells lose their focal adhesion points and these integrins can later be ligated with the antibody in the bath. Since the antibody is in solution, the number of integrin binding sites is much greater than when ligand coated be ads are used. Besides, antibody can bind to more than one integrin subunit thus amplifying the cellular response and resulting in potent contraction. Ratio of the force along the long axis of the cell versus along the shorter axis is a means to determine the directional distribution of the majority of forces exerted by th e cells and the cell polarization. Addition of 1 integrin antibody decreases the traction force exerted by the cells almost immediately. This implies that the cell contracts almost instantaneously on ligating with the soluble antibody. But by the end of 30 minutes the VSMCs re vive slightly to increase the amount of force asserted. But this is still noticeably smaller than the force experienced at resting conditions. Studying the angular distribu tion of force under different experimental conditions helps us understand the force pattern in the

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161 cells. Lo and coworkers showed that intact fibroblasts the pattern remained sinusoidal and this was di srupted in myosin mutant cell types (Lo et al., 2004). It is interesting to note that in the experiments using FN-coated beads the peaks for each cell aligned under different conditions namely, cell under resting condition, after the addition of microbeads, during pull, after pull. Th is implies that the angle at which the cells are aligned with the elec tromagnet or the position of the beads on the cells do not alter the force experienced by the whole cell. In short, the cells respond globally to local stimuli. This supports Ingbers Tensegrity model (Ingber, 2003a; Ingber, 2003b) which explains that the whole cell is inte rconnected by its cytoskeleton and force experienced in one part of th e cell can be effectively transduced to inflict a global response. Treatment with 1 integrin antibody resulted in alleviation of the force indicating reduction in polariza tion. The lesser the polarization of the cells the more are they contracted. Addition of soluble ligand, RGD, caused fluctuation of the maximal force exhibited by the VSMCs. An earlier study (Chan et al., 2001) showed that RGD peptide triggers Ca 2+ oscillations in cultured VSMCs. Ca 2+ being an essential intracellular ion drives many proc esses within the cells including contraction as they are an integral part of cross bridge cycling event.

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162 External forces are often transmitte d to the intracellular cytoskeletal network which in turn determines th e properties of the membrane and also regulates calcium influx (G hosh and Greenberg, 1995; Sachs, 1988). Thus one can speculate that the Ca 2+ oscillations are reflected as fluctuations in the traction forc e. More studies to simultaneously determine the changes in [Ca 2+ ] i and traction force will shed light on this issue. We also calibrated the magnitude of the force experienced by the individual paramagnetic beads by dragging the beads in a viscous liquid as described in other studies (Alenghat et al., 2004; Lammerding et al., 2003; Matthews et al., 2004). This helps us determine the optimal distance to place the electromagnet in the studies involving the magnetic bead pu ll. It also gives us an idea about the quantity of the magnetic forc e experienced by the individual microbeads. AFM has been used to study the various aspects of cells including shear stress (Ohashi et al., 2002; Sato et al., 2000), cell spreading (Bhadriraju and Hansen, 2002) and differentiation (Collinsworth et al., 2002). From the AFM studies we char acterized the cultured VSMCs and further analyzed the effects of integr in binding peptides. The height of

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163 the cells increased significantly when RGD containing peptide was added. Ligation of integrins has been shown to trigger an increase in [Ca 2+ ] i in cultured VSMCs. Increase in [Ca 2+ ] i can trigger various downstream signals including contraction in smooth muscle cells. It can signify the changes in cytoskeleton which caused balling up of the cells which are otherwise very flat. There is not much change in the cell height between treatment with RGD or RGE. This needs to be looked at with caution as the sample size was low and the standard error was high for RGE treated cells. Another point to be considered is that cell height is measured as a si ngle highest point on the cell and not as an average of a selected area which can overlook subtle changes in cell height. Surface roughness was originally used on metal or other solid surfaces where the variability of the measurement was minimal. Studies using RBCs (Girasole et al., 2007) and osteoblasts quantitatively measured surface roughness of its plasma membrane with the help of AFM. On cells the heterogeneity of the surface membrane is a major contributor for the wider erro r margins. Our experiments revealed a significant increase when the VSMCs were treated with RGD peptide when co mpared to HBSS and RGE peptide treatments. This implies that ligatio n of integrins can cause ruffling or

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164 convolutions in the membrane surfac e. Integrin ligation is known to cause recruitment of kinases especia lly through integrin-linked kinase. Kinases are involved in many downstre am effects directly or indirectly including trafficking of proteins. So such increase in roughness can perhaps be due to insertion of membrane proteins, formation of caveoli or even due to recycling of transmembrane proteins. Although there was a significant difference between RGD and RGE treatments, one cannot neglect the fact that the control sample size was small with a large standard error. The potential of the AFM for stable imaging and acquisition of force curves on living cells for exte nded time periods facilitates the study of dynamic processes due to external stimuli like the effect of integrin binding peptide on the cytoskeleton. Modulation of the cytoskeletal stiffness is reflected in terms of how elastic the cells can be. Cellular elasticity measured us ing this technique showed that a compromised actin cytoskeleton leads to a less elastic cell membrane in HIV infected transgenic glomer ular podocytes (Tandon et al., 2007). AFM was used to show that the rigidi ty of cytoskeletal increased after transduction of external force into the fibroblasts with magnetic beads (Glogauer et al., 1997). They also showed that such pulling experiments triggered an increase in [Ca 2+ ] i (Glogauer et al., 1995). In

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165 our study there are no signific ant differences in the maximum deflection between the different treatment groups. The force curves themselves are generated by coming down on the cell at a point and then retracting back. They are ex tremely sensitive and dependant to the level of nanometers on the ex act point at which the probe makes the contact on the cell surface. Th e force curves will be flatter and sharper with lesser deflection between the two curves if the probe hits a cytoskeletal filament as oppose d to landing between two filaments where the cytoplasm is more compliant At this time, we do not have enough evidence through AFM to co nclude whether or not integrin mediated mechanotransduction mo dulated the cytoskeleton. More potent integrin ligation can be achieved using integrin specific antibodies. Perhaps using a mlange of such antibodies may show a greater degree of observable changes. FIEL (force integration to equal limits) mapping is another improvised method to measure relative elasticity. Hassan and co-workers (E et al., 1998) used FIEL to map relative elasticity in MDCK cells. Th is method is more robust and does not depend on the tip-sample contact.

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166 CHAPTER SEVEN INTEGRIN MEDIATED MYOGENIC RESPONSE IN INTACT AFFERENT ARTERIOLES INTRODUCTION A study by Platts and collegues showed that vasodilation, in vasculature from the cremaster muscle, seen on RGD ligation is linked to K + channels at least in part. This would result in hyperpolarization and prevent elevation of [Ca 2+ ] i (Platts et al., 1998). While another study showed that addition of exogenous RGD peptide triggered Ca 2+ dependent vasoconstriction in rat a fferent arterioles (Yip and Marsh, 1997). In cremaster muscle arterioles in crease in intraluminal pressure results in depolarization which leads to Ca 2+ entry. This is a significant contributor to myogenic tone (Kotecha and Hill, 2005). Changes in pressure can expose cryptic RGD bi nding sites on vasculature. Then the exogenous integrin binding peptides that bind to these cryptic sites trigger pathways to modulate myogenic response. As seen from the previous chapters we have shown that integrins can transduce mechanical force into the VSMCs and ligation of integrins elicits Ca 2+ sparks and VSMC contraction. So the next

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167 logical step would be to determine whether ligation of integrins modulates the myogenic response in intact vasculature. For this experiment we used isolated afferent arterioles from rats and cannulated it (Chapter 2e). RESULTS To test whether integrins might contribute to mechanotransduction in intact rena l VSMCs, we examined the effects of integrin-binding peptide on pre ssure induced myogenic constriction. Pressure induced myogenic constr iction was observed in perfused afferent arterioles as reported previously (Yip and Marsh, 1996). An increase in perfusion pressure fr om 80 mm Hg to 120 mmHg elicited an immediate dilatation, followed by myogenic constriction. The mean normalized inner diameter versus time is shown in figure 49 A. Preincubation of afferent arterioles wi th synthetic integrin-binding peptide GRGDSP (1 mM) for 20 min did not reduce the luminal diameter significantly. The mean luminal diameter before and after 25 min of GRGDSP incubation were 21.9 1.8 m and 22.1 1.8 m (n=10) respectively. It was consistent with the previous report that vasoconstriction induced by GRGDSP lasts only about 45 s (Yip and Marsh, 1997). However, an increase in perfusion pressure from 80

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168 mmHg to 120 mmHg induced only dilatation in the presence of GRGDSP. No myogenic constriction was detected (figure 49 B). Pre-incubating afferent arterioles with the control peptide GRGESP (Gly-Arg-Gly-Glu-Ser-Pro, 1mM), which does not have the RGD binding sequence to interact wi th integrins, had no effect on pressure induced myogenic cons triction (figure 49 C). These observations indicated that integrins function as mechanotransducers not only in freshly isolated VSMCs but also in intact renal VSMCs. Preincubating afferent arterioles with 50 M of ryanodine for 20 min dilated the afferent arterioles s lightly. The mean luminal diameter before and after ryanodine incubation were 21.2 1.6 m and 22.7 2.1 m (n=8, p<0.05, paired t-test) respectively. Pre-incubating afferent arterioles with ryanodine inhibited pressure induced myogenic constriction (figure 49 D), suggesting that pressure induced myogenic constriction is a ryanodine sensitive process.

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Figure 49. Time course of chan ges in lumen diameter of renal afferent arterioles A B C 0100200300Time,s 0.6 0.8 1.0 1.2 1.4Normalized diameter 0100200300Time,s 0.6 0.8 1.0 1.2 1.4Normalized diameter 010020030 0 Time,s 0.6 0.8 1.0 1.2 1.4Normalized diameter 010020030 0 Time,s 0.6 0.8 1.0 1.2 1.4Normalized diameter D 169

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170 Figure 49. Mean normalized time course of chan ges in lumen diameter of afferent arterioles when pressure was in creased from 80 mmHg to 120 mmHg. Pre-incubation of (A) buffer only, (B) 1 mM integrin binding peptide GRGDSP, and (C) 1 mM non-integrin binding peptide GRGESP, and (D) 50 M ryanodine. The mean prepressurized diameters are 21.9 1.8 m (n=10), 22.1 1.8 m (n=10), 22.2 3.1 m (n=5) and 22.7 2.1 m (n=8) respectively. Perfusion pressure was stepped up at time=0. Dotted lines are means SE. indicates that the diameter is significant from the pre-pr essurized baseline (p<0.05).

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171 DISCUSSION Myogenic constriction in the renal artery is associated with membrane depolarization and is attenuated by L-type Ca 2+ channel blockers (Harder et al., 1987). Membrane depolarization was shown to trigger Ca 2+ sparks in SMCs via current through L-type Ca 2+ channel (Collier et al., 2000; Morimura et al., 2006). 5 1 integrin is known to regulate the activity of L-type voltage-gated Ca 2+ channels (Cav1.2) activity by phosphorylating serine and tyrosine residues of 1C (Gui et al., 2006). Currents through L-type voltage-gated Ca 2+ channels are acutely potentiated following 5 1 integrin activation by fibronectin and anti5 1 integrin antibodies (both solu ble and coated on beads) in VSMCs of cremaster skeletal arteri ole (Gui et al., 2006; Wu et al., 1998). By imposing step change in arte rial pressure and monitoring the dynamics of whole kidney blood flow of rats, myogenic response is shown to be completed in the first 7-9 s, and reaches the maximum speed at 2.2 s (Just and Arendsho rst, 2003). The occurrence of Ca 2+ sparks (0.5-3 s delay as obtained from FN-bead pull experiments) seems to fit well with the finding that Ca 2+ sparks precede the myogenic constriction. The time delays of Ca 2+ sparks occurrence were

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172 probably overestimated because of the time gap be tween every 512 scan lines. As discussed before Ca 2+ sparks might induce relaxation or contraction in VSMCs, depending on which types of Ca 2+ -dependent channels are activated on the pl asma membrane. Depolarization induced by Ca 2+ sparks might trigger myog enic constriction via Ca 2+ influx through L-type Ca channels (Harder et al., 1987). Our observations that ryanodine inhibi ted pressure induced myogenic constriction are consistent with this hypothesis. It is also in line with a recent report by Loutzenhiser et al. (Loutzenhiser et al., 2006) that the initial fast constriction in th e myogenic response in afferent arteriole is ryanodine sensitive. Ryanodine only dilated afferent arteriole moderately in the present st udy, which is consistent with the low occurrence rate of spontaneous Ca 2+ sparks in renal VSMCs. Future studies are required to test whether Ca 2+ sparks induce membrane depolarization in renal VSMCs. Pre-incubation of synthetic inte grin binding peptides GRGDSP inhibited pressure induced myogenic co nstriction in afferent arterioles, but the non-integrin binding peptid e GRGESP had no inhibitory effect. The same peptide GRGDSP also inhibits pressure induced

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173 vasoconstriction in cremaster muscle resistance arterioles (MartinezLemus et al., 2005).These observati ons implicate that functional interactions between integrins and ECM are required in the signaling processes of myogenic response. Dilatation due to increase of transmural pressure and the subsequent myogenic constriction are probably associated with differential engagement between ECM and integrins in VSMCs. One possible interpretation is that th e signal transduction process of myogenic response requires form ation of new connections between ECM and integrins. The presence of integrin binding peptide interferes with the formation of such new co nnections, and thus inhibits the myogenic response. Evidences de rived from integrin mediated mechanotransduction in vascular endo thelial cells and NIH3T3 cells are in line with this interpretation. The mechanotransduction in vascular endothelial cells and NIH3T3 cells both require dynamic interactions and formation of new connections be tween specific ECM and integrins (Jalali et al., 2001; Katsumi et al., 2005). Blocking of ECM/integrin interactions with synthetic integrin binding peptide inhibited pressure induced myogenic constriction in renal arterioles, suggesting that integrins might function as mechanotransducers in situ

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174 CHAPTER EIGHT SUMMARY AND CONCLUSIONS The main purpose of my study was to show that integrins can transduce mechanical force in to renal VSMCs. Integrins bridging the cytoskeleton to the ECM transduc e mechanical force and trigger Ca 2+ sparks in renal VSMCs. They are st rategically located to integrate mechanical signals from the extrac ellular environment to result in structural modulations in the intr acellular milieu. RGD is a common integrin binding motif found in many extracellular proteins. To illustrate that integrins can transduce mechanical force in to the VSMCs we custom manufactured a miniaturized electromagnet to pull paramagnetic beads coated with an integrin binding ligand, FN. Previous studies showed that soluble RGD peptide can trigger an increase in [Ca 2+ ] i (Chan et al., 2001). I showed that RGD peptide can trigger contraction even in cultured VSMCs. I also demonstrated that in tact CSK is essential for mechanotransduction via integrins. Based on the tensegrity model (Ingber, 2003a; Ingber, 2003b) we can thus predict that the integrins form a continuous mesh of lin kages with the CSK which helps to

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175 disperse the external force throughout the cell. Furthermore, my experiments illustrated that ligation wi th integrin specific antibodies ( 5 1 ) triggered Ca 2+ sparks which simulated th e response to FNbead pull experiments. The potential for VSMCs to remo del their cytoskeleton can be inferred from traction force micro scopy studies in which mechanical force was transduced via integrins in cultured VSMCs Atomic force microscopy studies revealed the in crease in surface roughness and height of the cultured VSMCs when treated with RGD-containing peptide. Active changes are thus observed as a result of ligation of integrins. RGD peptide was also shown to inhibit pressure-induced myogenic constriction in intact vascu lature from rat afferent arteriole. In summarizing my data we can conclude the following observations: a) RGDcontaining peptide triggers contraction in cultured VSMCs which can be measured using ECIS. b) Integrins transduce mechanical force into VSMCs isolated from renal arterioles. c) Such transduction triggers Ca 2+ sparks which often leads to global Ca 2+ increase.

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176 d) Integrin mediated mechanotransduction requires intact cytoskeleton. e) Ligation of integrins with anti5 or 1 integrin antibodies simulate mechanotransduction by pulling FN-coated beads in mobilizing intracellular Ca 2+ f) Ligation of integrins using antibodies trigger Ca 2+ sparks which are inhibitable by ryanodine. g) Only the integrin mediated mechanotransduction triggers cytoskeletal remodeling as measured by traction force microscopy. Non-integrin mediat ed mechanotransduction has no effect. h) The height of the VSMCs and their surface roughness were elevated after treatment with integrin binging ligand when monitored by AFM. i) Pre-incubating perfused afferent arterioles with ryanodine or integrin binding peptide inhibite d the pressure-induced myogenic constriction. Integrins have to be duly respected as they may be the first in line in the myogenic response path way. Remodeling of integrins and their connections to the ECM occurs in both health and disease. Thus

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177 integrins are perhaps the stepping stones to elucidate a more complicated mechanism.

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178 CHAPTER NINE PERSPECTIVES The next line of research should follow continuing and correlating the findings on mechanotransduct ion with real time myogenic response events in the intact vasculature. The newer generation confocal systems with longer colle ction duration enable continuous periods of vascular response to be monitored without intermittent data loss. Combining this te chnology with the technique of cannulated arterioles can provide a invaluable t ool for studying my ogenic response in intact system. The question still lingers about how the detected mechanical force is finally converted to ch anges in membrane polarization. Electrophysiological experiments to detect the changes in the membrane potential and to ascertain which ion(s) play a major role in myogenic response will give us more insight in to this phenomenon. The traction force microscopy is a robust technique to study the prospective variations of force ex erted by the VSMCs depending on ligation to integrins. Data from these experiments can be used to

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179 predict the outcome in intact vasculature. Further, the use of atomic force microscopy on cultured VSMCs has a long way to go with regard to maximizing the potential of the technology itself. Perhaps investigations to the scale of single ion channels and their difference in conformation based on integr in ligation can support the electrophysiological data.

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204 BIBLIOGRAPHY Berne RM, and Levy MN (1998) Physiology. St.Louis: Mosby Inc. Braga PC, and Ricci D (2004) Atomic Force Microscopy Biomedical Methods and Applications. Totowa: Humana Press. Brenner BM, and Rector FC (1 986) The Kidney: W.B.Saunders Company. Guyton AC, and Coleman TG (1967) Physical Basis of Circulatory Transport: Regulation and Exchange. Philadelphia: W.B.Saunders Company. Guyton AC, and Hall JE (1996) Textbook of Medical Physiology: W.B.Saunders Company. Koeppen BM, and Stanton BA ( 1997) Renal Physiology: Mosby. Orloff J, and Berliner RW (1973) Handbook of Physiology, Section 8 : Renal Physiology. Washington, D.C.: American Physiological Society. Valtin H, and Schafer JA (1995) Renal Function: Little, Brown and Company.

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205 APPENDICES

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206 APPENDIX A Abbreviations Used AFM atomic force microscopy BK Ca or K Ca channels large-conductance Ca 2+ -activated K + channels BSA bovine serum albumin [Ca 2+ ] i intracellular Ca 2+ concentration CICR Ca 2+ induced Ca 2+ release Cl Ca channels Ca 2+ activated chloride channels CSK cytoskeleton DAG diacyl glycerol ECIS electric cell-substrate impedance sensing ECM extracellular matrix ER endoplasmic reticulum F Ca 2+ fluorescence signal of a confocal image with or after Ca 2+ sparks F 0 baseline of fluorescence in a region of the image without Ca 2+ sparks FAK focal adhesion kinase FDHM full-duration at half the maximum intensity of Ca 2+ spark

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207 FN fibronectin FWHM full-width at half the maximum intensity of Ca 2+ spark HBSS Hanks Balanced Saline Solution IDL Interactive Data Language software ILK integrin linked kinase IP3 inositol 1,4, 5 trisphosphate IP3R inositol 1,4,5 trisphosphate receptor K v channels voltage-dependent K + channels LDL low density lipoprotein MLCK myosin light chain kinase PIP 2 phosphatidylinositol 4,5-bisphosphate PKC protein kinase C PLC phospholipase C R a arithmetic average of the absolu te values of the surface height deviations measured from the mean plane using AFM RGD Argine-Glycine-Aspartate (Arg-Gly-Asp) tripeptide RGE Argine-Glycine-Glutamate (Arg-Gly-Glu) tripeptide RyR Ryanodine receptor SERCA sarcoplasmic/endoplasmic reticular calcium ATPase SMC(s) smooth muscle cell(s) SOCs store operated channels SR sarcoplasmic reticulum

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208 STICs spontaneous tran sient inward currents STOCs spontaneous tran sient outward currents Sulfo-SANPAH Sulfosuccinimidyl-6(4-azido-2-nitrophenylamino) hexanoate VGCC voltage-gated Ca 2+ channels VSM vascular smooth muscle VSMC(s) vascular smooth muscle cell(s)

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209 APPENDIX B Source code for IDL program used to analyze Ca 2+ sparks n=1 ; number of sparks visually detected tscan=2. ;ms nscan=2048 ; number of line scanned pixel=0.0757 ; m filename='fn2048' infile1=filename+'.tif'; input file name sfactor =5; smoothing factor (1,3,5 ...) for smoothing the image bg=0; background dark current max1=fltarr(100) xmax=fltarr(100) ymax=fltarr(100) FTHM=fltarr(100) FWHM=fltarr(100) loadct ,3 window ,0,xs=512,ys=512

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210 file1=read_tiff(infile1) a1=float(file1) a1=smooth(a1-bg,sfactor,edge=1) b1=rebin(a1(*,*),512,512) wset ,0 & tvscl ,b1(*,*) ; pick regions that are used as background (no spark) print 'click to define the left margin' cursor x1,y1,3,/device print 'click to define the right margin' cursor x2,y2,3,/device print x1,y1 print x2,y2 an1=a1/rebin ( rebin (b1(*,y2:y1),512,1),512,nscan) an1(*,0)=0.5 & an1(*,nscan-1)=2.0 window 1, xs=nscan, ys=512 wset ,1 & tvscl rotate(an1(*,0:nscan-1),3) ; set output data file format close,2 openw ,2,filename+'.da1'

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211 printf 2, FORMAT='(" spark no", 4x,"frame-no",7x,"x-pos",7x,"ypos",2x,"event time",2x,"baseline-F",7x,"max-F",4x,"changeF",8x,"FDHM",8x,"FWHM")' close, 2 ; estimation of the peak, XY position s, duration of half maximum, fullwidth-half maximum. nss=nscan/256 spark=1 ss=1 sss=1 for ss=1, nss do begin print ss window 3, xs=512, ys=256 wset 3 ;set image window anss=an1(*,(((ss-1)*256)+0):(((ss-1)*256)+255)) tvscl anss while sss eq 1 do begin wset 3 ;set image window print 'click at the space right befo re the spark and right click to finish' cursor ,ix,iy,3,/dev

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212 if !err eq 4 then goto, jump2 if (!err eq 1) or (!err eq 2) then begin repeat cursor,ix,iy,0,/dev until !err eq 0 endif print ,'click to define the left and top margin' cursor ,xx1,yy1,3,/device print ,'click to define the right and bottom margin' cursor ,xx2,yy2,3,/device print ix, iy, xx1, yy1, xx2, yy2 max1=max(anss(xx1:xx2,yy2:yy1))& max1=max1(0) tmax=where(anss eq max(anss(xx1:xx2,yy2:yy1))) xmax=tmax mod 512 & ymax=t max/512 & xmax=xmax(0) & ymax=ymax(0) tv,[255,255,255,255,255,255,255],xmax-3,ymax print max1, xmax, ymax iy_a=iy-20 & if iy_a le 0 then iy_a=0 baseline=anss(xmax-3:xmax+3,iy_a:iy) mean_base=MOMENT(baseline)& mean_base=mean_base(0)

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213 print mean_base FTHMA=where(anss(xmax,yy2:yy1) ge (max1mean_base)/2+mean_base)& sz1=size(FTHMA) FTHM=(sz1(1)+1)*tscan FWHMB=where(anss(xx1:xx2,ymax) ge (max1mean_base)/2+mean_base)& sz2=size(FWHMB) FWHM=(sz2(1)+1)*pixel xmax_a=xmax-50 & if xmax50 le 0 then xmax_a=0 xmax_b=xmax+50 & if xmax+ 50 ge 511 then ymax_b=511 ymax_a=ymax-50 & if ymax50 le 0 then ymax_a=0 ymax_b=ymax+50 & if ymax+ 50 ge 255 then ymax_b=255 window 4, xs=512, ys=512 wset 4 & plot anss(xmax,ymax_a:ymax_b) close,2 openw ,2,filename+'.das', append =1 print FORMAT='(10f12.4)',spark, ss, xmax, ymax, ((((ss1)*256)+ymax)*tscan)/1000., mean_base, max1, max1-mean_base, FTHM, FWHM

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214 printf 2, FORMAT='(10f12.4)',spark, ss, xmax, ymax, ((((ss-1)*256)+ymax)*tscan)/1000., mean_base, max1, max1mean_base, FTHM, FWHM close,2 spark=spark+1 endwhile ss=ss+1 jump2: endfor end

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ABOUT THE AUTHOR Lavanya Balasubramanian grad uates with bachelors and masters degrees in Nutrition and Dietetics (Chennai, India). In 2002, she received M.S. in Biomedical Sc iences from the Eastern Virginia Medical School, VA. After acquirin g her M.S. in Medical Sciences (2003) from the University of South Florida she continued her doctoral education in the Department of Molecular Pharmacology and Physiology. She was awarded th e prestigious American Heart Predoctoral Fellowship (2005-07). She received the Caroline tum Suden/Frances Hellebrandt award for Professional Excellence by American Physiological Society and other recognitions in scientific meetings. As part of her dissert ation she has authored several publications: L. Balasubramanian, A. Ahmed, C.-M. Lo, J.S.K. Sham and K.P. Yip. Integrin-mediated mech anotransduction in renal vascular smooth muscle cells: activation of ca lcium sparks. Am J Physiol Regul Integr Comp Physiol (in publication). L. Balasubramanian, J.S.K. Sham and K.-P. Yip. Calcium signaling in vasopressin induced aq uaporin-2 trafficking. Pflugers Archiv (in publication).