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Intradermal delivery of plasmids encoding angiogenic growth factors by electroporation promotes wound healing and neovascularization
h [electronic resource] /
by Bernadette Ferraro.
[Tampa, Fla] :
b University of South Florida,
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Dissertation (Ph.D.)--University of South Florida, 2009.
Includes bibliographical references.
Text (Electronic dissertation) in PDF format.
ABSTRACT: Gene therapy techniques delivering exogenous angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), are currently being investigated as potential treatments for ischemia resulting from a variety of conditions, such as peripheral artery disease (PAD) and chronic wounds. Despite these intense efforts, a viable clinical option to promote therapeutic neovascularization remains elusive. Electroporation is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues including skin and muscle. This study investigated intradermal injection of plasmids encoding angiogenic growth factors with electroporation as a novel therapeutic approach to increase perfusion in areas of ischemia. Two common animal models of ischemia were employed: a skin flap model, used to study wound healing, and a hindlimb ischemia model, used to investigate potential therapies for PAD.In the skin flap model, delivery of plasmid VEGF with electroporation significantly increased VEGF expression for 5 days after delivery compared to injection of the plasmid alone. While the increase in VEGF expression was short-term, it significantly increased expression of the downstream angiogenic growth factor endothelial nitric oxide synthase, as well as perfusion and healing in the distal area of the skin flap. To facilitate the translation of electroporation to the clinic, a novel electrode configuration was previously designed for cutaneous delivery of plasmids to a large surface area. The design of the Multielectrode Array allows for delivery to a large surface area without the need to increase the applied voltage. Conditions for plasmid delivery with this electrode were optimized and it was then utilized to deliver plasmid FGF-2 (pFGF) to the hindlimb ischemia model. FGF-2 expression, perfusion, and angiogenesis were assessed.FGF-2 expression was significantly higher for 10 days after treatment with pFGF with electroporation compared to injection of pFGF alone. This increase in FGF-2 expression induced a significant increase in perfusion and angiogenesis in the ischemic limb. The research presented here suggests intradermal injection of plasmids encoding angiogenic factors by electroporation is a novel potential therapeutic approach to increase perfusion to areas of ischemia and promote wound healing.
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Co-advisor: Richard Heller, Ph.D.
Co-advisor: Kenneth Ugen, Ph.D.
Vascular endothelial growth factor (VEGF)
Fibroblast growth factor-2 (FGF-2)
x Molecular Medicine
t USF Electronic Theses and Dissertations.
Intradermal Delivery o f Plasmids Enco ding Angiogenic Growth Factors b y Electroporati on Promotes Wound Healing and Neovascularization by Bernadette Ferraro A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Co Major Professor: Richard Heller, Ph.D. Co Major Professor: Kenneth Ugen, Ph.D. Burt Anderson, Ph.D William G. Marshall, Jr., M.D. Larry Solomonson, Ph.D Date of Approval: March 2 0 2009 Keywords: Gene t herapy, vascular endothelial growth factor (VEGF), fibroblast growth f actor 2 (FGF 2), angiogenesis, wound healing peripheral artery disease, i schemia Co pyright 2009, Bernadette Ferraro
A CKNOWLEDGMENTS I am sincerely grateful to Dr. Richard Heller for his expert guidance and financial support of the research presented in this dissertation. This work would not have been possible without his r emarkable patience understanding and steadfast encouragement. I am also exceptionally grateful to my co major professor, Dr. Kenneth U gen, for his guidance, encouragement and support, especially during t he last year of completing this research. I am also sincerely grateful to my committee members for their scientific guidance, especially Dr. William Marshall for his insight and advi ce r egarding skin flaps and limb ischemia I would also like to thank Margi Baldwin, for her invaluable technical guidance in creating the hindlimb ischemia model and Yolmari Cruz who enthusiastically assisted in completing the rodent surgeries for both th e skin flap and hindlimb ischemia models This work could not have been completed without Dr. Mark Jaroszeski who constructed both the Four Plate E lectrod e and the Multi E lectrode A rray I would also like to thank Dr. Michele Glozak for her sage advice through my graduate career and for editing this manuscript. I will be forever grateful for the encouragement of my mother in law, father in law and my close friends while completing this research and I am especially grateful to my husband, Tony, for his unwavering support patience and inspiration
i T ABLE OF C ONTENTS L IST OF F IGURES ................................ ................................ ................................ ................. vi L IST OF A BBREVIATIONS ................................ ................................ ................................ ... viii A BSTRACT ................................ ................................ ................................ .......................... xi G ENERAL INTRODUCTION ................................ ................................ ................................ ..... 1 Neovascularization ................................ ................................ ................................ ... 1 VEGF regulation neovascularization ................................ ........................... 3 FGF regulation of neovascularization ................................ .......................... 4 Methods of therapeutic gene delivery ................................ ................................ ...... 6 In vivo electroporation for gene delivery ................................ ..................... 9 Wound healing ................................ ................................ ................................ ....... 11 Skin flaps ................................ ................................ ................................ ... 14 Therapeutic angiogenesis to promote wound healing ................................ 16 Peripheral artery disease ................................ ................................ ........................ 17
ii Preclinical models of limb ischemia ................................ .......................... 19 Therapeutic angiogenesis for t he treatment of limb ischemia ................... 21 C ENTRAL HYPOTHESIS ................................ ................................ ................................ ........ 24 I NTRADERMAL DELIVERY OF PLASMID VEGF 165 BY ELECTROPORATION P ROMOTES WOUND HEALING ................................ ................................ ................................ ................ 25 Introduction ................................ ................................ ................................ ............ 25 Results ................................ ................................ ................................ .................... 26 In vitro plasmid VEGF expressio n ................................ ........................... 26 VEGF expression kinetics in the gastrocnemius muscle .......................... 27 VEGF expression kine tics in the random skin flap ................................ .. 28 Delivery of p lasmid VEGF with electroporation increases eNOS expression ................................ ................................ ........................ 31 Effect of plasmid VEGF with electroporation on distal skin flap blood flow ................................ ................................ ................... 32 Plasmid VEGF with electroporation promotes distal skin flap healing ..... 34 Discussion ................................ ................................ ................................ .............. 39
iii Future directions ................................ ................................ ................................ .... 4 2 O PTIMIZATION OF DELIV ERY CONDITIONS FOR T HE M ULTIE LECTRODE A RRAY .................. 4 5 Introduction ................................ ................................ ................................ ............ 4 5 Results ................................ ................................ ................................ .................... 4 6 Effect of field strength and pulse width on transgene expression ............. 4 6 Transg ene expression kinetics ................................ ................................ ... 48 Control of tran s ge ne expression by plasmid dose ................................ ...... 5 0 Discussion ................................ ................................ ................................ .............. 5 3 Future directions ................................ ................................ ................................ .... 5 5 I NTRADERMAL DELIVERY OF PLASMID FGF 2 BY ELECTROPORATION INCREASES PERFUSION AND ANGIOGENESIS IN AN ISCHEMIC HINDLIMB ................................ ................. 5 7 Introduction ................................ ................................ ................................ ............ 5 7 Results ................................ ................................ ................................ .................... 58 In vitro plasmid FGF 2 expression ................................ ............................ 58 Optimization of electro poration conditions for plasmid FGF 2 delivery ................................ ................................ ........................... 59
iv FGF 2 expression kinetics ................................ ................................ ......... 6 1 Plasmid FGF 2 with electroporation increases blood flow in the ischemic hindlimb ................................ ................................ ............ 6 1 Plasmid FGF 2 with e lectroporation increases angiogenesis in the ischemic hind limb ................................ ................................ .......... 6 5 Discussion ................................ ................................ ................................ .............. 6 7 Future directions ................................ ................................ ................................ .... 69 M ATERIALS AND M ETHODS ................................ ................................ ................................ 7 3 Plasmids ................................ ................................ ................................ ................. 7 3 Cell culture and transfection ................................ ................................ .................. 7 3 Animals and anesthesia ................................ ................................ .......................... 7 4 The random skin flap model ................................ ................................ .................. 7 4 The hindlimb ischemia model ................................ ................................ ................ 7 5 In vivo electroporation ................................ ................................ ........................... 7 5 Plasmid delivery to muscle ................................ ................................ ........ 7 5 Plasmid delivery to skin ................................ ................................ ............. 7 6
v In vitro luciferase assays ................................ ................................ ........................ 7 7 In vivo luciferase assays ................................ ................................ ......................... 77 Determination of protein expression ................................ ................................ ...... 78 Laser Doppler Perfusion Imaging ................................ ................................ .......... 79 Random skin flap model ................................ ................................ ............ 79 Hindlimb ischemia model ................................ ................................ .......... 79 Assessment of skin flap healing ................................ ................................ ............. 79 Histology ................................ ................................ ................................ ................ 8 0 I mmunohistochemistry ................................ ................................ .......................... 8 0 Statistical Analysis ................................ ................................ ................................ 8 1 R EFERENCES ................................ ................................ ................................ ....................... 8 2 A BOUT THE AUTHOR ................................ ................................ ............................... End P age
vi L IST OF F IGURES Figure 1 The Four Plate Electrode ................................ ................................ .......... 10 Figure 2 The random skin flap model ................................ ................................ ...... 25 Figur e 3. In vitro plasmid VEGF expression ................................ ............................ 27 Figure 4. pVEGFE+ increases VEGF expression in the gastrocnemius muscle ................................ ................................ ................ 28 Figure 5. VEGF expression kinetics in the random skin flap ................................ ... 30 Figure 6. pVEGFE + increases eNOS expression ................................ ...................... 31 Figure 7. pVEGFE+ increases distal skin flap blood flow ................................ ........ 33 Figure 8. p VEGFE+ increases distal skin flap healing ................................ ............. 35 Figure 9. pVEGFE+ decreases inflammation and myonecrosis ............................... 36 Figure 1 0. pVEGFE+ treatment on POD 2 increases healing more than treatment at POD 0 or multiple time points .............................. 38 Figure 1 1. The MultiElectrode A rray ................................ ................................ .......... 45
vii Figure 1 2. Effect of field strength and pulse width on luciferase expression ............. 47 Figure 1 3. Luciferase expression kinetics ................................ ................................ ... 49 Figure 1 4. Control of lucifera se expression by plasmid dose ................................ ..... 51 Figure 1 5. Increasing plasmid injection volume does not affect luciferase expression ................................ ................................ .................. 5 2 Figure 1 6. In vitro pFGF expression ................................ ................................ ........... 59 Figure 1 7. Comparison of FGF 2 expression aft er pFGF delivery with the 4PE or M E A ................................ ................................ ......................... 60 Figure 1 8. FGF 2 expression k inetics ................................ ................................ ......... 62 Figure 19 a p FGF E+ increases hindlimb blood flow ................................ .................... 63 Figure 19b. p FGF E+ increases hindlimb blood flow ................................ .................... 64 Figure 20. p FGF E+ increases angiogenesis in the ischemic hindlimb ................................ ................................ ................................ ..... 66
viii L IST OF A BBREVIATIONS 4PE Four Plate E lectrode ABI Ankle brachial index ANOVA Analysis of variance ATP Adenosine 5' triphosphate CLI Critical limb ischemia DTT Dithiothreitol EC Endothelial cell ECM Extracellular matrix EDTA Ethylene diamine tetraacetic acid EGTA E thylene glycol tetraacetic acid ELISA Enzyme Linked Immunos orbent Assay eNOS Endothelial nitric oxide synthase EP Electroporation ERK E xtracellular signal regulated kinase FGFs Fibroblast growth factors FGF 1 F ibroblast growth factor 1 FGF 2 Fibroblast growth factor 2 FGFR1 FGF receptor 1 FGFR2 FGF receptor 2
ix H&E H ematoxylin and eosin HGF Hepatocyte growth factor HPF High power field (400X magnification) IGF 1 Insulin like growth factor 1 IL 12 Interleukin 12 I/NI In the hindlimb ischemia model, the rati o of blood flow in the ischemic limb to the blood flood in the non ischemic limb IC Intermittent claudation KGF 1 Keratinocyte growth factor 1 KPO 4 Potassium phosphate LDPI Laser Doppler Perfusion Imaging MEA Mu lti Electrode Array MCP 1 Monocyte chemoattractant protein 1 MAPK Mitogen activated protein kinase MEK Mitogen activated protein Erk kinase MgSO 4 Magnesium sulfate MMP Matrix metalloproteinase NaCl Sodium Chloride NO Nitric oxide NP 40 N onyl phenoxylpolyethoxylethanol PAD Peripheral artery disease PBS Phosphate buffered saline PDGF Platelet derived growth factor
x P E No treatment pFGF Plasmid encoding human FGF 2 pFGFE Intradermal injection of pFGF without EP pFGFE+ Intradermal injection of pFGF with EP PI3K Phosphoinositide 3 kinase PKC Protein kinase C pLuc Plasmid encoding the luciferase reporter gene pLucE Intradermal injection of pLuc without EP pLucE+ Intradermal injection of pLuc with EP PO D Post operative day pVAXE+ Intradermal injection of the pVAX plasmid with EP pVEGF Plasmid encoding human VEGF 165 pVEGFE Intradermal injection of pVEGF without EP pVEGFE+ Intradermal injection of pVEGF with EP RSF Random skin flap SD Standard deviation SEM Standard error of the mean SMC Smooth muscle cell TGF uPA Urokinase type plasminogen activator VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor
xi I NTRADERMAL D ELIVERY OF P LASMIDS E NCODING A NGIOGENIC G ROWTH F ACTORS BY E LECTROPORATION P ROMOTES W OUND H EALING AND N EOVASCULARIZATION Bernadette Ferraro ABSTRACT Gene therapy techniques delivering exogenous angioge nic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (F GF 2 ), are currently being investigated as potential treatments for ischemia resulting from a variety of co nditions, such as peripheral artery disease (PAD) and chronic wounds Despite the se intense efforts, a viable clinical option to promote therapeutic neovas cularization remains elusive. Electroporation is a simple in vivo method to deliver normally impermeable molecules, such as plas mid DNA, to a variety of tissues including skin and muscle. Th is study investigated intradermal injection of plasmids encoding an giogenic growth factors with electroporation as a novel therapeutic approach to increase perfusion in areas of ischemia. Two common animal models of ischemia were employed: a skin flap model, used to study wound healing, and a hindlimb ischemia model, u sed to investigate potential therapies for PAD I n the skin flap model, delivery of plasmid VEGF with electroporation significantly increased VEGF expression for 5 days after delivery compared to injection of the plasmid alone While the increase in VEGF expression was short term, it significantly increased expression of the downstream angiogenic growth factor endothelial nitric oxide synthase as well as perfusion and
xii healing in the distal area of the skin flap. To facilitate the translation of electrop oration to the clinic, a novel electrode configuration was previously designed for cutaneous delivery of plasmids to a large su rface area. The design of the Multielectrode Array allows for delivery to a large surface area without the need to increase the applied voltage. C onditions for plasmid delivery w ith this electrode were optimiz ed and it was then utilized to deliver p lasmid FGF 2 (pFGF) to the hindlimb ischemia model. FGF 2 expression, perfusion and angiogenesis were assessed. FGF 2 expression was significantly higher for 10 days after treatment with pFGF with electroporation compared to injection of pFGF alone. This increase in FGF 2 expression induced a significant increase in perfusion and a ngiogenesis in the ischemic limb. T h e research presented here suggests intradermal injection of plasmids encoding angiogenic factors by electroporation is a novel potential therapeutic approach to increase perfusion to areas of ischemia and promote wound healing.
1 G ENERAL I NTRODUCTION Neovascularization Neovascularization plays a major role in both health and disease. The formation of new blood vessels occurs during normal physiological processes such as embryonic development and wound healing, but excessive neovascularization contributes to the pathology of several diseases, notably cancer as well as ocular and inflammatory disorders. Aberrant neovascularization is also associated with many othe r disease s including diabetes mellitus asthma, psoriasis and arthritis. In contrast, in sufficient neovascularization is characteristic of other diseases including ischemic heart and limb diseases, neurodegeneration and osteoporosis  N ew vessel growth occurs by t hree main mechanisms: angiogenesis, a rteriogenesis and vasculogenesis. In embryonic development, vasculogenesis refers to the formation of the capillaries by the mobilization of bone marrow derived endothelial stem cells that will eventually form the vasculature of the adult  In the adult, vasculogenesis is simply a term for angiogenesis characterized by intussusception of progenitor cells in and around the new vascular structures  Angiogenesis refers to the sprouting of new capillary like structures from existing vasculature initiated in response to tissue hypoxi a  In contrast, a rteriogenesis occurs in response to c hronic or acu te occlusion of a major artery, such as the coron ary or
2 femoral artery In arteriogenesis, p reexisting arteriolar connections are recruited to bypass the sit e of occlusion in response to an increase in fluid shear stress  It is current ly not well understood how the stimulus of increased shear stress is transmitted from the endothelial cell (EC) membrane to the nucleus but increase s in fluid shear stress result in the production of the vasodilator nitric oxide (NO) and the activation of ECs and transcription factors including early growth response factor 1 (Egr 1) and activator protein 1 (AP 1)  Once activated, transcription factors increase the expression of chemokines, notably MCP 1 and adhesion molecules such as intracellular adhesion molecule 1 (ICAM 1) [2, 6] The second stage of arteriogenesis is m ediated by an inflammatory response. The presence of MCP 1 and other chemokines on the cell surface of the activated ECs results in a chemotactic gradient that recruits circulating monocytes. Adherence of monocytes to the vascular endothelium is enhanced by the presence of selectins, intracellular adhesion molecules and vascular adhesion molecules. M onocytes or after maturation macrophages, then migrate into the deeper areas of the collateral vessel wall where they induce EC and smooth muscle cell ( SMC ) proliferati on This process results in the release of growth factors and cytokines that promote vascular wall remodeling [3, 5] In s everal months the new collateral artery is almost indistinguishable from a normal artery, except for a slightly higher collagen content between the smooth muscle layers  T he initiation and progression of angiogenesis is mediated through the temporal and spatial production of multiple growth factors and cytokines. Consistent with hypoxia being the driving force of angiogenesis, the transcription of multiple genes that play a role in angiogenesis is induced by hypoxia, primarily through the action of h ypoxia
3 inducible transcription factors HIF Angiogene sis initiates with a hypoxia mediated increase in nitric oxide synthases leading to increased NO production and subsequently vasodilation [8, 9] Following vasodilation, v ascular permeabil ity increases in response to VEGF signal ing allowing for the extravasation of plasma proteins that lay down a provisional scaffold for migrating ECs The destabilization of mature vessels by matrix metalloproteinases ( MMPs ) and other proteases then libera tes ECs that migrate to the distal site of neovascularization The ECs then proliferate to form an immature vessel lumen which is stabilized by a covering of pericytes and vascular SMCs recruited to the new vessels by the chemotactic action of PDGF  VEGF regulation of neovascularization VEGF is a highly specific mitoge n for vascular ECs and exerts its effects mainly during angiogenesis, not arteriogenesis VEGF does not naturally associate with or induce arteriogenesis in vivo because it occurs in a normoxic environment  VEGF 165 (referred to as simply VEGF throughout this manuscript) is the predominant isoform of 5 isoforms (VEGF 121 VEGF 14 5 VEGF 165 VEGF 1 89 and VEGF 206 ) generated by alter n ative splicing of the VEGF A gene These isoforms differ not only in their molecular mass but also in their biological properties [11, 12] VEGF expression levels are increased in response to hypoxia by both transcriptional mechanisms, thr ough the actions of HIF 1 and by stabilization of and increased translation of VEGF mRNA [13 15] Several angiogenic growth factors, including epiderm al growth factor, PDGF and FGFs and inflammatory cytokin e s, such as interleukin 2, also upregulate VEGF mRNA  In vi tro and in vi vo studies have demonstrated that the level of VEGF expression
4 must be finely regulated to induce successful angiogenesis [17 20] High levels of constitutive VEGF expression can result in edema  formation of hemangiomas and unstable immature vessels [17 20, 22] In contrast, low levels or premature cessation of VEGF expression results in regression of the newly formed vess els  The downstream effects of VEGF are mediated by two tyrosine kinase receptors VEGF Receptor 1 (VEGFR1) and VEGF Receptor 2 (VEGFR2). VEGFR1 has a high affinity for VEGF, however its tyrosine kinase activity is approximately 10 fold weaker than VEGFR2 V EGF binding to VEGF R1 weakly induces EC proliferation, simulates the migration of monocytes and induces MMPs and proteases that degrade the ECM resulting in the release of angiogenic growth factors [16, 24] It is generally accepted that VEGFR2 is major mediator of the mitogenic, angiog enic and vessel permeabil ity enhancing effects of VEGF. VEGF binding of VEGFR2 also induces the phosphorylatio n of PI3K to promote EC survival and activates the Raf MEK ERK pathway to increase EC proliferation  VEGF mediated increases in vascular permeability are largely due to an upregulat ion of endothelial nitric oxide synthase (eNOS) that stimulates the production and release of NO [25 27] FGF regulation of neovascularization In comparison to the VEGF family, the FGF family is much more complex containing 23 known isoforms that share approx imately 30 70% homolog y in their amino acid sequences. FGF 1 and FGF 2 differ from other members of the FGF family in that they lack a signal sequence for extracellular transport but are instead exported by an alternative pathway during active angiogenesis  FGF 1 (also known as acidic
5 FGF) FGF 2 (also known as basic FGF) and FGFs 4, 5 and 8 have been shown to play a role in neovascularization but much of the in vivo and in vitro research centers on the actions of FGFs 1 and 2 After FGF 1 and FGF 2 are e xported from ECs they bind FGF re ceptor 1 (FGFR1) or FGF receptor 2 (FGFR2) to promote both angiogenesis and arteriogenesis by increasing vascular EC proliferation, migration and degradation of the ECM Activation of FGFR1 and FGFR2 by the binding of FGF 1 or FGF 2 increase s EC proliferation through activation of MAPK signaling pathway  I n addition to activ ation of the MAPK signaling cascade sustained activation of PKC is required for FGF 2 to exert its full mitogenic effect in ECs  Activation of both the MAPK pathway and PKC are also required for FGF 1 and FGF 2 me diated cell migration  During arteriogenesis, the release of FGF 2 from macrophages enhances both EC and SMC proliferation in the vascular wall  FGF 1 and FGF 2 promote ECM degradation by upregulating the expression of uPA and the expression of the uPA receptor on the surfac e of ECs. uPA converts plasmino gen to the serine protease plasmin. Plasmin d egrades fibrin, as well as other ECM proteins and activates MMPs Expression of the uPA receptor on ECs localizes prot e olytic degradation to leading edge of the front of EC migration While FGF 1 and FGF 2 initially promote ECM degradation, during the later steps of neovascularization they promote the formation of the ECM to stabilize the newly formed vessels  FGF 2 also regulates EC morphogenesis, the reorganization and assembly of EC s into three dimensional tubes by signaling through VEGFR1  There is a considerable degree of cross talk between FGF 2 and VEGF/VEGFRs. Several studies have shown t hat FGF 2 may require the activation of VEGF/VEGFRs to promote
6 angiogenesis and, under some conditions VEGF may also require FGF 2 to exert its angiogenic effects [34, 35] Methods of therapeutic gene deliver y Therapeutic delivery of exogenous angiogenic growth factors and cytokines to increase neovas cularization, or ther apeutic angiogenesis, has been investigated as a potential treatment for ischemia related diseases and to promote wound healing. The main obstacle for wi despread use of gene therapy approaches for the treatment of disease s in general is the lack of a safe and efficient system for delivery of transgenes. The simplest gene transfer approach is d irect injection of naked plasmid DNA This method has had some success, but is limited by low expression levels due to inefficient uptake of the plasmid by cells  Thus, methods to increase the expression of plasmids encoding genes of therapeutic interest for the treatment of a particular disease are actively being explored. In vivo gene therapy approaches can broadly be divided into two categories viral and non viral. Viral approaches utilize a variety of genetically engineered viral vectors for gene delivery while non viral approaches focus o n the use of chemical or physical meth ods to increase the uptake and expression of plasmid DNA Direct injection to the target tissue or intravenous administration of recombinant proteins has also been investigated as a therapeutic approach Recombinant proteins can also be topically applied to promote wound healing. The efficacy of recombinant protein delivery is often hampered by the short half life, and subsequently low bioavailability, of many proteins of therapeutic interest [37 41]
7 V iral vectors have been employed to deliver genes to provide either transient or permanent transgene expression Viral vectors that do not integrate into the host genome, such as adenoviral and vaccinia vectors, confer transient expression th at is relatively short term (1 to 2 weeks) compared to expression levels achieved with viral vectors that do integrate into the host gene. Vectors that do integrate into the host genome, such as retroviral and adeno associated viral vectors, confer high levels of p ermanent transgene expression  Through 2007, v iral vectors have been used in approximately 67 % of gene therapy clinical tria ls worldwide, with aden oviral (25%) and retroviral (23 %) vectors being the most common  The safety of viral vectors has been ques tioned as a result of adverse e vents in human clinical trials In clinical trials a denoviral vectors have caused severe inflammatory responses, formation of antibodies to the adenoviruses, transient fever hepatotoxicity and in rare cases death [44 46] The use of r etroviral vectors was questioned after patients developed leukemia in a clinical trial treating a denosine deami nase deficiency, a fatal autosomal recessive form of severe combined immunodeficiency (SCID)  The preference of retroviral vectors to integrate into the genome at transcriptional start sites and introns is the most probable cause of the a dverse events reported in the first SCID trial  but t he frequency of integration was also likely to be higher than normal due to a number of se condary risk factors associated with SCID  In contrast to earlier reports, a more recent SCID gene therapy clinical trial reported positive results and no adverse ef fects in patients transfused with autologous bone marrow cells transduced with a retroviral vector containing the adenosine deaminase gene  This recent success may lead to renewed interest and more investigations of therapeutic gene deli very using retroviral vectors
8 As mentioned above, non viral approaches for therapeutic gene delivery encompass chemical or physical methods to increase transfection of plasmid DNA. Chemical methods for non viral gene delivery are largely based on the us e of positively charged cat i onic liposome complexes Combining the cationic liposomes with negatively charged plasmid DNA facilitates the uptake of the plasmid by the cell membrane and subsequently plasmid internalization Cationic liposome mediated gene transfer is technical ly simple and can be used for the delivery of a large amount of DNA by direct injection or intravenous administration, but this approach general ly does not confer high levels of transgene expression [51, 52] Interestingly, the transfection of plasmid DNA with cationic liposomes was not enhanced in mouse skin with the addition of in vivo EP (discussed below) indicating other physical methods of delivery may not be efficacious for en hancing the effects of these reagents  A variet y of physical methods have been explored to increase plasmid gene transfer including ultra sound, laser, particle bombardment, magnetic fields, hydrodynamic pressure and in vivo EP The use of ultra sound, focused lasers, particle bombardment (also known as the gene gun) and magnetic fields share the common advantage of being non invasive approaches, but generally do not confer a substantial increase in transgene expression compared to direct injectio n of plasmid DNA alone  Hydrodynamic pressure, rapidly injecting a large volume of plasmid DNA in to the tail vein of rodent models, has been successfully used to tra nsfect hepatocytes throughout the majority of the live r [55, 56] This approach has also been successful for gene delivery to the kidney  W hi le useful for studies in rodent models however, it is currently not directly applicable to humans
9 In vivo electroporation for gene delivery In vivo EP involves the application of a controlled pulsed external electric field that slightly surpasses the capacitance of the cell membrane Application of the electric pulse transient ly increase s cell membrane permeabil ity allowing for an increase in the uptake of plasmid DNA. The first si gnificant demonstrations of successful deliver y of plasmid DNA with in vivo EP were conducted in rat liver  murine brain tumor s  and murine skeletal muscle  Since these initial experiments, in vivo EP has also been used for plasmid delivery to a wide variety of tissues inc luding skin [61 77] brain [ 78 81] kidney  and lung [83 86] EP delivery of plasmids encoding genes with therapeutic potential to the skin could potentially be used to treat a variety of diseases The skin is an attractive target for delivery of plasmid DNA because it is easily accessible, which allows for enhanced control over expression levels I f higher expression levels are needed, the area treated or number of treatments can be increased. Further, p lasmid DNA has been successfully delivered to the ski n with in vivo EP for both systemic and tissue specific expression [61 63, 67, 69] This approach has shown promise in several fields including vaccines [69 73] and wound healing [74 77] A recent publication by Daud et al.  r eported the results of the first human trial utilizing in vivo EP for plasmid delivery The trial investigated intra tumoral delivery of a plasmid encoding interleukin 12 (IL 12) with in vivo EP as a possible therapeutic option for the treatment of metastatic melanoma. The r esults of this Phase I trial indicated the pote ntial treatment to be promising as well as safe, effective, and reproducible Importantly, intra tumoral delivery of plasmid IL 12 by in vivo EP allowed for control of IL 12 expression levels.
10 Figure 1. The Four Plate Electrode The 4PE in the open ( a ) and closed ( b ) positions. P revious work in the Heller laboratory led to the development of a novel electrode configuration, the Four Plate E lectrode (4PE) that was optimiz e d for cutaneous plasmid delivery with EP with minimal tissue damage  As the name suggests, t h e 4PE is composed of 4 metal plates that grip the injection site and a placed in the center of the 4 plates (Figure 1). The use of an electric field to increase plasmid uptake and expression is more efficient when the electric field is applied in more than one direction [88 90] This approach permeabilizes a greater area of the cell surface and a larger number of cells in the target tissue  The design of the 4PE allows for the application of 2 sets of electric pulses with the second set of pulses rotat ing 90 o relative to the first set of pulses, without removing the electrode from the delivery site Prior to the development of the 4PE, elec trically mediated delivery of plasmids to skin was typically facilitated with a 2 plate caliper electrode. One drawback to this approach was the requirement of the electrode plates to be manipulated around the treatment site to administer 2 sets of pulses. Removal and replacement of the caliper electrode from the delivery site resulted in variation in the distance between the electrode plates and thus variation in the applied voltage. The that is placed in the center of the 4 plates of the 4PE ensures a constant distance between the plates and thus constant voltage across the delivery site.
11 W ound Healing Wound healing is frequently defined as the repair or reconstitution of a defect in an organ or tissue, commonly t he skin. Cutaneous wounds are classified as acute, having occurred within the past 3 to 4 weeks, and chronic or delayed healing persist ing 6 to 8 weeks or longer  Surgical intervention to facilitate wound healing can be desc ribed as healing by primary intentio n, delayed primary closure or secondary intention. Primary intention and delayed primary closure are achieved by suturing or stapling to join the wound edges but the latter refers to wounds that are not immediately clo sed due to infection or other complications Secondary intention refers to allowing the wound to close throu gh the normal healing process. The process of wound healing occurs in three general overlapping phases: (1) an inflammatory phase which encompasse s coagulation and the initial migration of inflammatory cells to the wound site (2) a prolifer ative phase (also known as the migration phase) involving the migration and proliferation of keratinocytes, fibroblasts and endothelial cells, leading to re epithelialization and granulation tissue formation and (3) an extended remodeling phase which results in a reduction of both cell content and blood flow in the scar tissue [92, 93] There is a significant temporal overlap in the progression through these phases and the later phases of wound repair are critically dependent on the initial events of the healing process A deficiency in one or more essential growth factors or cytokines may result in asynchronous progression through the phases of wound healing lead ing to the development of a chronic wound. Several systemic conditions or diseases, such as malnutrition, immunodeficiency or diabetes
12 mellitus are frequently the underlying cause of th e cytokine or growth factor deficiency and are often associated with impaired healing [94 96] The inflammatory phase of wound healing begins immediately following injury with the formation of a fibrin clot to maintain tissue homeostasis and prevent infection The fibrin clot consists of platelets embedded in a mesh composed largely of polymerized fibrin and smaller amounts of fibronectin, vitronectin and thrombospondin  In addition to serving as a temporary cover of the wound, the fibrin clot also functions as a scaffold for inflammatory cells Inflamm atory cells are recruited by cytokines and growth factors released by platelet degranulation and by peptides cleaved from bacterial proteins [97, 98] F or the first 48 hours following injury neutrophils are the predominant inflammatory cell type in the woun d. Neutrophils serve several functions in the wound bed including the removal of dead tissue prevent ion of infection and degradation of the ECM to prepare the wound for the later phases of healing Additionally, neutrophils are also a source of proinflammatory cytokines that may provide the earliest signals to activate keratinocytes the major constituent of the epidermis, and fibroblasts which synthesize the ECM  Between 48 to 72 hours post injury monocytes / macrophages replace neutrophils as the predominant inflammatory cell type in the wound area. Circulating monocytes are recruited to the wounded through the chemo tactic effects of growth factors and cytoki nes, primarily MCP 1  M onocytes / macrophages are essent ial for effective wound healing. I f macrophage infiltration is prevented wound healing will be severely impaired  The hypoxic environment of the wound also plays a key role in mediating the early stages of healing. While prolo nged ischemia is deleterious to the healing process
13  hypoxia serves a positive role during the early stages of injury. Hypoxia increases the proliferation of fibroblasts, migration of keratinocyte s for re epithelialization and promotes early angio genesis. Moreover, hypoxia induces the transcription of growth factors, such as VEGF, that are integral to the wound healing process [95, 102] Due to the temporal overlap of the phases of wound healing there is some discrepancy in the time post injury that the pr oliferative phase begins. In general the on se t is thought to occur within 2 to 4 days after injury and persist through days 14 to 21 after injury [92, 93] The proliferative phase is marked by the formation of granulation tissue and ECM, entrance of macrophages into the wound, angiogenesis and completion of re epithelializ ation Approximately 4 days after injury the provisional fib rin clot is gradually replaced by granulation tissue which provides a framework for angiogenesis and cell migration. Granulation tissue is composed of three cell types: ECs macrophages and fibroblasts, which drive the production of the ECM Macrophages in the granulation tissue continue to secrete growth factors and cytokines to induce fibroblast proliferation, migration and ECM deposition Release of angiogenic growth factors, such as VEGF and FGF 2, by macrophages also facilitates EC morp hogenesis and new vessel formation An giogenesis is integral to the wound healing process. If angiogenesis is inhibited, the healing of a wound will be severely impaired [92, 103] Another key event that begins during the proliferative phase is wound contraction. Wound contraction occu rs if a wound was not surgically closed and functi ons to decrease the wound area. This process is mediated by fibroblast s that have acquired a contracti le phenotype and transformed in to myofibroblasts Wound contraction begins approximately 5 days after injury and continues into the onset of the remodeling phase of healing 
14 T he remodeling phase is the longest phase in the wound healing process It begins approximately 14 to 21 days after injury and persists for as long as a year, depending on the size and location of the wound. Currently, remodeling is the least characterized phase of healing and its reg ulation is poorly understood At the onset o f the remodeling phase new blood vessels that are no longer required to supply nutrients to the injured tissue regress and re epithelializ ation is complete d During the remodeling phase the t ype III collagen that was synthesized by fibroblasts in the granulation tissue is slowly degraded by the action of MMPs and replaced by type I collagen. The replacement of type III collagen by the stronger type I collagen coupled with an increase in collagen cross linking slowly improves t he tensile strength of the wound [92, 93, 96] At the beginning of the remodeling phase the strength of the wound is approximately 20 % of intact skin but it continues to increase during the phase to reach a maximum of 80 % at 1 year following injury  Skin flaps Skin flaps are frequently used in plastic and reconstruct ive operations to repair large skin defects and deep wounds formed from injuries, operations ulce rations or congenital defects. In skin flap surgery, a piece of full thickness skin is transferred to the affected area while remaining attached to the donor site by a vascular pedicle. Because skin flaps receive their blood supply from the donor site they are preferable over skin grafts to cover recipient beds of poor vascularity such as areas of exposed bone and tendons  Moreover, skin flaps often provide bett er cosmesis w hen repairing
15 delicate areas such as facial structures and are less prone to wound contraction than skin grafts [106, 107] Skin flaps are grouped into two general categories axial and random. The pedicle of a xial pattern skin flaps is based on a defined vascular territory and the blood is supplied through the vascular pedicle by a direct subcutaneou s artery In contrast, the pedicle of random skin flaps (RSFs) is based on random, non dominant vessels and the vascular pedicle contains only small musculocutaneous or septocutaneous perforators  In both axial and RSFs insufficient arterial supply and inad equate venous drainage can lead to ischemia and necrosis of the distal portion of the skin flap. RSFs are considered more at ri sk for necrosis tha n axial skin flaps due to the low level of blood supplied to t he flap by the small vessels in the pedicle and the i nstability of RSFs further increases when the length to pedicle width ratio exceeds 2.5 to 1  The tendency of the distal region of skin flaps to suffer severe ischemia and necrosis remains a significant issue in the clinic, but h as also provides researchers with a valuable tool to study wound healing in animal models in the laboratory  Currently, the use of the technique of surgical delay is the st andard method used clinically to increase skin flap survival. Surgical delay consists of partially elevating and undermining the skin flap 7 to 14 days prior to full elevation and inset. Investigation s of the molecular mechanisms by which the delay procedure increases skin flap viability have found that the proce dure increases the expression of both FGF 2 and VEGF, but not TGF [111 113] Gene therapy techniques deliv ering exogenous growth factors, such as VEGF and FGF 2, may therefore mimic the endogenous response to
16 surgical delay and are currently being investigated to increase skin flap perfusion and healing. T herapeutic angiogenesis to promote wound healing Pr eclinical studies have shown that t herapeutic angiogenesis is a promising option for increasing wound healing, but as of 2007 there were only a small number of clinical trials investigating gene transfer approaches targeting wound healing [41, 51] A multitude of growth factors have been inve stigated to promote all phases of wound healing in both skin flap and incisional wound models [51, 98] For example, therapeutic delivery of members of the FGF family has shown promise for increasing wound healing. I n vivo EP delivery of a plasmid encoding KGF 1, (also known as FGF 7) increased healing in both a diabetic mouse  and septic rat model  Also, l iposome mediated delivery of plasmid KGF 1, in combination with a plasmid encoding IGF 1 accelerated re epithelialization and increased expression of VEGF in an acute wound model  Further transfer of FGF 2 by recombinant protein injection  or plasmid with in vivo EP increased skin flap survival  PDGF and VEGF have been the most intensely studied growth factors for promoting therapeutic angiogenesis to increase wound healing. In preclinical studies, delivery of exogenous PDGF by viral mediated transfer [117 119] and by direct injection  or liposome mediated  delivery of a plasmid encoding PDGF successfully increased wound healing. In addition, a topical gel containing recombinant PDGF Regranex (Ortho McNeil), is currently prescribed for the treatment o f diabetic neuropathic foot ulcers  In several multicenter trials daily application of Regranex
17 only modestly increased ulcer closure. Specifically, ulcer closure ranged from 25% to 36% with the placebo gel and from 36% to 50 % with Regranex  T opical application of recombinant VEGF protein has also been investigated as potential treatment to increase healing of diabetic neuropathic foot ulcers In a Phase I clinical trial, application of a cream containing recombinant VEGF protein every 2 days for 6 weeks showed a positive, but not statistically significant, trend in ulcer healing  In contrast, preclinical studies have shown direct injec tion or systemic delivery of recombinant VEGF protein improves skin flap survival [124 127] One study demonstrated that i ntravenous delivery of VEGF prote in was more effective than localized injection  but this approach rais es the concern for pathological angiogenesis that may result from systemic VEGF expression [17 20] Also in preclinical models v iral mediated [128 131] transfer of VEGF and direct injection [132 134] liposome mediated  or transfer in a fibrin matrix [135, 136] of plasmid VEGF have all increased skin flap survival. Nonetheless, non protein methods for gene transfer of PDGF or VEGF ha ve yet to be explore d as a method to increase healing in clinical trials. Periph eral Artery Disease Peripheral artery disease (PAD) commonly res ulting from atherosclerosis, is one of the leading cause s of morbidity and mortality in the western world [137, 138] Th e primary pathophysiology of PA D is a reduction in blood flow to the lower extremities caused by stenosis or occlusion of a collateral artery. The reduction in blood flow g enerally results in on e of two clinical presentations: intermittent claudication (IC) or critical limb ischemia (CLI). IC is the most common presentation of PAD and is
18 characterized by muscular leg discomfort induced by exercise that is relieved by rest Patients with IC are limited in walking speed and distance and in other measures of quality of life  CLI, a less common but more severe presentation of PAD, results from insufficient blood flow to the a ffected limb even at rest. The decrease in blood flow t o the limb with CLI is so severe that limb loss is likely without therapeutic revascularization. There are a myriad of symptoms associated with CLI including : rest pain in the extremity, m etatarsalgia (pain and inflammation of the m etatarsal region of the foot), ulcers or wounds that fail to heal and gangrene  Current treatment options for PAD include risk factor reduction, physical therapy and training, and pharmacological treatment of the underlying atherosclerosis. Also, in patients with severe IC or CLI, interventional (catheter based) or open surgical procedures may be used to r evascularize the ischemic limb. A pharmacotherapy to increase perfusion to the effected limb is not availabl e and treatments targeti ng the underlying atherosclerosis are largely i neffective. Cilostazol, a reversible phosphodiesterase 3 inhibitor has shown some clinical benefit for treatment of IC but cannot be used in patients with any degree of heart failure  Pentoxifylline a pharmacological trea tment that decreases blood viscosity and thereby increases circulation in the limb, is widely prescribed in the United States for the treatment of IC but has little or no clinical benefit [141, 142] As mentioned above, r evascularization of the lower extremity for patients with severe IC or CLI can be accomplished by interventional catheter based techniques as well as open surgical procedures However, direct revascularization is often impossible d ue to the anatomic extent of PA D and is also limited by associa ted co morbidities, such as d iabetes, coronary artery disease or stroke
19 requiring a majority of patients to undergo limb amputation [138, 143 145] The lack of an e ffective pharmacological treatment and the limits of interventional and surgical procedures have spurred an intense investigation of alternative a pproaches such as therapeutic angiogenesis, for the treatment of PAD Preclinical models of limb ischemia Animal models of hindlimb ischemia are the standard preclinical model for evaluating potential therapies for limb ischemia. The first characterization of an animal model of hindlimb ischemia was in the 1950 s with studies in the rabbit  Since this initi al work experimental hindlimb ischemia models have been developed in both large and small animals. In general, large animal models, such as swine and canine, are advantageous because the limb vessels and associated branches are easily identifiable while small animal models, such as mouse and rat, are advantageous because of their low cost and the availability of transgenic lines The design of preclinical limb ischemia models vari es and the level of ischemia a chieved by each design depends greatly on t he species and even the strain of animal  The most common approaches to induce hindlimb ischemia are the tourniquet technique, simple ligation of the common femoral or common iliac arteries and ligation with excision of these arteries and their distal branches. The tourniquet technique is a non invasive approach that has been used in both large and small animal models to interrupt all arterial and venous blood flow to the limb, including blood flow from potential collateral vessels  This approach results in a rapid onset of severe muscle necrosis and is more appropriate for studying acute ischemic injury than the chronic
20 occlusive disease characteristic of PAD. The level and duration of ischemia achieved by l igation of the common femoral or common iliac arter y depends greatly on the level of collateral blood flow in the species. In general this technique fails to produce chronic ischemia at rest, as seen in patients with CLI, but in some species and transgenic models it does result in a reduction in blood flow with exercise characteristic of patients with IC [147, 149, 150] In animals with an extensive network of collateral vessels, such as the rat  ligation of the common femoral  or common iliac artery  only transiently decrease s blood flow to the li mb However, a study by Angersbach et al.  suggested that prolonged ligation of the femoral artery in the rat will eventually result in similar symptoms to IC. In comparison to simple ligation, ligation and excision of the most distal point of the external iliac or common femoral and associated distal branches reduces blood flow to the limb at rest, even in model systems with extensive coll ateral networks such a s the rat [147, 155 157] Thus, t he ligation and excision method is con sidered an appropriate model to study potential therapies for CLI [147, 155 157] Although there are differences in the methodology and resulting ischemia in the above mentioned preclinical models all of these models are associated with some degree of perfusion recovery even without t herapeutic intervention. Thus, the tim ing of delivery of potential therapeutics and assessment of limb perfusion is critical Interventions delivered at the time of surgery, or shortly thereafter, will effectively measure the ability of the intervention to improve limb perfusion. If the intervention is delivered at later time points, usually no later than post operative day (POD ) 7 the ability to detect the effectiveness of the intervention will be hampered because perfusion in the ischemic limb
21 will be naturally nearing that found in the non ischemic limb, which is often used as a control  There are multiple methods to determine the effect of a potential treatment on limb blood flow in preclinical models. H istological assessment of capillary density alone is not an accurate measure of perfusion and should be supplemented by a method that detects limb blood flow directly such as calf blood pressure ratio laser Dop pler perfusion imaging ( LDPI ) or magnetic resonance imaging Therapeutic angiogenesis for the treatment of limb ischemia Gene therapy techniques deliver ing angiogen ic growth factors are emerging as promising option s for the treatment of IC and CLI in patients when direct revascularization is not possible. A large number of angiogenic growth factors and cytokines have been explore d as therapeutic candidates in both preclinical models and clinical trials  Of these, FGF 1, FGF 2 and members of the VEGF family have been the mo st actively investigated but s everal other gro wth factors have successfully promoted therapeutic angiogenesis in preclinical models including HIF [159, 160] MCP 1 [161, 162] and eNOS [163, 164] H epatocyte growth factor (HGF) has also shown promise for therapeutic angiogenesis in both pre clinical models [165, 166] and Phase I clinical trial as a treatment for CLI  In clinical trial s several end points are measured to determine the efficacy of the t reatment. In patients with IC leg pain induced by exercise is the main clinic al manifestation. Thus, quality of life questionnaires peak walking time and measurements of limb blood flow, such as ankle brachial index (ABI) or LDPI are considered appropriate clinical trial end points. Because the clinical manifestations and prognosis of limb ische mia are more severe with CLI, trials
22 investigating therapeutic angiogensis for the treatment of CLI often include additional end points, such as the size and healing of ulcers and when appropriate loss of tissue  There have been multiple Phase I and II clinical trials investigating VEGF as a therapeutic agent for the treatment of PAD. Phase I clinical trials investigating intramuscular injection [168 170] and intra arterial infusion  of plasmid VEGF 165 showed the treatment increased angiographic score ABI, and ulcer healing and decreased the i ncidence of limb loss in patients with CLI In a Phase II trial conducted in patients with both CLI and diabetes mellitus intramuscular injection of plasmid VEGF 165 increase d ABI and ulcer healing  Also in a Phase II trial intra arterial administration of adenoviral VEGF 165 in combination with plasmid VEGF 165 increased angiographic score in patients with CLI  Clinical trials have also been conducted with adenoviral VEGF 121 In Phase I trials, intramuscular injection of adenoviral VEGF 121 increased peak walking time  but when moved into Phase II trials there was not a significant improvement in the e nd points measured (quality of life and peak walking time)  Of the FGF family members FGF 1 and FGF 2 have been the most intensely studied and most frequently utilized in emerging therapeutic angiogenesis approaches. In a Phase I I clinical trial intramuscular injection of plasmid FGF 1 was well tolerated and decreased the incidence of limb amputation in patients with CLI  Patients for a Phase III trial are currently being recruited to continue the study of intramuscular injection of plasmid FGF 1 for the treatment of PAD  There have been several clinical trials investigating the use of recombinant FGF 2 protein to promote therapeutic
23 angiogenesis in patients with PAD In a Phase I trial i ntra arterial del ivery of recombinant FGF 2 significant ly increase d calf blood flow  In a nother Phase I / II trial, recombinant FGF 2 suspended in a biodegradable gelatin hydrogel to promote sustained release was inject ed into the gastrocnemius muscle in patients with CLI. In comparison to baseline, the patients in this study showed significant improvements in peak walking time, limb perfusion and healing of foot ulcers  In the Phase II Therapeutic Angiogenesis with FGF 2 (TRAFFIC) trial intra arterial injection of recombinant FGF 2 protein significant ly improved peak walking time and ABI, but no differences were seen in claudic ation onset ti me or quality of life measurements  Non protein methods delivering FGF 2 to promote therapeutic angiogen esis have yet to be explored in clinical trials. Given the success intramuscular injection of plasmid FGF 1 in CLI patients, it would be of interest to explore other gene therapy approaches to deliver FGF 2 in clinical trials
24 C ENTRAL H YPOTHESIS The end goal of therapeutic angiogenesis is to increase the amount of nutrients supplied to and the rate of waste product removal from ischemic tissue by increasing vasodila tion, vascular permeability, neovascualarization and, subsequently, blood flow As a whole, this research explored the hypothesis that intradermal injection of plasmids enc oding angiogenic growth factors followed by non invasive in vivo EP is a feasible approach for promoting therapeutic angiogenesis in ischemic tissue to increase perfusion
25 I NTRADERMAL DELIVERY OF PLASMID VEGF 165 BY ELECTROPORATION PROMOTES WOUND HEALING Introduction In addition to direct injection of plasmid DNA, the most prevalent methods of therapeutic delivery of VEGF to skin flaps documented in the literature are adenovir al mediated gene transfer and recombinant protein injection. These methods have shown promis e for increasing skin flap healing [124 134, 181] but have draw backs (see General Introduction Methods of therapeutic gene delivery ) Moreover, in comparison to non viral approaches, transgene expression with a d enoviral mediated delivery persists longer increasing the potential for the occurrence of the adverse side effects associated with continuous unregulated VEGF expression [17 20] Thus, the development of a gene t ransfer approach that allows for tight control over expression level and duration would be advantageous for VEGF mediated therapeutic angiogenesis. The research presented Figure 2. The random skin flap model Left Approximate placement of the RSF Center Location of the distal area monitored for perfusion and healing Right a pproximate location of the treatment sites
26 in this Chapter investigated intradermal injection of pVEGF followed by in vivo EP, using the 4PE as potential therapy to increase wound healing This strategy was explored using a modified McFarlane [182, 183] 8 cm x 3 cm RSF model (Figure 2). Specifically, pVEGFE+ or control treatments were delivered at 4 sites to the RSF on POD 2 VEGF expression kin et ics and the ability of the treatment to increase an endogenous angiogenic response, perfusion and healing were evaluated Results In vitro plasmid VEGF expression The ability of pVEGF to increase VEGF expression was tested in vitro prior to use in vivo The promoter and coding region of VEGF was removed from pBLAST VEGF and cloned into the backbone of pVAX1 to allow for more efficient preparation of large quantities of the pl asmid. The level of VEGF expression with pVEGF was equi valent to that with pB LAST VEGF in both the cell lysate and cell culture supernatant 48 hours af ter lipid mediated transfection (Figure 3).
27 VEGF expression kinetics in the gastrocnemius muscle Plasmids encoding a wide variety of genes with therapeutic potential, including VEGF, have been successfully delivered to skeletal muscle by intramuscular injection and in vivo EP [54, 184] resulting in increased transgene expression compared to injection of the plasmid alone. After determining pVEGF increases VEGF expression in vitro in vivo expr ession was confirmed by delivering pVEGFE+ and pVEGFE to the rat gastr ocne mius mu scle. On days 2 5 and 7 after pVEGF delivery, muscle was excised Figure 3. In vitro plasmid VEGF expression Average VEGF expression SD was determined by ELISA 48 hours after lipid mediated transfection of B16F10 cells with pVEGF or pBLAST VEGF. For the mock transfection cells were treated with the transfection reagent without plasmid DNA. n=3 for all groups.
28 from the delivery site s and assayed for VEGF expression using an ELISA that specifically detects human VEGF 165 Compared to pVEGFE VEGF expression was significantly greater with pVEGFE+ for 5 days after delivery (Figure 4 ) VEGF expression kinetics in the random skin flap Because pVEGFE+ successfully increased VEGF expression in gastrocnemius muscle it was then inv estigated whether the same increase could be achieved with pVEGFE+ in the skin of the RSF model. Skin flaps were tre ated on POD 2 O n PODs 4 7 10 and 14 skin samples from the treatment sites were excised and assayed for VEGF Figure 4. pVEGFE+ increases VEGF expression in the gastrocnemius muscle pVEGFE+ increased VEGF expression in gastrocnemius muscle c ompared to pVEGFE Day 2 and day 7: n=6 per group, Day 5: pVEGFE n=5; pVEGFE+ n=4. p< 0.05.
29 expression using an ELISA that speci fically detects human VEGF 165 Necrosis prevented some sites from being harvested, especially in the pVEGFE group at later time points. On both POD s 4 and 7 VEGF expression was significantly higher in the pVEGFE+ treatment group compared to the pVEGFE treatment group (p< 0.05, both time points) (Figure 5) VEGF expression was determin ed for each treatment group in the proximal area, closest to the RSF pedicle, and distal area, farthest from the RSF pedicle. Interestingly, on POD 4 VEGF expression wa s significantly higher in the distal area of the RSF, compared to the proximal area, for the pVEGFE+ treatment group (p<0.05). There was a 2 fold increase in VEGF express ion in the distal area compared to the proximal area, with pVEGFE but this increas e was not statistical significant (p= 0.157). The level of VEGF expression achieved with pVEGFE+ in the distal area of the RSF was similar to the level and duration observ ed in the gastrocnemius (Figures 4 and 5 ). Compared to POD 4 there was an overall significant decr ease in VEGF expression on POD 7 (p< 0.05, both treatment groups in both the proximal and distal areas) but levels were still significant ly higher with pVEGFE+ compared to pVEGFE in each area (p< 0.05). At POD 10 there was not a signifi cant increase in VEGF expression with pVEGFE+ in either the proximal or distal area of the RSF, and by POD 14 only background levels of VEGF were detec ted for both pVEGFE+ and pVEGFE VEGF expression was not detected when the vector backbone lacking the VEGF cDNA insert was delive red to the RSF with EP (pVAXE+) or in the skin of untreate d controls (P E ). Also, VEGF expression was not detected in serum samples at any time point du ring the study (data not shown) indicating pVEGFE+ only increases VEGF expr ession locally, not systemically.
30 Figure 5. VEGF expression kinetics in the random skin flap On PODs 4 and 7 VEGF expression was signif icantly greater with pVEGFE+ tha n pVEGFE in the RSF. VEGF expression was further increased in the distal region of the RSF pVEGFE : POD 4 proximal n=8, distal n=6; POD 7 proximal n=3, distal n=4; POD 10 proximal n=3, distal n=2; and POD 14 proximal and distal n=2. pVEGFE+: POD 4 proximal n=4, distal n=7; POD 7 proximal n=7, distal n=6; POD 10 proximal n=3, distal n=4; and POD 14 both proximal >3 the mean SEM is reported. p< 0.05.
31 Delivery of plasmid VEGF with electroporation increases eNOS expression Both in vitro and in vivo studies [26, 185] have shown increases in VEGF can increase levels of eNOS. To det ermine if pVEGFE+ and pVEGFE increased eNOS in the RSF, skin samples from the distal area of the RSF were assayed for eNOS by ELISA at both PODs 4 and 7. On POD 4 eNOS was significantly higher following pVEGFE+ treatment c ompared to pVEGFE treatment (p < 0.05), but by POD 7 eNOS levels were similar in the pVEGFE+ and pVEGFE treatment groups (Figure 6 ). The application of an electric field to tissues alone can transiently induce expression of some genes [186 188] but pVAXE+ did not in crease eNOS in the RSF model (p < 0.01). Figure 6. pVEGFE+ increases eNOS expression pVEGFE+ significantly increased eNOS expression in the distal area of the RSF on POD 4 n=7 per group per time point. p< 0.05 compared to pVEGFE 0.01 compared to pVAXE+.
32 Effect of plasmid VEGF with electroporation on distal skin flap blood flo w Because RSF survival shows little cha nge after POD 15  skin flap perfusion and healin g were monitored through POD 14 pVEGFE+ resulted in the highest level of VEGF expression in the distal area of RSFs which is more at risk for ischemia and necrosis since it is farthe st from the vascular pe dicle. Thus, it was determined if pVEGFE+ treatment increased pe rfusion more than control treatments in the distal area of the RSF, corresponding to approximately 2 5% of the total RSF area. LDPI was used to measure perfusion preoperatively (baseline), immediately postoperatively, and on PODs 2, 4, 7, 10, and 14 A sh arp decrease in distal perfusion was observed immediately postoperatively through POD 4 indicating the RSF was effectively rendered ischemic. Perfusion for each treatment group is reported as the percen t recovery of baseline (Figure 7 a) and postoperative perfusion (Figure 7 b). At PODs 10 and 14 perfusion was significantly different between all treatment groups for both recovery of baseline and postoperative perfusion (p< 0.01). Perfusion in the pVEGFE+ treatment group was significantly greater than all c ontrols on both PODs 10 and 14 for both recovery of b aseline (p< 0.01) and postoperative (p< 0.05) perfusion.
33 Figure 7. pVEGFE+ increases distal skin flap blood flow Perfusion was measured in the distal region of the RSF by LDPI at the indicated time points. pVEGFE+ significantly increased recovery of baseline ( a ) and postoperative ( b ) perfusion on PODs 10 and 14 compared to all controls. n=7 per group, per time point. ** p< 0.01, *p < 0.05 compared to all controls. a b
34 Plasmid VEGF with electroporation promotes distal skin flap healing Visual assessment of RSF viability over the time course of the study showed a clear decrease in distal necrosis in the pVEGFE+ treatment group compa red to control groups (Figure 8, top panel ). The surviving region of the distal area of the RSFs was quantitated on PODs 7 a nd 14 (Figure 8, bottom panel ). To quantitate the percent survival of the distal area, the area of skin with normal texture and color was expressed as a percent of the total distal area. Non surviving areas were defined as dark, rigid necrotic skin or scabbed necrotic lesions and surviving areas were defined as skin normal in texture and color. The pVEGFE+ treatment increased the percent of distal RSF sur vival on POD 7 ( 8 9.4 4.3) ( p < 0.05) compared to pVEGFE (56.6 10.6), pVAXE+ (59.3 9.8) and P E (60.2 9.6). The surviving area in all treatment g roups increase d approximately 5% to 10% on POD 14 but healing was still greater and more consistent in th e pVE GFE+ group (95.2 2.2) (p < 0.01) compared to pVEGFE (65.6 9.3), pVAXE+ (70. 6 7.4) and P E (73.5 4.1). Adverse side effects associated with increased VEGF expression levels, such as edema, [17 20] were not observed by gross visual assessment in any of the treatment groups. The mean percent surviv al of the pVEGFE group on POD 7 in this study is similar to previously reported values delivering a plasmid encoding VEGF to a similar RSF model [120, 134] On POD 14 skin samples were excised 2 cm from the distal end of the RSFs for histological analysis by Hematoxylin Eosin (H&E) staining ( Figure 9). If the skin was necrotic at 2 cm from the distal end samples were excised near the edge of the surviving border. Skin from the pVEGFE+ treatment group appeared healthy (Figure 9 a b) while skin from the
35 pVEGFE treatment group showed evidence of acute inflammation, nec rosis and myonecrosis (Figure 9 c f). Figure 8. pVEGFE+ increases distal skin flap healin g ( Top ) Representative images of RSFs from each treatment group over the time course of the study. ( Bottom ) Quantification of distal survival on PODs 7 and 14 n=7 per group, per time point. ** p< 0.01, p < 0.05 compared to all controls
36 Figure 9. pVEGFE+ decreases inflammation and myonecrosis Representative images of skin excised 2 cm from the distal end of RSF s on POD 14 and stained with H&E from the pVEGFE+ ( panels a and b) and pVEGFE ( panels c through f) treatment groups. Open arrows indicate areas of epidermal lifting characteristic of necrosis with pVEGFE (panels c and d) compared to a normal epidermis with pVEGFE+ (panels a and b). Panel f, solid arrow indicates an example of an area of myonecrosis of the panniculus c arnosus muscle observed with pVEGFE Open circles (panels d, e and f ) indicate areas of inflammation characterized by the dark staining of numerous inflammatory cells. Inflammatory infiltrate was not observed with pVEGFE+ (panels a and b).
37 Preliminary studies were completed to explore if alterative treatment time points would increase RSF healing more than pVEGFE+ treatment on POD 2 I t was first confirmed that pVEGFE+ and pVEGFE increase d VEGF expression when delivered to the RSF on POD 0 (Figure 10a ). Wh ile immediate postoperative delivery of pVEGFE+ increased VEGF expression on POD 2 compared to pVEGFE the increase was not as robust as on POD 4 when treating on POD 2 (Figures 5 and 10a ). It is possible the lower level of VEGF expression when tr eating on POD 0 was due to the need for the tissue to recover prior to treatment. Also, because VEGF primarily mediates the proliferative phase of wound healing  high levels of VEGF expression during the inflammatory phase may have be en recognized as deleterious to the endogenous wound healing process Thus, treatment on POD 0 may have resulted in the exogenous VEGF mRNA or protein being targeted for degradation Because of the exploratory nature of these experiments the use of experimental animal s was minimized Thus, the small number of treatment sites assayed resulted in a large variation in VEGF expression ( Figure 10a ) RSF healing was also evaluated for the alternative treatment time points (Figure 10b). RSFs treated wit h pVEGFE on both PODs 0 and 2 showed the largest increase in healing of the alternative treatment time points, but healing with pVEGFE+ treatment on POD 2 was still greater. Interestingly, RSF healing was much lower with pVEGFE+ treatment on bo th PODs 0 and 2 compared to treatment on POD 2 alone. This may be be cause excessively h igh levels of VEGF expression can have negative effects on the process of angiogenesis [17 22]
38 Figure 10 pVEGFE+ treatment on POD 2 increases healing more than treatment at POD 0 or multiple time points (a) pVEGFE+ treatment on POD 0 increases VEG F expression on POD 2 n=2 per group for both proximal and distal areas Mean SD is reported (b) Quantification of distal RSF survival on PODs 7 and 14 when treating on POD 0, POD 2 or both POD 0 and 2. Treatment on POD 0 and POD 0 and 2 n=2 per group. Tre atment on POD 2 n= 7 per group. a b
39 D iscussion The data presented in this chapter demonstrate a novel therapeutic approach to increase VEGF expression, induce an endogenous angiogenic response, and increase perfusion and healing of the distal area of RSFs. pVEGFE+ is an attractive alternative to viral and recombinant protein gene transfer approaches to promote healing because it allows for the control of localize d expression level s and duration. This approach also circumvents t he adverse side effects and practicality issues associated with viral mediated and recombinant protein approaches. Previous gene therapy studies delivered VEGF to skin flaps either preoperatively or intraoperatively [120, 124 133, 135, 136] This study wa s the first to deliver pVEGF on the second POD VEGF modulates the proli ferative phase of wound healing that begins 2 to 4 days after the initial injury  By administering pVEGFE+ on POD 2 VEGF expression was timed to peak during the beginning of the proliferative phase of healing. Preliminary st udies treating the RSFs on POD 0 and on both POD 0 and 2 suggested that pVEGFE+ treatment on POD 2 increases healing more than trea tment at these alternative time points (Figure 10b) Al so, by administering treatment on POD 2 when the distal area of the skin flap is most hypoxic  it was determined that electrically mediated delivery of plasmid DNA to a large area of ischemic skin is in fact possible. These results demonstrate that pVEGFE+ significantly increased VEGF expression, compared to pVEGFE and that VEGF expr ession further increased with distance fro m the vascular pedicle. Also in the distal area of the RSF pVEGFE+ resulted in similar levels of VEGF expression as delivery to the g astrocnemius muscle
40 (Figure s 4 and 5 ). Higher VEGF expression in the distal area of the RSF, compared to proximal region, could be due to hypoxia induced stabilization of the VEGF mRNA or increased translation  The cur rent known mechanisms to increase VEGF mRNA stability and translation involve genetic regions not contained within the plasmid used in this study. It is possible other unknown mechanisms could enhance VEGF levels and may be responsible for the increase in VEGF expression in the distal area, compared to the less hypoxic proximal region. Importantly, VEGF expression was transient and not detected systemically, indicating that the increase was localized to the delivery area Moreover, pVEGFE+ confers a short term localized increase in VEGF expression thereby decreasing the possibility of the occurrence of the adverse side effects that are associated with long term high levels of VEGF expression [17 22] The i ncrease in eNOS expression after the pVEGFE+ and pVEGFE treatments indicate s that the observed increase in exogenous VEGF successfully stim ulated an endogenous angiogenic response. pVEGFE+ induces an earlier and more robust increase in eNOS expression compared to pVEGFE (Figure 6 ). These results are consistent with reports that in vitro VEGF induced upregulation of eNOS is time and dose de pendent  and with a study by Huang et al.  that demonstrated adenoviral delivery of VEGF 165 to RSFs increased eNOS. Relative to pVEGFE there was a 2.2 fold increase in eNOS protein with pVEGFE+ in the distal area of the RSF on the fourth POD that was mediated by a 6.6 fol d increase in VEGF protein (Figure s 5 and 6 ). Significant progress has been made elucidating the mechanisms underlying VEGF mediated increases in eNOS but many aspects of the complete mechanism remain unclear despite the well established role of this pat hway in wound healing and angiogenesis [163, 193, 194] I n
41 vitro [26, 192] studies utilizing the optimal VEGF dosage, and i n vivo  studies have shown that VEGF mediated increases in eNOS protein levels are typically not greater than 2 to 5 fold over background leve ls. In the work presented here, eNOS levels with p VEGFE+ remain si milar from POD 4 to POD 7 but increase with pVEGFE over this time period, suggesting that maximal level of eNOS has been reached in the pVEGFE+ group on POD 4 The small increase in eNOS levels, relative to VEGF levels, may be due to inhibition of eNOS production by high levels of NO  Also, the persistence of eNOS expression in the pVEGFE+ and pVEGFE treatment groups at POD 7 despite a decrease in VEGF expression, may be the result of a VEGF mediated increase in eNOS mRNA stability  eNOS mRNA has a normal half life of 48 hours  and further extending the half life would allow for the increase in eNOS protein to persist despite a decrease in VEGF levels. The ea rly increase in eNOS observed with pVEGFE+ could contribute to the increased perfusion and healing observed at the later time points. eNOS production of NO is important in the early stages of skin flap healing  and contributes to maintaining peripheral skin flap cir culation  Another possible mechanism for the increased perfusion and healing of the RSF after treatment with pVEGFE+ is increased neovascularization There was not a significant increase in vessel density (data not shown) in skin samples harvest ed 2 cm from the distal end of the RSFs on POD 14. Other studies delivering exogenous VEGF to wound healing models have reported the same phenomenon of increased healing without an associated increase in vessel density [127, 133] while other studies do not discuss changes in capillary density. To prevent ischemic necrosis there must be adequate perfusion pressure to the distal region of the
42 skin flap  In this study, increased eNOS, and the subsequent increase in vasodilation, could be the p rimary mechanism of the pVEGFE+ mediated increase in perfusion and healing, but d etermination of the exact mechanism will require further studies. In summary, the pVEGFE+ treatment significantly in creased VEGF expression for 5 days after treatment ( through POD 7) and this short term increase was sufficient to significantly increase eNOS, compared to pVEGFE treatment 2 days after treatment Further, pVEGFE+ significantly increased recovery of baseline and postoperative perfusion and as well as healing compared t o all control treatment groups. Thus, electrically mediated intradermal delivery of pVEGF is a potential non invasive therapeutic approach to increase perfusion and wound healing Future directions T o move this approach forward into a clinical setting, t he efficacy of pVEGFE+ to increase perfusion and healing of RSFs should next be examine d in a large animal model. Porcine skin physiologically resembles that of human skin Also, direct injection of plasmid DNA in porcine skin results in a similar express ion pattern as in human skin, suggesting the swine as an appropriate large animal model for future work  Also, because wound healing is impaired in patients with diabetes mellitus [92, 95] it would be interesting to explore if pVEGFE+ increases the healing of RSFs in a diabetic model. Diabetes has been successfully induced in both the rat  and swine  with s treptozotocin Also, transgenic rat models of diabetes are available that could be used for these future experiments [203, 204]
43 E xperiments investigating the mechanism by which pVEGFE+ significantly increases VEGF expression in the distal area, compared to the proximal area, of the RSF could elucidate a novel mechanism of VEGF mRNA stability The ELISA used in these studies specifically detects human VEGF 165 and becaus e only background levels of VEGF were detected with P E and pVAXE+ it is unlike ly that the increase in VEGF in the distal area of the RSF is due to non specific detection of endogenous VEGF. The expression of endogenous VEGF is increased and attenuated during hypoxia by both transcriptional regulation and mRNA stabilization HIF 1 increases VEGF transcription throu gh binding the hypoxia response element in the endogenous VEGF promoter a nd VEGF and untranslated regions of the endogenous mRNA [13 15] N either of these genetic regions is present in pVEGF suggesting the increase i n VEGF expression occurs through an unknown mechanism. Several genes such as c fos, c myc and tubulin contain RNA regulatory elements within their coding regions  and it is possible the increase in VEGF expression in the distal area of the RSF occurs through a similar mechanism E ndogenous VEGF is increased during hypoxia by transcriptional mechanisms, but it has been shown that the increase in VEGF protein occurs mainly through an increase in m RNA stability  In vitro experiments could be used to determine if the increase in VEGF with pVEGFE+ in the distal area of the RSF results from increased mRNA stabilization through a regulatory region in the coding sequence To evaluate this hypothesis assays similar to those reported by Shima et al.  could be used Briefly, pVEGF would be transfected into a non human cell line in order to distinguish endogenous and recombinant VEGF, and then cultured under both hypoxic and normoxic conditions After 15 hours, t he culture media would be replaced
44 with new media containing actinomycin D to inhibit new RN A synthesis and cells would then be cultured under normal conditions. VEGF mRNA levels would then be determined by northern blot using a human VEGF 165 specific probe, at time points up to 8 hours after the addition of actinomycin D Changes in endogenou s mRNA could be used as a positive control. If VEGF 165 mRNA levels are increased in cells cultured under hypoxic conditions it would indicate that there is a putative hypoxia regulatory element within VEGF mRNA coding region Further experiments could then be c arried out t o determine the location of the regulatory region and the RNA binding proteins that bind it It would also be of interest to determine if increased healing with pVEGFE+ is primarily mediated through an early increase in eNOS expression. In a rat model of hindlimb ischemia Namba, et al.  demonstrated increased p erfusion with intramuscular injection of a plasmid encoding eNOS was mediated through VEGF. In their study t hey i nhibit eNOS activity by administering N (G) nitro L arginine methyl ester (L NAME) a general inhibitor of nitric oxide synthase activity, thr ough the animals drinking water. Employing the same strategy, eNOS could be inhibited and RSF perfusion and survival could be assessed Effective inhibition of the VEGF mediated increase in eNOS should first be confirm ed on the fourth POD when the increase is first seen, by ELISA. Although L NAME is a general inhibitor of nitric oxide synthase activity, and not eNOS specifically, these experiments would elucidate if inhibiting the VEGF mediated increase eNOS effects RSF healing.
45 O PTIMIZATION OF DELIV ERY CONDITIONS FOR T HE M ULTI E LECTRODE A RRAY Introduction The design of the MultiE lectrode Array ( M E A ) (Figure 11) expands on the configuration of the 4PE. The MEA is composed of an array of 16 small electrodes, in a 4 by 4 configuration, with a 2 mm gap width between electrodes. Similar to the 4PE, the design of the MEA allows for multiple sets of electrical pulses to be applied in 2 electric field orientations at a 90 angle without removing the electrode from the delivery site One drawback to current electrode designs is that deliver y of plasmid to a large surface area requires the gap width between the electrode pairs to be increased thereby the applied voltag e must also be increased for cell permeabilization to be effective. The design of the MEA allows for its dimensions to be expanded by incorporating a larger number of electrode pairs thus enabling a larger surface area to be treated without the need to in crease the distance between electrodes. Further, the small gap width between the electrode pairs decreases the depth of the electric field penetration and thus should decrease the amount of nerve stimulation and pain. Figure 11. The Multi Electrode A rray
46 The goal of the work presented in this chapter was to further evaluate and optimize delivery parameters for the MEA This work was completed using a plasmid encoding the luciferase reporter gene (pLuc) delivered by intradermal injection in a guinea pig model followed by in vivo EP with the MEA Specifically, expression kinetics and the effect s of varying field strength, pulse duration and plasmid dose were evaluate d. Results Effect of field strength and pulse width on transgene expression For in vivo EP the voltage applied and pulse width are considered the main determinants of the level of transgene expression while other pulse parameters, such as pulse number, have a lesser impact [65, 206] Previous work determined that a field strength of 300 V/cm and 150 ms pulse width applied with the MEA resulted in similar luciferase expression levels achieved with the 4PE at 200 V/cm and 20 ms [Heller, R et al ., manuscript in preparation] To further evaluate t he effect s of field strength and pulse width for plasmid delivery with the MEA 3 field strengths ( 300 V/cm, 250 V/cm and 225 V/cm ) were examined with varying pulse width s ranging from 350 ms to 50 ms (Figure 1 2 ) Two days after plasmid delivery the delivery sites were excised and luciferase activity determined by a standard in vitr o luciferase assay. For all 3 field st rengths and pulse widths, except for 250 V/cm, 50 ms pulses, there was a significant increase in luciferase expression with pLucE+ compared to pLuc E (p< 0.001). In general, a pulse duration of longer than 150 ms resulted in slightly higher levels of luciferase expression, but 150 ms provided the highest level of expression with the least variation and minimal visual damage to the delivery area.
47 Figure 12. Effect of field strength and pulse width luci ferase expression Luciferase expression was determined 48 hours after delivery using an in vitro luciferase assay for the indicated field strengths (V/cm) and pulse widths (ms). p< 0.001 compared to pLucE
48 Transgene expression kinetics Because a pulse width of 150 ms for each of the field strengths examined in Figure 1 2 (300 V/cm, 250 V/cm and 225 V/cm) resulted in the highest level of transgene expression with the least variati on and visible tissue damage the duration of transgene expr ession was then evaluated using all 3 field strengths with 150 ms pulses. There was a significant increase in luci ferase expression with all 3 field strengths beginning 24 hours after delivery. For 300 V/cm and 250 V/cm, luciferase expression remained si gnificantly higher than pLucE through day 17 after delivery (Figure 13) (p < 0.001) With all field strengths luciferase expression decreased to background levels 21 days after delivery (data not shown). For 225 V/cm, luciferase expression remained signif icantly higher with pLucE + t hrough day 10 after delivery (p < 0.001 ) Fourteen day s after delivery there was no significant increase in luciferase expression with 225 V/ cm compared to pLucE (p=0.103). However, o n day 17 luciferase expression with 225 V/c m increased slightly and expression was significantly higher compared to pLucE at that time point (p < 0.001) Although there was a slight increase in expression on day 17 with 225 V/cm (3.4 x 10 5 photons / s) compared to day 14 (1.6 x 10 5 photons / s), the significant difference on day 17 between pLucE+ and pLucE for 225 V/cm is most likely due to the decrease in l uciferase expression with pLucE on day 17 ( 3.8 x 10 4 photons / s) compared to day 14 ( 1.2 x 10 5 photons / s) Luciferase expression for all field strengths did not significantly decrease from the initial peak expression at 24 hours until day 10 after delivery (p< 0.001). In contras t, luciferase expression with pLuc E significantly decrease d 96 hours after delivery (p< 0.001).
49 Figure 13 Luciferase expression kinetics Luciferase expression was determined at the indicated time points by in vivo luciferase assay for the indicated field strengths (V/cm) with 150 ms pulse widths. pLucE and 225 V/cm n=16 per group per time point. 250 V/cm n=15 per group per time point. 300 V/cm n=14 per group per time point. p< 0.001 compared to pLucE for that time point.
50 Control of tran s gene expression by plasmid dose C ontrol over t ransgene expression levels is desirable for delivery of plasmids encoding genes of therapeutic potential As such, it was next determined whether a dose response relationship existed between the amount of plasmid delivered and the resulting level of transgene expression. A range of pLuc doses ( 20 g, 50 g, 10 0 g, 150 g and 200 g ) was delivered at 250 V/cm and 150 ms pulses. Two days after delivery luciferase expression was determined by in vivo luciferase assay (Figure 14) For pLuc E+, compared to 100 g of pLuc ( the dose delivered in Figures 1 2 and 13 ) a significant decrease in luc iferase expression was seen when delivering 20 g of pLuc (p< 0.01) and a significant increase was seen when delivering 200 g of pLuc (p< 0.01). While altering the pLuc dose required altering the intradermal injection volume varying injection volume did not significantly affect transgene expression with pLuc E+ (Figure 15)
51 Figure 14. Control of luciferase expression by plasmid dose Luciferase expression was determined 48 hours after pLuc delivery by in viv o luciferase assay. pLucE+ = 250 V/cm 150 ms. For 20 g, 150 g and 200 g, n=6 per group for both pLucE and pLucE+; For 100 g n=8 per group for both pLucE and pLucE+; For 50 g pLucE n=6 and pLucE+ n=5. 0.05 comp ared to expression with pLucE+ delivering 100 g
52 Figure 15. Increasing plasmid injection volume does not affect luciferase expression Luciferase expression was determined using an in vivo luciferase assay 48 hours after delivery of 100 g pLuc in the indicated injection volume. pLucE+ = 250 V/cm 150 ms. n=8 per group. p< 0.001 compared to pLucE for that injection volume.
53 Discussion As a whole, the data presen ted in this chapter demonstrate that the level and duration of transgene expression achieved for pLucE+ delivered with the MEA can be adjusted by altering either electroporation parameters or plasmid dose. The duration and magnitude of the voltage applied has a significant impact on th e level of electropermeabilization of the cell membrane in the target tissue  Delive ry of pLuc at 300 V/cm compared to 250 V/cm and 225 V/cm, resulted in slightly higher luciferase expression at all pulse widths examined, but there was frequently visible tissue damage after delivery. It is possible that damage to the tissue is the reaso n for increased variability with 300 V/cm. Compared to 225 V/cm, luciferase expression was higher with 250 V/cm at all pulse widths. The lack of a significant increase in luciferase expression with 50 ms pulses delivered at 250 V/cm (Figure 12) indicates that 100 ms may be the minimum pulse duration to reach the necessary level of cell membrane permeabilization to increase plasmid uptake at a field strength of 250 V/cm or less The results presented in Figure 1 2 utilized in vitro luciferase ass ays to evaluate transgene expression. In vivo bioluminescen se imaging allows for the use of fewer experimental animal s and was used to determine luciferase expression in Figures 13, 14 and 15 Prior to using in vivo luciferase assays, it was determined t hat t he results obtained by the in vivo assay were highly correlated with the results obtained by the in vitro assay (r 2 = 0.94, n=8). It was previously reported  transgene expression decreases 3 days after plasmid DNA deliver y by intradermal injection  C onsistent with these findings, in this study luciferase expression with pLucE significantly decrease d between days 3 and
54 4 after delivery compared to the initial peak expression at 24 hours (Figure 12 ). For all pLucE+ conditions there was a significant decrease in luciferase expression 10 days after delivery compared to the initial peak at 24 hours, and expression with 300 V/cm and 250 V/cm remained greater than pLucE for 17 days L uciferase expression with 225 V/cm was not significantly greater then pLucE 14 days after delivery but o n day 17 expression with 225 V/cm slightly increased and was significantly higher than expression with pLucE+. T he significant difference in expression with 225 V/cm, compared to pLucE at day 17 is most l ikely due to the decrease in luciferase expression with pLucE Also, 225 V/cm resulted in a n overall lower level of luciferase expression then 300 V/cm and 250 V/cm beginning at day 10 after delivery Preliminary data not reported here determined that a field strength of 200 V/cm and 100 150 ms pulses increases luciferase expression approximately 2 fold compared to pLucE 2 days after delivery. Overall, these results indicate that if a lower level of expression is desired it could be achieved by decrea sing the field strength. Adjusting the level of transgene expression can also be accomplished by increasing or decreasing the dose of plasmid delivered. Compared to delivery of 100 g pLuc ( 7 .3 x 10 8 photons / s) delivery of 200 g ( 4 .5 x 10 9 photons / s) resulted in a 6 fold increase and delivery of 20 g a 3. 4 fold decrease (2.1 x 10 8 photons / s) in luciferase expression. L uciferase expression continued to increase up to 200 g of pLuc and there was a linear correlation between the amount o f plasmid delivered and luciferase expression (r 2 = 0.88) indicating the maximum expression level was not reached. Further experiments will be needed to determine the dose required to achieve the maximum level of expression
55 In summary, for pLucE+, the level of luciferase expression increased with the magnitude of the voltage applied F or each of the field strengths examined a pulse duration of 150 ms achieved the highest level of expression with the least variation and visible tissue damage Also, fo r all 3 field strengths, luciferase expression did not decrease from initial peak expression at 24 hours until 10 days after delivery an d with 300 V/cm and 250 V/ cm remained significantly higher than pLucE for at least 17 days. In addition to adjusting t ransgene expression levels by altering fielding strength, levels can also be controlled by adjusting the plasmid dose Future directions While the MEA was designed f or plasmid delivery to the skin, it may also be a useful non penetrating electrode design for the delivery of plasmid to other tissues. Plasmid DNA can be delivered to internal organs using both penetrating (needle arrays) and non penetrating electrodes. Generally, if a non penetrating electrode design is selected to deliver plasmid DNA to internal organs such as the liver, in vivo EP would be carried out with a paddle electrode The concern s wit h this approach are similar to those when delivering plasmid to the skin with the caliper electrode. In ord e r to apply 2 sets of pulses the electrode plates need to b e manipulated around the treatment site and removal and replace ment of the electrode plates results in variation in the distance between the plates and thus a variation in the applied voltage. The design of the MEA would eliminate this variation. In addition to being effective for plasmid delivery to the skin, t he MEA has already been shown to be effective for in vivo EP plasmid delivery to
56 cardiac muscle [ Marshall, WG et al. submitted for p ublication ] and could easily be explored for delivery to other organs such as the liver. Experiments optimizing delivery conditions for the MEA were initially planned to be carried out in a hairless guinea pig model because the structural characteristics and vascularity of hairless guinea pig skin closely resemble human skin [207, 208] Unfort unately, hairless guinea pigs were not available at the time these experiments were completed or for the foreseeable future. Confirmation of the data presented here generated in Hartley guinea pig s in the hairless guinea pig if available, would be helpful when moving this technology into clinical trials. If hairless guinea pigs are not available then confirmation of this data in a porcine model which also have skin similar to human skin  would be an alternative option. The MEA was designed to allow for its dimensions to be expanded by incorporating a larger number of electrode pairs to treat a larger surface area without the need to increase the distance between electrodes. While the data presented here confirms the principle of the MEA design experiments employing a larger electrode (composed of more electrode pairs) than the one utilized in these experiments have not yet been completed. A further understanding of the expression levels and kinetics that can be achieved with a larger MEA woul d be useful for future studies and for translation into clinical applications.
57 I NTRADERMAL DELIVERY OF PLASMID FGF 2 BY ELECTROPORATION I NCREASES PERFUSION AND ANGIOG ENESIS IN AN ISCHEMI C HINDLIMB Introduction Gene therapy approaches deliver ing plasmid FGF 2 by i ntramuscular injection of plasmid FGF 2 alone  or followed by intramuscular in vivo EP [210, 211] have sho wn promise as a treatment for PAD in preclinical models Also, delivery of recombinant FGF 2 has been shown to be beneficial in patients with PAD in clinical trials [178 180] Currently, a simple non invasive technique to deliver plasmid FGF 2 is not available. The primary focus of the research presented in this chapter was to determine if delivery of a plasmid encoding human FGF 2 ( pFGF ) to the skin with the MEA would increase FGF 2 expression, blood flow and angiogenesis in a rat model of hindlimb ischemia. In addition, this was the first study utilizing the MEA to deliver a plasmid encoding a gene with therapeutic potential and expression levels were compared to those achieved with the 4PE. To create the rat model of hindlimb ischemia the most distal end of the external iliac artery and the femoral artery proximal and distal to the bifurcation of the superficial epigastric artery and vein were ligated The femoral artery wa s then cut between the ligation of the external iliac, adjacen t to the inguinal ligament and dissected free to the bifurcation of the superficial artery and vein (Materials and M ethods for complete
58 description) This method of inducing hindlimb ischemia t he ligation and excision method effectively decreased blood flow to the limb at res t for the duration of the study ( 14 days ) and is considered an appropriate model to study potential therapies for CLI [147, 155 157] After postoperative perfusion assessment with LDPI, pFGF was delivered to the ischemic hindlimb at 2 sites on the medial aspect of the ischemic limb by intradermal injection f ollowed by EP with the MEA Results In vitro plasmid FGF 2 expression The a bility of pFGF to increase FGF 2 expression was tested in vitro prior to use in vivo The promoter and coding region of FGF 2 was removed from pBLAST bFGF and cloned into the backbone of the pVAX1 to allow for more efficient preparation of large quantitie s of the plasmid. The level of FGF expression with pFGF was slightly lower compared to pBLAST bFGF but pFGF still effectively increased FGF 2 expression. As expected, FGF 2 was detected in the cell lysate and not the cell culture supernatant because FGF 2 is only secreted from cells during active angiogenesis  (Figure 16).
59 Optimization of electrop oration conditions for plasmid FGF 2 delivery The work described in the previous chapter utilized a reporter plasmid (pLuc) in a guinea pig model to evaluate delivery conditions for the MEA. It was expected that delivery of a plasmid encoding a gene of t herapeutic potential (pFGF) in a different animal model would result in different expression kinetics. Thus, it was next evaluated whether pFGFE+ delivered with the MEA increased FGF 2 expression in the skin of the rat flank and if the level of expression achieved was similar to that achieved when Figure 16. In vitro pFGF expression Average FGF 2 exp ression SD was determined by ELISA 48 hours after lipid mediated transfection of HaCat cells with pFGF, pBLAST bFGF or the pVAX 1 vector backbone without a cDNA insert. pFGF n=3 pBLAST FGF 2 n=1, pVAX1 n=1.
60 delivered with the 4PE. Two days after pFGFE or pFGFE+, skin sampl es from the delivery sites were excised and assayed for FGF 2 expression by ELISA ( Figure 17 ). The level of FGF 2 expression was approximately e qual for pFGFE+ delivered with the 4PE at 200 V/cm, 20 ms ( 4 763 1 105 pg / sample) and the MEA at 250 V/cm, 150 ms (4 ,257 81 5 pg / sample) FGF 2 expression was slightly greater when pFGF was delivered with the MEA at 300 V/cm, 150 ms (5,482 901 pg / sample) Figure 17. Comparison of FGF 2 expression after pFGF delivery with the 4PE or M E A FGF 2 expression was determined by ELISA 48 hours after delivery of pFGF to the ra t flank with either the 4PE or M E A at the indicated field strengths and pulse widths. n=4 per group. Average expression S.D. is reported. p< 0.0 2 compared to pFGFE
61 FGF 2 expression kinetics The duration of FGF 2 expression was next determined for pFGFE+ delivered at 300 V/ cm, 150 ms with the MEA At the time points indicated in Figure 1 8 skin from the treated areas w er e excised and assayed for FGF 2 expression by ELISA. F or 10 days after plasmid delivery pFGFE+ significantly increased FGF 2 protein levels compared to pFGFE (p < 0.05) before decreasing to background levels at days 14 and 17 after delivery The application of an electric field to tissues alone can transiently induce expression of some genes, including angiogenic growth factors [186 188] however, pVAXE+ resulted in similar levels of FGF 2 expression as P E (n=4, 1416 326 total pg / sample ). P lasmid FGF 2 with electroporation increases blood flow in the ischemic hindlimb It was next evaluated whether the p FGF E+ treatment could increase blood flow in a rat model of hindlimb ischemia Im mediately postoperatively pFGFE + or control tre atments were administered at 2 sites on the medial aspect of the ischemic limb. LDPI was used to measure blood flow in the distal area of both the ischemic and non ischemic limbs preoperatively (baseline), immediately postoperatively and on PODs 1 3 7 and 14 ( Figure 19a) I mmediately postoperatively perfusion decre ased to approximately 40% of baseline indicating the hindlimb was effectively rendered ischemic. In Figure 19b blood flow is reported for each treatment group as the ratio of blood flow in the isch emic hindlimb to the non ischemic hindlimb (I/NI) (Figure 19b, top panel) and as a percent of the perfusion recorded at baseline (Figure 19b, bottom panel). There was significant
62 difference in limb blood flow begi nning on the thir d POD between all treatment groups for I/NI (p< 0.02) and as a percentage of baseline blood flow (p< 0.001). Also on POD 3 blood flow in the pFGFE+ treatment group was significantly greater than all of the control groups for I/NI and percentage of baseli ne bl ood flow (p< 0.001). Perfusion in the ischemic limb continued to be higher in the pFGFE+ treatment group compared to the control treatment groups for all subsequent time points in the study (p< 0.05, all time points for I/NI and percent of baseline b lood flow ) The pFGFE treatment group showed Figure 18 FGF 2 expression kinetics At the indicated time points skin samples were harvested from the delivery site s and assayed for FGF 2 protein expression by ELISA. To determine FGF 2 expression resulting from pFGFE+ and pFGFE the average FGF 2 expression in untreated skin (n=4, 1416 326 total pg / sample) was su btracted from the total pg/sample determined for each treatment site. Day 2 n=10, days 4, 7, and 10 n=6 and days 14 and 17 n=8 per group per time point. pFGFE+= 300 V/cm, 150 ms. ** p< 0.01, p< 0.05.
6 3 a slight, but not significant, increase in perfusion compared to the pVAXE+ and P E treatment groups on PODs 7 and 14. Figure 19a pFGFE+ increases hindlimb blood flow Representa tive LDPI images of the ischemic limb f r om the pFGFE+ and pFGFE treatment groups at baseline and on PODs 0, 3, 7 and 14 As indicated in the color legend on the right, a reas red or white in color represent higher perfusion and areas in blue lower perfusion. The white box indica tes the approximate area where perfusion was assessed. The black arrows indicate the absence of perfusion in the area of the femoral artery after the operation to induce hindlimb ischemia.
64 Figure 19b pFGFE+ increases hindlimb blood flow Perfusi on was determined by LDPI. ( Top) Ratio of perfusion in the ischemic limb to the non ischemic limb (I/NI) for all tr eatment groups. (Bottom) Postoperative perfusion at the indicated time points relative to b aseline perfusion for all t reatment groups Percent of baseline perfusion was calculated using the I/NI determine during the baseline perfusion measurements. n=6 per group per time point. p< 0.05 compared to all controls.
65 Plasmid FGF 2 with electroporation increases angiogenesis in the isc hemic hind limb Next, it was determined whether the increase in limb perfusion in t he pFGFE+ treatment group result ed from an increase in angiogenesis. In rodent models of hindlimb ischemia as well as in patients with PAD, angiogenesis typically occurs in the gastrocnemius muscle or distal to the arterial occlusion while arteriogenesis occurs higher in the limb near the site of the occlusion [147, 213] Thus, on POD 14 samples were harvested from the gastrocnemius muscle of the ischemic limb and capillary densit y was visualized by immunohistochemical staining for the endothelial cell marker factor VIII associated antigen Representative fie lds (400X) from each of the 4 treatment groups are shown in Figure 20 Also i n F igure 20, the average number of vessels in 5 randomly selected high power fields (HPFs) (400 X) for 5 animals in each treatment group is reported. There was approximately a 2 fold increase in capillary number in the pFGFE+ treatment group compared to all control groups (p< 0.001) indicating the increase in perfusion to the ischemic limb with pFGFE+ was in fact due to an increase in angiogenesis
66 Figure 20. pFGFE+ increases angiogenesis in the ischemic hindlimb (Left) Representative cross sections (400X) of Factor VIII immunohistological staining of samples excised from the gastrocnemius on POD 14 (Right) quantification of capillary density. The average number of capillaries in 5 fields, for 5 animals in each treatme nt group. p< 0.01 compared to all controls.
67 Discussion In addition to direct injection of naked plasmid DNA, the most prevalent methods of FGF 2 delivery do cumented in the literature are viral mediated gene transfer and recombinant protein injectio n. These methods are promising for treatment of PAD but have draw backs (See General introduction, Methods of t herapeutic gene delivery ) Further the unregulated expression and long term expression conferred by some viral vectors relative to plasmid DNA vectors, increases the possib i l it y of the occurrence of the adverse side effects associated with high levels of FGF 2 expression, such as potential toxicity and proteinuria [140, 214] In both animal models [21, 215, 216] and clinical tr ials [178 180] intramuscular injection or int ra arterial administration of recombinant FGF 2 protein improved perfusion to the ischemic limb or PA D related symptoms, respectively Similar to VEGF, the use of recombinant FGF 2 in a clinical setting is not prac tical due to its short half life and poor bioavailability. C onsequently frequent administration of the recombinant protein is often required to sustain lasting effects [37, 38, 140] The strategy presented here is a simple, non invasive approach that effectively increases FGF 2 protein expression, ischemic lim b blood flow and angiogenesis. FGF 2 expression was similar for pFGFE+ delivered with the 4PE and MEA (Figure 17 ) and was significantly higher with pFGFE+ than pFGFE for at least 10 days after delivery (Figure 18) Beginning on POD 3 there was a signif icant difference in blood flow in the ischemic hindlimb among all treatment groups and blood flow in the pFGFE+ treatment group was significantly greater than all control groups (p< 0.05) The increase in
68 hind limb perfusion with pFGFE+ treatment persiste d from POD 3 through POD 14, the end point of the study (Figure 19 b ) There was also a significant increase in the number of capillaries in the gastrocnemius muscle with pFGFE+ treatment indicati ng the increase in limb blood f low is, a least in part, the result of an increase angiogenesis (Figure 20) (p< 0.001) The increases in perfusion and angiogenesis in the pFGFE+ treatment group may be occurring by several known in vivo mechanisms. FGF 2 is normally confined to the cell compartment bu t during active angiogenesis it is exported to the extracellular space to exert downstream effects  Therefore, a lthough pFGFE+ treatment is administered to the skin, increased levels of FGF 2 protein after treatment and subsequent export could activate downstream angiogenic pathways. For example, a recent study by Fujii, et al.  demonstrated that in a model of hindlimb ischemia intramuscular injection of a plasmid encoding FGF 2 increase d the expression of placental growth factor via the up regulation of VEGF expression. P lacental growth factor alone is known to increase angiogenesis in models of hindlimb ischemia, and it is possible a similar mechanism may be occurring with pFGFE+ [217, 218] Another poss ible mechanism for the increase in angiogenesis and hindlimb blood flow with pFGFE+ treatment is the up regulation of HGF FGF 2 stimulates HGF ex pression, which itself has also been successful in increasing perfusion in preclinical models [165, 166] and in a clinical trial  Determination of the exact mechanism of pFGFE+ mediated angiogenesis will require further studies. In summary, e lectrically mediated intradermal delivery of pFGF is a potential non invasive non viral therapeutic approach to increase perfusion and angiogenesis in a
69 hindlimb ischemia model and warrants further investigation as a possible treatment for PA D. Future Dir ections Unlike humans, the rat has an abundant network of collateral vessels in the lower limb s due to the physiological absence of the profunda femoris artery  Prior to investigating this approach as a potential therapy for limb ischemia in the clinic it should be further tested in a larger a nimal model with a vascular anatomy that more closely resembles that of humans, such as the rabbit or swine  If pFGFE+ is explored as a potential treatment for PAD in a larger a nimal model the field strength and pulse width should first be optimized for that animal model. In this study, a lower field strength was used for pFGF delivery to the hindlimb (250 V/cm, 150 ms) to achieve FGF 2 expression levels that were comparable to the levels observed after pFGF delivery to the flank at a higher field strength (300 V/cm, 150 ms ). Relative to the skin of rat flank, the skin of the hindlimb is markedly thinner. Th erefore, it is expected that a higher field strength would be required to ach ieve equivalent expression in a larger animal mod el, such as the swine, with thicker skin t han that of the rat hindlimb. Optimally, both angiogenesis and arteriogenesis should be stimulated to increase perfusion to the lower limb [144, 219] At the time these experiments were carried out the technology used to assess collateral vessel formation in a small animal model was not available. When moving t his appr oach to a larger animal model it would also be beneficial to determine if pFGFE+ increases arteriogenesis in addition to angiogenesis. Gross observation of the skeletal muscle in the ischemic limbs on POD 14 of this study
70 suggested there may be an increase in arteriogenesis with pFGFE+ as indicated by the healthier color of the muscle and the presence of superficial immature collaterals identified by an increase in calibres and their typical corkscrew pattern  The presence of immatur e collaterals would be expected as a result of inducing ischemia in the hindlimb alone and therefore is not necessarily indicative of pFGFE+ increasing collateral vessel growth. In this study, pFGFE+ increased blood flow in the ischemic hindlimb but t he mechanism was not defined One possible pathway for FGF 2 to increase blood flow and angiogenesis is through the upregulation of VEGF. Other studies have shown in models of hindlimb ischemia that delivery of exogenous FGF 2 increases VEGF expre ssion a nd that blocking VEGF abrogates the therapeutic effects of FGF 2 [21, 209] Future studies could be conducted using a VEGF neutralizing antibody to block endogenous VEGF expression, similar to those conducted by Masaki et al.  to determine if pFGFE + mediates its therapeutic effects through VEGF. Briefly, a VEGF neutralizing antibody would be administered the same day as pFGFE+ treatment, hindlimb perfusion would be monitored through POD 14 and capill ary density would be determined in gastr ocnemius muscle samples excised at the end point of the study. Successful n eutralization of endogenous VEGF expression and the duration of the neutralizing effects of the antibody, should first be determined in tis sue samples from the gastrocnemius muscle using a VEGF ELISA. If pFGFE+ exerts its angiogenic effects by upreguating VEGF perfusion and capillary density should be similar in the pFGFE+ group receiving the VEGF neutralizing antibody and the pVAXE+ and P E control groups.
71 The skin is an attractive target for transgene delivery with in vivo EP because it is easily accessible and thus frequency and / or number of treatments can be easily increased. For delivery to the rat hindlimb (250 V/cm, 150 ms) and the flank ( 30 0 V/cm, 150 ms), pFGFE+ increased FGF 2 expression approximately 2 fold compared pFGFE This increase was sufficient to induce a biological effect, but it would be of interest to evaluate if tre atm ent at more than 2 sites on the ischemic hindlimb or if increasing the plasmid dose would further increase limb perfusion and neovascularization. Of note, the observed increase in FGF 2 with pFGFE+ may have been diminished by the presence of high levels o f endogenous FGF 2 in the rat skin ( 1416 326 total pg / sample ) The skin of the medial aspect of the hindlimb is exceptionally thin and increasing the field strength above 250 V/cm resulted in visual tissue damage. Thus increasing the field strength is not a viable option to increase FGF 2 expression with pFGFE+ treatment in the rat model of hindlimb ischemia and increasing the dose of plasmid and number of treatments should be further explored As mentioned above, increasing the field strength will likely be necessary to increase the level of FGF 2 expression achieved with pFGFE+ in a larger animal model. Also, in a larger animal model with thicker skin, a larger range of EP parameters could be examined and it is possible that increasing the field s trength or pulse width would result in a larger fold increase in FGF 2 expression with pFGFE+, compared to pFGFE The establishment of new functional vascular networks is a complex process orchestrated by a myriad of growth factors and cytokines. Transf er of a single angiogenic growth factor or cytokine has shown promise in preclinical and clinical studies, but the efficacy of these therapeutic approaches may be improved by delivering a
72 combination of angiogenic growth factors and / or cytokines An ele gant study by Cao, et al determined that delivery of both FGF 2 and PGDF to a rat and rabbit model of hindlimb ischemia synergistically incre ases neovascularization  Synergistic i ncreases in neovascularizat ion have also be en observed with combined delivery of FGF 2 and VEGF in rabbit  and mouse  models of hindlimb ischemia. The combination of FGF 2 with HGF has also been shown to synergistic ally increase neovascularization more than either growth factor alone in a mouse model of hindlimb ischemia  pVEGFE+ and pVEGFE did not increase VEGF expression above background levels in this or a more severe hindlimb ischemia model (data not shown). Despite the decrease in blood flow to the muscles of the ischemic limb, the skin most likely remained normoxic and did not allow for VEGF mRNA stabilization and expression. If the combination of FGF 2 and VEGF were to be explored, VEGF could be delivered to the gastrocnemius muscle while still delivering pFGFE+ to the skin of the ischemia limb, but the potential treatment would no longer be non invasive. Future experiments exploring if pFGFE+ in combination with a plasmid encoding PDGF or HGF increases limb blood flow more than either treatment alone would be useful for moving this approach toward clinical trials, especially if the experiments were conducted in a larger animal model as suggested above.
73 M ATERIALS AND M ETHODS Plasmids The CMV promoter was removed from the pVAX1 plasmid (Invitrogen) to create a promoterless pVAX1. The human VEGF 165 plasmid was cloned by sub cloning the hEF1 HTLV hVEGF165 sequence from pBLAST49 hVEGF (Invivogen ) into the promoterless backbone of pVAX1. The human FGF 2 plasmid (pFGF) was cloned by sub cloning the hEF1 IF4g hbFGF sequence from pBLAST45 hbFGF2 (Invivogen) into the promoterless backbone of pVAX1. The lucif erase plasmid used in this research was gWiz Luc (Aldevron). For in vivo use, all plasmids were commercially prepared (Aldevron). Cell culture and transfection Experiments confirming i n vitro expression of pVEGF were performed using the B16F10 mouse melanoma cell line maintained in McCoy's Media 5A with 10% FBS and 0.2% Gen tamycin In vitro expression of pFGF was confirmed in the immortalized keratinocyte HaCaT cell line maintained in DMEM m edia with 10% FBS and 0.2% Gentamycin Twenty four hours prior to transfection, 2 x 10 5 cells w ere plated per well in 6 well cell culture dishes. Cell s the TransIT eight
74 hours after transfection cells were harvested and mM NaCl, 20 mM sodium phosphate pH 7.4, 10% glycerol, 1% NP 40 and protease inhibitors (Roche )) for 30 min on ice. Lysates were then centrifuged at 4 C for 10 minu tes at 10,000 rpm and protein expression determined by ELISA. Animals and anesthesia All pr ocedures were approved by the Animal Use and Care Committee of the University of South Florida College of Medicine Male Sprague Dawley rats (250 300 g) (Harlan) were used for both the RSF model and hindlimb ischemia model. Female Hartley guinea pigs (25 0 300 g) were purchased from Charles River. Prior to operations and all procedures, animals were placed in an induction chamber and anesthetized with 5% isoflurane in oxygen After the a nimals were anesthetized they were fitted with a standard rodent mas k and maintained under anesthesia with 3.0% isoflurane in oxygen. All operations were performed using standard aseptic technique. At indicated time points animals were humanely euthanized by exposure to CO 2. The random skin flap mode l To create the rostral based single pedicle RSF on the left lateral flank the rat was shaved and an 8 cm by 3 cm template was traced with a surgical marker. Full thickness incisions were made along the traced template lines. The skin and subcutaneous tis sue were then elevated and sutured back to its bed using simple interrupted and running stitches with 4 0 non absorbable sutures.
75 The hindlimb ischemia model Hindlimb ischemia was induced in right hindlimb of the Sprague Dawley rats. Through an approximately 2 cm long incision parallel to the inguinal ligament the saphenous artery was ligated distally of the bifurcation of the femoral artery into the saphenous and popliteal arteries. T he most distal end of the external iliac was ligated immediat ely adjacent to the inguinal ligament and the femoral artery was ligated proximal and distal to the bifurcation of the superficial epigastric artery and vein. The superficial epigastric artery a nd vein were also ligated at 2 sites adjacent to the femoral bifurcation and cut between these sites. The femoral artery was then cut between the ligations adjacent to th e inguinal ligament and distally of the superficial epigastric artery and vein The femoral artery was then dissec ted free from the point of the distal ligation of the saphenous artery to the ligation of the femoral placed distally to the bifurcation of the superficial artery and vein. In vivo electroporation For all in vivo experiments all plasmids were at a conc sterile injectable saline and all injections were 50 L in volume unless otherwise noted. All injections were performed using a 25 gauge, 5/8 inch length needle. Plasmid delivery to muscle The rat gastrocnemius muscle was exposed u sing a 1 cm incision. pVEGF was then injected into the center of the muscle and th e injection site marked with a single stitch using a 4 0 non absorbable suture to later identify the delivery site to be excised for
76 prote in extraction. For pVEGFE+, a 4 needle elect rode array, with a 6 mm gap width between needles, was then place d ar ound the delivery site and 8 20 ms pulses were delivered at 200 V/cm. Plasmid delivery to skin For delivery to the RSF with the 4PE: Injections were centered along the midlin e of the RSF at 1, 3, 5, and 7 cm from the pedicle. Injection sites were marked with a surgical marker if they were to be excised later for protein extraction. Where noted, proxim al delivery sites were placed at 1 cm and 3 cm from the RSF pedicle and dis tal d elivery sites were placed at 5 cm and 7 cm from the RSF pedicle. After injection of plasmid DNA, the 4PE  was used to deliver 8 20 ms electric pulses at 200 V/cm to pVAXE+ and pVEG FE+ treatment groups. For delivery of pLuc to guinea pig skin with the M E A : Plasmid was intradermally injected at sites on the guinea pig flank. The MEA was then placed over the in jection bubble and an electric field applied at the indicated field strengths and pulse widths. Delivery sites were marked with a surgical mark er for later identification for quantification of luciferase expression. For delivery of pFGF with the MEA : pFGF was delivered to 2 sites on the medial aspect of the ischemic hind limb immediately after assessment of postoperative perfusion. After injection of plasmid DNA the MEA was placed over the injection bubble and 150 ms pulses were applied at 300 V/cm (delivery to skin of rat flank for initial expression kinetics) and 250 V /cm (delivery to hindlimb). FGF 2 expression levels from pFGFE+ and pFGFE were equivalent at 300 V/cm for delivery to the rat flank and 250 V/cm for delivery to the
77 hindlimb. FGF 2 expression with pFGFE+ and pFGFE did not differ between the ischemic an d non ischemic hindlimb In vitro luciferase assays At the indicated time points animals were humanely euthanized and the delivery sites were excised and snap frozen on dry ice. Tiss ue samples were homogenized in 1 to 2 mL of homogenization buffer (25 mM Tris, pH 7.8, 2 mM EDTA, 1 mM DTT and 10% glycerol). Homogenat es were then centrifuged at 4C for 2 minutes and the supernatants were removed and assayed for luciferase activity in Luciferase Assay Buffer (25 mM glycylgl ycine, pH 7.8, 15 mM KPO 4 pH 7.8, 15 mM MgSO 4 4 mM EGTA, 2 mM ATP, 1 mM DTT and 100 M Luciferin). Luciferase activity was quantitated using a MLX microtiter plate luminometer (Dynex Technologies). Using a standard preparation of recombinant luciferase (Promega) relative light unit values were converted to pg of luciferase and luciferase activity is reported as total pg luciferase per tissue sample. In vivo luciferase assays In vivo bioluminescence imaging was completed using the IVIS 100 system (Xeno gen Corp.) in conjunction with the Living Image acquisition and analysis software (Xenogen Corp.). Luciferin (Xenogen Corp.) was dissolved to 7.5 mg/ml in PBS, filter sterilized, and stored in single use aliquots at 20C. At the indicated time points t he guinea pigs were anesthetized and received a 50 L intradermal injection of Luciferin solution at the deliver y site. Images were acquired 2 minutes after Luciferin injection.
78 Quantitation of luciferase activity was based on the total flux (photons / sec) of emitted light from the delivery site Determination of protein expression At the indicated time points the delivery sites were excised and snap frozen on dry ice. Tissue samples were homogenized in 1 to 2 mLs of lysis buffer (150 mM NaCl, 20 mM sodium phosphate pH 7.4, 10% glycerol, 1% NP 40 and protease inhibitors (Roche)). Homogenates were then centrifuged at 4 C for 10 minutes at 10,000 rpm. The supernatants were removed and assayed for protein express ion using commercially available ELISAs for human VEGF 165 (R&D Systems, DY293B), eNOS (R&D Systems, DEN00) and FGF instructions. Laser Doppler Perfusion Imaging A Laser Doppler Perfusion Imager (Moor Instruments Ltd.) was used to record perfusion at the indicated time points. A low intensity (2 nm) scanned the surface of the skin without contact at a standardized working distance of 28 cm. Scan modus was set at 10 ms/pi xel and resolution at 256 x 256 pixels. To control for varia bility that may result from fluct uations in temperature and ambient light, animals were placed on a heating pad and measurements were completed in a low lit room.
79 Random skin flap model Three scans were completed per time point for each animal and average perfusion in arbitrary units (flux) for the distal region of the skin flap (5 cm 2 area) was determined for each time point. Baseline perfusion was assessed immediately preoperatively and post operative perfusion was assessed immediately after the operation to create the RSF Perfusion results are reported as the percentage of perfusion units relative to baseline or postoperative perfusion (normalized to 100%). Hindlimb ischemia model Three scans were completed per time point for each animal for both the ischemic and non ischemic limbs and average perfusion in arbitrary units (flux) was determined separately for each limb. Perfusion in the ischemic limb was normalized to the contra lateral n on ischemic limb to minimize variation due to ambient light and temperature. Baseline perfusion was assessed immediately preoperatively and postoperative perfusion as sessed immediately after completion of the operation to induce hindlimb ischemia The av erage I/NI was used to calculate percentage of baseline perfusion at the postoperative time points. Assessment of skin flap healing The p ercent survival of the distal 5 cm 2 area of t he RSF was quantitated on PODs 7 and 14. Areas dark in color and / or cov ered in scabs were defined as necrotic / non surviving areas. Areas with normal skin texture and color were defined as surviving
80 areas. The surviving areas were calculated using the Moor imaging software (Moo r Instruments Ltd.) and images were recorded u sing a digital camera (Nikon). Histology On POD 14 animals were humanely euthanized. Tissue samples were excised 2 cm from the distal end in the center of the RSF, fixed in 10% neutral buffered formalin for at least 12 hours at room temperature, embedd ed in paraffin and sectioned (4 If the tissue was necrotic at 2 cm from the distal end, samples were excised at the border of the necrotic and surviving tissue. Sections were then stained with H&E using a standard protocol. Stained sections were analy zed using a standard light microscope (Olympus BX51) and representative fields photographed with a Spot Insight 2 digital camera (Pathtech). Immunohistochemistry On POD 14 animals were humanely euthanized and tissue samples from the gastrocnemius musc le we re excised an d fixed in 10% neutral buffered formalin for at least 12 hours at room temperature before embeddin g in paraffin and sectioning (4 Capillaries in the cross sections were visualized using the Blood Vessel Staining Kit (Chemicon) according to manufactures instructions for Factor VIII with Hematoxylin counterstaining. The number of Factor VIII positive vessels was manually c ounted in a blinded fashion in 5 random ly selected fields (400X) for 5 animals from each treatment group. Stained sect ions were analyzed using a standard light microscope (Olympus BX51). The average capillary density per HPF (400X) for each treatment group is
81 reported. Representative fields from each treatment group were photographed at 400X with a digital camera (Spot Insight 2). Statistical Analysis All values are reported as the mean SEM unless otherwise noted. Statistical Statistical Package for the So cial Sciences (SPSS ). Analysis of VEGF expression was comple ted using a 2 test. Analysis of eNOS expression and distal RSF perfusion was completed using ANOVA with a post a single control group (pVEGFE+ ). Analysis of distal RSF survival was completed using the Mann Whitney Wilcoxon test with a post hoc Bonferroni Dunn correction to adjust for multiple comparisons. Analysis of luciferase activity was comple ted using a 2 test when comp aring 2 groups. Analysis of 3 or more groups was completed by ANOVA with a post s Least Significant Difference adjustment for multiple comparisons. Analysis of FGF 2 expression and capillary density in the ischemic hindlimb was comple ted using a 2 test. A post hoc Bonferroni correction was used to adjust for multiple comparisons to pFGFE+ for analysis of capillary density. Analysis of hindlimb perfusion was completed by ANOVA with post nce a djust ment for multiple comparisons.
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A BOUT THE A UTHOR Bernadette Ferraro grew up in the Hudson V alley area of New York State. She began her undergraduate studies at the Universit y of South Florida in 1996 and graduated with a Bachelor of S cience in Microbiology in May 2000. S he returned to USF in the fal l semester of 2003 to pursue a Master of Public Health in Epidemiology while working the laboratory of Eric Haura, M.D. Upon d e ciding to advance to a career in basic science research, Bernadette began graduate work in the USF Department of Biochemistry and Molecular Biology in 2004 and later enrolling in the PhD Plus program to complete her MPH and PhD concurrently While in the program, Bernadette received several awards to present her work at both national and international conferences and she filed a provisional patent as a co inventor with her co major professor Richard Heller, Ph.D . Bernadette plans to pursue a career as an independent investigator