DeWitt Daughtry Family Department of Surgery Division of Vascular Surgery University of Miami
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1 31 st Annual Florida Vascular Society Scientific Session Orlando, FL April 26 th, 2018 Intramuscular E-selectin/AAV Gene Therapy Promotes Wound Healing in a Critical Limb Ischemia Mouse Model Punam P. Parikh MD, Roberta M. Lassance-Soares PhD, Hongwei Shao PhD, Manuela Regueiro PhD, Yan Li PhD, Zhao-Jun Liu PhD, Omaida C. Velazquez MD DeWitt Daughtry Family Department of Surgery Division of Vascular Surgery University of Miami 1
2 DISCLOSURES Punam Parikh, MD University of Miami Miller School of Medicine and Research team filed an intellectual property (IP) patent for E- selectin gene therapy for PAD, CLI, and wound healing 2
3 BACKGROUND Atherosclerosis-mediated critical limb ischemia (CLI) represents the most severe form of peripheral artery disease (PAD) PAD affects 8-12 million Americans Incidence of CLI: ~160,000 Main cause of rest pain, gangrene, and nonhealing wounds Affects 1.1 per 1000 population 3
4 BACKGROUND Impaired wound healing due to reduced tissue perfusion and oxygenation Current standard of therapy for CLI: surgical bypass or endovascular revascularization 20-40% patients unsuitable candidates High operative risk Unfavorable anatomy Ultimately require amputation 4
5 BACKGROUND One alternative for no-option patients may be gene therapy Attempts by prior multi-center RCTs: failed to meet primary endpoint to lower amputation rate VEGF, FGF, HGF Novel gene therapy using E-selectin/AAV Cell surface adhesion molecule Pre-clinical phase of testing 5
6 INFLAMED VASCULAR BED Activated Endothelial cells Quiescent Endothelial cells E-selectin EPCs EPCs Cytokines 6
7 PURPOSE To test the efficacy of E-selectin gene therapy on wound healing in an ischemic mouse model 7
8 METHODS: HINDLIMB ISCHEMIC* WOUND MODEL Adult FVB male mice Preoperative: Gene therapy priming on -4, -2, 0 days E-selectin/AAV (treatment, N=11) or LacZ/AAV (control, N=10) viral particles/injection 5x medial thigh *Parikh PP, Castilla D, Lassance-Soares RM, Shao H, Regueiro M, Li Y, Vazquez-Padron R, Webster KA, Liu ZJ, Velazquez OC. A Reliable Mouse Model of Hindlimb Gangrene. Annals of Vascular Surgery. PMID
9 METHODS: HINDLIMB ISCHEMIC* WOUND MODEL Hindlimb surgery Femoral artery and vein ligation (FAL) 4-mm full-thickness punch biopsy wound created Left anterior hindlimb *Parikh PP, Castilla D, Lassance-Soares RM, Shao H, Regueiro M, Li Y, Vazquez-Padron R, Webster KA, Liu ZJ, Velazquez OC. A Reliable Mouse Model of Hindlimb Gangrene. Annals of Vascular Surgery. PMID
10 EI Inguinal ligament FA LCI Transverse Incision EI- External Iliac Artery LCI- Lateral Circumflex SEA Iliac Artery SEA PF PA FA-Femoral Artery SEA-Superficial Epigastric Artery SA PF-Profunda Femoris Artery PA-Popliteal Artery SA-Saphenous Artery 10
11 THNIQUES & ENDPOINTS 1. Immunofluorescence E-selectin transgene expression 2. Daily wound surface area Percent wound healing 3. Laser Doppler Imaging (LDI) Foot perfusion 4. DiI Perfusion and confocal microscopy Wound angiogenesis 11
12 12 1. IMMUNOFLUORESCENCE
13 GREATER E-SELTIN AND CD31 IN TREATED MICE E-Selectin LacZ Overlay Overlay CD31 CD31 E- selectin E-selectin CD31 E-selectin DAPI Overlay Figure 1. E-selectin and CD31 Immunofluorescence DAPI Overlay
14 Quantification of E-selectin and CD31 Percent Area 3 2 ** * CD31 E-selectin 1 0 Treatment Control Figure 2. E-selectin Immunofluorescence: *P= for CD31 in treatment and control groups; **P= for E-selectin in treatment and control groups 14
15 15 2. DAILY WOUND SURFACE AREA
16 E-selectin POD0 POD1 POD5 POD10 LacZ 16
17 Percent Area Wound Healed GREATER E-SELTIN WOUND SURFACE AREA HEALED *P< POD0 POD1 POD2 POD3 POD4 POD5 POD6 POD7 POD8 POD9 POD10 Eselectin LacZ E-selectin, n=11 LacZ, n=10 17
18 18 3. LASER DOPPLER IMAGING
19 E-selectin Pre-surgery Post-surgery POD 3 POD 10 LacZ 19
20 Ischemia Index: Ligated/Non-ligated GREATER E-SELTIN FOOT PERFUSION ON LDI Eselectin Lacz *P<0.05 E-selectin, n=11 LacZ, n= Pre-surgery Post-surgery POD 3 POD 10 20
21 21 4. DiI PERFUSION
22 E-selectin LacZ
23 Percent Area of Capillary Density E-SELTIN PROMOTES WOUND NEOVASCULARIZATION *P<0.05 E-selectin, n=5 LacZ, n=5 0 E-selectin Eselectin Lacz LacZ
24 SUMMARY Intramuscularly injected E-selectin gene therapy improves hindlimb perfusion and wound neovascularization Accelerating wound healing Future studies: Role of E-selectin in stem cell recruitment CD133 and CD34 24
25 ACKNOWLEDGEMENTS Dr. Omaida C. Velazquez Dr. Zhao-Jun Liu Dr. Hongwei Shao Dr. Yan Li Dr. Manuela Regueiro Dr. Roberta Lassance-Soares Dr. Adriana Artiles Dr. Diego Castilla Marcia Boulina Melissa Carballosa 25
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