Increased perfusion and angiogenesis in a hindlimb ischemia model with plasmid FGF-2 delivered by noninvasive electroporation

Abstract

Gene therapy approaches delivering fibroblast growth factor-2 (FGF-2) have shown promise as a potential treatment for increasing blood flow to ischemic limbs. Currently, effective noninvasive techniques to deliver plasmids encoding genes of therapeutic interest, such as FGF-2, are limited. We sought to determine if intradermal injection of plasmid DNA encoding FGF-2 (pFGF) followed by noninvasive cutaneous electroporation (pFGFE+) could increase blood flow and angiogenesis in a rat model of hindlimb ischemia. pFGFE+ or control treatments were administered on postoperative day 0. Compared to injection of pFGF alone (pFGFE-), delivery of pFGFE+ significantly increased FGF-2 expression for 10 days. Further, the increase in FGF-2 expression with pFGFE+ was sufficient to significantly increase ischemic limb blood flow, measured by laser Doppler perfusion imaging, beginning on postoperative day 3. Ischemic limb blood flow in the pFGFE+ treatment group remained significantly higher than all control groups through the end point of the study, postoperative day 14. Immunohistochemical staining of gastrocnemius cross sections determined there was a twofold increase in capillary density in the pFGFE+ treatment group. Our results suggest that pFGFE+ is a potential noninvasive, nonviral therapeutic approach to increase perfusion and angiogenesis for the treatment of limb ischemia.

Figure 1. FGF-2 expression kinetics

At the indicated time points skin samples were harvested from the delivery site 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 subtracted from the total pg/sample 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. pFGFE-: intradermal injection of plasmid FGF-2; pFGFE+: intradermal injection of plasmid FGF-2 followed by electroporation.

Figure 2. Effect of pFGFE+ on ischemic limb blood flow

A, Representative laser Doppler perfusion images for the pFGFE+ and pFGFE- treatment groups at day 14 postoperatively. The white box indicates the approximate area where blood flow was assessed. The arrows indicate the absence of perfusion in the area of the femoral artery after the operation to induce hindlimb ischemia. B, Quantification of blood flow as determined by laser Doppler perfusion imaging. (Top panel) Ratio of blood flow in the ischemic limb to the non-ischemic limb (I/NI). (bottom) Postoperative blood flow as a percentage of baseline line blood flow. n=6 per group per time point. * p<0.05 compared to all controls. pFGFE+: intradermal injection of plasmid FGF-2 followed by electroporation; pFGFE-: Intradermal injection of plasmid FGF-2; pVAXE+: intradermal injection of vector backbone followed by electroporation; P-E-: no treatment.

Figure 2. Effect of pFGFE+ on ischemic limb blood flow

A, Representative laser Doppler perfusion images for the pFGFE+ and pFGFE- treatment groups at day 14 postoperatively. The white box indicates the approximate area where blood flow was assessed. The arrows indicate the absence of perfusion in the area of the femoral artery after the operation to induce hindlimb ischemia. B, Quantification of blood flow as determined by laser Doppler perfusion imaging. (Top panel) Ratio of blood flow in the ischemic limb to the non-ischemic limb (I/NI). (bottom) Postoperative blood flow as a percentage of baseline line blood flow. n=6 per group per time point. * p<0.05 compared to all controls. pFGFE+: intradermal injection of plasmid FGF-2 followed by electroporation; pFGFE-: Intradermal injection of plasmid FGF-2; pVAXE+: intradermal injection of vector backbone followed by electroporation; P-E-: no treatment.

Figure 3. Effect of pFGFE+ on neovascularization

A, Representative cross-sections (400X) of factor VIII-asscociated antigen immunohistological staining of cross-sections excised from the gastrocnemius on POD 14. Arrows indicate examples of stained vessels. B, Quantification of capillary density. The average number of capillaries in 5 fields, for 5 animals in each treatment group. * p<0.01 compared to all controls. HPF: High power field (400 X). pFGFE+: intradermal injection of plasmid FGF-2 followed by electroporation; pFGFE-: Intradermal injection of plasmid FGF-2; pVAXE+: intradermal injection of vector backbone followed by electroporation; P-E-: no treatment.

Figure 3. Effect of pFGFE+ on neovascularization

A, Representative cross-sections (400X) of factor VIII-asscociated antigen immunohistological staining of cross-sections excised from the gastrocnemius on POD 14. Arrows indicate examples of stained vessels. B, Quantification of capillary density. The average number of capillaries in 5 fields, for 5 animals in each treatment group. * p<0.01 compared to all controls. HPF: High power field (400 X). pFGFE+: intradermal injection of plasmid FGF-2 followed by electroporation; pFGFE-: Intradermal injection of plasmid FGF-2; pVAXE+: intradermal injection of vector backbone followed by electroporation; P-E-: no treatment.