Abstract 17955: Thereapeutic Angiogenesis Induced by Human Trx-1 Gene in Mice Hind Limb Ischemia Model: Preclinical Study for Treatment of Peripheral Artery Disease

Abstract

Introduction: Peripheral artery disease affects 12-20% Americans over the age of 60. Thioredoxin-1 (Trx-1) is a class of small redox proteins. We have demonstrated earlier that Trx-1 reduces oxidative stress resulting in less inflammation and increased angiogenesis in cardiac muscle via heme oxygenase-1 (HO-1) and VEGF after myocardial infarction. In the current study, we evaluate the effect of Trx-1 on post-ischemic hindlimb recovery. Methods: Peripheral artery disease was mimicked using a hindlimb ischemia (HLI) model. Wild type (WT) and Trx-1 transgenic (Trx-1Tg/+) mice (8-12 weeks old) were subjected to femoral artery ligation. Following surgery, mice were observed for 5 weeks. Serial laser doppler images were obtained, and perfusion ratios between the ischemic and non-ischemic limbs were calculated at set time intervals. The perfusion ratios were compared between WT and Trx-1Tg/+ groups. Immunohistochemical analysis of the skeletal muscle was performed to quantify the extent of fibrosis, capillary and arteriolar density 35 days after surgery. In addition, another set of experiments was designed with Ad.Trx-1 gene therapy after femoral artery ligation to study the molecular mechanism of neovascularization with Trx-1. Results: The recovery of hind limb perfusion was significantly increased in Trx-1Tg/+ mice at day 7 (0.19 ± 0.03 vs. 0.36 ± 0.07 (n=12-9), day-21 (0.37 ± 0.05 vs. 0.62 ± 0.03 (n=12-9), and day 28 (0.40 ± 0.04 vs. 0.79 ± 0.04 (n=10-9); p<0.05). Capillary density [1265 ± 87.8 vs. 762.4 ± 86.6 counts/mm2 ; (n=5); p<0.05] and arteriolar density [36.2 ± 2.96 vs. 22± 1.33 counts/mm2 ; (n=5); p<0.05] staining showed significant increase in Trx-1Tg/+ mice as compared to WT mice. Picrosirrus Red and immunofluorescence staining showed decreased fibrosis [8.3 ± 0.46 vs. 22.2 ± 1.04 (n=5); p<0.0001] and increased HO-1 expression respectively in Trx-1Tg/+ mice group as compared to WT mice, respectively. Trx-1 gene therapy study also revealed by Western blot analysis, increased Trx-1 (4.2 fold) and HO-1 (8.2 fold) expression in Ad.Trx-1-HLI as compared to Ad.LacZ-HLI. Conclusions: Our results suggest that Trx-1 is a potential therapeutic agent to increase blood perfusion and angiogenesis for the treatment of critical limb ischemia patients.