M. T. Rishi1,2, I. A. Shaikh1,2, V. Selvaraju1, M. Thirunavukkarasu1, J. Palesty2, N. Maulik1 1University Of Connecticut Health Center,Molecular Cardiology And Angiogenesis Laboratory, Department Of Surgery,Farmington, CT, USA 2Saint Mary’s Hospital,Stanley J. Dudrick Department Of Surgery,Waterbury, CT, USA
Introduction: There is a growing interest in developing novel therapeutic modalities at a molecular level for the treatment of peripheral arterial disease (PAD). We have previously shown that Thioredoxin -1 (Trx-1), which is a cytosolic 12-kDa redox protein, enhances neovascularization and reduces ventricular remodeling during chronic myocardial infarction. Here we aim to investigate the role of this protein in a murine hind limb ischemia (HLI) model.
Methods: Adult 8-12 week old C57Bl/6 mice were divided into two groups: (1) adeno-thiroedoxin-1 gene treatment group (Ad-Trx-1) and (2) control group (Ad-LacZ). The mice in Ad-Trx-1 group underwent right femoral artery ligation to create a murine model of HLI. Immediately after surgery, adeno-thioredoxin -1 in a concentration of 1×10⁹ PFU was injected in both semimembranosus and gastrocnemius muscles of the right leg whereas the left leg was used as an internal control. The mice in Ad-LacZ group also underwent right femoral artery ligation but instead received similar concentration of Ad-LacZ at the same muscle sites. The two groups underwent serial laser doppler imaging (LDI) pre-operatively and post-operatively for 28 days to assess hind limb perfusion. Immunohistochemistry and ELISA were performed on post-operative day 4 to determine the expression for various key angiogenic proteins.
Results: Mice in the Ad-Trx-1 group showed a significantly increased perfusion ratio on postoperative day 21 [0.893±0.067 (n=10) vs. 0.593±0.065 (n=10); p<0.05] and day 28 [0.908±0.081 (n=08) vs. 0.660±0.057 (n=10); p<0.05] [Figure 1] and a higher motor function score on post-operative day 7 [3.1±0.233 (n=10) vs. 2.1±0.276 (n=10); p<0.05], day 14 [3.9±0.233 (n=10) vs. 2.8±0.249 (n=10); p<0.05], day 21 [4.9±0.1 (n=10) vs. 3.7±0.213 (n=10); p<0.05] and day 28 [4.875±0.125 (n=8) vs. 3.8±0.249 (n=10); p<0.05] as compared to mice in Ad-LacZ group. Four days after femoral artery ligation, Ad-Trx-1 group showed increased Vascular Endothelial Growth Factor (VEGF) expression by immunohistochemical analysis and ELISA [38.48±7.165 (n=4) vs. 21.70±2.560 (n=5); p<0.05] as compared to Ad-LacZ group. Ad-Trx-1 group also showed increase expression of Flt-1 and Angiopoietin -1 proteins as compared to Ad-LacZ group by immunohistochemical analysis.
Conclusion: Taken together, our study demonstrates that the adeno-thioredoxin-1 gene delivery enhances blood perfusion and increases angiogenic protein expression in a murine hind limb ischemia model. We hope that this molecule can be a future potential target for clinical trials and subsequently drug therapy in peripheral vascular disease management.
