Liposomal Hsp90 cDNA induces neovascularization via nitric oxide in chronic ischemia

Objective: Induction of angiogenesis has been reported subsequent to eNOS overexpression or activation, the latter involving Hsp90 as a chaperone protein. Here, we investigated the potential of regional Hsp90 overexpression to induce therapeutic neovascularization in vivo in a chronic rabbit hindlimb ischemia model. Methods: In rabbits (n=7 per group), the external femoral artery was excised at day 0 (0). At d7, liposomes containing eGFP (control group) or Hsp90 were retroinfused into the anterior tibial vein. At day 7 and day 35, angiographies were obtained and analyzed for collateral formation and perfusion velocity (frame count score) (% of d7 values). Capillary/muscle fiber (C/MF) ratio was calculated from five muscle areas of the ischemic limb. L-NAME and Geldanamycin were co-applied, where indicated. Results: Compared to mock-treated controls, Hsp90 transfected increased C/MF ratio at day 35 (1.78 +/- 0.15 vs. 1.19 +/- 0.13, p<0.05), an effect blunted by L-NAME (1.39 +/- 0.11). Hsp90 transfection increased collateral formation (157 +/- 11% vs. 110 +/- 13%) and frame count score (174 +/- 18% vs. 117 +/- 10%), both sensitive to inhibition by L-NAME coapplication (135 +/- 17% and 134 +/- 14%, respectively). Of note, C/MF ratio was found elevated 3 days after Hsp90 transfection (1.61 +/- 0.16 at d10), at a time point when collateral formation was unchanged (106 6%), and tended to remain elevated in the presence of L-NAME applied thereafter (1.64 +/- 0.35 at d35), though L-NAME blocked subsequent changes in collateral growth or increase in perfusion at d35. Conclusions: We conclude that Hsp90 is capable of inducing angiogenesis and arteriogenesis via nitric oxide (NO) in a rabbit model of chronic ischemia. Our findings describe the capillary level as an initial site of Hsp90-cDNA-induced neovascularization, followed by growth of larger conductance vessels, resulting in an improved hindlimb perfusion. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reseved.