Liposomal Hsp90 cDNA induces neovascularization via nitric oxide in chronic ischemia

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. In rabbits (n=7 per group), the external femoral artery was excised at day 0 (d0). 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. 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. 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.