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 (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.


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.
Pfosser
Achim
A
Internal Medicine I, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, Marchioninistr. 15, 81377 Munich, Germany.
Thalgott
Mark
M
Büttner
Kerstin
K
Brouet
Agnès
A
Feron
Olivier
O
Boekstegers
Peter
P
Kupatt
Christian
C
D000818:Animals; D002196:Capillaries / Q000473:pathology; D002908:Chronic Disease; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D018841:HSP90 Heat-Shock Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics*; D007511:Ischemia / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D008081:Liposomes; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D011817:Rabbits; D014162:Transfection
D018076:DNA, Complementary; D018841:HSP90 Heat-Shock Proteins; D008081:Liposomes; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester