Abstract 5111: Endothelial Neuregulin Expression is Essential for Angiogenesis in Response to Hindlimb Ischemia
Neuregulins (NRG) are a family of growth factors synthesized by endothelial cells and shown to promote cell survival and growth via activation of erbB receptors. We have previously shown that NRG/erbB signaling may play a role in angiogenesis. Here, we tested whether endothelial-specific NRG deletion in vivo would affect angiogenesis in response to hindlimb ischemia induced by femoral artery ligation. A mouse expressing tamoxifen-inducible Cre-recombinase under the control of the vascular endothelial cadherin promoter with homozygously floxed NRG sites (CFF) was generated. CFF or Cre only expressing mice (C) were treated daily with vehicle or tamoxifen beginning three days before femoral artery ligation (1 mg, ip to knock out endothelial NRG expression) and continuing for 7 days after surgery (n=5– 8 per group). To assess angiogenesis, mice were injected with a 99mTc-labeled Arg-Gly-Asp (RGD) peptide (99mTc-NC100692, GE Healthcare) that specifically binds to activated αv integrins, followed by microSPECT imaging at 60 minutes post injection. Nuclear images were analyzed and the ratio of ischemic-to-nonischemic (I/N) NC100692 uptake within the distal hindlimb was calculated. The I/N uptake ratio was comparable between C tamoxifen and CFF vehicle treated animals (1.90±0.07 vs. 1.71±0.1, NS), but was significantly reduced in the CFF tamoxifen treated animals (1.29±0.1, P<0.05 vs. both controls), indicating a decrease in angiogenesis. These findings were further validated using anti-PECAM stained sections of muscles taken from ischemic and non-ischemic hindlimbs to quantify capillaries. The ratio of capillary numbers per muscle fiber in the ischemic to nonischemic area was comparable between the C tamoxifen and CFF vehicle (1.52±0.28 vs. 1.46±0.18, NS). However, this ratio was significantly reduced in the CFF tamoxifen treated animals (1.07±0.09, P<0.05 vs. both controls), confirming an impaired angiogenic response. These findings demonstrate a central role for NRG in mediating ischemia-induced angiogenesis and highlight the NRG/erbB pathway as a potential clinical target in the treatment of pathological and non-pathological angiogenesis.