D023 CHOP-10 Deletion improves neovascularization and stem/progenitor cells pro-angiogenic potential in type I diabetic mice with hindlimb ischemia

Abstract

Diabetes-induced reactive oxygen species overproduction impairs neovascularization. CHOP 10 is a novel developmentally regulated nuclear protein that emerges as critical transcriptional integrator among pathways regulating differentiation, proliferation and survival. Of interest, CHOP-10 has been shown to trigger oxidative stress-induced β cells apoptosis in the setting of diabetes. Here, we analyzed the role of CHOP-10 in postnatal neovascularization and bone-marrow-derived mononuclear cells (BMC) pro-angiogenic potential in type I diabetic mice with hindlimb ischemia. Ischemia was induced by right femoral artery ligation in C57/ Bl6 animals (WT, n = 8), diabetic C57/Bl6 animals (diab WT, n = 8, Streptozotocin 40 mg/kg) and diabetic CHOP-10-deficient animals (diab CHOP-10 KO, n = 8). Two days after ischemia, CHOP-10 mRNA and protein levels were increased by 7- (p < 0.001) and 4-fold (p < 0.01), respectively in ischemic muscle of WT diab compared to WT. Angiographic score, capillary density and foot perfusion were increased by 3.3- (p < 0.01), 1.8- (p < 0.001) and 2.2-fold (p < 0.001)in diab CHOP-10 KO compared to WT diab, 21 days after ischemia. This effect was associated with a reduction in the number of apoptotic cells and an increase in eNOS levels in diab CHOP- 10 KO compared to WT diab. We next analyzed the role of CHOP- 10 in post-natal vasculogenesis. Injection of BMC isolated from diab CHOP-10 KO in WT mice with hindlimb ischemia improved neovascularization by around 1.8-fold when compared to WT diab BMC (p < 0.05). BMC isolated from diab CHOP-10 KO mice showed an upregulation of eNOS protein levels and an increase in their ability to differentiate into cells with endothelial phenotype in vitro differentiation assay. Finally, treatment of cultured HUVEC with homocysteine increased CHOP-10 mRNA levels and repressed eNOS gene expression. Consistent with these results, eNOS protein expression was significantly upregulated in the CHOP-10 siRNA- transfected endothelial cells. Additionally, overexpression of CHOP-10 inhibited the basal transcriptional activation of the eNOS promoter as assessed by a reporter gene assay using a 3,500-bp fragment of the human eNOS gene. Our study unravels an important inhibitory role of CHOP-10 in the regulation of vessel formation in the setting of diabetes.