Abstract 5441: The Roles for MicroRNA-221 and IGF-1 Signaling in Regulation of High Glucose-Induced Endothelial Dysfunction and Apoptosis

Abstract

Persistent hyperglycemia represents a major metabolic complication of diabetes, which may cause endothelial cell dysfunction and apoptosis. microRNAs are novel biomarkers, modulators and therapeutic targets for disease. The microRNA miR-221 is expressed in human umbilical vein endothelial cells (HUVEC). Insulin-like growth factor 1 (IGF-1) is an important survival factor for multiple cell types. This study was to investigate whether high glucose (HG) induces endothelial dysfunction and apoptosis through altering the microRNA expression and IGF-1 signaling. The mirVana qRT-PCR miRNA detection assay showed that exposure to glucose for 24 h dose-dependently (up to 25 mM) increased miR-221 expression by 2.6 ± 0.5 folds (p<0.05). HG treatment decreased c-kit protein expression (58 ± 7 % decrease vs. control at 48 h) assessed by flow cytometry. The treated HUVEC had a reduced capacity for wound healing responded to stem cell factor (SCF, the ligand of c-kit). SCF-induced HUVEC migration potential was also reduced significantly by exposure to high dose of glucose. As estimated by prediction algorithms, c-kit, but not IGF-1, was identified as the target of miR-221. Transfection of HUVEC with 2′-O-methyl antisense inhibitory oligonucleotide (AMO-221) downregulation of miR-221 led to attenuation of the glucose’s effect on the c-kit expression, SCF-induced migration and wound healing in HUVEC. Prolonged glucose exposure (25 mM, 96 h) induced apoptosis of HUVEC assessed by Death ELISA (O.D. values = 0.49 ± 0.06 in glucose group vs. 0.12 ± 0.07 in control, p<0.01). Addition of IGF-1 (30 ng/ml) blocked the glucose-induced apoptosis, whereas AMO-221 did not attenuate the apoptosis. In addition, the anti-apoptotic effects of IGF-1 was attenuated by PI3 kinase inhibitor LY294002 (15 uM) (IGF-1 + glucose + PI3 kinase inhibitor vs. IGF-1 + glucose = 0.45 ± 0.07 vs. 0.15 ± 0.05 O.D. values). Our data demonstrate that downregulation of miR-221 attenuates HG-induced reduction of c-kit, wound healing and migration of HUVEC, and enhanced IGF-1 signaling prevents HG-induced apoptosis. Thus, reducing miR-221 expression and activating IGF-1 signaling pathway may be a useful strategy to prevent diabetic-induced endothelial dysfunction and apoptosis. This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).