Abstract 16023: Hyperhomocysteinemia Aggravated Hyperglycemia-Induced Endothelial Dysfunction: Role of Calpain

Abstract

Accumulative evidence indicates that hyperhomocysteinemia (HHcy) is a strong risk factor of cardiovascular diseases in diabetes. We investigated effects of HHcy on vascular function in hyperglycemia (HG) using mouse model of HHcy and/or HG. HG (524 to 675 mg/dl) was induced by streptozotocin (STZ) injection (i.p., 40 mg/kg bw, 5 days). Severe HHcy was induced in cystathionine β-synthase heterozygous deficient (CBS^-/+) mice by fed a high methionine diet for 8 weeks (plasma Hcy, 88 to 172 µmol/l). HHcy synergistically impaired endothelium-dependent vascular relaxation to acetylcholine (ACh, maximal relaxation from 82.9 to 62.7%) in HG. Vascular relaxation to ACh was completely abolished by L-NAME, indicating that nitric oxide (NO) plays a major role in HHcy/HG-induced endothelial dysfunction (ED) in the aorta. HHcy/HG-impaired relaxation to ACh was prevented by pre-incubating aortas with calpain inhibitors MDL28170 (MDL), ALLM and calpeptin, antioxidants polyethylene glycol-superoxide dismutase (PEG-SOD) and Tempol. Chronic treatment with MDL (1 mg/kg, i.p., 2 weeks) improved vascular relaxation to ACh in CBS^-/+ mice with or without STZ. HHcy and HG synergistically increased calpain activity in mouse aortic endothelial cells (MAEC) isolated from CBS^-/+ mice HM diet and STZ injection. Moreover, HHcy and HG synergistically increased proteolytic activity of µ-calpain isoform, but not m-calpain, in mouse aorta extracts. Further, HHcy synergistically promoted superoxide (O₂^-) and nitrotyrosin levels in the endothelium of aorta, and urinary 8-isoprostan levels in CBS^-/+ mice with HG. L-Hcy (250 µM) and D-Glucose (D-Glu, 25mM) synergistically increased calpain activity and O₂^- production in cultured human aortic endothelial cells (HAECs), which were reversed by PEG-SOD and MDL, respectively. Finally, L-Hcy (250µM) and D-Glu (25mM) synergistically reduced NO production and eNOS activity in HAECs, which were restored by MDL and µ-calpain siRNA. Our results indicate that HHcy aggravated HG-induced ED in aorta, at least in part, by calpain-dependent eNOS inactivation and O₂^- production. Our study suggests that calpain system may be a novel target for prevention and treatment of vascular disease in diabetic patients with HHcy.