title: Hyperhomocysteinemia and hyperglycemia induce and potentiate endothelial dysfunction via μ-calpain activation.
abstract: Plasma homocysteine (Hcy) levels are positively correlated with cardiovascular mortality in diabetes. However, the joint effect of hyperhomocysteinemia (HHcy) and hyperglycemia (HG) on endothelial dysfunction (ED) and the underlying mechanisms have not been studied. Mild (22 µmol/L) and moderate (88 µmol/L) HHcy were induced in cystathionine β-synthase wild-type (Cbs(+/+)) and heterozygous-deficient (Cbs(-/+)) mice by a high-methionine (HM) diet. HG was induced by consecutive injection of streptozotocin. We found that HG worsened HHcy and elevated Hcy levels to 53 and 173 µmol/L in Cbs(+/+) and Cbs(-/+) mice fed an HM diet, respectively. Both mild and moderate HHcy aggravated HG-impaired endothelium-dependent vascular relaxation to acetylcholine, which was completely abolished by endothelial nitric oxide synthase (eNOS) inhibitor N(G)-nitro-L-arginine methyl ester. HHcy potentiated HG-induced calpain activation in aortic endothelial cells isolated from Cbs mice. Calpain inhibitors rescued HHcy- and HHcy/HG-induced ED in vivo and ex vivo. Moderate HHcy- and HG-induced μ-calpain activation was potentiated by a combination of HHcy and HG in the mouse aorta. μ-Calpain small interfering RNA (μ-calpsiRNA) prevented HHcy/HG-induced ED in the mouse aorta and calpain activation in human aortic endothelial cells (HAECs) treated with DL-Hcy (500 µmol/L) and d-glucose (25 mmol) for 48 h. In addition, HHcy accelerated HG-induced superoxide production as determined by dihydroethidium and 3-nitrotyrosin staining and urinary 8-isoprostane/creatinine assay. Antioxidants rescued HHcy/HG-induced ED in mouse aortas and calpain activation in cultured HAECs. Finally, HHcy potentiated HG-suppressed nitric oxide production and eNOS activity in HAECs, which were prevented by calpain inhibitors or μ-calpsiRNA. HHcy aggravated HG-increased phosphorylation of eNOS at threonine 497/495 (eNOS-pThr497/495) in the mouse aorta and HAECs. HHcy/HG-induced eNOS-pThr497/495 was reversed by µ-calpsiRNA and adenoviral transduced dominant negative protein kinase C (PKC)β2 in HAECs. HHcy and HG induced ED, which was potentiated by the combination of HHcy and HG via μ-calpain/PKCβ2 activation-induced eNOS-pThr497/495 and eNOS inactivation.
authors.affiliation: Center for Metabolic Disease Research, Temple University School of Medicine, Philadelphia, PA Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Zhongjian
authors.initials: Z
authors.lastname: Cheng
authors.affiliation: Center for Metabolic Disease Research, Temple University School of Medicine, Philadelphia, PA Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA Center for Cardiovascular Research, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Center for Metabolic Disease Research, Temple University School of Medicine, Philadelphia, PA Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Meghana
authors.initials: M
authors.lastname: Pansuria
authors.affiliation: Center for Metabolic Disease Research, Temple University School of Medicine, Philadelphia, PA Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Pu
authors.initials: P
authors.lastname: Fang
authors.affiliation: Center for Metabolic Disease Research, Temple University School of Medicine, Philadelphia, PA Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Jietang
authors.initials: J
authors.lastname: Mai
authors.affiliation: Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Karthik
authors.initials: K
authors.lastname: Mallilankaraman
authors.affiliation: Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Gandhirajan
authors.affiliation: Center for Cardiovascular Research, Temple University School of Medicine, Philadelphia, PA Department of Physiology, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Satoru
authors.initials: S
authors.lastname: Eguchi
authors.affiliation: Center for Cardiovascular Research, Temple University School of Medicine, Philadelphia, PA Department of Physiology, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Rosario
authors.initials: R
authors.lastname: Scalia
authors.affiliation: Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Muniswamy
authors.initials: M
authors.lastname: Madesh
authors.affiliation: Center for Metabolic Disease Research, Temple University School of Medicine, Philadelphia, PA Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA Center for Cardiovascular Research, Temple University School of Medicine, Philadelphia, PA Center for Thrombosis Research, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Yang
authors.affiliation: Center for Metabolic Disease Research, Temple University School of Medicine, Philadelphia, PA Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA Center for Cardiovascular Research, Temple University School of Medicine, Philadelphia, PA Center for Thrombosis Research, Temple University School of Medicine, Philadelphia, PA hongw@temple.edu.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003541:Cystathionine beta-Synthase; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism* / Q000503:physiopathology*; D020138:Hyperhomocysteinemia / Q000378:metabolism* / Q000503:physiopathology*; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D001786:Blood Glucose; D013481:Superoxides; D052250:Nitric Oxide Synthase Type III; D002154:Calpain; C449002:mu-calpain; D003541:Cystathionine beta-Synthase