TY - JOUR
T1 - Hyperhomocysteinemia and hyperglycemia induce and potentiate endothelial dysfunction via μ-calpain activation
AU - Cheng, Zhongjian
AU - Jiang, Xiaohua
AU - Pansuria, Meghana
AU - Fang, Pu
AU - Mai, Jietang
AU - Mallilankaraman, Karthik
AU - Gandhirajan, Rajesh Kumar
AU - Eguchi, Satoru
AU - Scalia, Rosario
AU - Madesh, Muniswamy
AU - Yang, Xiaofeng
AU - Wang, Hong
N1 - Publisher Copyright:
© 2015 by the American Diabetes Association.
PY - 2015/3
Y1 - 2015/3
N2 - 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 highmethionine (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 NG-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 μmol) 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.
AB - 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 highmethionine (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 NG-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 μmol) 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.
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U2 - 10.2337/db14-0784
DO - 10.2337/db14-0784
M3 - Article
C2 - 25352635
AN - SCOPUS:84962054016
SN - 0012-1797
VL - 64
SP - 947
EP - 959
JO - Diabetes
JF - Diabetes
IS - 3
ER -