OBJECTIVE: A risk factor for cardiovascular disease, hyperhomocystinemia (HHcy), is associated with endothelial dysfunction. In this study, we examined the mechanistic role of HHcy in endothelial dysfunction. METHODS AND RESULTS: Through the use of 2 functional models, aortic rings and intravital video microscopy of the cremaster, we found that arterial relaxation in response to the endothelium-dependent vessel relaxant, acetylcholine or the nitric oxide synthase (NOS) activator (A23187), was significantly impaired in cystathionine beta-synthase null (CBS(-/-)) mice. However, the vascular smooth muscle cell (VSMC) response to the nitric oxide (NO) donor (SNAP) was preserved in CBS(-/-) mice. In addition, superoxide dismutase and catalase failed to restore endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) activity was significantly reduced in mouse aortic endothelial cells (MAECs) of CBS(-/-) mice, as well as in Hcy-treated mouse and human aortic endothelial cells (HAECs). Hcy-mediated eNOS inhibition--which was not rescued by adenoviral transduction of superoxide dismutase and glutathione peroxidase, or by tetrahydrobiopterin, sepiapterin, and arginine supplementations in MAEC--was associated with decreased protein expression and increased threonine 495 phosphorylation of eNOS in HAECs. Ultimately, a protein kinase C (PKC) inhibitor, GF109203X (GFX), reversed Hcy-mediated eNOS inactivation and threonine 495 phosphorylation in HAECs. CONCLUSIONS: These data suggest that HHcy impairs endothelial function and eNOS activity, primarily through PKC activation.
OBJECTIVE: A risk factor for cardiovascular disease, hyperhomocystinemia (HHcy), is associated with endothelial dysfunction. In this study, we examined the mechanistic role of HHcy in endothelial dysfunction. METHODS AND RESULTS: Through the use of 2 functional models, aortic rings and intravital video microscopy of the cremaster, we found that arterial relaxation in response to the endothelium-dependent vessel relaxant, acetylcholine or the nitric oxide synthase (NOS) activator (A23187), was significantly impaired in cystathionine beta-synthase null (CBS(-/-)) mice. However, the vascular smooth muscle cell (VSMC) response to the nitric oxide (NO) donor (SNAP) was preserved in CBS(-/-) mice. In addition, superoxide dismutase and catalase failed to restore endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) activity was significantly reduced in mouse aortic endothelial cells (MAECs) of CBS(-/-) mice, as well as in Hcy-treated mouse and human aortic endothelial cells (HAECs). Hcy-mediated eNOS inhibition--which was not rescued by adenoviral transduction of superoxide dismutase and glutathione peroxidase, or by tetrahydrobiopterin, sepiapterin, and arginine supplementations in MAEC--was associated with decreased protein expression and increased threonine 495 phosphorylation of eNOS in HAECs. Ultimately, a protein kinase C (PKC) inhibitor, GF109203X (GFX), reversed Hcy-mediated eNOS inactivation and threonine 495 phosphorylation in HAECs. CONCLUSIONS: These data suggest that HHcy impairs endothelial function and eNOS activity, primarily through PKC activation.
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