Induction of oxidative stress by homocyst(e)ine impairs endothelial function*

Abstract

Previous studies have demonstrated a relationship between hyperhomocysteinemia and endothelial dysfunction, reduced bioavailability of nitric oxide, elastinolysis and, vascular muscle cell proliferation. In vivo decreased nitric oxide production is associated with increased matrix metalloproteinase (MMP) activity and formation of nitrotyrosine. To test the hypothesis that homocysteine neutralizes vascular endothelial nitric oxide, activates metalloproteinase, causes elastinolysis and vascular hypertrophy, we isolated aortas from normotensive Wistar rats and cultured them in medium containing homocysteine, and calf serum for 14 days. Homocysteine-mediated impairment of endothelial-dependent vasodilatation was reversed by co-incubation of homocysteine with nicotinamide (an inhibitor of peroxinitrite and nitrotyrosine), suggesting a role of homocysteine in redox-mediating endothelial dysfunction and nitrotyrosine formation. The Western blot analysis, using anti-nitrotyrosine antibody, on aortic tissue homogeneates demonstrated decreased nitrotyrosine in hyperhomocysteinemic vessels treated with nicotinamide. Zymographic analysis revealed increased elastinolytic gelatinase A and B (MMP-2, -9) in homocysteine treated vessels and the treatment with nicotinamide decreases the homocysteine-induced MMP activation. Morphometric analyses revealed significant medial hypertrophic thickening (1.4 +/- 0.2-fold of control, P = 0.03) and elastin disruption in homocysteine-treated vessels as compared to control. To determine whether homocysteine causes endothelial cell injury, cross-sections of aortas were analyzed for caspase activity by incubating with Ac-YVAD-AMC (substrate for apoptotic enzyme, caspase). The endothelium of homocysteine treated vessels, and endothelial cells treated with homocysteine, showed marked labeling for caspase. The length-tension relationship of homocysteine treated aortas was shifted to the left as compared to untreated aortas, indicating reduced vascular elastic compliance in homocysteine-treated vessels. Co-incubation of homocysteine and inhibitors of MMP, tissue inhibitor of metalloproteinase-4 (TIMP-4), and caspase, YVAD-CHO, improved vascular function. The results suggest that alteration in vascular elastin/collagen ratio and activation of MMP-2 are associated with decreased NO production in hyperhomocysteinemia.

Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

“…Previously, we demonstrated that Hcy-mediated endothelial dysfunction was ameliorated by caspase inhibitor [Mujumdar et al, 2001]. In this study, we show that Hcy induces ROS …”

“…Previously, we demonstrated that Hcy-mediated endothelial dysfunction was ameliorated by caspase inhibitor [Mujumdar et al, 2001]. In this study, we show that Hcy induces ROS production, which in part instigates the intrinsic apoptotic pathway during hyperhomocysteinemia leading to programmed cell death.…”

“…The activated caspase-9 cleaves downstream caspases such as caspase-3 and caspase-6. Effector caspases like caspase-3, 6, and 7 are responsible for initiating the events that lead to the hallmarks of apoptosis, including chromatin condensation, DNA fragmentation, cleavage of poly (ADPribose) polymerase (PARP), and formation of apoptotic bodies [Porter and Janicke, 1999].Previously, we demonstrated that Hcy-mediated endothelial dysfunction was ameliorated by caspase inhibitor [Mujumdar et al, 2001]. In this study, we show that Hcy induces ROS …”

Role of matrix metalloproteinase-9 in endothelial apoptosis in chronic heart failure in mice

“…TUNEL detection alone is not enough to confirm apoptotic death (12,25,30). Therefore, we measured the levels of caspase-3 and showed that a caspase-3 inhibitor ameliorated the endothelial cell apoptosis (22). Here, we suggested that caspase-3 was significantly induced in AVF hearts compared with the sham or the MMP-9Ϫ/Ϫ mouse hearts with and without AVF (Fig.…”

“…Previously, our laboratory demonstrated that tissue inhibitor of metalloproteinase-4 (TIMP-4) ameliorated the endothelial cell apoptosis in part by inhibiting MMPs (22). To demonstrate a direct role of MMP in apoptosis, we tested the hypothesis that ablation of the MMP-9 gene can also alleviate the endothelial cell apoptosis in heart failure.…”