Nitrative Stress in Cerebral Endothelium is Mediated by mGluR5 in Hyperhomocysteinemia

Abstract

Hyperhomocysteinemia (HHcy) disrupts nitric oxide (NO) signaling and increases nitrative stress in cerebral microvascular endothelial cells (CMVECs). This is mediated, in part, by protein nitrotyrosinylation (3-nitrotyrosine; 3-NT) though the mechanisms by which extracellular homocysteine (Hcy) generates intracellular 3-NT are unknown. Using a murine model of mild HHcy (cbs(+/-) mouse), we show that 3-NT is significantly elevated in cerebral microvessels with concomitant reductions in serum NO bioavailability as compared with wild-type littermate controls (cbs(+/+)). Directed pharmacology identified a receptor-dependent mechanism for 3-NT formation in CMVECs. Homocysteine increased expression of inducible NO synthase (iNOS) and formation of 3-NT, both of which were blocked by inhibition of metabotropic glutamate receptor-5 (mGluR5) with the specific antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride. Activation of mGluR5 is both sufficient and necessary to drive the nitrative stress because direct activation using the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine also increased iNOS expression and 3-NT formation while knockdown of mGluR5 receptor expression by short hairpin RNA (shRNA) blocked their increase in response to Hcy. Nitric oxide derived from iNOS was required for Hcy-mediated formation of 3-NT because the effect was blocked by 1400W. These results provide the first evidence for a receptor-dependent process that explains how plasma Hcy levels control intracellular nitrative stress in cerebral microvascular endothelium.

Cerebral Venous Thrombosis with Subarachnoid Hemorrhage: a Case Report

“…This mechanism also could have been present in our patient, who had proinflammatory status because of his altered metabolism of homocysteine. In fact, hyperhomocysteinemia is thought to lead to endothelial dysfunction, probably produced by reduction in the availability of nitric oxide, 17 and to an increase of the oxidative stress with alteration of the redox state of the endothelium. 18 Management of SAH secondary to CVT is quite different from that of arterial SAH.…”

The connexin 43/ZO-1 complex regulates cerebral endothelial F-actin architecture and migration

“…In-cell ELISAs were performed in 96-well plates as previously described (1,2,19). Anti-Cx43 primary antibody (catalog no.…”

Attenuation of Conducted Vasodilation in Skeletal Muscle Arterioles during Hyperhomocysteinemia

“…We used wild-type (C57BL/6J) and hyperhomocysteinemic cystathionine beta synthase heterozygous (CBS-/+) animals aged 12 weeks and weighing approximately 25-30 g. CBS heterozygous knockout mice on standard chow are a well-known model for HHcy [28]. Animals were used for various experiments and euthanized in accordance with National Institute of Health Guidelines for animal research.…”