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INTRODUCTION: We previously demonstrated that N-methyl-D-aspartate (NMDA) receptor exists in vascular smooth muscle and endothelial cells, and that homocysteine may act via NMDA receptor to increase neointimal hyperplasia. In the current study, we investigated first, the expression profile of the constituent NMDA receptor subunits following vascular surgical procedure in an environment of hyperhomocysteinemia in rat model; and second, evaluated the expression of NR2D-containing NMDA receptor at transcriptional and protein level following vascular injury in a rat model.
METHODS: Rats were fed homocysteine supplemented diet or control for 2 weeks prior to carotid endarterectomy (CEA) and continued on preoperative diets. Animals were sacrificed at defined time points (3, 8 or 14 days) postoperatively and carotid arteries were processed for molecular studies (quantitative real-time reverse transcriptase-PCR, Western blot) and histomorphometry.
RESULTS: All five known NMDA receptor subunits (NR1, NR2A-2D) mRNAs were expressed in carotid arteries of homocysteinemic rats at 2 weeks post-CEA, but at different levels. Receptor subunit NR2D mRNA rose 6-fold versus NR2A, 4-fold versus NR2B or NR1 and 3 times versus NR2C; all P < 0.05. Protein bands corresponding to NR1, NR2A-2D also showed immunoreactivity at 2 weeks post-injury. Receptor subunit NR2D protein band was prominently expressed in the carotid artery compared to other subunits. This robust NR2D receptor subunit protein expression in the post-injury carotid artery is mimicking the high level of mRNA expression observed using qRT-PCR. Homocysteine supplementation for 8 or 14 days but not 3 days post-CEA triggered significant upregulation of both NR2D mRNA and protein versus control-CEA; all P < .0004. Furthermore, rats on control diet for 8 or 14 days but not 3 days post-CEA revealed remarkable increase in both NR2D mRNA and protein expressions versus control non-CEA (intact) arteries; all P < .019. Although damage to the carotid artery appears to upregulate the expression of NR2D receptor subunit, there was a quantitative difference in response to vascular injury alone versus injury supplemented with homocysteine at three defined time points (3, 8 or 14 days postoperatively), with the latter being higher. Neointimal hyperplasia, expressed as percentage lumenal stenosis of carotid artery was significantly increased in CEA plus homocysteine versus CEA on control diets at 2 weeks;P < .0001.
CONCLUSIONS: This preliminary study is the first to show that mRNA and protein for NR2D-containing NMDA receptor were upregulated in the carotid artery at 8 or 14 days post-CEA in a time dependent manner with or without homocysteine supplementation. Carotid endarterectomy with homocysteine supplementation for 2 weeks triggered significant increase in lumenal stenosis. These morphological and biochemical evidence represent the basis for our assumption that NR2D receptor subunit may be involved in homocysteine-mediated hyperplastic response. We suggest that NR2D receptor subunit may constitute a useful therapeutic target to prevent/attenuate neointimal proliferation following vascular injury.