Identification of a homocysteine receptor in the peripheral endothelium and its role in proliferation Hongjiang Chen, MD, PhD, Ryan Fitzgerald, BS, Aliza.

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Identification of a homocysteine receptor in the peripheral endothelium and its role in proliferation Hongjiang Chen, MD, PhD, Ryan Fitzgerald, BS, Aliza T. Brown, PhD, Irfan Qureshi, MD, Julie Breckenridge, Rafi Kazi, Yunfang Wang, Yongmei Wu, Xianjian Zhang, MD, Perkins Mukunyadzi, MD, John Eidt, MD, Mohammed M. Moursi, MD Journal of Vascular Surgery Volume 41, Issue 5, Pages 853-860 (May 2005) DOI: 10.1016/j.jvs.2005.02.021 Copyright © 2005 The Society for Vascular Surgery. Terms and Conditions

Fig 1 Immunodetection of NMDAr NR1 subunits in rat carotid artery and brain. Rat brain and carotid artery from previously endarterectomized rats were analyzed for the NR1 NMDAr subunits. Right panel figures are negative controls, left panel figures are positive for NR1 antibody and used to immunolocalize NMDAr subunits, rat carotids, rat carotids were photographed at ×200 magnification. Positive-bound complexes are indicated by increasing intensity of color. Rat brain sections were used for positive controls and were photographed at ×400 magnification. Journal of Vascular Surgery 2005 41, 853-860DOI: (10.1016/j.jvs.2005.02.021) Copyright © 2005 The Society for Vascular Surgery. Terms and Conditions

Fig 2 Expression of NMDAr subunits in RAECs. Positive identification of NMDAr subunits was confirmed in RAECs by RT-PCR. NMDA receptor subunit sizes are given by base pair (bp) (NR1 333, NR2A 224, NR2B 222, NR2C 204, and NR2D 224 bp). An analysis of the receptor subunit bands indicated 99% to 100% identification sequence homology to NMDAr cDNA subunits in GenBank, shown in Table II. Journal of Vascular Surgery 2005 41, 853-860DOI: (10.1016/j.jvs.2005.02.021) Copyright © 2005 The Society for Vascular Surgery. Terms and Conditions

Fig 3 Homocysteine (Hcys) stimulation of the protein expression for the NR1 NMDAr in RAEC over time (A) and by dose (B). Results are representative of three separate experiments. A, Homocysteine-treated endothelial cells (50 μM) increased protein (size, 130 kD) concentrations of the NMDAr. Three to 12 hours of treatment indicated increased protein levels compared with controls or zero time. Six hours of treatment provided the highest increase in NR1 protein. The housekeeping gene β-actin was used as a loading control (size, 42 kD). B, Increasing concentrations of homocysteine increased endothelial cellular NR1 subunit protein after 6 hours of treatment. Homocysteine treatment at 50 μM produced the highest concentration of NMDAr protein (130 kD) as indicated by Western blot analysis. The housekeeping gene β-actin was used as a loading control (size, 42 kD). Journal of Vascular Surgery 2005 41, 853-860DOI: (10.1016/j.jvs.2005.02.021) Copyright © 2005 The Society for Vascular Surgery. Terms and Conditions

Fig 4 Increases in homocysteine 3H-thymidine incorporation are attenuated by MK-801. Cultured RAECs were treated with either control (CON), CON + 50 μM of MK-801, 50 μM homocysteine (HCYS), or HCYS + 50 μM of MK-801 for 48 hours. Homocysteine treatment of the endothelial cells increased 3H-thymidine incorporation vs all groups, which was attenuated with the addition of 50 μM of MK-801 to the homocysteine-supplemented cells for a decreased rate of cellular replication. N = 6/treatment group. Means are ±SEM. *P value of <.001 vs other treatment groups. Journal of Vascular Surgery 2005 41, 853-860DOI: (10.1016/j.jvs.2005.02.021) Copyright © 2005 The Society for Vascular Surgery. Terms and Conditions

Fig 5 Proliferative effects of homocysteine on aortic endothelial cells. RAECs were cultured and treated with increasing concentrations of homocysteine. A 50-μM homocysteine group was cotreated with MK-801 (50 μM), which blocked cellular proliferation when compared with 50 μM homocysteine only. All groups had an N = 6/treatment. *P value of <.05 vs control or homocysteine + 50-μM MK-801-treated cells. Journal of Vascular Surgery 2005 41, 853-860DOI: (10.1016/j.jvs.2005.02.021) Copyright © 2005 The Society for Vascular Surgery. Terms and Conditions