Oak Min-Ho, El Bedoui Jasser, Anglard Patrick, Schini-Kerth Valérie B
Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Illkirch, France.
Circulation. 2004 Sep 28;110(13):1861-7. doi: 10.1161/01.CIR.0000142617.52881.F4. Epub 2004 Sep 13.
Regular consumption of moderate amounts of red wine is associated with a reduced risk of coronary disease. Matrix metalloproteinases (MMPs) that participate in extracellular matrix degradation have been involved in atherosclerotic plaque growth and instability. The present study examined whether red wine polyphenolic compounds (RWPCs) inhibit activation of MMP-2, a major gelatinase, in vascular smooth muscle cells (VSMCs).
Expression of pro-MMP-2 was assessed by Western and Northern blot analyses; MMP-2 activity was assessed by zymography and cell invasion by a modified Boyden's chamber assay. High levels of pro-MMP-2 and low levels of MMP-2 activity were found in conditioned medium from unstimulated VSMCs. Thrombin induced cell-associated pro-MMP-2 protein expression and MMP-2 activity in conditioned medium of VSMCs. The stimulatory effect of thrombin on MMP-2 activation was prevented by RWPCs in a concentration-dependent and reversible manner. Thrombin markedly increased cell-associated membrane type 1 (MT1)-MMP activity, the physiological activator of pro-MMP-2, and this response was not affected by RWPCs. However, addition of RWPCs directly to MT1-MMP abolished its metalloproteinase activity in a reversible manner. Finally, matrix invasion of VSMCs was stimulated by thrombin, and this response was prevented by RWPCs as efficiently as a broad-spectrum MMP inhibitor.
The present findings demonstrate that RWPCs effectively inhibit thrombin-induced matrix invasion of VSMCs, most likely by preventing the expression and activation of MMP-2 via direct inhibition of MT1-MMP activity. The inhibitory effect of RWPCs on the activation of pro-MMP-2 and matrix degradation might contribute to their beneficial effects on the cardiovascular system.
经常适量饮用红酒与冠心病风险降低有关。参与细胞外基质降解的基质金属蛋白酶（MMPs）与动脉粥样硬化斑块的生长和不稳定性有关。本研究检测了红酒多酚化合物（RWPCs）是否能抑制血管平滑肌细胞（VSMCs）中主要明胶酶MMP-2的激活。
通过蛋白质免疫印迹法和Northern印迹分析法评估前MMP-2的表达；通过酶谱分析法评估MMP-2活性，通过改良的Boyden小室分析法评估细胞侵袭能力。在未受刺激的VSMCs的条件培养基中发现高水平的前MMP-2和低水平的MMP-2活性。凝血酶可诱导VSMCs条件培养基中细胞相关的前MMP-2蛋白表达和MMP-2活性。RWPCs以浓度依赖性和可逆的方式阻止了凝血酶对MMP-2激活的刺激作用。凝血酶显著增加了细胞相关的1型膜（MT1）-MMP活性，即前MMP-2的生理性激活剂，而这种反应不受RWPCs的影响。然而，将RWPCs直接添加到MT1-MMP中可使其金属蛋白酶活性以可逆的方式丧失。最后，凝血酶刺激了VSMCs的基质侵袭，而RWPCs能像广谱MMP抑制剂一样有效地阻止这种反应。
本研究结果表明，RWPCs能有效抑制凝血酶诱导的VSMCs基质侵袭，很可能是通过直接抑制MT1-MMP活性来阻止MMP-2的表达和激活。RWPCs对前MMP-2激活和基质降解的抑制作用可能有助于其对心血管系统的有益作用。
