Riches Kirsten, Morley Michael E, Turner Neil A, O'Regan David J, Ball Stephen G, Peers Chris, Porter Karen E
Multidisciplinary Cardiovascular Research Centre (MCRC), University of Leeds, Leeds LS2 9JT, UK.
J Mol Cell Cardiol. 2009 Sep;47(3):391-9. doi: 10.1016/j.yjmcc.2009.06.002. Epub 2009 Jun 11.
Cardiac myofibroblasts are pivotal to adaptive remodelling after myocardial infarction (MI). These normally quiescent cells invade and proliferate as a wound healing response, facilitated by activation of matrix metalloproteinases, particularly MMP-2. Following MI these reparative events occur under chronically hypoxic conditions yet the mechanisms by which hypoxia might modulate MMP-2 activation and cardiac myofibroblast invasion have not been investigated. Human cardiac myofibroblasts cultured in collagen-supplemented medium were exposed to normoxia (20% O(2)) or hypoxia (1% O(2)) for up to 48 h. Secreted levels of total and active MMP-2 were quantified using gelatin zymography, TIMP-2 and membrane-associated MT1-MMP were quantified with ELISA, whole cell MT1-MMP by immunoblotting and immunocytochemistry and MT1-MMP mRNA with real-time RT-PCR. Cellular invasion was assessed in modified Boyden chambers and migration by scratch wound assay. In the human cardiac myofibroblast, MT1-MMP was central to MMP-2 activation and activated MMP-2 necessary for invasion, confirmed by gene silencing. MMP-2 activation was substantially attenuated by hypoxia (P<0.001), paralleled by inhibition of myofibroblast invasion (P<0.05). In contrast, migration was independent of either MT1-MMP or MMP-2. Reduced membrane expression of MT1-MMP (P<0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2. In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI. Regulation of these MMPs remains an attractive target for therapeutic intervention.
心肌成纤维细胞在心肌梗死（MI）后的适应性重塑中起关键作用。这些通常处于静止状态的细胞作为伤口愈合反应而侵入并增殖，这一过程由基质金属蛋白酶（尤其是MMP-2）的激活所促进。MI后，这些修复事件在慢性缺氧条件下发生，但缺氧可能调节MMP-2激活和心肌成纤维细胞侵入的机制尚未得到研究。在补充胶原蛋白的培养基中培养的人心脏成纤维细胞暴露于常氧（20% O₂）或缺氧（1% O₂）环境中长达48小时。使用明胶酶谱法定量总MMP-2和活性MMP-2的分泌水平，用ELISA法定量TIMP-2和膜相关MT1-MMP，通过免疫印迹和免疫细胞化学法定量全细胞MT1-MMP，并用实时RT-PCR法定量MT1-MMP mRNA。在改良的博伊登小室中评估细胞侵入，并通过划痕试验评估迁移。在人心脏成纤维细胞中，MT1-MMP对MMP-2激活至关重要，而激活的MMP-2是侵入所必需的，基因沉默证实了这一点。缺氧显著减弱了MMP-2的激活（P<0.001）, 同时抑制了成纤维细胞的侵入（P<0.05）。相比之下，迁移与MT1-MMP或MMP-2均无关。MT1-MMP膜表达的降低（P<0.05）导致了MMP-2激活受缺氧影响而降低，而总MMP-2或TIMP-2均无变化。总之，缺氧通过降低MT1-MMP的膜表达减少了MMP-2的激活以及随后人心脏成纤维细胞的侵入，可能会延迟MI后的愈合。这些基质金属蛋白酶的调节仍然是治疗干预的一个有吸引力的靶点。
