Rao Velidi H, Kansal Vikash, Stoupa Samantha, Agrawal Devendra K
Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, 68178, Nebraska.
Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, 68178, Nebraska ; Center for Clinical and Translational Science, Creighton University School of Medicine, Omaha, 68178, Nebraska.
Physiol Rep. 2014 Feb 10;2(2):e00224. doi: 10.1002/phy2.224. eCollection 2014 Feb 1.
Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)-mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP-9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP-9, MMP-1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP-9 activity and mRNA transcripts for MMP-9, MMP-1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP-9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF-treated VSMCs of both groups. Additionally, the magnitude in decreased MMP-9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP-9 gene with siRNA in EGF-treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP-9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.
动脉粥样硬化斑块破裂是心肌梗死和中风发生发展的关键因素，但其潜在机制尚未明确。在此，我们研究了表皮生长因子（EGF）介导的基质金属蛋白酶（MMP）对从有症状和无症状颈动脉狭窄患者的颈动脉内膜切除术组织中分离出的血管平滑肌细胞（VSMC）间质胶原稳定性的作用。对从无症状和有症状患者的颈动脉斑块中分离出的VSMC进行EGF处理。通过明胶酶谱法定量MMP-9活性，并分别通过qPCR和免疫荧光分析MMP-9、MMP-1、EGFR以及I型胶原（Col I(α1)）和III型胶原（Col III(α1)）的mRNA转录本和蛋白质。在有症状患者颈动脉斑块分离出的VSMC中, EGF处理增加MMP-9活性以及MMP-9、MMP-1和EGFR的mRNA转录本并减少Col I(α1)和Col III(α1)的mRNA转录本的作用，是无症状患者的三到四倍。EGFR抑制剂（AG1478）和MMP-9小分子抑制剂降低了两组EGF处理的VSMC中的MMP9表达，并上调了Col I(α1)和Col III(α1)。此外，在EGF处理的VSMC中，由于用siRNA敲低MMP-9基因导致的MMP-9 mRNA减少以及Col I(α1)和Col III(α1)增加的幅度，有症状组明显大于无症状组。因此，选择性阻断EGFR和MMP-9可能是一种稳定颈动脉狭窄患者易损斑块的新策略和有前景的靶点。
