Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME, 04074, USA.
Department of Developmental, Molecular and Chemical Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA, 02111, USA.
Lab Invest. 2019 Mar;99(3):290-304. doi: 10.1038/s41374-018-0072-1. Epub 2018 May 23.
Atherosclerosis is the most common cause of heart disease and stroke. The use of animal models has advanced our understanding of the molecular signaling that contributes to atherosclerosis. Further understanding of this degenerative process in humans will require human tissue. Plaque removed during endarterectomy procedures to relieve arterial obstructions is usually discarded, but can be an important source of diseased cells. Resected tissue from carotid and femoral endarterectomy procedures were compared with carotid arteries from donors with no known cardiovascular disease. Vascular smooth muscle cells (SMC) contribute to plaque formation and may determine susceptibility to rupture. Notch signaling is implicated in the progression of atherosclerosis, and plays a receptor-specific regulatory role in SMC. We defined protein localization of Notch2 and Notch3 within medial and plaque SMC using immunostaining, and compared Notch2 and Notch3 levels in total plaques with whole normal arteries using immunoblot. We successfully derived SMC populations from multiple endarterectomy specimens for molecular analysis. To better define the protein signature of diseased SMC, we utilized sequential window acquisition of all theoretical spectra (SWATH) proteomic analysis to compare normal carotid artery SMC with endarterectomy-derived SMC. Similarities in protein profile and differentiation markers validated the SMC identity of our explants. We identified a subset of differentially expressed proteins that are candidates as functional markers of diseased SMC. To understand how Notch signaling may affect diseased SMC, we performed Jagged1 stimulation of primary cultures. In populations that displayed significant growth, Jagged1 signaling through Notch2 suppressed proliferation; cultures with low growth potential were non-responsive to Jagged1. In addition, Jagged1 did not promote contractile smooth muscle actin nor have a significant effect on the mature differentiated phenotype. Thus, SMC derived from atherosclerotic lesions show distinct proteomic profiles and have altered Notch signaling in response to Jagged1 as a differentiation stimulus, compared with normal SMC.
动脉粥样硬化是心脏病和中风的最常见原因。动物模型的使用促进了我们对导致动脉粥样硬化的分子信号的理解。进一步了解人类的这种退行性过程需要人体组织。在缓解动脉阻塞的动脉内膜切除术过程中去除的斑块通常被丢弃，但它可能是患病细胞的重要来源。比较了颈动脉内膜切除术和股动脉内膜切除术切除的组织与无已知心血管疾病的供体颈动脉。血管平滑肌细胞 (SMC) 有助于斑块形成，并且可能决定易发生破裂的倾向。Notch 信号在动脉粥样硬化的进展中起作用，并在 SMC 中发挥受体特异性调节作用。我们使用免疫染色来定义 Notch2 和 Notch3 在中层和斑块 SMC 中的蛋白定位，并使用免疫印迹法比较了总斑块中的 Notch2 和 Notch3 水平与整个正常动脉。我们成功地从多个内膜切除术标本中获得了用于分子分析的 SMC 群体。为了更好地定义患病 SMC 的蛋白特征，我们利用序贯窗口采集所有理论光谱 (SWATH) 蛋白质组学分析来比较正常颈动脉 SMC 与内膜切除术衍生的 SMC。蛋白谱和分化标志物的相似性验证了我们外植体的 SMC 身份。我们确定了一组差异表达的蛋白，它们是患病 SMC 功能标志物的候选蛋白。为了了解 Notch 信号如何影响患病的 SMC，我们对原代培养物进行了 Jagged1 刺激。在显示出显著生长的群体中，Jagged1 通过 Notch2 抑制增殖；生长潜力低的培养物对 Jagged1 无反应。此外，Jagged1 不会促进收缩性平滑肌肌动蛋白，也不会对成熟分化表型产生显著影响。因此，与正常 SMC 相比，源自动脉粥样硬化病变的 SMC 显示出明显的蛋白质组谱，并在 Jagged1 作为分化刺激时表现出改变的 Notch 信号。
