Lee Taeseung, Esemuede Nowokere, Sumpio Bauer E, Gahtan Vivian
Section of Vascular Surgery, Yale University School of Medicine and the VA Connecticut Healthcare System, New Haven, CT 06520, USA.
J Vasc Surg. 2003 Jul;38(1):147-54. doi: 10.1016/s0741-5214(02)75468-2.
Thrombospondin-1 (TSP-1), an extracellular matrix (ECM) glycoprotein, is associated with a variety of cellular processes relevant to atherosclerosis and intimal hyperplasia, including vascular smooth muscle cell (VSMC) migration. Matrix metalloproteinase-2 (MMP2) is associated with basement membrane and ECM degradation, important processes for cell migration. We hypothesized that TSP-1 modulates MMP2 activity in VSMCs and is critical for VSMC migration.
Quiescent bovine aortic VSMCs (48 hours) were incubated in serum-free media (SFM) with or without TSP-1 (10 or 20 microg/mL). Gelatinase activity was measured with zymography to determine pro-MMP2 and MMP2 activity. MMP2 messenger RNA expression was determined with Northern blot analysis. Invasion assays were performed. A binding assay was used to determine the specificity of TSP-1 binding to MMP2. Blots were quantified with densitometry, and all comparisons were made with a paired t test.
TSP-1 induced production of activated forms of MMP2, as well as upregulation of pro-MMP2. MMP2 mRNA was upregulated 1.7-fold by TSP-1 at 10 and 20 microg/mL. GM6001, an MMP inhibitor, inhibited VSMC migration across the matrix barrier, whereas migration that occurred in the absence of the matrix barrier was unaffected. With a binding assay, TSP-1 interacted physically with MMP2, and TSP-1-bound MMP2 showed the strongest binding activity in comparison with collagen I, fibronectin, and elastin.
TSP-1 induced MMP2 activation through transcriptional and posttranslational mechanisms. These findings imply that MMP2 activation is relevant to the mechanism of TSP-1-induced VSMC migration.
血小板反应蛋白-1（TSP-1）是一种细胞外基质（ECM）糖蛋白，与多种与动脉粥样硬化和内膜增生相关的细胞过程有关，包括血管平滑肌细胞（VSMC）迁移。基质金属蛋白酶-2（MMP2）与基底膜和ECM降解有关，这是细胞迁移的重要过程。我们假设TSP-1调节VSMC中MMP2的活性，并且对VSMC迁移至关重要。
将静止的牛主动脉VSMC（48小时）在含或不含TSP-1（10或20μg/mL）的无血清培养基（SFM）中孵育。用酶谱法测量明胶酶活性以确定前MMP2和MMP2活性。用Northern印迹分析确定MMP2信使核糖核酸表达。进行侵袭试验。使用结合试验确定TSP-1与MMP2结合的特异性。用密度测定法定量印迹，所有比较均采用配对t检验。
TSP-1诱导MMP2活化形式的产生以及前MMP2的上调。在10和20μg/mL时，TSP-1使MMP2 mRNA上调1.7倍。MMP抑制剂GM6001抑制VSMC跨基质屏障的迁移，而在无基质屏障时发生的迁移不受影响。通过结合试验，TSP-1与MMP2发生物理相互作用，与I型胶原、纤连蛋白和弹性蛋白相比，与TSP-1结合的MMP2表现出最强的结合活性。
TSP-1通过转录和翻译后机制诱导MMP2活化。这些发现表明MMP2活化与TSP-1诱导的VSMC迁移机制有关。
