Shah P K, Falk E, Badimon J J, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon J T, Regnstrom J, Fuster V
Atherosclerosis Research Center, Cedars-Sinai Medical Center, Los Angeles, CA 90077, USA.
Circulation. 1995 Sep 15;92(6):1565-9.
Rupture of the fibrous cap of the atherosclerotic plaque is a key event that predisposes to coronary thrombosis, leading to acute coronary syndromes. Recent studies have shown that the fibrous caps of vulnerable and ruptured atherosclerotic plaques have reduced collagen and glycosaminoglycan content in association with an increased macrophage density and a reduced smooth muscle cell density. Since collagen breakdown in the fibrous caps may contribute to a thinning and weakening of the cap, increasing its vulnerability to rupture, we tested the hypothesis that monocyte-derived macrophages, by producing matrix-degrading metalloproteinases (MMPs), could induce collagen breakdown in human atherosclerotic fibrous caps.
Monocytes were isolated from human blood by Ficoll-Paque density gradient and allowed to grow in cell culture until phenotypic and staining characteristics indicated transformation into macrophages (4 to 7 days). Fibrous caps were dissected from human aortic or carotid plaques and incubated for 48 hours with macrophages in serum-free medium without (n = 21) and with (n = 10) an MMP inhibitor or with cell- and serum-free medium only (n = 9). Hydroxyproline released in the culture medium was measured by a spectrophotometric method and used as evidence of collagen breakdown in the fibrous caps. Immunocytochemistry with specific monoclonal antibodies was used to identify expression of MMP-1 (interstitial collagenase) and MMP-2 (72-kD gelatinase) in cell culture, and zymography was used to detect MMP activity in the culture supernatant. The amount of hydroxyproline released was significantly greater when fibrous caps were incubated with macrophages than when incubated with cell-free medium (0.4 +/- 0.16 micrograms.mL-1.mg-1 versus 0.02 +/- 0.03 micrograms.mL-1.mg-1 of tissue; P < .04 by Mann-Whitney test). There was no hydroxyproline release when fibrous caps were incubated with macrophages in the presence of an MMP inhibitor. Immunocytochemistry demonstrated MMP-1 and MMP-2 expression by macrophages between days 4 and 7, and zymography confirmed the presence of MMP-2 activity in the supernatant.
In this study, human monocyte-derived macrophages were shown to induce collagen breakdown in fibrous caps of human atherosclerotic plaques associated with cellular expression and zymographic evidence of MMP activity; no evidence of collagen breakdown was found in the presence of an MMP inhibitor. These findings support the hypothesis that increased macrophage density and/or activation in the atherosclerotic plaque may induce collagen breakdown in the fibrous cap by secreting MMPs and possibly other proteases, thus contributing to vulnerability to plaque rupture.
动脉粥样硬化斑块纤维帽破裂是引发冠状动脉血栓形成进而导致急性冠状动脉综合征的关键事件。近期研究表明，易损及破裂的动脉粥样硬化斑块纤维帽中，胶原蛋白和糖胺聚糖含量减少，同时巨噬细胞密度增加，平滑肌细胞密度降低。由于纤维帽中的胶原蛋白分解可能导致帽变薄和变弱，增加其破裂的易感性，我们检验了以下假设：单核细胞衍生的巨噬细胞通过产生基质降解金属蛋白酶（MMPs），可诱导人动脉粥样硬化纤维帽中的胶原蛋白分解。
通过Ficoll-Paque密度梯度从人血液中分离单核细胞，并在细胞培养中使其生长，直至其表型和染色特征表明已转化为巨噬细胞（4至7天）。从人主动脉或颈动脉斑块中分离出纤维帽，并在无血清培养基中与巨噬细胞一起孵育48小时，其中一组不添加MMP抑制剂（n = 21），一组添加MMP抑制剂（n = 10），另一组仅与无细胞和无血清的培养基孵育（n = 9）。通过分光光度法测量培养基中释放的羟脯氨酸，并将其用作纤维帽中胶原蛋白分解的证据。使用特异性单克隆抗体进行免疫细胞化学，以鉴定细胞培养中MMP-1（间质胶原酶）和MMP-2（72-kD明胶酶）的表达，并使用酶谱法检测培养上清液中的MMP活性。与无细胞培养基孵育相比，纤维帽与巨噬细胞孵育时释放的羟脯氨酸量显著更高（组织中为0.4±0.16μg·mL-1·mg-1 对0.02±0.03μg·mL-1·mg-1；Mann-Whitney检验，P <.04）。当纤维帽在MMP抑制剂存在下与巨噬细胞孵育时，没有羟脯氨酸释放。免疫细胞化学显示巨噬细胞在第4至7天表达MMP-1和MMP-2，酶谱法证实上清液中存在MMP-2活性。
在本研究中，人单核细胞衍生的巨噬细胞被证明可诱导人动脉粥样硬化斑块纤维帽中的胶原蛋白分解，这与细胞表达和MMP活性的酶谱证据相关；在MMP抑制剂存在下未发现胶原蛋白分解的证据。这些发现支持以下假设：动脉粥样硬化斑块中巨噬细胞密度增加和/或激活可能通过分泌MMPs以及可能的其他蛋白酶诱导纤维帽中的胶原蛋白分解，从而导致斑块破裂的易感性增加。
