Calpain-1 Activity and Central Arterial Aging

血管紧张素II（Ang II）信号传导，包括基质金属蛋白酶II型（MMP 2）活化，已与血管平滑肌细胞（VSMC）的迁移/侵袭和增殖能力或衰老能力的年龄相关增加以及动脉老化的促炎标志相关。钙蛋白酶-1激活是成纤维细胞中MMP 2表达所必需的，并且在心肌细胞中由Ang II诱导。本研究记录了钙蛋白酶-1与其底物在控制动脉壁内与年龄相关的促炎状态中的作用结果。目前的研究结果表明，转录，翻译和活性的钙蛋白酶-1显着上调，在大鼠腹主动脉或早期通过主动脉VSMC从老年（30个月）FXBN大鼠相比，年轻（8个月）。双重免疫标记的动脉壁表明，共定位的钙蛋白酶-1和Ang II蛋白增加内的老年动脉壁。为了进一步探讨钙蛋白酶-1与血管紧张素Ⅱ的分子关系，我们将血管紧张素Ⅱ长期注入幼鼠，或用血管紧张素Ⅱ处理培养的主动脉环和VSMC。血管紧张素Ⅱ诱导钙蛋白酶-1蛋白和活性表达的主动脉壁在体内和体外和VSMC。钙蛋白酶抑制剂1 calpastatin可阻断血管紧张素Ⅱ介导的年龄相关的MMP 2活性增加和VSMC迁移。钙蛋白酶-1在年轻VSMC中的过表达导致完整波形蛋白的裂解，以及增加的迁移能力，以及MMP活性模仿老年VSMC的MMP活性，其被MMP抑制剂GM 6001阻断。 此外，年龄相关的中央动脉壁硬化与细胞外基质（ECM）重塑有关，包括纤维化、弹性蛋白溶解和钙化。血管紧张素Ⅱ诱导MMP 2和钙蛋白酶-1的表达和活性在动脉壁。但钙蛋白酶-1在MMP 2激活和ECM重塑中的作用仍不清楚。因此，我们进一步调查。双重免疫标记表明，与年轻大鼠相比，老年大鼠VSMC和老年动脉壁内的钙蛋白酶-1和MMP 2的共定位分别增加。 钙蛋白酶-1的过表达诱导MMP 2转录物、蛋白水平和活性，部分地通过增加膜型1 MMP（MT 1-MMP）（MMP 2的激活剂）与金属蛋白酶组织抑制剂2（TIMP 2）（TIMP 2的抑制剂）的比率。钙蛋白酶-1过表达诱导的MMP 2活化的作用与增加的胶原蛋白I、II和III的产生和血管钙化有关。此外，钙蛋白酶-1的过表达还诱导转化生长因子-β 1/ Sma和Mad（母亲对decapentaplegic）SMAD信号传导，弹性蛋白降解（弹性蛋白溶解），碱性磷酸酶激活和钙沉积，但降低钙化抑制剂，骨桥蛋白和骨连接素的表达，在体外培养的VSMC中，并在离体颈动脉环。 TIMP 2可部分降低这些效应。 有趣的是，钙蛋白酶-1和II型胶原（钙化的一种成分）在老化的人主动脉内膜中增加。在老年人主动脉壁中，与大体正常区域相比，钙蛋白酶-1和胶原蛋白II在动脉钙化斑块区域中高度表达。两种蛋白酶，钙蛋白酶-1和MMP 2的相互作用导致活性MMP 2的分泌，其通过增强胶原蛋白产生、弹性蛋白降解、生物活化和促进血管钙化的矿化来调节ECM重塑。 最近的研究表明，calpain-1在人动脉粥样硬化斑块的肩部、基底部和冠部有明显表达，与炎症（CD 68+巨噬细胞浸润）和VSMCs的衰老炎症密切相关，并影响动脉粥样硬化斑块的易损性。钙蛋白酶-1蛋白在人类大体正常的主动脉壁中显著增加，特别是在内膜中随着年龄的增长。有趣的是，与年龄相关的乳脂球EGF-8（MFG-E8）片段，medin，一种淀粉样蛋白的增加，显着增强钙蛋白酶-1蛋白的表达。这些结果确立了钙蛋白酶-1作为一种新的候选分子，以促进年龄相关的ECM重塑，钙化，淀粉样变性及其伴随的动脉粥样硬化的风险。年龄相关性高血压、动脉粥样硬化和动脉粥样硬化斑块不稳定性以及阿尔茨海默病（血管性痴呆）背后的钙蛋白酶-1的详细分子和细胞机制正在进行调查。

Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in either migration/invasion and proliferation capacity or senescent capacity of vascular smooth muscle cells (VSMCs), and to proinflammatory hallmarks of arterial aging. Calpain-1 activation is required for MMP2 expression in fibroblasts and is induced in cardiomyocytes by Ang II. The consequences of engagement of calpain-1 with its substrates in governing the age-associated proinflammatory status within the arterial wall are documented in this study. The present findings have demonstrated that transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) FXBN rats compared to young (8-mo). Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II protein increases within the aged arterial wall. To further explore the molecular relationship of calpain-1 to Ang II, we chronically infused Ang II into young rats, or treated cultured aortic rings and VSMC with Ang II. Ang II induces calpain-1 protein and activity expression in the aortic walls in vivo and ex vivo and VSMC in vitro. The Ang II mediated, age-associated increased MMP2 activity and migration in VSMC are both blocked by calpain inhibitor, 1 calpastatin. Over-expression of calpain-1 in young VSMC results in the cleavage of intact vimentin, and an increased migratory capacity, and MMP activity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001. Furthermore, age-associated central arterial wall stiffening is linked to extracellular matrix (ECM) remodeling, including fibrosis, elastolysis, and calcification. Ang II induces both MMP2 and calpain-1 expression and activity in the arterial wall. But the role of calpain-1 in MMP2 activation and ECM remodeling remains unknown. Thus, we investigated further. Dual immunolabeling demonstrates increased co-localization of calpain-1 and MMP2 within old rat VSMCs and old arterial walls compared as young ones, respectively. Over-expression of calpain-1 induces MMP2 transcripts, protein levels and activity, in part, by increasing the ratio of membrane-type 1 MMP (MT1-MMP), an activator of MMP2, to tissue inhibitor of metalloproteinases 2 (TIMP2), an inhibitor of TIMP2. The effect of calpain-1 over-expression-induced MMP2 activation is linked to increased collagen I, II and III production and vascular calcification. In addition, over-expression of calpain-1 also induces transforming growth factor-beta1/ Sma and Mad (Mothers against decapentaplegic) SMAD signaling, elastin degradation (elastolysis), alkaline phosphatase activation and calcium deposits, but reduces the expression of calcification inhibitors, osteopontin and osteonectin, in cultured VSMCs in vitro, and in carotid artery rings ex vivo. These effects are partially reduced by TIMP2. Interestingly, both calpain-1 and collagen II, an element of calcification, increase within the aging human aortic intima. In the aged human aortic wall, both calpain-1 and collagen II are highly expressed in arteriosclerotic calcific plaque areas compared to grossly normal areas. Crosstalk of two proteases, calpain-1 and MMP2, leads to secretion of active MMP2, which modulates ECM remodeling via enhancing collagen production, elastin degradation, bioactivation and mineralization facilitating vascular calcification. Recent studies indicate that calpain-1 is markedly expressed in the shoulder, base, and cap of human atherosclerotic plaques, which is closely associated with inflammation (CD68+ macrophage infiltration) and senescent inflamed VSMCs and impacts on the vulnerability of atherosclerotic plaque. Calpain-1 protein is significantly increased in the human grossly normal aortic walls, particularly in the intima with aging. Interestingly, age-associated increases of the milk fat globule EGF-8 (MFG-E8) fragment, medin, an amyloid protein, markedly enhances calpain-1 protein expression. These results establish calpain-1 as a novel molecular candidate to facilitate age-associated ECM remodeling, calcification, amyloidosis and its attendant risk for atherosclerosis. The detailed molecular and cellular mechanisms of calpain-1 behind age-associated hypertension, atherosclerosis and atherosclerotic plaque instability, and Alzheimers disease (vascular dementia) are under the ongoing investigation.