Calpain-1 Activity and Central Arterial Aging

血管紧张素II(Ang II)信号，包括基质金属蛋白酶II(MMP2)的激活，已被认为与年龄相关的血管平滑肌细胞(VSMCs)迁移/侵袭和增殖能力的增加有关，并与动脉老化的促炎标志有关。在成纤维细胞中，MMP2的表达需要Calain-1的激活，在Ang II诱导的心肌细胞中，Calain-1的激活是必需的。本研究记录了Calain-1与其底物在调节动脉壁内与年龄相关的促炎状态中的作用。目前的研究结果表明，老年(30mo)FXBN大鼠主动脉或早期通道型VSMC中calain-1的转录、翻译和活性显著高于青年(8mo)。动脉壁的双重免疫标记表明，Calain-1和Ang II在老年动脉壁内的共存增加。为了进一步探讨血管紧张素转换酶-1与血管紧张素转换酶II的分子关系，我们将血管紧张素转换酶II慢性注入幼年大鼠体内，或用血管紧张素转换酶II处理培养的主动脉环和VSMC。Ang II介导的、与年龄相关的MMP2活性增加和VSMC迁移均可被Calain抑制剂1所阻断。在年轻的VSMC中过表达calain-1导致完整的Vimentin被切割，迁移能力增加，这类似于老年VSMC，这一过程可被基质金属蛋白酶抑制剂GM6001阻断。 此外，与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、弹性溶解和钙化。Ang II可诱导MMP2和calain-1在动脉壁的表达和活性。但Calain-1在MMP2激活和ECM重塑中的作用尚不清楚。因此，我们进行了进一步的调查。双重免疫标记显示，与年轻大鼠相比，Calain-1和MMP2在老年大鼠VSMCs和老年动脉壁中的共同定位分别增加。Calain-1的过表达诱导MMP2的转录、蛋白水平和活性，部分是通过增加MMP2的激活剂-1型膜基质金属蛋白酶(MT1-MMPs)与金属蛋白酶组织抑制因子2(TIMP2)的比率。Calain-1过度表达诱导MMP2激活的作用与增加I型、II型和III型胶原的产生和血管钙化有关。此外，在体外培养的VSMCs和体外培养的颈动脉环中，Calain-1的过表达还可诱导转化生长因子-β1/Sma和Mad信号通路、弹性蛋白降解(弹性溶解)、碱性磷酸酶激活和总钙含量，但降低钙化抑制因子骨桥蛋白和骨联素的表达。TIMP2可部分降低这些影响。有趣的是，钙化的一种成分Calain-1和胶原II在老化的人主动脉内膜中都增加了。在老年人的主动脉壁中，钙化斑块中钙化斑块中钙化钙化斑块的钙化钙化斑块中，钙蛋白酶-1和II型胶原蛋白的表达高于大体正常区域。两种蛋白水解酶-1和MMP2的相互作用导致活性MMP2的分泌，MMP2通过促进胶原的产生、弹性蛋白的降解、生物激活和促进血管钙化来调节ECM的重塑。 最近的研究表明，Calain-1在人类动脉粥样硬化斑块的肩部、基底部和帽部显著表达，与炎症(CD68+巨噬细胞浸润)密切相关。CaPain-1蛋白在人体正常的主动脉壁中显著增加，特别是在随着年龄增长的内膜中。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，显著增强Calain-1蛋白的表达。这些结果确立了Calain-1作为一种新的候选分子，用于促进与年龄相关的ECM重塑、钙化、淀粉样变性及其伴随的动脉粥样硬化风险。Calain-1在老年性高血压、动脉粥样硬化和动脉粥样硬化斑块不稳定以及阿尔茨海默病(血管性痴呆)背后的详细分子和细胞机制仍在研究中。

Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in migration/invasion and proliferation 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 demonstrate 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 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 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 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 total calcium content, 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 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). 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 still under investigation.