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型（MMP2）激活，与血管平滑肌细胞（VSMCs）迁移/侵袭和增殖能力的年龄相关增加以及动脉衰老的促炎标志有关。在成纤维细胞中MMP2的表达需要Calpain-1的激活，并且在心肌细胞中由Ang II诱导。本研究记录了calpain-1及其底物在动脉壁内控制与年龄相关的促炎状态的后果。目前的研究结果表明，与年轻的FXBN大鼠（8个月）相比，老年（30个月）大鼠主动脉或早期通道主动脉VSMC中calpain-1的转录、翻译和活性显著上调。动脉壁的双重免疫标记表明calpain-1和Ang II在老年动脉壁内的共定位增加。为了进一步探讨calpain-1与Ang II的分子关系，我们将Ang II长期注入幼龄大鼠，或用Ang II处理培养的主动脉环和VSMC。Ang II诱导体内、离体主动脉壁和体外VSMC中calpain-1的表达。在VSMC中，Ang II介导的年龄相关的MMP2活性和迁移增加都被calpain抑制剂1 calpastatin阻断。calpain-1在年轻VSMC中的过度表达导致完整vimentin的切割，并增加模仿老年VSMC的迁移能力，这被MMP抑制剂GM6001阻断。

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.