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、增殖和炎症的年龄相关增加有关。Calpain-1激活心肌成纤维细胞中MMP2的表达，并在心肌细胞中被angii诱导。calpain-1与其底物在控制年龄相关的促炎状态和动脉壁重塑中的作用的后果在本项目中得到了解决。我们的研究结果表明，与幼龄动物（8个月）相比，老年（30个月）Fisher 344杂交褐挪威（FXBN）大鼠主动脉或早期主动脉VSMC中calpain-1的转录、翻译和激活显著上调。动脉壁的双重免疫标记表明calpain-1和Ang II蛋白在老年主动脉壁内共定位增加。为了探索calpain-1与Ang II之间的分子关系，我们长期给幼鼠注入Ang II，或用Ang II处理培养的主动脉环和VSMCs。结果表明，Ang II可诱导体内、离体主动脉壁和体外VSMCs中calpain-1蛋白的表达和激活。Ang II介导的年龄相关的MMP2激活和VSMCs的迁移均被calpain-1抑制剂calpastatin阻断。年轻VSMC中calpain-1的过度表达导致完整vimentin的分裂，迁移能力增加，MMP2活性模仿未治疗的老年VSMC， MMP2活性被MMP抑制剂GM6001阻断。

Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, is linked to age-associated increases in the migration, invasion, proliferation, and inflammation of vascular smooth muscle cells (VSMCs). Calpain-1 activates MMP2 expression in myocardial fibroblasts and is induced by Ang II in cardiomyocytes. The consequences of engagement of calpain-1 with its substrates in governing the age-associated proinflammatory status, and remodeling within the arterial wall are addressed in this project. Our findings have demonstrated that the transcription, translation, and activation of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) Fisher 344 crossbreed Brown Norway (FXBN) rats when compared to young (8-mo) animals. Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II protein increases within the aged aortic wall. To explore the molecular relationship between calpain-1 and Ang II, we chronically infused young rats with Ang II, or treated cultured aortic rings and VSMCs with Ang II. The results indicate that Ang II induces calpain-1 protein expression and activation in the aortic walls in vivo and ex vivo and VSMCs in vitro. The Ang II mediated, age-associated increased MMP2 activation and migration of VSMCs are both blocked by the calpain-1 inhibitor, calpastatin. Over-expression of calpain-1 in young VSMCs results in the cleavage of intact vimentin, an increased migratory capacity, and MMP2 activity mimicking that of old untreated VSMC, which is blocked by the MMP inhibitor, GM6001. Age-associated changes of the central arterial endothelial dysfunction and stiffening are 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. We have found that calpain-1 plays an important role in MMP2 activation and ECM remodeling in the arterial walls or VSMCs. Dual immunolabeling demonstrates increased co-localization of calpain-1 and MMP2 within old rat VSMCs and old arterial walls when compared with young animals or cells. Over-expression of calpain-1 upregulates MMP2 mRNA, and protein levels and its 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 associated with increased collagen I, II and III production and vascular calcification in the arterial wall with aging. Notably, over-expression of calpain-1 induces transforming growth factor-beta1 (TGF-1)/ Sma and Mad (Mothers against decapentaplegic 2/3) (SMAD2/3) signaling, elastin degradation (elastolysis), alkaline phosphatase activation, osteocalcin, and calcium deposits, and 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 inhibited by TIMP2. Interestingly, both calpain-1 and collagen type II, an element of cartilage calcification, increase within the aging human aortic wall. Both calpain-1 and collagen II are highly expressed in arteriosclerotic calcific plaque regions compared to grossly normal areas in the aged human aortic wall. Crosstalk of two proteases, calpain-1 and MMP2, lead to the increased secretion of active MMP2, which modulates ECM remodeling via increased collagen production, elastin degradation, the breakdown of milk fat globule EGF VIII (MFG-E8), an arterial inflammatory molecule, into medin, a small fragmented amyloidogenic protein, and the bioactivation and mineralization that facilitates vascular calcification with advancing age. Here we also report distinct material properties of primary VSMCs isolated from the thoracic aorta of young (8 months) vs. old (30 months) F344XBN rats, which are linked with calpain-1/MMP2/TGF-1 activation. Individual VSMCs derived from old animals showed an internal network of the actin cytoskeleton, exhibiting increased stiffness and frictional moduli than those derived from the adult animals. This discrete mechanical response was long-lived in culture and persistent across a physiological range of matrix rigidity. Strikingly, the pro-fibrotic molecule, TGF-1, emerged as a specific modifier of age associated VSMC stiffening in vitro. TGF-1 reinforced the mechanical phenotype of arterial aging in VSMCs on multiple time and length scales through the clustering of mechanosensitive 51 and v3 integrins. These studies identify a novel nodal point for the long-range regulation of VSMC stiffness, serving as a proof-of-concept that the broad-based inhibition of TGF-1 expression or TGF-1 signal transduction in VSMCs, may be a useful therapeutic approach to mitigate the pathophysiologic progression of central arterial wall stiffening associated with aging. Calpain-1 protein is significantly increased in aging human grossly normal aortic walls, particularly in the intima. Importantly, 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, which impacts the growth and necrotic core enlargement (vulnerability) of atherosclerotic plaques. Interestingly, age-associated increases of medin, a fragment of MFG-E8 and a common arterial amyloid protein, markedly enhances calpain-1 protein expression. These novel results suggest that calpain-1 as a novel molecular candidate to facilitate age-associated increases in atherosclerosis. The detailed molecular and cellular mechanisms of calpain-1 behind age-associated atherosclerotic progression and atherosclerotic plaque frailty are under ongoing investigation. In summary, aging is a major risk factor for quintessential cardiovascular diseases, which are closely related to calpain-associated arterial proinflammation. The age-related alterations of the amount, distribution, and properties of the collagen fibers, such as cross-linking and degradation in the arterial wall, are the major sequelae of proinflammation. In the aging arterial wall, collagen types I, II, and III are predominant, and are mainly produced by stiffened VSMCs, governed by proinflammatory signaling molecules, leading to profibrosis. Profibrosis is regulated by an increase in the proinflammatory molecules Ang II, MFG-E8, MMP2, and the TGF-1 signaling cascade. The release and activation of calpain-1 triggers the activation of MMP2 and activates profibrogenic TGF-1 signaling, contributing to profibrosis. The age-associated increase in activated MMP2 cleaves latent TGF- and subsequently increases TGF-1 activity leading to collagen deposition in the arterial wall. Notably, a blockade of the proinflammatory signaling pathway alleviates fibrotic signaling, reduces profibrotic, elastolysis, calcification, and prevents arterial stiffening with aging. Thus, age-associated increases in proinflammation, fibrosis, elastolysis, amyloidosis, and calcification are the underlying molecular mechanisms of arterial stiffening with advancing age.