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(VSMC)迁移能力的增加有关，并与动脉老化的其他促炎特征有关。在成纤维细胞中，MMP2的表达需要Calain-1的激活，并由Ang II诱导心肌细胞中的MMP2的表达。然而，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尚不清楚。 目前的研究结果表明，与年轻大鼠(8个月)相比，老年(30个月)大鼠的主动脉或早期通过主动脉的VSMC中Calain-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，Calain-1和Ang II在老年动脉壁内的共存增加。为了进一步探讨Calain-1与Ang II的关系，我们将Ang II长期注入幼年大鼠体内，用Ang II处理培养的主动脉环或VSMC，并构建了携带Calain-1(CANP1)或其内源性抑制物calastatin(CAST)的腺病毒，并将其感染VSMC。血管紧张素转换酶II可诱导血管内皮细胞、血管内皮细胞和血管内皮细胞中Calain-1的表达。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或CAST阻断。在年轻的VSMC中过表达calain-1导致完整的Vimentin被切割，并增加迁移能力，这类似于老年VSMC，这一点可被基质金属蛋白酶抑制剂GM6001阻断。 钙化细胞外基质(ECM)是老年动脉壁的一个显著特征，其主要原因是血管平滑肌细胞(VSMC)表型的综合改变。老年VSMC分泌的基质为血管钙化(VC)提供了良好的微环境：矿物质代谢异常，磷酸酶活性增强，骨桥蛋白(OPN)和骨连素(ON)减少。在本研究中，双重免疫染色显示Calain-1与I型胶原(Col I)或I型胶原(Col III)共定位，在传代早期的VSMC中，随着年龄的增长，Col-1的表达增加。此外，通过重组腺病毒(PAD/CANP1)在年轻的VSMC中过表达calain-1，与对照病毒(Pad/GFP)相比，Col I增加了2.18倍，Col III增加了1.99倍，达到了老年对照细胞的水平。在培养的年轻VSMC中，与pAD/GFP感染相比，高表达calain-1的VSMC分泌的碱性磷酸酶活性增加了35%，细胞内钙含量(7.9-0.4比4.6-0.3 ug/mg总蛋白，N=3)增加。有趣的是，在年轻的VSMC中过表达Calain-1使OPN减少了65%，比模拟衰老的对照细胞减少了30%。相反，年轻的VSMC感染含有calastatain cDNA的重组腺病毒(pAD/CAST)后，OPN增加了48%，比对照组增加了54%。 因此，在与年龄相关的动脉Ang II/MMP2信号级联中，Calain-1的激活是一个关键的分子事件，与细胞骨架蛋白重组、VSMC迁移和前钙化有关。因此，靶向calain-1有可能延缓或逆转动脉重塑和动脉钙化，这些都是与年龄相关的疾病，即动脉粥样硬化的基础。

Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in migration capacity of vascular smooth muscle cells (VSMC), and to other proinflammatory features 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, however, in governing the age-associated proinflammatory status within the arterial wall, remains unknown. 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) 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 relationship of calpain-1 to Ang II, we chronically infused Ang II into young rats, and treated cultured aortic rings or VSMC with Ang II. We also constructed adenoviruses harboring calpain-1 (CANP1) or its endogenous inhibitor calpastatin (CAST) and infected these into VSMC. 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 or CAST. Over-expression of calpain-1 in young VSMC results in cleavage of intact vimentin, and an increased migratory capacity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001. It is known that calcified extracellular matrix (ECM) is a salient feature of the aged arterial wall, which is contributed mainly by a synthetic shift of vascular smooth muscle cells (VSMC) phenotype. Secreted matrices from aged VSMC produce a favorable microenviroment of vascular calcification (VC): abnormal mineral metabolism with enhanced phosphatase activity while osteopontin (OPN) and osteonectin (ON) are decreased. In this study, dual immunostaining shows that calpain-1 colocalized with collagen I (Col I) or III (Col III), which increases in early passage VSMC with aging. Further, over-expression of calpain-1 by a recombinant adenovirus (pAd/CANP1) infection in young VSMC increased Col I by 2.18- fold and Col III by 1.99-fold compared to control virus (pAd/GFP), up to levels of old control cells. In cultured young VSMC, over-expression of calpain-1 increased both secreted alkaline phosphotase activity by 35% and intracellular calcium content (7.9 0.4 vs 4.6 0.3 ug Ca2+/mg total protein, N=3) compared with pAd/GFP infection. Interestingly, over-expression of calpain-1 in young VSMC decreases the OPN by 65% and ON by 30% over control cells, which mimics aging. Conversely, young VSMC infected with recombinant adenovirus containing calpastatain cDNA (pAd/CAST), an endogenous inhibitor of calpain-1, increases OPN by 48% and ON by 54% over control cells. Thus, Calpain-1 activation is a pivotal molecular event in the age-associated arterial Ang II/MMP2 signaling cascade that is linked to cytoskeleton protein restructuring and VSMC migration and to procalcification. Therefore, targeting calpain-1 has the potential to delay or reverse the arterial remodeling and arterial calcification that underlies age-associated diseases i.e. atherosclerosis.