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）激活，与血管平滑肌细胞（VSMC）迁移能力的年龄相关性增加以及动脉老化的其他促炎特征有关。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。然而，钙蛋白酶-1与其底物的接合在动脉壁内与年龄相关的促炎状态中的后果仍然未知。 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通主动脉VSMC从老年（30个月）大鼠相比，年轻（8个月）。动脉壁的双重免疫标记表明，钙蛋白酶-1和Ang II的共定位在老年动脉壁内增加。为了进一步探讨钙蛋白酶-1与血管紧张素Ⅱ的关系，我们将血管紧张素Ⅱ长期输注到幼年大鼠体内，并用血管紧张素Ⅱ处理培养的主动脉环或VSMC。我们还构建了携带钙蛋白酶-1（CANP 1）或其内源性抑制剂钙蛋白酶抑制蛋白（CAST）的腺病毒，并将其感染到VSMC中。血管紧张素II诱导钙蛋白酶-1在体内和离体的主动脉壁和VSMC在体外的表达。Ang II介导的年龄相关的VSMC中MMP 2活性和迁移增加均被钙蛋白酶抑制剂1或CAST阻断。钙蛋白酶-1在年轻的VSMC中的过度表达导致完整的波形蛋白的裂解，以及模仿老年VSMC的迁移能力的增加，这被MMP抑制剂GM 6001阻断。 钙化的细胞外基质（ECM）是老年动脉壁的一个显著特征，其主要是由血管平滑肌细胞（VSMC）表型的合成性转变引起的。老年VSMC分泌的基质为血管钙化（VC）提供了有利的微环境：矿物质代谢异常，磷酸酶活性增强，骨桥蛋白（OPN）和骨连接素（ON）减少。在这项研究中，双重免疫染色显示钙蛋白酶-1与胶原蛋白I（Col I）或III（Col III）共定位，其在早期传代VSMC中随衰老而增加。 此外，与对照病毒（pAd/GFP）相比，在年轻VSMC中通过重组腺病毒（pAd/CANP 1）感染过表达钙蛋白酶-1使Col I增加2.18倍，Col III增加1.99倍，达到老年对照细胞的水平。 在培养的年轻VSMC，钙蛋白酶-1的过度表达增加了35%的分泌碱性磷酸酶活性和细胞内钙含量相比，pAd/GFP感染。有趣的是，与对照细胞相比，年轻VSMC中钙蛋白酶-1的过表达使OPN降低65%，ON降低30%，这模拟了衰老。相反，年轻的VSMC感染含有calpastatain cDNA的重组腺病毒（pAd/CAST），一种内源性calpain-1抑制剂，与对照细胞相比，OPN增加48%，ON增加54%。 因此，钙蛋白酶-1激活是与年龄相关的动脉血管紧张素II/MMP 2信号级联中的关键分子事件，其与细胞骨架蛋白重构和VSMC迁移以及前钙化有关。因此，靶向钙蛋白酶-1具有延迟或逆转动脉重塑和动脉钙化的潜力，动脉重塑和动脉钙化是年龄相关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 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.