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与其底物结合在调节动脉壁内与年龄相关的促炎和前钙化状态中的后果尚不清楚。 在这项研究中，我们的结果首先证明了在旧的主动脉壁中，Calain-1基因的转录水平、蛋白水平和活性随年龄增加而增加；Ang II在体内和体外的主动脉壁和体外VSMC中诱导了Calain-1的表达；Ang II介导的与年龄相关的MMP2活性的增加被Calain抑制剂1或CAST阻断，这表明Ang II处理后的MMP2激活需要Calain的活性。在年轻的VSMC中，Calain-1的过度表达模拟了老年VSMC迁移能力的增加。总而言之，这些发现表明Calain-1活性增加，作为被夸大的Ang II信号通路的中心组成部分，该通路参与了与年龄相关的动脉重构。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和VSMC迁移有关。 此外，与年龄相关的动脉改变的特点是VSMC从收缩表型转变为合成表型，即细胞骨架重构和迁移能力增强，以及细胞外基质重塑(ECM)，即I型和III型胶原，可被蛋白酶降解，成为未成熟的胶原，形成前钙化生态位。我们最近的研究表明，Calain-1在VSMC内增加，并协调血管紧张素II级联反应，介导VSMC在老化的主动脉壁内的表型转变，并在导致前钙化的动脉基质改变中发挥重要作用。我们的结果表明，携带全长cDNA的重组腺病毒(pAD/CANP1)在年轻的VSMC中过表达异位Calain-1，使Col I和Col III增加到未处理的老年对照细胞的水平。此外，在年轻的VSMC中，Calain-1的过度表达减少了模拟衰老的反钙化分子骨桥蛋白(OPN)和骨连蛋白(ON)。相反，年轻的VSMC感染含有calastatain cDNA的重组腺病毒(pAD/CAST)，OPN和ON增加。随着年龄的增长，VSMCs内Calain-1活性增加，导致胶原生成增加，抗钙化分子减少，有利于脱钙化状态，这是随着年龄增长动脉基质重塑的潜在分子机制。因此，靶向calain-1是延缓或逆转与年龄相关的动脉前炎症和钙化状态的潜在方法，该状态也与高血压、动脉粥样硬化和中风等疾病有关。

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 and procalcification status within the arterial wall, remain unknown. In this study, our results first demonstrate an age-associated increase of calpain-1 gene transcripts, protein levels and activity in the old aortic wall; increased 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, is blocked by calpain inhibitor 1 or CAST, indicating that calpain activity is required for MMP activation by Ang II treatment. An increase in old VSMC migratory capacity is mimicked in young VSMC by over-expression of calpain-1. Collectively, these findings point to increased calpain-1 activity, as a central component of the exaggerated Ang II signaling pathway, which is involved in age-associated arterial remodeling. 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. Furthermore, age-associated arterial alterations are characterized both by a shift of VSMC from a contractile to synthetic phenotype, i.e., cytoskeleton restructuring and enhancement of migratory capability, and extracellular matrix remodeling (ECM), ie., collagen types I and III, which could be degenerated by proteases and become immature collagen to form a procalcification niche. Our recent study indicates that calpain-1, increases within VSMC; and orchestrates an angiotensin II cascade which mediates a VSMC phenotype shift within the aged aortic wall, and plays an important role in generating arterial matrix alterations that lead to procalcification. Our results show that over-expression of ectopic calpain-1 by a recombinant adenovirus harboring full length cDNA (pAd/CANP1) in young VSMC increased Col I and III up to the levels of untreated old control cells. Further, over-expression of calpain-1 in young VSMC decreases the counter-calcification molecules osteopontin (OPN) and osteonectin (ON), which mimics aging. In contrast, young VSMC infected with recombinant adenovirus containing calpastatain cDNA (pAd/CAST), an endogenous inhibitor of calpain), increases OPN and ON. An age-associated increase of calpain-1 activity within VSMCs results in enhanced collagen production and reduced counter-calcification molecules, favoring a procacification status, which is a potential molecular mechanism of arterial matrix remodeling with aging. Thus, targeting calpain-1 is a potential approach to delay or reverse the age-associated arterial pro-inflammation and -calcification state that is also associated with diseases such as hypertension, atherosclerosis, and stroke.