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，在Ang II诱导的心肌细胞中，Calain-1的激活是必需的。本研究记录了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。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。为了进一步探讨血管紧张素转换酶-1与血管紧张素转换酶II的关系，我们将血管紧张素转换酶II慢性注入幼年大鼠体内，并用血管紧张素转换酶II处理培养的主动脉环或VSMC。血管紧张素转换酶II可诱导血管内皮细胞和体外培养的VSMC表达Calain-1。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。 此外，与年龄相关的中央动脉壁僵硬与细胞外基质(ECM)重塑有关，包括纤维化和血管钙化。Ang II可诱导MMP2和calain-1在动脉壁的表达和活性。但Calain-1在MMP2激活和ECM重塑中的作用尚不清楚。因此，我们做了进一步的调查。双重组织免疫标记显示Calain-1和MMP2在老年大鼠血管平滑肌细胞中共定位。Calain-1的过表达诱导MMP2的转录、蛋白水平和活性，部分是通过增加膜型1基质金属蛋白酶(MT1)与金属蛋白酶组织抑制因子2(TIMP2)的比率。Calain-1过度表达诱导MMP2激活的效果与增加I型和III型胶原的产生和血管钙化有关。此外，在体外培养的血管平滑肌细胞和体外培养的颈动脉环中，Calain-1过表达还诱导转化生长因子-β1/SMAD信号转导、弹性蛋白降解、碱性磷酸酶激活和总钙含量，但降低钙化抑制因子骨桥蛋白和骨联素的表达。这些作用被TIMP2部分消除。有趣的是，随着年龄的增长，人主动脉内膜中的钙蛋白酶-1和II型胶原蛋白都会增加。在老年人的主动脉壁中，钙蛋白酶-1和II型胶原在动脉硬化斑块区域与大体正常区域相比都高表达。Calain-1和MMP2两种蛋白酶的相互作用导致活性MMP2的分泌，MMP2通过促进胶原生成和促进血管钙化来调节ECM重塑。 最近的研究表明，Calain-1在人类动脉粥样硬化斑块的肩部、基底部和帽部显著表达。这些结果表明，Calain-1是一种新的候选分子，可促进年龄相关的ECM重塑及其导致动脉粥样硬化的风险。年龄相关的动脉粥样硬化和动脉粥样硬化斑块不稳定性背后的详细分子和细胞机制正在调查中。

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 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) 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. 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. Furthermore, age-associated central arterial wall stiffness is linked to extracellular matrix (ECM) remodeling, including fibrosis and vascular 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 did further investigation. Dual histo-immunolabeling demonstrates co-localization of calpain-1 and MMP2 within old rat vascular smooth muscle cells. Over-expression of calpain-1 induces MMP2 transcripts, protein levels and activity, in part, by increasing the ratio of membrane-type 1 MMP (MT1MMP) to tissue inhibitor of metalloproteinases 2 (TIMP2). The effect of calpain-1 over-expression-induced MMP2 activation is linked to increased collagen I and III production and vascular calcification. In addition, over-expression of calpain-1 also induces transforming growth factor-beta1/SMAD signaling, elastin degradation, alkaline phosphatase activation and total calcium content, but reduces the expression of calcification inhibitors, osteopontin and osteonectin, in cultured vascular smooth muscle cells in vitro and in carotid artery rings ex vivo. These effects are partially abolished by TIMP2.Interestingly, both calpain-1 and collagen II increase with aging within human aortic intima. In the aged human aortic wall, both calpain-1 and collagen II are highly expressed in arteriosclerotic plaque areas compared to grossly normal areas. Cross-talk of two proteases, calpain-1 and MMP2, leads to secretion of active MMP2, which modulates ECM remodeling via enhancing collagen production and facilitating vascular calcification. Recent studies indicate that calpain-1 is markedly expressed in the shoulder, base, and cap of human atherosclerotic plaques. These results establish calpain-1 as a novel molecular candidate to facilitate age-associated ECM remodeling and its attendant risk for atherosclerosis. The detailed molecular and cellular mechanisms behind age-associated atherosclerosis and atherosclerotic plaque instability are under investigation.