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。 1. 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）。 2. 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。 3. 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。 此外，年龄相关的中央动脉壁硬度与细胞外基质（ECM）重塑有关，包括纤维化和血管钙化。血管紧张素II诱导基质金属蛋白酶2（MMP 2）和钙蛋白酶-1的表达和活性在动脉壁。但钙蛋白酶-1在MMP 2激活和ECM重塑中的作用仍不清楚。 4. 双重组织免疫标记显示老年大鼠血管平滑肌细胞内钙蛋白酶-1和MMP 2的共定位。 钙蛋白酶-1的过度表达诱导MMP 2的转录，蛋白质水平和活性，部分通过增加膜型1 MMP的金属蛋白酶2的组织抑制剂的比例。 5. 钙蛋白酶-1过表达诱导的MMP 2活化的作用与I型和III型胶原的产生增加和血管钙化有关。 6. 此外，钙蛋白酶-1的过度表达还诱导转化生长因子-β 1/Smad信号传导、弹性蛋白降解、碱性磷酸酶活化和总钙含量，但降低体外培养的血管平滑肌细胞和离体颈动脉环中钙化抑制剂骨桥蛋白和骨连接素的表达。 7. 有趣的是，钙蛋白酶-1和II型胶原蛋白在人主动脉内膜中随着年龄的增长而增加。在老年人主动脉壁中，与大体正常区域相比，钙蛋白酶-1和胶原蛋白II在动脉粥样硬化斑块区域中高度表达。 两种蛋白酶，钙蛋白酶-1和MMP 2的相互作用导致活性MMP 2的分泌，其通过增强胶原蛋白的产生和促进血管钙化来调节ECM重塑。这些结果确立了钙蛋白酶-1作为一种新的候选分子，以延缓与年龄相关的ECM重塑及其伴随的高血压和动脉粥样硬化的风险。

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, remain unknown. 1. 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). 2. 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. 3. 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. Angiotensin II induces both matrix metalloproteinase type 2 (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. 4. 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 MMPs to tissue inhibitor of metalloproteinases 2. 5. The effect of calpain-1 over-expression-induced MMP2 activation is linked to increased collagen I and III production and vascular calcification. 6. 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. 7. Interestingly, both calpain-1 and collagen II increase with aging within human aortic intima. In 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. These results establish calpain-1 as a novel molecular candidate to retard age-associated ECM remodeling and its attendant risk for hypertension and atherosclerosis.