Calpain-1 Activity and Central Arterial Aging

National Institute On Aging

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Abstract

Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, is linked to age-associated increases in the migration/invasion vascular smooth muscle cells (VSMCs), their proliferation or senescent capacity and other proinflammatory hallmarks of arterial aging. Calpain-1 activates MMP2 expression in fibroblasts and is induced by Ang II in cardiomyocytes. The consequences of engagement of calpain-1 with its substrates in governing the age-associated proinflammatory status, and remodeling within the arterial wall are documented in this study. The present findings have demonstrated that the transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) FXBN rats when compared to young (8-mo). Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II protein increases within the aged arterial wall. To further explore the molecular relationship between calpain-1 and Ang II, we chronically infused young rats with Ang II, or treated cultured aortic rings and VSMCs with Ang II. Ang II induces calpain-1 protein and activity expression in the aortic walls in vivo and ex vivo and VSMCs in vitro. The Ang II mediated, age-associated increased MMP2 activity and migration of VSMCs are both blocked by the calpain-1 inhibitor, calpastatin. Over-expression of calpain-1 in young VSMCs results in the cleavage of intact vimentin, an increased migratory capacity, and MMP2 activity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001. Furthermore, age-associated changes of the central arterial wall, endothelial dysfunction and stiffening are linked to extracellular matrix (ECM) remodeling, including fibrosis, elastolysis, and 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 investigated further. Dual immunolabeling demonstrates increased co-localization of calpain-1 and MMP2 within old rat VSMCs and old arterial walls when compared to young. Over-expression of calpain-1 induces MMP2 mRNA, protein levels and activity, in part, by increasing the ratio of membrane-type 1 MMP (MT1-MMP), an activator of MMP2, to tissue inhibitor of metalloproteinases 2 (TIMP2), an inhibitor of TIMP2. The effect of calpain-1 over-expression induced MMP2 activation is linked to increased collagen I, II and III production and vascular calcification. In addition, over-expression of calpain-1 also induces transforming growth factor-beta1 (TGF-1)/ Sma and Mad (Mothers against decapentaplegic) (SMAD) signaling, elastin degradation (elastolysis), alkaline phosphatase activation, osteocalcin, and calcium deposits, and reduces the expression of calcification inhibitors, osteopontin, and osteonectin, in cultured VSMCs in vitro, and in carotid artery rings ex vivo. These effects are partially reduced by TIMP2. Interestingly, both calpain-1 and collagen type II, an element of cartilage calcification, increase within the aging human aortic wall. Both calpain-1 and collagen II are highly expressed in arteriosclerotic calcific plaque regions compared to grossly normal areas in the aged human aortic wall. Crosstalk of two proteases, calpain-1 and MMP2, lead to the secretion of active MMP2, which modulates ECM remodeling via increased collagen production, elastin degradation, the breakdown of milk fat globule EGF VIII (MFG-E8) into medin, a small fragment amyloidogenic protein, and the bioactivation and mineralization that facilitates vascular calcification with advancing age. Here we report distinct material properties of primary VSMCs isolated from the thoracic aorta of adult (8 months) vs. aged (30 months) F344XBN rats, which are linked with calpain-1/MMP2/TGF-beta 1 activation. Individual VSMCs derived from aged animals showed an internal network of the actin cytoskeleton, exhibiting increased stiffness and frictional moduli than those derived from the adult animals. This discrete mechanical response was long-lived in culture and persistent across a physiological range of matrix rigidity. Strikingly, the pro-fibrotic molecule, TGF-beta 1, emerged as a specific modifier of age associated VSMC stiffening in vitro. TGF-1 reinforced the mechanical phenotype of arterial aging in VSMCs on multiple time and length scales through the clustering of mechanosensitive alpha 5 beta 1 and alpha v beta 3 integrins. These studies identify a novel nodal point for the long-range regulation of VSMC stiffness and served as a proof-of-concept that the broad-based inhibition of TGF-beta 1 expression, or TGF-beta 1 signal transduction in VSMCs, may be a useful therapeutic approach to mitigate the pathologic progression of central arterial wall stiffening associated with aging. Recent studies indicate that calpain-1 is markedly expressed in the shoulder, base, and cap of human atherosclerotic plaques, which is closely associated with inflammation (CD68+ macrophage infiltration) and senescent inflamed VSMCs, which impacts the vulnerability of atherosclerotic plaque. Calpain-1 protein is significantly increased in aging human grossly normal aortic walls, particularly in the intima. Interestingly, age-associated increases of medin, a fragment of MFG-E8 and an amyloid protein, markedly enhances calpain-1 protein expression. These results establish calpain-1 as a novel molecular candidate to facilitate age-associated ECM remodeling, calcification, amyloidosis, and its attendant risk for atherosclerosis. The detailed molecular and cellular mechanisms of calpain-1 behind age-associated hypertension, atherosclerotic progression and atherosclerotic plaque instability, and Alzheimer's disease (vascular dementia) are still under ongoing investigation. In summary, aging is a major risk factor for quintessential cardiovascular diseases, which are closely related to arterial proinflammation. The age-related alterations of the amount, distribution, and properties of the collagen fibers, such as cross-linking and degradation in the arterial wall, are the major sequelae of proinflammation. In the aging arterial wall, collagen types I, II, and III are predominant, and are mainly produced by stiffened VSMCs governed by proinflammatory signaling molecules, leading to profibrosis. Profibrosis is regulated by an increase in the proinflammatory molecules Ang II, MFG-E8, and TGF-beta 1 signaling and a decrease in the vasorin signaling cascade. The release of these proinflammatory factors triggers the activation of MMP2 and activates profibrogenic TGF- beta 1 signaling, contributing to profibrosis. The age-associated increase in activated MMP2 cleaves latent TGF-beta and subsequently increases TGF-beta 1 activity leading to collagen deposition in the arterial wall. Furthermore, a blockade of the proinflammatory signaling pathway alleviates fibrogenic signaling, reduces profibrosis, and prevents arterial stiffening with aging. Thus, age-associated proinflammatory-profibrosis coupling is the underlying molecular mechanism of arterial stiffening with advancing age.