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, proliferation, profibrosis, procalcification, and proinflammation of vascular smooth muscle cells (VSMCs). Calpain-1 activates MMP2 activity in myocardial fibroblasts and is induced by Ang II in cardiomyocytes. The consequences of engagement of calpain-1 activation with its substrates such as vitronectin in governing the age-associated proinflammatory status, and remodeling within the arterial wall are addressed in this project. Our findings have demonstrated that the transcription, translation, and activation of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC in primary culture from old (30-mo) Fisher 344 crossbreed Brown Norway (FXBN) rats when compared to young (8-mo) animals or young cells. Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II protein increases within the aged aortic wall and VSMCs. To 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. The results indicate that Ang II induces calpain-1 protein expression and activation in the aortic walls in vivo and aortic rings ex vivo and VSMCs in vitro. The Ang II mediated, age-associated increased MMP2 activation, migration, and synthetic phenotypic shift of VSMCs are all 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 untreated VSMC, which is blocked by the MMP inhibitor, GM6001. Age-associated changes of the central arterial system include endothelial dysfunction and stiffening which are linked to extracellular matrix (ECM) remodeling, including fibrosis, elastolysis, and calcification. Ang II induces both MMP2 and calpain-1 expression and activation in the arterial wall and VSMCs. We have found that calpain-1 plays an important role in MMP2 activation and ECM remodeling in the arterial walls or VSMCs. Dual immunolabeling demonstrates increased co-localization of calpain-1 and MMP2 within old rat VSMCs and old arterial walls when compared with young animals or cells. Over-expression of calpain-1 upregulates MMP2 mRNA and protein levels, and its 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 associated with increased collagen I, II and III production and vascular calcification in the arterial wall and VSMCs with aging. Notably, over-expression of calpain-1 induces transforming growth factor-beta1 (TGF-1)/ Sma and Mad (Mothers against decapentaplegic 2/3) (SMAD2/3) 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 inhibited 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 increased secretion of active MMP2, which modulates ECM remodeling via increased collagen production, elastin degradation, the breakdown of milk fat globule EGF VIII (MFG-E8), an arterial inflammatory molecule, into medin, a small fragmented amyloidogenic protein, and the bioactivation and mineralization that facilitates vascular wall or cell calcification with advancing age. Here we also report distinct material properties of primary VSMCs isolated from the thoracic aorta of young (8 months) vs. old (30 months) F344XBN rats, which are linked with calpain-1/MMP2/transforming growth factor-beta 1 (TGF-1) activation. Individual VSMCs derived from old 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-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 51 and v3 integrins. These studies identify a novel nodal point for the long-range regulation of VSMC stiffness, serving as a proof-of-concept, that the broad-based inhibition of TGF-1 expression or TGF-1 signal transduction in VSMCs, may be a useful therapeutic approach to mitigate the pathophysiologic progression of central arterial wall stiffening associated with aging. Calpain-1 protein is significantly increased in aging human grossly normal aortic walls, particularly in the intima. Importantly, calpain-1 is markedly expressed in the shoulder, base, and cap of human atherosclerotic plaques, which is closely associated with inflammation (CD68+ macrophage infiltration),senescent, inflamed and dead VSMCs, which impacts the growth and necrotic core expansion of atherosclerotic plaques. Interestingly, age-associated increases of medin, a fragment of MFG-E8 and a common arterial amyloid protein in old upper bodies, markedly enhances calpain-1 protein expression. These novel results suggest that calpain-1 is a novel molecular candidate to facilitate age-associated increases in atherosclerosis and arterial amyloidosis. The detailed molecular and cellular mechanisms of calpain-1 behind age-associated atherosclerotic progression, atherosclerotic plaque frailty, and arterial amyloidosis is under ongoing investigation. 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 aged, stiffened VSMCs, governed by proinflammatory signaling molecules, leading to profibrosis. Profibrosis is regulated by an increase in the proinflammatory molecules Ang II, MFG-E8, MMP2, and the TGF-1 signaling cascade. The release and activation of calpain-1 triggers the activation of MMP2 and activates profibrogenic TGF-1 signaling, and medin aggregation, contributing to profibrosis. The age-associated increase in activated MMP2 cleaves latent TGF- and subsequently increases TGF-1 activity leading to collagen deposition in the arterial wall. Notably, a blockade of the proinflammatory signaling pathway alleviates fibrotic signaling, reduces profibrosisc, elastolysis, calcification, and prevents arterial stiffening and amyloidosis with aging. Thus, age-associated increases in proinflammation, fibrosis, elastolysis, amyloidosis, and calcification are the underlying molecular mechanisms of arterial stiffening with advancing age. In summary, aging is a major risk factor for quintessential cardiovascular diseases such as hypertension, atherosclerosis, heart failure, vascular related cognitive decline, and impairment, which are closely related to calpain-associated arterial proinflammation.