Matrix Metalloproteinase 2 Activation of Transforming Growth Factor-β1 (TGF-β1) and TGF-β1–Type II Receptor Signaling Within the Aged Arterial Wall

Abstract

Objective-To study matrix metalloproteinase 2 (MMP-2) effects on transforming growth factor-␤1 (TGF-␤1) activation status and downstream signaling during arterial aging. Methods and Results-Western blotting and immunostaining showed that latent and activated TGF-␤1 are markedly increased within the aorta of aged Fisher 344 cross-bred Brown Norway (30 months of age) rats compared with adult (8 months of age) rats. Aortic TGF-␤1-type II receptor (T␤RII), its downstream molecules p-similar to mad-mother against decapentaplegic (SMAD)2/3 and SMAD4, fibronectin, and collagen also increased with age. Moreover, TGF-␤1 staining is colocalized with that of activated MMP-2 within the aged arterial wall and vascular smooth muscle cell (VSMC) in vitro, and this physical association was confirmed by coimmunoprecipitation. Incubation of young aortic rings ex vivo or VSMCs in vitro with activated MMP-2 enhanced active TGF-␤1, collagen, and fibronectin expression to the level of untreated old counterparts, and this effect was abolished via inhibitors of MMP-2. Interestingly, in old untreated rings or VSMCs, the increased TGF-␤1, fibronectin, and collagen were also substantially reduced by inhibition of MMP-2. Conclusions-Active TGF-␤1, its receptor, and receptor-mediated signaling increase within the aortic wall with aging.TGF-␤1 activation is dependent, in part at least, by a concomitant age-associated increase in MMP-2 activity. Thus, MMP-2-activated TGF-␤1, and subsequently T␤RII signaling, is a novel molecular mechanism for arterial aging.

Calorie Restriction Curbs Proinflammation That Accompanies Arterial Aging, Preserving a Youthful Phenotype

“…Hematoxylin and eosin, Picrosirius Red, Verhoeff's Elastic‐van Gieson, and Masson's trichrome stains (American MasterTech Scientific, Inc, Lodi, CA) were performed per the manufacturer's instructions as described previously 5, 6, 18…”

“…Zymography was performed according to modified protocols, as previously reported 5, 6, 7, 18. Sodium dodecyl sulfate polyacrylamide gel electrophoresis zymography was performed according to the manufacturer's protocol (ThermoFisher).…”

“…Immunoblotting of supernatant from thoracic aortae homogenates or VSMC lysates were performed as previously described 5, 6, 19, 20. Primary antibodies used for Western blotting are listed in Table.…”

“…VSMCs were enzymatically isolated as previously described 5, 6, 19, 20. Briefly, young and old AL and CR F344 rat thoracic aortae (n=3 animals/group) were rinsed in Hanks balanced salt solution containing 50 μg/mL penicillin, 50 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (ThermoFisher).…”

“…Increases in the local arterial wall proinflammatory molecules, platelet‐derived growth factor (PDGF), matrix metalloproteinase type II (MMP2) and transforming growth factor beta 1 (TGF‐β1), promote cellular and matrix phenotypic shifts that contribute to aortic wall thickening and stiffening that accompany advancing age in mammals, a prominent arterial phenotype of biological aging 4, 5, 6, 7, 8, 9. Interestingly, arterial proinflammatory conditions reduces the threshold or increases the susceptibility for pernicious inflammatory arterial events under hostile conditions such as hyperlipidemia 1, 10…”