MFG-E8 Signaling Promotes Elastolysis and Calcification in the Aging Aortic Wall

Objective: The proinflammatory phenotypic shift of vascular smooth muscle cells (VSMCs) is closely linked to the elastolysis and calcification in the aging arterial wall. Degradation of tropoelastin (TPELN) is a key molecular event which often accompanies the proinflammatory phenotypic shift of VSMCs, arterial elastolysis and calcification with aging. Milk fat globule-EGF factor 8 (MFG-E8), secreted mainly from VSMCs, predominantly binds to degenerated elastin fibers, and alters VSMCs phenotypes. Here, we investigated how MFG-E8 proinflammatory signaling in the arterial wall, in vivo, and VSMCs, in vitro, affects arterial elastolysis and calcification with advancing age. Methods and Results: In vivo immunostaining and immunoblotting studies indicated that MFG-E8, elastic lamina breaks, and calcium-phosphorus products were markedly increased while intact TPELN (~70Kda) protein was dramatically decreased in aortic walls or isolated aortic VSMCs harvested from old (30-month-old) vs. young (8-month-old) Fischer 344 crossbred Brown Norway rats (FXBN). In vitro studies demonstrated that (1) treating either young or old VSMCs to recombinant human MFG-E8 (rhMFG-E8) for 24 hours significantly reduced intact TPLEN levels and this effect was reduced by SB203580, a p38 mitogen-activated protein kinase inhibitor; (2) activated MMP-2 levels were significantly increased in both young and old VSMCs treated with rhMFG-E8, and this activation was also inhibited by SB203580; (3) MMP-2 physically interacted with TPLEN and cleaved intact TPLEN from VSMCs; (4) downregulation of either MFG-E8 or MMP-2 in VSMCs via siRNA significantly increased the levels of intact TPELN; (5) rhMFG-E8 treatment markedly reduced contractile protein, smooth muscle 22-alpha, and anti-calcification protein, fetuin-A, levels in both young and old VSMCs, which were blocked by SB203580. Notably, MMP-2 activity, elastic laminae degeneration, and calcium deposits were significantly increased while fetuin-A levels are markedly decreased in old WT vs. MFG-E8 mice. Conclusions: Taken together, our current findings suggest that MFG-E8, via p38 signaling in VSMCs, promotes MMP-2-associated elastolysis and calcification in the aging arterial wall.