Inflammation Drives Retraction, Stiffening, and Nodule Formation via Cytoskeletal Machinery in a Three-Dimensional Culture Model of Aortic Stenosis

In calcific aortic valve disease, the valve cusps undergo retraction, stiffening, and nodular calcification. The inflammatory cytokine, tumor necrosis factor (TNF)-alpha, contributes to valve disease progression; however, the mechanisms of its actions on cusp retraction and stiffening are unclear. We investigated effects of TNF-alpha on murine aortic valvular interstitial cells (VICs) within three-dimensional, free-floating, compliant, collagen hydrogels, simulating their natural substrate and biomechanics. TNF-alpha increased retraction (percentage of diameter), stiffness, and formation of macroscopic, nodular structures with calcification in the VIC-laden hydrogels. The effects of TNF-alpha were attenuated by blebbistatin inhibition of myosin 11 mediated cytoskeletal contraction. Inhibition of actin polymerization with cytochalasin-D, but not inhibition of Rho kinase with Y27632, blocked TNF-alpha induced retraction in three-dimensional VIC hydrogels, suggesting that actin stress fibers mediate TNF-alpha-induced effects. In the hydrogels, inhibitors of NF-kappa B blocked INF-alpha induced retraction, whereas simultaneous inhibition of c-Jun N-terminal kinase was required to block TNF-alpha-induced stiffness. INF-alpha also significantly increased collagen deposition, as visualized by Masson's trichrome staining, and up regulated mRNA expression of discoidin domain receptor tyrosine kinase 2, fibronectin, and a-smooth muscle actin. In human aortic valves, calcified cusps were stiffer and had more collagen deposition than noncalcified cusps. These findings suggest that inflammation, through stimulation of cytoskeletal contractile activity, may be responsible for valvular cusp retraction, stiffening, and formation of calcified nodules.