Interactions between TGFß1 and cyclic strain in modulation of myofibroblastic differentiation of canine mitral valve interstitial cells in 3D culture.

ABSTRACT

OBJECTIVES: The mechanisms of myxomatous valve degeneration (MVD) are poorly understood. Transforming growth factor-beta1 (TGFß1) induces myofibroblastic activation in mitral valve interstitial cells (MVIC) in static 2D culture, but the roles of more physiological 3D matrix and cyclic mechanical strain are unclear. In this paper, we test the hypothesis that cyclic strain and TGFß1 interact to modify MVIC phenotype in 3D culture. ANIMALS, MATERIALS AND METHODS: MVIC were isolated from dogs with and without MVD and cultured for 7 days in type 1 collagen hydrogels with and without 5 ng/ml TGFß1. MVIC with MVD were subjected to 15% cyclic equibiaxial strain with static cultures serving as controls. Myofibroblastic phenotype was assessed via 3D matrix compaction, cell morphology, and expression of myofibroblastic (TGFß3, alpha-smooth muscle actin - αSMA) and fibroblastic (vimentin) markers. RESULTS: Exogenous TGFß1 increased matrix compaction by canine MVIC with and without MVD, which correlated with increased cell spreading and elongation. TGFß1 increased αSMA and TGFß3 gene expression, but not vimentin expression, in 15% cyclically stretched MVIC. Conversely, 15% cyclic strain significantly increased vimentin protein and gene expression, but not αSMA or TGFß3. 15% cyclic strain however was unable to counteract the effects of TGFß1 stimulation on MVIC. CONCLUSIONS: These results suggest that TGFß1 induces myofibroblastic differentiation (MVD phenotype) of canine MVIC in 3D culture, while 15% cyclic strain promotes a more fibroblastic phenotype. Mechanical and biochemical interactions likely regulate MVIC phenotype with dose dependence. 3D culture systems can systematically investigate these phenomena and identify their underlying molecular mechanisms.