Fibroblast growth factor 2 regulation of mitral valve interstitial cell repair in vitro

Objective: Because elongated mitral valve interstitial cells have features of myofibroblasts, it is likely that these cells are essential for the repair of injured valve leaflets. We characterized the cellular morphology and pattern of repair of these interstitial cells in wounds produced in vitro and tested the hypothesis that fibroblast growth factor 2 enhances interstitial cell repair. Methods: Mitral valve interstitial cells were plated onto glass coverslips, reached confluence after 1 week, and were wounded by passage of a spatula along the center of a monolayer, which created a linear wound with two edges. The wounds were observed from 0 to 96 hours by phase-contrast microscopy. Wounds were also fixed at 0, 2, and 24 hours and stained for fibroblast growth factor 2 and fibroblast growth factor receptor 1 by means of immunofluorescence and laser confocal microscopy. Results: Cells in confluent monolayers and in the monolayer behind the wound edge formed a multilayered orthogonal pattern of elongated cells similar to fibroblasts. Cells along the wound edge migrated into the wound after 4 hours, and at 24 hours single cells with prominent lamellipodia and tails were present within the wound. There was overlapping of cells as well, similar to smooth muscle cells. Fibroblast growth factor 2 and fibroblast growth factor receptor 1 were present in the cells of the undisturbed confluent monolayer. They were upwardly regulated relative to the unwounded monolayer in the cells along the wound edge at 2 hours and in the monolayer behind the wound edge at 24 hours. In single cells that migrated into the wound, fibroblast growth factor 2 and fibroblast growth factor receptor 1 were prominent. Fibroblast growth factor 2 showed a 6-fold increase in concentration relative to unwounded cultures in conditioned medium from wounded cultures at 2 hours after wounding. Addition of a neutralizing antibody to fibroblast growth factor 2 significantly delayed wound closure at 24 to 96 hours. Addition of exogenous fibroblast growth factor 2 to cultures at the time of wounding did not enhance wound repair. Conclusion: Mitral valve interstitial cells have the ability to repair wounds, and fibroblast growth factor 2 is a modulator of these repair processes.