Abstract
Wound contraction closes open wounds by the generation of contractile forces within granulation tissue. We investigated the mechanism of wound contraction using the in vitro fibroblast-populated collagen lattice (FPCL) contraction model. The contraction of the free-floating (FF)-FPCL is through rapid myosin ATPase activity, while the contraction of the attached-delayed-released (ADR)-FPCL is through sustained myosin ATPase activity. All FPCLs were cast identically and the contraction of FF-FPCLs was recorded daily for 4 days and the contraction of ADR-FPCLs was recorded 1 hour after release on day 4. At day, 4 cell numbers were determined and cells undergoing apoptosis were identified and counted. Differences in sustained and rapid myosin ATPase activity were shown by added inosine triphosphate-induced cell contraction in permeabilized fibroblast monolayer preparations. At 2 days, the FF-FPCLs were mostly contracted, while an ADR-FPCL completed contraction 1 hour after release at day 4. Contracted myofibroblasts, identified by alpha-smooth muscle actin-stained stress fibers, were identified in contracted ADR-FPCL, whereas elongated fibroblasts were identified in contracted FF-FPCLs. Vanadate inhibited both inosine triphosphate-induced cell contraction and ADR-FPCL contraction, but neither inhibited ATP-induced cell contraction or FF-FPCL contraction. Genistein inhibited FF-FPCL contraction, but not ADR-FPCL contraction. Advancing tyrosine phosphorylation in fibroblasts promotes rapid myosin ATPase activity, while advancing tyrosine dephosphorylation in myofibroblasts promotes sustained myosin ATPase. The ADR-FPCL had a reduced cell count and a greater proportion of cells had entered apoptosis compared with FF-FPCL. These experiments show that FF-FPCL contraction is through elongated fibroblasts and rapid myosin ATPase, requiring tyrosine phosphorylation. In contrast, the mechanism for ADR-FPCL contraction is through cell contraction by sustained myosin ATPase, involving tyrosine dephosphorylation.
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