Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization.
Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair.
Lee
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TY
Vascular Research Laboratory, The Toronto Hospital Research Institute, Department of Laboratory Medicine and Pathobiology, Banting and Best Diabetes Center, Center for Cardiovascular Research, University of Toronto, 200 Elizabeth Street, Ontario, Canada.
Gotlieb
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AI
D008841:Actin Cytoskeleton / Q000473:pathology; D000199:Actins / Q000502:physiology; D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000502:physiology*; D010766:Phosphorylation; D013552:Swine; D014443:Tyrosine; D014945:Wound Healing
D000199:Actins; D014443:Tyrosine