title: Molecular mechanisms regulating the vascular endothelial cell motile response to injury.
abstract: Vascular endothelial cell (EC) wound healing was characterized on an EC-synthesized extracellular matrix (ECM) previously treated with enzymes and antibodies specific for ECM components. Using a computer-assisted video-microscope recording system capable of automatic EC recognition, we learned whether components of the EC-synthesized matrix influenced post-injury migration and wound healing in vitro. Localization of actin and its encoded mRNA using isoform-specific antibodies and labeled cDNA probes allowed for a direct correlation of living-cell behavior with cytoskeletal form and distribution. Results of these studies indicate that the computer-assisted EC tracking system allows for an automatic and reproducible analysis of EC behavior following injury in vitro. EC migrate fastest immediately following injury and then achieve a new, slower migration rate that is maintained until EC from one edge of 200- to 300-microns-wide wound zone contact EC from the other wound face. Treatment of EC-synthesized matrices with antibodies against fibronectin and laminin has no effect on EC migration following injury (-0.25 microns/min) or on cytoskeletal array. Similarly, digestion of these matrices with heparinase and hyaluronidase has no effect on wound healing rates. Slowly spreading EC cytoplasm, which borders the intact and antibody-treated EC matrices, is rich in actin but lacks myosin II. Two different preparations of collagenase (bacterial and mammalian) each potentiate EC wound healing in vitro. Bacterial collagenase treatment of the EC-synthesized matrices potentiates EC migration fivefold (1 micron/min) while treatment of EC-matrices with mammalian cell collagenase stimulates EC migration following injury some twofold (0.4 micron/min) over control values. Whereas EC on control matrices migrate in unison as a tissue-like sheet, EC on the collagenase-treated EC matrices migrate as individuals. Concomitant with the increased rates of migration following injury on the collagenase-treated EC-matrices is a two- to fourfold increase in the steady-state levels of beta-actin mRNA. This increase in actin mRNA abundance is observable by its preferential localization (seen by in situ hybridization) in the lamellae bordering the wound edge in association with beta-actin, which is exclusively localized there. Because beta-actin and its encoded mRNA are positioned together in association with the plasma membrane in regions of moving cytoplasm, it seems likely that beta-actin filament assembly is required for motility following endothelial injury.
authors.affiliation: Program in Cell, Molecular, and Developmental Biology, Tufts University Health Science Schools, Boston, Massachusetts 02111.
authors.forename: I M
authors.initials: IM
authors.lastname: Herman
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D017364:Collagenases / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005109:Extracellular Matrix / Q000502:physiology; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects / Q000502:physiology
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; D015342:DNA Probes; D012333:RNA, Messenger; D017364:Collagenases