The endothelium is a highly metabolic monolayer of cells regulating numerous physiological and pathological functions that maintain the permeability and thromboresistant functions of the endothelium. The structure and function of the endothelial cytoskeleton prevents vascular disease by regulating the structure of the endothelium to act as a resting molecular barrier to atherogenic proteins and by becoming an activated layer of migrating cells to repair denuding injuries. The purpose of this review is to examine the structure of the endothelial cytoskeleton and its roles in cell-cell and cell-substratum adhesion, cell signaling, and regulation of wound repair. Studies focused on the cellular and molecular biology of the structure and function of the endothelial cytoskeleton and in wound repair are reviewed. The cytoskeleton is a key regulator in maintaining endothelial integrity and in restoring integrity following injurious denudation, such as those that occur in the pathogenesis of atherosclerosis. Actin microfilaments and their associated adherens junctions and focal adhesions are important regulators of cell signaling, cell locomotion, cell adhesion, and wound repair mechanisms. Various proteins have been implicated in controlling cytoskeletal-based endothelial function and repair such as tyrosine kinases/phosphatases and the Rho family of proteins. The normal function of the endothelium is highly dependent on the endothelial cytoskeleton. Disruption and dysfunction of the cytoskeleton may result in impairment of endothelial function, subsequently tipping the balance towards vascular disease. Thus, an understanding of the cellular and molecular biology of the endothelial cytoskeleton is essential in our understanding of the pathogenesis of vascular disease, especially atherosclerosis.

The endothelial cell is unique because it must undergo a transition from a resting cell with a cytoskeleton organized for barrier function to one which promotes cell translocation following denuding endothelial injury. Since actin microfilaments are critical for both maintaining the integrity of the resting monolayer and for optimum reendothelialization, we carried out a detailed study of the organization of microfilaments as the cell undergoes the transition from a resting to a translocating cell. We used an in vitro model in which a linear wound was made in a confluent monolayer of porcine aortic endothelial cells. The complex reorganization of actin microfilament bundles following injury and their relationship to microtubules and vinculin was studied in cells at the wound edge using immunofluorescent scanning laser confocal microscopy and time-lapse videomicroscopy. In the resting confluent monolayer, microfilaments were present as a dense peripheral band (DPB) located toward the upper part of the cell and as central microfilament bundles at the substratum. Three distinct stages of microfilament reorganization occurred sequentially during early repair. Stage 1 followed wounding and involved the reduction of the DPBs of microfilaments and associated peripheral cell-cell vinculin plaques. This was associated with rapid forward actin-based lamellipodia extrusions and cell elongation. Low-dose cytochalasin, which did not disrupt the morphology of microfilament bundles, reduced elongation. Stage 2 was characterized by central microfilaments behind the lamellipodia distributed parallel to the wound edge with vinculin plaques at their tips. This was associated with prominent spreading at the front of the cell which enhanced the extent of coverage of the denuded wound area. Stage 3 was characterized by the orientation of central microfilaments perpendicular to the wound edge with vinculin plaques at their tips and was associated with the initiation of cell translocation. There was no specific structural association between central microfilaments and microtubules as the former were toward the substratum while the latter were toward the center and upper part of the cell. Thus, the sequential appearance of the three patterns of microfilament distribution define the cytoskeletal events that regulate the reestablishment of endothelial integrity following denuding endothelial injury.

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.