Avrum I. Gotlieb

Transient and Steady-State Effects of Shear Stress on Endothelial Cell Adherens Junctions

Endothelial cells exhibit profound changes in cell shape in response to altered shear stress that may require disassembly/reassembly of adherens junction protein complexes that mediate cell-cell adhesion. To test this hypothesis, we exposed confluent porcine aortic endothelial cells to 15 dyne/cm(2) of shear stress for 0, 8.5, 24, or 48 hours, using a parallel plate flow chamber. Cells were fixed and stained with antibodies to vascular endothelial (VE) cadherin, alpha-catenin, beta-catenin, or plakoglobin. Under static conditions, staining for all proteins was intense and peripheral, forming a nearly continuous band around the cells at cell-cell junctions. After 8.5 hours of shear stress, staining was punctate and occurred only at sites of continuous cell attachment. After 24 or 48 hours of shear, staining for VE-cadherin, alpha-catenin, and beta-catenin was intense and peripheral, forming a band of dashes (adherens plaques) that colocalized with the ends of stress fibers that inserted along the lateral membranes of cells. Staining for plakoglobin was not observed after 24 hours of shear stress, but returned after 48 hours. Western blot analysis indicated that protein levels of VE-cadherin, alpha-catenin, and plakoglobin decreased, whereas beta-catenin levels increased after 8.5 hours of shear stress. As cell shape change reached completion (24 to 48 hours), all protein levels were upregulated except for plakoglobin, which remained below control levels. The partial disassembly of adherens junctions we have observed during shear induced changes in endothelial cell shape may have important implications for control of the endothelial permeability barrier and other aspects of endothelial cell function.

In vivo modulation of endothelial F-actin microfilaments by experimental alterations in shear stress.

F-actin microfilament reorganization in response to alterations in shear stress has not been experimentally tested in vivo. In the current study, we analyzed changes in F-actin distribution in endothelial cells around the site of a coarctation performed in the midabdominal aorta of rabbits. The coarctation caused a 60% decrease in luminal diameter and produced three distinct zones: 1) a high shear region immediately upstream of the coarct (Zone I); 2) a region of low, fluctuating shear immediately downstream of the coarct (Zone II); and 3) an annular vortex characterized by high shear extending 0.5 to 3 mm downstream of the coarct (Zone III). Endothelial cells of control abdominal aortas were ellipsoid in shape and aligned in the direction of blood flow. They displayed a prominent circumferential band of microfilaments and short, thin stress fibers. Near coarctations, cells of Zone I were much more elongate, and stress fibers were markedly thicker and longer than in control abdominal aortas. In Zone II, cells were polygonal in shape and showed a prominent peripheral band of microfilaments and central stress fibers. Zone III cells were similar in shape to control abdominal aortic endothelial cells but showed very striking central stress fibers. These findings indicate that in vivo F-actin microfilament distribution can be modulated by experimentally altering flow conditions. F-actin redistribution in response to elevated shear stresses may increase cell-substrate adhesion and thus maintain endothelial integrity.

Patterns of endothelial microfilament distribution in the rabbit aorta in situ.

The available data on F-actin microfilament distribution in vascular endothelial cells in vivo is limited. In this study, the appearance and distribution of endothelial cell microfilaments in the rabbit thoracic aorta, the abdominal aorta and its major arterial branch points, and the aortic bifurcation were examined. Perfusion fixed rabbit aortas were stained in situ for F-actin by infusing rkodamine phalloidin via a peristaltic pump into the aortas at a slow flow rate. This new technique resulted in excellent visualization of branch points and allowed for a precise description of the actin microfilament bundles in endothelial cells along flow dividers. In the thorack and abdominal aorta, away from branch ostia, actin microfilaments were localized in two regions of the endothelial cells, as a prominent band that completely outlined the cell periphery, and also as short central stress fibers. The central stress fibers were more frequent and prominent in cells of the abdominal aorta. At branch sites and at the aortic bifurcation, long, thick microfilament bundles were present in endothelial cells extending from the tip of the flow divider to a few millimeters along the branch arteries, the aorta, and the iliac arteries. Peripheral actin, however, no longer completely surrounded the cells. The thick bundles were not prominent in endothelial cells located adjacent to the proximal Up of branches or at the iliac arteries opposite the flow divider. This study shows that endothelial cell F-actin microfilament distribution in vivo is well defined along the aortic-arterial system. The prominent central microfilament bundles and the reduced peripheral microfilaments seen at localized regions may reflect an adaptive response to elevated shear stress at these sites.

Dynamics of shear-induced redistribution of F-actin in endothelial cells in vivo.

The steady-state responses of endothelial cell F-actin distribution to changes in in vivo shear stress have been well documented. The purpose of the current work was to define the dynamics of redistribution of F-actin in the period immediately after experimental changes in shear. We used abdominal aortic coarctation in rabbits to experimentally increase shear stress downstream from the coarctation by approximately twofold. In situ staining was employed to track subsequent F-actin redistribution. Within 12-15 hours, the number of stress fibers in the central regions of the cells decreased, and some separation of junctional actin in adjacent cells occurred. Long, central stress fibers of variable thickness were evident at 24 hours, but the band of actin normally seen at the periphery of the cells could no longer be distinguished. The redistribution of F-actin was completed over the next 24 hours by an increase in thickness of central stress fibers. Restoration of normal F-actin distribution after coarctations were removed proceeded more slowly. The long, thick stress fibers that were induced by high shear were replaced by thinner or shorter microfilament bundles 48 hours after the coarctations were removed. At 72 hours, central stress fibers were primarily long, thin structures. Peripheral F-actin was not fully restored at this time. Peripheral F-actin was restored at 1 week after removal of the coarctation, but there were still more and longer stress fibers at this time than were observed in control aortas.(ABSTRACT TRUNCATED AT 250 WORDS)

ENDOTHELIAL INTEGRITY AND REPAIR

Large artery endothelial integrity and repair is an important area of investigation in atherosclerosis research. The development of an atherosclerotic plaque (fibrofatty plaque) is a dynamic and complex process that is closely associated with the structure and dysfunction of endothelial cells. Although the sequence of events that lead to the initiation and growth of fibrofatty atherosclerotic plaques is not well understood, there is much experimental and clinical support for the concept that disruption of structural and functional endothelial integrity plays an important role in atherogenesis.

Vascular Tissue Response to Experimentally Altered Local Blood Flow Conditions

Local factors related to shear stress may influence atherogenesis through several mechanisms. It is probably for this reason that lesion development does not show a consistent relation to shear stress when different experimental models are compared. Thus, there is currently emphasis on the correlation that has been observed between low shear and lesion formation in several experimental models, for example, the human carotid bifurcation studied by Ku and co-workers (1985).As these investigators point out, however, it is often difficult to divorce low shears from shears that fluctuate rapidly in magnitude and especially in direction. Furthermore, atherosclerosis occurs in high shear regions in some models, although it now appears that this is not commonplace and tends to be species specific. Indeed, frequent sparing of high shear regions has raised speculations of adaptive responses to shear. Finally, it frequently appears that reproducible lesions may be distributed at sites not well correlated with shear stress. Thus, the distribution of lesions within the aorta of animals or humans is not readily related to available maps of aortic shear in mammals.

Control of Reendothelialization: The Importance of Endothelial Microfilaments, Microtubules and Centrosomes in Endothelial Locomotion

It appears then that the F-actin microfilaments and the centrosome and associated microtubules of the endothelial cytoskeleton are important in the repair of endothelial denudation. These dynamic cytoskeletal systems are able to act rapidly and can be regulated by a variety of factors including neighboring cells, extracellular matrix, and soluble factors in the environment. One testable hypothesis is that atherogenic agents which have been shown to enhance atherosclerosis may in fact act by perturbation of the endothelial cytoskeleton resulting in abnormal repair of the endothelial monolayer.

In vivo Responses of Endothelial Cells to Hemodynamic Stress

We have examined endothelial cell integrity and cytoskeleton structure at sites of flow disturbances in the vicinity of mid-abdominal aortic coarctations in rabbits. Coarctations produce high shear stresses in the inflow region and shears that fluctuate rapidly in direction immediately downstream. Both zones are characterized by elevated endothelial cell death and turnover. Furthermore, repair of endothelial injury was inhibited in both areas. Studies of cytoskeleton in the high shear regions demonstrated expression of very large and long actin microfilament bundles (“stress fibres”). We interpret this response as cellular adaptation that may limit shear-related trauma to endothelium.