Effect of differential shear stress on platelet aggregation, surface thrombosis, and endothelialization of bilateral carotid-femoral grafts in the dog.

PURPOSE: The purpose of this study was to determine the effects of increased shear stress on the aggregability of platelets as they traverse a long, small-caliber (6 mm) Dacron graft in the dog and on the surface thrombosis and endothelialization of such a graft. METHODS: Each of nine dogs received bilateral carotid-femoral artery grafts, approximately 75 cm long, for 3 months; one graft of each pair had a distal femoral arteriovenous fistula to produce a higher shear rate than the contralateral graft. Platelet aggregation scores were determined on blood withdrawn from the external jugular vein and from the proximal and distal ends of the grafts in each animal. Graft flow rates, which were used in the computation of shear stress, and luminal pressure gradients through grafts were measured during surgery and specimen retrieval. Specimens were studied with light microscopy after hematoxylin and eosin and immunocytochemical staining and by scanning electron and transmission electron microscopy to evaluate the nature, composition, and thickness of the flow surface lining, as well as the transmural healing. RESULTS: Two high-shear stress and two low-shear stress grafts occluded unilaterally; five dogs had bilaterally patent grafts, allowing comparative analyses. All subjects had low platelet aggregability with aspirin. Platelet aggregation scores taken from proximal and distal ends of the grafts were not significantly different. The high-shear stress grafts had significantly more endothelial-like cell coverage (p < 0.0371) than the low-shear stress grafts, less flow-surface thrombus (p < 0.0056), and a thinner surface lining (p < 0.0029), on both the neointima and pseudointima. CONCLUSIONS: In subjects with low platelet aggregation scores, long Dacron grafts do not elevate platelet aggregability of blood flowing through them. High-shear stress grafts have less flow surface thrombus, more endothelialization, and a thinner surface lining than do low-shear stress grafts.

Mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress.

An in vitro flow apparatus in combination with cultured endothelium was used to determine the effects of fluid-generated shear stress on cells undergoing mitosis and cytokinesis. Cell responses were recorded by time-lapse video microscopy under phase contrast or Hoffman modulation contrast optics. Completion of cell division in mitotic cells was dependent upon both the initial presence of intercellular attachments and the magnitude of fluid wall shear stress. In nonisolated populations, 95.3%, 69.5%, and 57.1% of the cells completed cell division as opposed to 66.6%, 20.4%, and 11.9% in the isolated cell groups at 2.8, 14.1, and 33 dynes/cm2, respectively. Prestressing cells for 14 h prior to monitoring failed to increase retention of isolated mitotic cells. The presence of neighboring cells facilitated replication by providing an anchoring attachment or a luminal surface for completion of division. Cell detachment most commonly occurred at the onset of cytokinesis when substrate contact areas were minimal and focal contacts were absent. A comparison between no flow controls and shear stress specimens indicated no significant differences in transit times for mitosis and cytokinesis. Thus, subconfluent endothelial cells may be more susceptible to detachment during cell division due to increases in shear stress, the absence of intercellular attachments, and reduced cell-substrate contacts.

Effect of shear stress upon localization of the Golgi apparatus and microtubule organizing center in isolated cultured endothelial cells.

Despite substantial evidence to suggest that directed cell migration is dependent upon positioning of the Golgi apparatus (GA) and the microtubule organizing center (MTOC), some controversy exists about whether such a relationship is relevant to endothelial cells under flow. The present study was undertaken to provide an indepth investigation of the relationship between shear stress, GA/MTOC localization, cell migration and nuclear position. Bovine carotid endothelial cells were exposed to 22 or 88 dynes/cm2 for 0.5, 2, 8 or 24 h, and localization of their GA/MTOCs was determined relative to the direction of flow. In no-flow control specimens, (0, 0.5, 2, 8 and 24 h) there was no change in the equally distributed GA/MTOCs. In contrast, during the first 8 h at 88 dynes/cm2 and by 2 h at 22 dynes/cm2 there was a significant increase in the number of cells with GA/MTOCs localized upstream to flow direction. The effect was temporary, however, and by 24 h there was no significant difference between the no-flow, 22 and 88 dynes/cm2 specimens. Analysis of GA/MTOC localization with respect to the direction of cell migration determined that 72.5% of no-flow cells possessed GA/MTOCs localized to the sides of nuclei nearest the direction of migration. In contrast, 64% of the specimens shear stressed over the same time period had GA/MTOCs localized to the sides of nuclei opposite the direction of migration. These results suggest that positioning of the GA/MTOC in endothelial cells is not dependent completely upon the direction of migration.(ABSTRACT TRUNCATED AT 250 WORDS)

Dextran increases survival of subconfluent endothelial cells exposed to shear stress.

In vitro devices in combination with cultured cells have been used to study the relationship between shear stress and endothelial injury. Almost exclusively, these investigations have used confluent monolayers and conventional culture media as perfusates and reported little cell loss over a wide range of shear stress conditions. In this investigation when subconfluent endothelial cells were exposed to 22 and 88 dyn/cm2 for 2, 8, and 24 h in a perfusate of medium and 5% serum, a progressive cell loss was observed. Lower cell densities were a product of decreased cell proliferation as measured by bromodeoxyuridine (BrdU) incorporation and loss of the initial cell population. Video recordings indicated that cells characteristically detached by proximal cell peeling from the substrate and an aneurysmal rupture of the cell membrane. Cell retention was increased by including 250 and 475 microM neutral dextran (70 kDa) in perfusates. Experimental evidence suggests dextran does not directly stimulate proliferation or correct an osmotic imbalance. This investigation has substantiated that fluid-generated shear stress can cause endothelial denudation and that conditions (subconfluency, time, and perfusate supplementation) under which shear stress is applied are as important for cell survival as shear stress magnitude.

Endothelial adherence under shear stress is dependent upon microfilament reorganization.

In response to externally applied shear stress, cultured endothelial monolayers develop prominent, axially-aligned, microfilamentous bundles, termed "stress fibers" (Dewey: Journal of Biomechanical Engineering 106:31-35, 1984; Franke et al.: Nature 81:570-580, 1984; Franke et al.: Klin. Wochenschr 64:989-992, 1986; Wechezak et al.: Laboratory Investigation 53:639-647, 1985). It is unclear, however, whether similar stress fibers develop in noncontiguous endothelial cells and whether these structures are necessary for adherence of individual cells under shear stress. It also is unknown what alterations occur in microtubules, intermediate filaments, and focal contacts as a consequence of shear stress. In this study, endothelial cells, free of intercellular contact, were exposed to 93 dynes/cm2 for 2 hr. With the aid of specific labeling probes and interference reflection microscopy, the distributional patterns of microfilaments, microtubules, intermediate filaments, and focal contacts were examined. Following shear stress, microfilament bundles and their associated focal contacts were concentrated in the proximal (relative to flow direction) cell regions. In contrast, microtubules were distributed uniformly within cell contours. Intermediate filaments displayed only an occasional tendency for accumulation at proximal edges. When cells were shear-tested in the presence of cytochalasin B to inhibit microfilament assembly, considerable cell loss occurred. Following inhibition of tubulin polymerization, no increase was observed in the percentage of cells lost due to shear over nontreated controls. Nocodazole-treated cells, however, were characterized by prominent stress fibers throughout the cell. These results indicate that stress fiber and focal contact reorganization represent major responses in isolated endothelial cells exposed to shear stress and that these cytoskeletal structures are necessary for adherence.

An apparatus to study the response of cultured endothelium to shear stress.

An apparatus which has been developed to study the response of cultured endothelial cells to a wide range of shear stress levels is described. Controlled laminar flow through a rectangular tube was used to generate fluid shear stress over a cell-lined coverslip comprising part of one wall of the tube. A finite element method was used to calculate shear stresses corresponding to cell position on the coverslip. Validity of the finite element analysis was demonstrated first by its ability to generate correctly velocity profiles and wall shear stresses for laminar flow in the entrance region between infinitely wide parallel plates (two-dimensional flow). The computer analysis also correctly predicted values for pressure difference between two points in the test region of the apparatus for the range of flow rates used in these experiments. These predictions thus supported the use of such an analysis for three-dimensional flow. This apparatus has been used in a series of experiments to confirm its utility for testing applications. In these studies, endothelial cells were exposed to shear stresses of 60 and 128 dynes/cm2. After 12 hr at 60 dynes/cm2, cells became aligned with their longitudinal axes parallel to the direction of flow. In contrast, cells exposed to 128 dynes/cm2 required 36 hr to achieve a similar reorientation. Interestingly, after 6 hr at 128 dynes/cm2, specimens passed through an intermediate phase in which cells were aligned perpendicular to flow direction. Because of its ease and use and the provided documentation of wall shear stress, this flow chamber should prove to be a valuable tool in endothelial research related to atherosclerosis.

Fibronectin and F-actin redistribution in cultured endothelial cells exposed to shear stress.

Cultured endothelial cells exposed to shear stresses in vitro undergo a reorganization of their F-actin-containing cytoskeletons which culminates in realignment with flow direction. Since a close transmembrane association exists between actin microfilaments and extracellular fibronectin, this study was undertaken to examine whether the actin reorganization induced by shear stress is accompanied by perturbations in the underlying fibronectin matrix. In a closed circulatory loop, bovine endothelial monolayers were exposed to steady, laminar flows corresponding to shear stress levels of 6 and 26 dynes/cm2 for 2, 6, 12, and 24 hours. The co-distribution of fibronectin and F-actin was determined in specimens which were double-labeled with antiserum to fibronectin and rhodamine phalloidin, respectively. Under the influence of shear stress, cells underwent coordinate shape changes resulting in varying degrees of alignment with flow direction. Reorientation at these shear stress levels was dependent on both the time of exposure and the magnitude of shear stress and was accompanied by a reorganization in cellular fibronectin and F-actin. In controls (no flow) correspondence between the two proteins was limited to similarly arranged, radial foci of fibronectin and F-actin filaments at the basal cell surfaces. In flow specimens, coincidence was detected only between occasional fibronectin fibrils and F-actin stress fibers. As a consequence of shear stress, fibronectin became more uniformly distributed beneath monolayers and frequently was organized into bands of densely packed fibrils. Despite this extensive reorganization, rearrangement of fibronectin did not result in the formation of identical, linear structures with F-.

Endothelial cell rounding associated with long-term implantations of left ventricular assist devices.

Culture endothelial cells have been utilized experimentally to inhibit thrombosis on cardiovascular prostheses. Grown on the internal surfaces of selected synthetic materials and implanted into autologous calves, prelinings have also proven to be a useful means to examine the influence of the in vivo environment on the endothelium. Initial short-term (less than 7 days) studies with left ventricular assist devices (LVADs) have demonstrated severe cell loss in endothelial prelinings as a result of continuous substrate flexure. These observations now have been extended to two chronic LVAD implantations of 28 and 125 days. By 28 days substantial regeneration of the endothelial layer had occurred to overlay the thrombus deposited as a result of prelining denudation. The morphologic characteristics of the endothelial linings in these two specimens have been studied. The most striking feature of the LVAD linings was the rounded appearance of the endothelial cells. These observations have led to the speculation that endothelial cells exposed to continuous mechanical strain assume an altered appearance. In addition, preliminary results utilizing a specially-built in vitro apparatus have demonstrated that endothelial rounding accompanied by intercellular separations may result from periodic stretching of the substrate material supporting the cells.