Changes in organization and composition of the extracellular matrix underlying cultured endothelial cells exposed to laminar steady shear stress.
BACKGROUND
In blood vessels, the extracellular matrix (ECM) underlying the endothelium supports endothelial cell (EC) attachment, spreading, migration, and proliferation. The structure and composition of the ECM may be modulated by hemodynamic shear stress, which may play a role in the pathogenesis of vascular diseases such as atherosclerosis.


METHODS
In this study, in vitro effects of fluid shear stress on the ECM of EC were investigated. Cultured bovine aortic EC (BAEC) were exposed to a steady laminar shear stress of 30 dyn/cm2 from 3 to 48 hours, using a parallel-plate flow chamber. Parallel control cultures were maintained under static conditions. The organization of fibronectin (Fn), laminin (Ln), collagen type IV (Col IV), and vitronectin (Vn) was analyzed by immunofluorescence microscopy. Changes in the profile of proteins present in the deoxycholate-insoluble ECM fraction of EC were determined using two-dimensional gel electrophoresis, and the levels of Fn, Ln, and Vn were determined by Western blotting.


RESULTS
Fn, Ln, and Col IV exhibited both a granular pattern in cell perinuclear areas and a fibrillar pattern localized underneath EC. On exposure of bovine aortic EC to shear stress, Fn fibrils grouped into thicker tracts of fibrils, and there was a tendency for some of these tracks of fibrils to align with the direction of flow. Ln and Col IV also grouped into thicker fibers, which, in contrast to Fn, were randomly oriented. Vn exhibited a diffuse granular pattern, which did not change in response to shear stress. Consistent increases in the levels of four unidentified acidic proteins (mol wt/pI = 52/4.9, 70/4.7, 70/5.5, and 110/4.4) were observed after 3 to 6 hours of exposure to flow. The level of Fn present in the ECM was decreased twofold 12 hours after exposure of the cell monolayer to flow, and then increased after 24 and 48 hours. The level of Ln showed a twofold increase after 24 and 48 hours of flow, whereas the level of Vn was not altered by shear stress.


CONCLUSIONS
These changes in organization and composition observed in the ECM of cultured EC may play a significant role in shear stress-induced morphologic alterations in EC and may represent relevant events in the initiation of atherosclerotic lesions by influencing both EC and smooth muscle cell function.
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002455:Cell Division / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D005353:Fibronectins / Q000032:analysis*; D006439:Hemodynamics; D007797:Laminin / Q000032:analysis*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D013314:Stress, Mechanical; D013997:Time Factors; D014652:Vascular Diseases / Q000209:etiology / Q000473:pathology; D019096:Vitronectin / Q000032:analysis*
D005353:Fibronectins; D007797:Laminin; D019096:Vitronectin; D003094:Collagen