AIMTo investigate the effects of different level of laminar shear stresses on the vasodilator-stimulated phosphoprotein (VASP) location and expression changes associated with actin reorganization and it's mechanism.

METHODSA parallel-plate flow chamber device was used to create laminar shear stress in vitro on cultured human umbilical endothelial cells (HUVECs). The distribution of VASP and microfilament were observed by double immunofluorescence staining. RT-PCR was used to test VASP mRNA level, while VASP parameters were analyzed quantitatively with Western blot.

RESULTSAfter exposure to a flow of 10 dyn/cm2 flow for 24 h, HUVECs were elongated and oriented gradually to the direction of the flow. The microfilaments were recruited and oriented to the direction of flow with thicker VASP, specially targeted to the ends of stress fibres. RT-PCR result indicated shear could induce VASP mRNA increase. Western blotting data showed a dynamic reversible phosphorylation of VASP during 24 h, and total VASP expression increased rapidly, peaked at 2 h, then recovered at 8h followed by a slow increase again. H89, a cAMP inhibitor could inhibit shear induced VASP expression increase and phosphorylation.

CONCLUSIONVASP is an potential important component which participates in the regulation of cell cytoskeleton reorganization and morphology modification induced by shear flow via a cAMP/cAK pathway.

Cell Adhesion Molecules ; metabolism ; Cells, Cultured ; Cytoskeleton ; physiology ; Human Umbilical Vein Endothelial Cells ; physiology ; Humans ; Microfilament Proteins ; metabolism ; Phosphoproteins ; metabolism ; Phosphorylation ; Shear Strength

To investigate the effects of physiological shear stress on the vasodilator-stimulated phosphoprotein (VASP) location and expression changes associated with actin remodeling, we isolated and cultured human umbilical endothelial cells(HUVECs) with trypsin digestion. A parallel-plated flow chamber device was used to create laminar shear stress in vitro. The distributions of VASP and microfilaments in cells were observed by double staining with Alexa488 and rhodamine-phalloidin. Changes of VASP expression and phosphorylation were analyzed quantitatively with Western blot before and after exposure to shear flow for different times. We found that, under a shear stress of 10 dyn/cm2, HUVECs were elongated and oriented gradually to the flow direction. Microfilaments were recruited and oriented also to the flow direction with thicker VASP, specially targeted to their extremities. Western blotting data showed a rapid phosphorylation of VASP, and an increase of total VASP expression which peaked at 2 h (2 folds), then recovered until 8 h, followed by a slow increase again. These results suggest that VASP is a potential component which participates in the regulation of cell actin remodelling induced by shear flow.
Cell Adhesion Molecules ; biosynthesis ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Microfilament Proteins ; Phosphoproteins ; biosynthesis ; Stress, Mechanical ; Umbilical Veins ; cytology ; metabolism