Wall shear stress generated by blood flow may regulate the expression of fibrinolytic proteins by endothelial cells. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor, type 1 (PAI-1) secretion by cultured human endothelial cells were not affected by exposure to venous shear stress (4 dynes/cm2). However, at arterial shear stresses of 15 and 25 dynes/cm2, the tPA secretion rate was 2.1 and 3.0 times greater, respectively, than the basal tPA secretion rate. PAI-1 secretion was unaffected by shear stress over the entire physiological range.

The goal of the present study was to determine if voltage-sensitive calcium channels are present in bovine aortic endothelial cell plasmalemma and if they contribute to the rise in cytosolic calcium produced by bradykinin. After bradykinin (100 nM) exposure, endothelial cell associated fura-2 fluorescence peaked within 10-20 seconds and then declined to a steady level 2- to 3-fold above resting values. Pretreatment with lanthanum (20 microM) abolished the steady level produced by bradykinin but had little effect on the initial, transient rise in cytosolic calcium. Chelation of extracellular calcium with EGTA before addition of bradykinin resulted in a substantial decrease in the fura-2 transient and elimination of the long-lasting component. Nimodipine (3 microM) and nitrendipine (1 microM) were without effect on either phase of the bradykinin-induced response. Moreover, elevation of extracellular potassium failed to produce a rise in intracellular calcium. With the use of the tight seal technique to voltage clamp the cells, inwardly rectifying and calcium-activated potassium currents were found to exist in the endothelial cells. Addition of bradykinin (100 nM) elicited a calcium-activated potassium current that was eliminated in the absence of intracellular potassium. No voltage-sensitive calcium currents were activated when the cells were exposed to 10 mM or 110 mM calcium chloride in the presence or absence of bradykinin. The binding of [3H](+)PN200-110 to endothelial cell membrane preparations was 1-3 orders of magnitude lower than that observed in PC-12, GH3, or BC3H1 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function.

The CD11/CD18 family of glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to inflammatory responses. Using a monoclonal antibody (MoAb) against CD18 (TS1/18), the role of these molecules in polymorphonuclear neutrophil (PMNL) adhesion to cultured primary human umbilical vein endothelial cells (HUVEC) was examined under venous flow conditions. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to interleukin-1 (IL-1)- treated HUVEC at 2.0 dynes/cm2 (TS1/18-treated 305 +/- 58 PMNL/mm2 v 334 +/- 63 PMNL/mm2 on control). Furthermore, incubation of HUVEC with R6.5.D6, an MoAb against intercellular adhesion molecule-1 (ICAM-1) did not significantly inhibit PMNL adhesion to IL-1-treated HUVEC at 2.0 dynes/cm2 (P greater than .3). In contrast to the lack of inhibition of adhesion under conditions of flow, incubation of PMNL with TS1/18 reduced PMNL adherence in static adhesion assays. PMNL migration beneath HUVEC monolayers has been shown to be stimulated by 4-hour IL-1 treatment. TS1/18 and R6.5.D6 significantly inhibited migration of PMNL beneath IL-1-treated HUVEC monolayers under flow conditions by slightly more than 80% (P less than .005). In flow experiments with CD18- deficient PMNL, virtually no transendothelial migration was observed. The effect of FMLP (10(-8) mol/L) on PMNL adhesion to untreated HUVEC at wall shear stresses ranging from 0.25 to 2.0 dynes/cm2 was also investigated. FMLP had little effect on PMNL adherence at shear stresses above 0.5 dynes/cm2 (P greater than .45). In response to FMLP exposure at lower wall shear stresses, PMNL adherence to untreated HUVEC increased 6.9-fold at 0.5 dynes/cm2 (P less than .001). At 0.25 dynes/cm2, FMLP stimulation increased PMNL adherence to untreated HUVEC 6.5-fold compared with controls (P less than .005), and FMLP failed to make CD18-deficient PMNL more adherent. In experiments with PMNL pretreated with TS1/18 (anti-CD18), there was a 67% inhibition of FMLP- stimulated adhesion at 0.5 dynes/cm2 (P less than .025). The upper threshold of CD18-mediated PMNL adhesion appears to be between 0.5 and 1.0 dyne/cm2. Above these wall shear stresses, the initial attachment of PMNL to cultured endothelium was mediated almost exclusively by CD18- independent mechanisms. By simulating some of the flow parameters in the microcirculation with well-characterized shear forces, PMNL adhesion by CD18-independent and dependent mechanisms can be differentiated. These data also indicate that CD18 is an important mediator of transendothelial migration by PMNL, which have attached to the endothelium by a CD18-independent mechanism.

The effect of hemodynamic flow on apparent cytosolic free Ca2+ concentration ([Ca2+]i) of cultured bovine aortic endothelial cells was examined in the absence and presence of adenine nucleotides using microfluorimetric analysis of fura-2 fluorescence. In the absence of adenine nucleotides, flow-induced shear stress produced little change (less than 10 nM) in [Ca2+]i. Similar results were obtained using calf pulmonary artery and human umbilical vein endothelial cells. However, addition of the adenine nucleotides ATP, ADP, or AMP under flow conditions produced a transient peak increase in [Ca2+]i that was followed by a sustained elevation. The rank order of potency for the peak response was ADP greater than or equal to ATP much much greater than AMP. Adenosine was without effect on [Ca2+]i. Washout of ATP resulted in the immediate return of [Ca2+]i to basal values, indicating that the effect of ATP was rapidly reversible. Decreasing the flow rate to zero during the sustained phase also resulted in an immediate decrease of [Ca2+]i. Similar results were obtained with ADP and AMP but not with the nonhydrolyzable adenine nucleotide analogues alpha,beta-methyleneadenosine-5'-diphosphate, beta,gamma-imidoadenosine-5'-triphosphate, or beta,gamma-methyleneadenosine-5'-triphosphate. Furthermore, the rate of [Ca2+]i decrease upon cessation of flow during the sustained phase of the response to ATP was inversely proportional to the ATP concentration. These results suggest that hydrolysis of ATP to adenosine by the ectonucleotidase is responsible for the termination of the ATP response under zero-flow conditions. Evaluation of the dose- and flow-dependent response of the cells to ATP indicates that convective-diffusive transport of ATP may play an important role in regulation of endothelial cell [Ca2+]i in presence of ectonucleotidase activity and could have important consequences for the regulation of blood flow in the vasculature.