GPIbα-vWF rolling under shear stress shows differences between type 2B and 2M von Willebrand disease.

Both type 2B and type 2M von Willebrand disease result in bleeding disorders; however, whereas type 2B has increased binding affinity between platelet glycoprotein Ibα and von Willebrand factor (vWF), type 2M has decreased binding affinity between these two molecules. We used R687E type 2B and G561S type 2M vWF-A1 mutations to study binding between flowing platelets and insolubilized vWF mutants. We measured rolling velocities, mean stop times, and mean go times at 37°C using high-speed video microscopy. The rolling velocities for wt-wt interactions first decrease, reach a minimum, and then increase with increasing shear stress, indicating a catch-slip transition. By changing the viscosity, we were able to quantify the effects of force versus shear rate for rolling velocities and mean stop times. Platelet interactions with loss-of-function vWF-A1 retain the catch-slip bond transition seen in wt-wt interactions, but at a higher shear stress compared with the wt-wt transition. The mean stop time for all vWF-A1 molecules reveals catch-slip transitions at different shear stresses (gain-of-function vWF-A1 < wt vWF-A1< loss-of-function vWF-A1). The shift in the catch-slip transition may indicate changes in how the different mutants become conformationally active, indicating different mechanisms leading to similar bleeding characteristics.

Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells.

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.

Flow effects on prostacyclin production by cultured human endothelial cells.

Endothelial cell functions, such as arachidonic acid metabolism, may be modulated by membrane stresses induced by blood flow. The production of prostacyclin by primary human endothelial cell cultures subjected to pulsatile and steady flow shear stress was measured. The onset of flow led to a sudden increase in prostacyclin production, which decreased to a steady rate within several minutes. The steady-state production rate of cells subjected to pulsatile shear stress was more than twice that of cells exposed to steady shear stress and 16 times greater than that of cells in stationary culture.

Unusually large von Willebrand factor multimers increase adhesion of sickle erythrocytes to human endothelial cells under controlled flow.

The interactions of normal erythrocytes and erythrocytes from patients having hemoglobin S hemoglobinopathies with normal human endothelial cells (EC) were investigated under flow conditions. When EC supernatant, containing 2.8-11.0 U/dl of von Willebrand factor (vWF) antigen and vWF multimeric forms larger than those present in normal plasma, was the red blood cell (RBC)-suspending medium instead of serum-free medium (SFM), the adhesion of sickle RBC, but not normal RBC, to endothelial cells was greatly increased (range of enhancement of sickle RBC adhesion, 2- to 27-fold). Adhesion of sickle RBC to endothelial cells was reduced to near serum-free levels when EC supernatant was immunologically depleted of vWF forms. Sickle RBC suspended in SFM containing 200 U/dl of purified vWF multimers of the type found in normal human plasma or 300 micrograms/ml human fibronectin were only slightly more adhesive to endothelial cells than sickle RBC suspended in SFM alone. These data indicate that unusually large vWF multimers produced by endothelial cells are potent mediators of the adhesion of sickle erythrocytes to endothelial cells. Vaso-occlusive crises in sickle cell anemia may be caused, at least in part, by adhesive interactions between the abnormal surfaces of sickle RBC and the endothelium after the release of unusually large vWF multimeric forms from stimulated or damaged endothelial cells.

The effect of shear stress on the uptake and metabolism of arachidonic acid by human endothelial cells.

The uptake and metabolism of arachidonic acid (AA) by human umbilical vein endothelial cells was studied for cells in stationary culture and for cells exposed to physiological levels of shear stress. For cells grown in stationary culture, the initial incorporation of arachidonic acid was primarily into diacylglycerol and phospholipids. Cells exposed to flow incorporated labeled arachidonic acid at a similar rate as cells maintained in stationary culture; however, the distribution of the label was altered by flow. The incorporation of arachidonic acid into diacylglycerol and phosphatidylinositol was increased in cells exposed to flow. The largest increase occurred for cells exposed to arterial levels of shear stress for the shortest time period studied, 0.5 h. Prostacyclin (PGI2) and PGF2 alpha were the principal arachidonic acid metabolites formed. Shear stress-stimulated cells preferentially produced PGI2 relative to other eicosanoid products. The initiation of flow caused a burst of AA metabolism which was highly specific for PGI2. This might represent an increase in the turnover of phosphatidylinositol-bound arachidonic acid which is specifically converted to PGI2 as a result of flow-induced membrane stresses.

Angiogenesis induced by tumor necrosis factor-agr; is mediated by alpha4 integrins.

Tumor necrosis factor-alpha (TNF-alpha) and fibroblast growth factor-2 (FGF-2 or bFGF) are potent stimulators of angiogenesis. TNF- alpha, but not FGF-2, can induce the expression of vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells. The soluble form of VCAM-1 has recently been demonstrated to function as an angiogenic mediator. Here we demonstrate that monoclonal antibodies directed against VCAM-1 or its alpha4 integrin counter- receptor inhibited TNF-alpha-induced endothelial cell migration in vitro. Angiogenesis induced in vivo in rat corneas by TNF-alpha was inhibited by a neutralizing antibody directed against the rat alpha4 integrin subunit. A peptide antagonist of the a4 integrins blocked TNF-alpha-induced endothelial cell migration in vitro and angiogenesis in rat corneas in vivo. No inhibition by the antibodies or peptide antagonist was observed either in vitro or in vivo when FGF-2 was used as the stimulus. The peptide antagonist did not inhibit TNF-a binding to its receptor nor did it block the function of alphavbeta3, an integrin previously implicated in TNF-a and FGF- 2 mediated angiogenesis. These results demonstrate that angiogenic processes induced by TNF-alpha are mediated in part by agr;4 integrins possibly by a mechanism involving the induction of soluble VCAM-1.

Degradation of polydispersed poly(L-lactic acid) to modulate lactic acid release.

Polydispersed poly(L-lactic acid) (PLLA) membranes comprised of blends of monodispersed PLLA of weight average molecular weight of 82,500 and 7600 were fabricated to investigate the effect of polydispersity on degradation characteristics. The PLLA blends exhibited large spherulites of high molecular weight chains embedded in a low molecular weight matrix. During degradation in phosphate buffer at pH 7.4 and 37 degrees C for 28 d, the release rate of lactic acid increased as the percentage of the low molecular weight component in the blend was increased. For low molecular weight compositions larger than 50%, voids were created in the degrading blends due to the degradation of low molecular weight chains and the concurrent dissolution of lactic acid, and also the release of undegraded particles of high molecular weight. These studies demonstrate the feasibility of modulating lactic acid release during in vivo degradation of PLLA implants by adjusting the polymer polydispersity.

Permeability of human endothelial monolayers: effect of vasoactive agonists and cAMP.

Permeability coefficients of human umbilical vein endothelial cell monolayers cultured on polycarbonate filters were determined by monitoring transendothelial albumin transport. Permeability was determined as a function of time in culture and in the presence of vasoactive agonists. Permeability decreased with increasing time in culture. All agonist experiments were performed with 15-day cultures because this time point best modeled the in vivo permeability barrier function. Permeability of endothelial monolayers decreased significantly in the presence of the stable prostacyclin analogue iloprost (6 nM), dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP, 0.5 mM)-3-isobutyl-1-methylxanthine (IBMX, 0.1 mM), 8-bromo cAMP (0.5 mM)-IBMX, dibutyryl cAMP-theophylline (0.5 mM), or IBMX. A 9.6-fold increase in permeability resulting from thrombin [0.15 U/ml (1 nM)] treatment was inhibited by pretreating the monolayers with dibutyryl cAMP-IBMX, 8-bromo cAMP-IBMX, dibutyryl cAMP-theophylline, dibutyryl cAMP, IBMX, iloprost, or D-Phe-Pro-Arg-CH2-alpha-thrombin (1 nM). The thrombin-induced permeability increase was not significantly altered by pretreating monolayers with aspirin (5 microM) or indomethacin (50 microM). Inactivated forms of thrombin, diisopropylflurophosphate-alpha-thrombin (1 nM) and D-Phe-Pro-Arg-CH2-alpha-thrombin, did not significantly affect permeability. Monolayer permeability was not altered in response to bradykinin (1 microM). These results suggest a mediating role for intracellular cAMP in the permeability barrier function of endothelial monolayers.

Fluid shear stress as a regulator of gene expression in vascular cells: possible correlations with diabetic abnormalities.

Diabetes mellitus is associated with increased frequency, severity and more rapid progression of cardiovascular diseases. Metabolic perturbations from hyperglycemia result in disturbed endothelium-dependent relaxation, activation of coagulation pathways, depressed fibrinolysis, and other abnormalities in vascular homeostasis. Atherosclerosis is localized mainly at areas of geometric irregularity at which blood vessels branch, curve and change diameter, and where blood is subjected to sudden changes in velocity and/or direction of flow. Shear stress resulting from blood flow is a well known modulator of vascular cell function. This paper presents what is currently known regarding the molecular mechanisms responsible for signal transduction and gene regulation in vascular cells exposed to shear stress. Considering the importance of the hemodynamic environment of vascular cells might be vital to increasing our understanding of diabetes.

Effects of fluid shear stress on gene regulation of vascular cells.

Hemodynamic forces such as fluid shear stress play an active role in many physiological and pathophysiological processes of the cardiovascular system. Shear stress resulting from blood flow and transmural plasma flux alters the function of vascular cell (primarily endothelial cells), leading to both rapid and slower adaptive tissue responses. Transmission of the shear stress signal throughout the vascular cell involves a complex interplay between cytoskeletal and biochemical elements and results in changes in structure, metabolism, and gene expression. Herein we review current knowledge on flow-induced mechanotransduction in the vascular endothelial cell and the molecular mechanisms believed responsible for shear-induced endothelial and smooth muscle cell gene regulation with an emphasis on signal transduction.

Immunochemical identification of human endothelial cells on the lining of a ventricular assist device.

We are studying the biologic (pseudointimal) lining that forms in the HeartMate (Thermo Cardiosystems, Inc.; Woburn, Massachusetts, USA), a left ventricular assist device with a pusher-plate blood pump, housed in solid titanium with uniquely textured blood-contacting surfaces. Sintered titanium microspheres cover the rigid surface, and integrally textured polyurethane lines the flexing diaphragm. The texture of the blood-contacting surfaces is designed to encourage formation of a biologic pseudointimal lining, which greatly reduces the risk of thromboembolic complications. We performed immunochemical analyses to characterize precisely the pseudointimal lining. Samples were taken from 2 explanted pumps; 1 had supported a patient for 132 days and the other, 189 days. The samples were cultured to detect factor-VIII-related antigen (von Willebrand factor), acetyl low-density lipoprotein receptors, smooth-muscle-cell actin, and surface adhesion molecules specific for monocytes/macrophages. Macrophage cells were predominant in both pumps, but in the 2nd pump, cultures from the center of the diaphragm were positive for acetyl low-density lipoprotein receptor and von Willebrand factor, indicating the presence of endothelial cells. We believe that blood-borne endothelial cells or endothelial cell precursors were deposited on the blood-contacting surfaces, which is an important clinical finding with regard to lowering the risk of thromboembolic complications and reducing the need for systemic anticoagulation in long-term left ventricular assist device patients.

Mechanical and biochemical aspects of leukocyte interactions with model vessel walls.

Leukocytes in flowing blood are continually undergoing collisions with the blood vessel walls. Whether these collisions result in adherence depends on a delicate balance between the fluid mechanical drag force, which tends to dislodge the PMNL, and the adhesive force generated at the area of contact with the endothelium. Local blood flow rate controls the first of these forces, with the important parameter being the velocity gradient at the wall (wall shear rate). The detailed morphology of the endothelial cell and the PMNL upon collision and the biochemical state of these cells determine the adhesive force, because this force is a product of the strength of interaction times the area of contact. If a leukocyte flattens out or spreads on the vessel wall surface, it will reduce the hydrodynamic drag and increase the area of contact, leading to a more stable adhesion. In the normal circulation, a significant fraction of the PMNL appear to be attached to the endothelium, particularly in the low flow venules. This leukocyte fraction is what hematology texts refer to as the marginal pool. The adherent PMNL are a dynamic population in the sense that some return to the circulation, some move through the endothelial cell mono-layer into the extravascular space, and others remain "attached" to the endothelial cells but roll along the surface in the flow direction. An equilibrium number is maintained under normal conditions by recruitment from the circulating pool. Increased blood flow rate will increase the hydrodynamic force, tending to dislodge the PMNL, but will also (at least in large vessels) increase the number of collisions of circulating PMNL with the vessel wall by increasing the effective "diffusion" coefficient of the leukocyte. Thus if one studied the kinetics of PMNL adhesion on an initially clean endothelial monolayer surface under flow, increasing the whole blood flow rate would probably increase in initial rate of attachment but may decrease the equilibrium number of adherent leukocytes (Fig. 5). The degree of cooperation in vivo between PMNL and endothelial cells required to enable the leukocytes to perform their bactericidal functions is fascinating. This type of interdependency also appears to be necessary in the inflammatory response [Wedmore and Williams, 1981]. Fluid mechanical forces in addition to biochemical events are crucial in regulating these interaction processes in vivo. In vitro models for examining PMNL adhesion should include control of the local fluid mechanics.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion.

The effect of flow on the adhesion of polymorphonuclear leukocytes (PMNL) to vascular endothelium was investigated using a parallel plate chamber with a well-defined flow field. Washed PMNL were perfused over a monolayer of primary human umbilical vein endothelial cells (HUVEC) pretreated with formyl-methionyl-leucyl-phenylalanine (FMLP, 1 X 10(-7) mol/L) for five minutes. In other experiments HUVEC were pretreated with interleukin 1 (IL1,2 U/mL) for four hours. PMNL adhesion to stimulated and control HUVEC was measured over a physiologic range of wall shear stresses. PMNL adhesion to nylon-coated surface was also studied. At a wall shear stress of 0.98 dynes/cm2,283 +/- 37.3 PMNL/mm2 (mean +/- SEM) adhered to FMLP-treated HUVEC while 195 +/- 20.3 PMNL/mm2 adhered to control HUVEC. At 1.96 dynes/cm2, 68 +/- 14.1 PMNL/mm2 adhered to FMLP-treated HUVEC and 42 +/- 6.0 PMNL/mm2 adhered to control HUVEC. At 3.92 dynes/cm2, virtually no PMNL adherence was noted on either control or FMLP-treated HUVEC. On IL 1-treated HUVEC at 1.96 dynes/cm2, 371 +/- 25.8 PMNL/mm2 adhered while 28 +/- 2.9 PMNL/mm2 adhered to control HUVEC. PMNL adhesion to IL 1-treated and control HUVEC dropped to 10.2 +/- 3.8 and 6.8 +/- 3.5 PMNL/mm2, respectively, at 3.01 dynes/cm2. The effect of flow on PMNL adhesion appears to be an important factor in determining the outcome of the PMNL/HUVEC adhesive interaction under these experimental conditions.

Shear stress induced stimulation of mammalian cell metabolism.

A flow apparatus has been developed for the study of the metabolic response of anchorage-dependent cells to a wide range of steady and pulsatile shear stresses under well-controlled conditions. Human umbilical vein endothelial cell monolayers were subjected to steady shear stresses of up to 24 dynes/cm(2), and the production of prostacyclin was determined. The onset of flow led to a burst in prostacyclin production which decayed to a long term steady state rate (SSR). The SSR of cells exposed to flow was greater than the basal release level, and increased linearly with increasing shear stress. This study demonstrates that shear stress in certain ranges may not be detrimental to mammalian cell metabolism. In fact, throughout the range of shear stresses studied, metabolite production is maximized by maximizing shear stress.

Cyclical strain effects on production of vasoactive materials in cultured endothelial cells.

Mechanical forces due to fluid flow and cyclical strain can alter endothelial cell morphology and function, including the release of vasoactive materials endothelin, prostacyclin (PGI2), and tissue plasminogen activator (t-PA). In this study, effects of cyclical strain were modeled by culturing bovine aortic endothelial cells on fibronectin-coated elastic membranes of silicone rubber (Silastic) or poly-etherurethane urea (Mitrathane). After growing to confluence under static conditions of 37 degrees C in humidified air with 5% CO2, cells were strained cyclically at membrane elongations of 5% or 10% for 24 hours at 1 Hz. Controls were maintained under static conditions or were exposed to fluid motions similar to the strained cells but without stretching. Secretion rates were constant throughout experiments in the strain chamber with no initial burst in metabolism associated with the initiation of strain. Secretion rates were not altered by choice of elastic membrane. At a physiological level of 10% cyclical strain, prostacyclin and endothelian secretion rates were increased by 2.5-fold and 1.7-fold, respectively, above stationary controls. Endothelin production demonstrated a dose-dependent response with cyclical strain, while PGI2 appeared to require a threshold strain before an increase in secretion occurred. No significant differences in t-PA levels were seen in cyclically strained cells compared with controls. These results indicate that endothelial cells respond metabolically to cyclical strain and suggest that mechanical strain may modulate secretion of selective vasoactive materials by vascular endothelial cells.