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.

Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells.

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

Integrin alpha(v)beta(3) is involved in stimulated migration of vascular adventitial fibroblasts by basic fibroblast growth factor but not platelet-derived growth factor.

We examined the effects of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) on the migration of vascular adventitial fibroblasts (VAFs) isolated from rat aortic adventitiae. Both bFGF and PDGF significantly stimulated VAF migration in vitro. An antibody to rat beta(3) integrin reduced bFGF-stimulated migration in a dose dependent manner. Moreover, VAF migration was inhibited in the presence of cyclic RGD (cRGD) peptide. However, PDGF-directed migration was blocked only by equivalent cRGD peptide but not by antibody to beta(3) integrin. These data suggest that alpha(v)beta(3) integrin mediates VAF migration stimulated by bFGF and that chemoattractant directed migration may be through distinct integrins.

DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells.

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes/cm(2) for 6 or 24 h (1 dyne = 10 microN), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF/GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine/spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells.

Shear stress reduces protease activated receptor-1 expression in human endothelial cells.

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells.

This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

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.

Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells.

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

Nitric oxide production by cultured human aortic smooth muscle cells: stimulation by fluid flow.

This study demonstrated that exposure of cultured human aortic smooth muscle cells (SMC) to fluid flow resulted in nitric oxide (NO) production, monitored by nitrite and guanosine 3',5'-cyclic monophosphate production. A rapid burst in nitrite production rate was followed by a more gradual increase throughout the period of flow exposure. Neither the initial burst nor the prolonged nitrite production was dependent on the level of shear stress in the range of 1.1-25 dyn/cm2. Repeated exposure to shear stress after a 30-min static period restimulated nitrite production similar to the initial burst. Ca(2+)-calmodulin antagonists blocked the initial burst in nitrite release. An inhibitor of nitric oxide synthase (NOS) blocked nitrite production, indicating that changes in nitrite reflect NO production. Treatment with dexamethasone or cycloheximide had no effect on nitrite production. Monoclonal antibodies directed against the inducible and endothelial NOS isoforms showed no immunoreactivity on Western blots, whereas monoclonal antibodies directed against the neuronal NOS gave specific products. These findings suggest that human aortic SMC express a constitutive neuronal NOS isoform, the enzymatic activity of which is modulated by flow.

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.

Inhibition of smooth muscle cell growth in vitro by an antisense oligodeoxynucleotide released from poly(DL-lactic-co-glycolic acid) microparticles.

We fabricated poly(DL-lactic-co-glycolic acid) (PLGA) 50:50 microparticles loaded with an antisense (AS) oligodeoxy-nucleotide (ODN) against the rat tenascin mRNA and determined the effect in vitro of the AS-ODN released on smooth muscle cell (SMC) proliferation and migration. AS-ODN was entrapped using a double-emulsion-solvent-extraction technique with high efficiency. Release of AS-ODN was characterized by a small initial-burst effect followed by a period of controlled AS-ODN release for up to 20 days. SMC proliferation studies exhibited dose-dependent growth inhibition with AS-ODN-loaded microparticles. Microparticles loaded with scrambled (SC) ODN showed less growth inhibition than AS-ODN. Moreover, only the AS-ODN-loaded microparticles inhibited migration. These results demonstrate the feasibility of entrapping an AS-ODN to rat tenascin in PLGA microparticles for controlled delivery to inhibit SMC proliferation and migration.

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.

Effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro.

After cardiovascular intervention, smooth muscle cells (SMC) are directly exposed to blood flow and thus their behavior might be affected by fluid hemodynamic forces. The aim of this study was to determine the effect of fluid shear stress on the growth rate of SMC. Human aortic smooth muscle cells (hASMC) were seeded on fibronectin-coated glass slides and were exposed to different levels of shear stress using parallel plate flow chambers. After 24 h, cell numbers in the stationary and sheared cultures were measured by a Coulter counter. Results demonstrated that increasing shear stress significantly reduces the proliferation rate of hASMC (P < 0.05). Comparable lactate dehydrogenase levels in the media of stationary and flow cultures provided evidence that the reduction of cell number was not due to cell injury. Proliferating cell nuclear antigen (PCNA) immunofluorescence studies indicated that the cell cultures were not growth arrested 24 h after exposure to shear stress, and that the differences in PCNA staining between stationary control and flow cultures were comparable to the cell counts.

Shear stress-mediated changes in the expression of leukocyte adhesion receptors on human umbilical vein endothelial cells in vitro.

Extensive monocyte recruitment is an early phenomenon associated with the development of atherosclerotic lesions, suggesting an active role for the involvement of adhesion receptors expressed by endothelial cells. In this study we describe the contribution of hemodynamic shear forces in regulating the expression of a few of the monocyte adhesion receptors, including intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin on endothelial cells. A parallel plate flow chamber and recirculating flow loop device was used to expose human umbilical vein endothelial cells (HUVECs) to different levels of shear (2-25 dyn/cm2). Subsequently the cells were analyzed either for shear induced changes in the mRNA levels of adhesion receptors by Northern blot analyses or for changes in the surface expression of ICAM-1 using flow cytometry. Results from the fluorescence analysis showed a transient increase in the surface expression of ICAM-1, 12 hr after exposure to 25 dyn/cm2 shear, returning to basal levels within 24 hr. This was quite different from the time dependent response of ICAM-1 to lipopolysaccharide (LPS), where ICAM-1 expression was maximally induced 18-24 hr post-stimulus. ICAM-1 mRNA level appeared slightly elevated after exposure to shear for 1 hr, compared to basal values, but dropped below basal levels within 6 hr. This biphasic response was seen irrespective of the magnitude of applied shear stress. VCAM-1 mRNA expression, in contrast, decreased below the baseline expression within an hour after onset of flow, and appeared to be considerably down-regulated within 6 hr. After exposure to shear for 24 hr, no increase in mRNA levels could be detected for either molecule, at any shear magnitude. E-selectin mRNA was less responsive to shear stress, especially at the lower magnitudes of shear. After an hour of exposure to flow E-selectin mRNA level appeared slightly reduced compared with control levels, but it remained at this level even after 6 hr of flow. These results indicate that the expression of adhesion receptors is sensitive to local shear stresses in a manner that is molecule specific in the short term even though prolonged exposure to flow results in similar down-regulation for both ICAM-1 and 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.

Modulation of secretion of vasoactive materials from human and bovine endothelial cells by cyclic strain.

The effects of cyclical expansion and elaxation of the vessel wall on endothelial cell metabolism have been modeled using a uniaxial strain device and cultured endothelial cell monolayers. Also, the effects of stopping and then restarting cyclic strain on metabolite secreation rates were determined. Secretion rates of prostacyclin (PGI(2)), endothelin, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-type 1 (PaI-1) by endothelial cells were constant over24-h periods The secreation of both PGI(2) and endothelin was enhanced in cells exposed to high physiological levels of cyclical strain (10% at 1Hz) compared with controls, while tPA production was unaltered. These results were true for both human and bovine endothelial cells. Characterization of the response of human endothelial cells to cyclical strain made evaluation of stretch effects on PAl-1 secretion possible. A nearly twofold increase in PAl-1 secretion by cells exposed to arterial levels of strain was observed. Endothelin secretion remained elevated even after strain was stopped for 12 h, while PGl(2) secretion returned to control values upon cessation of cyclic stretch. These results indicate that physiological levels of cyclic mechanical strain ca significantly modulate secretion of vasoactive metabolited form endothelial cells. The changes sen secretion are, in some cases, quite different from those caused by arterial levels of fluid shear stress exposure. (c) 1994 John Wiley & Sons, Inc.

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.