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L V McIntire

Publications and source records attributed to L V McIntire.

At least 37 records · Page 2Linked to original sources
PubMed2000

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.

Aorta↗
PubMed2000

Preferential sites for stationary adhesion of neutrophils to cytokine-stimulated HUVEC under flow conditions.

Neutrophils form CD18-dependent adhesions to endothelial cells at sites of inflammation. This phenomenon was investigated under conditions of flow in vitro using isolated human neutrophils and monolayers of HUVEC. The efficiency of conversion of neutrophil rolling to stable adhesion in this model was >95%. Neither anti-CD11a nor anti-CD11b antibodies significantly altered the extent of this conversion, but a combination of both antibodies inhibited the arrest of rolling neutrophils by >95%. The efficiency of transendothelial migration of arrested neutrophils was >90%, and the site of transmigration was typically <6 microm from the site of stationary adhesion. Approximately 70% of transmigrating neutrophils migrated at tricellular corners between three adjacent endothelial cells. A model of neutrophils randomly distributed on endothelium predicted a significantly greater migration distance to these preferred sites of transmigration, but a model of neutrophils adhering to endothelial borders is consistent with observed distances. It appears that stable adhesions form very near tricellular corners.

Adult↗
PubMed2000

Mutation in the leucine-rich repeat of platelet glycoprotein Ib alpha results in defects in its interaction with immobilized von Willebrand factor under flow.

OBJECTIVE: To characterize effects of the GP Ib alpha mutation (A156V) on its interaction with von Willebrand factor (vWf) under high fluid shear stress. METHODS: The residue A156 of GP Ib alpha was converted to a valine and the mutant expressed in CHO cells expressing wild-type GP Ib beta and GPIX. The transfected cells were tested for their interaction with a panel of GP Ib alpha antibodies and for rolling on immobilized vWf under high shear. RESULTS: The mutation led to surface expression of a GP Ib alpha polypeptide that adopted a different conformation at its N-terminus because binding of the GP Ib alpha antibody AN51, which has a binding epitope in the N-terminal 35 residues, was eliminated, whereas binding of the others (AK2, MB45, and SZ2, all of which bind to regions C-terminal to the AN51 epitope) was normal. Mutant-expressing cells could adhere and roll on immobilized vWf under high fluid shear stress and rolled significantly faster than wild-type cells. CONCLUSION: These studies demonstrate that the mutation A156V results in a conformation change at the N-terminus of GP Ib alpha, which leads to an increase in the dissociation rate of the bond between the GP Ib alpha mutant and vWf.

Animals↗
PubMed2000

[A mutation of platelet glycoprotein I balpha results in defects in its interaction with immobilized vWF].

OBJECTIVE: To study the importance of glycoprotein (GP) I balpha mutation (A156V) in interaction between mutant expressing cell and immobilized vWF under fluid shear stress. METHODS: Mutant GP I balpha cDNA was cloned into the EcoR I site of the mammalian expression vector pDX, mutant cDNA was then transfected into CHO betaIX cells. Human vWF was purified from blood cryoprecipitate by glycine and NaCl precipitation and subsequent separation on sepharose 4B column. The purified vWF was immobilized onto a coverslip. Cell rolling was induced in a parallel-plate flow chamber and observed by phase-contrast video microscope. RESULTS: CHO cells expressing GP I b-IX-V complex could adhere to and roll on immobilized vWF. The A156V mutant cells retained the ability to adhere and roll on vWF matrix, but the rolling speed was significantly faster than that of wild type cells, indicating that the off-rate of the ligand-receptor bond between the mutant and vWF was impaired. Binding of monoclonal antibody AN51 to mutant GP I balpha decreased significantly, indicating that the A156V mutation altered the conformation of the N-terminal ligand-binding region of GP I balpha. CONCLUSION: The mutant GP I balpha has a faster off-rate for its interaction with immobilized vWF. The mutant polypeptide adopts an altered conformation in N-terminal ligand-binding region of GP I balpha. The parallel-plate flow chamber is an extremely useful system in evaluating interaction between GP I balpha and vWF.

Animals↗
PubMed1999

The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.

We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibalpha, and GP Ibalpha-expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin-dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Novel optical methodologies in studying mechanical signal transduction in mammalian cells.

For the last 3 decades evidence has been accumulating that some types of mammalian cells respond to their mechanically active environment by altering their morphology, growth rate, and metabolism. The study of such responses is very important in understanding, physiological and pathological conditions ranging from bone formation to atherosclerosis. Obtaining this knowledge has been the goal for an active research area in bioengineering termed cell mechanotransduction. The advancement of optical methodologies used in cell biology research has given the tools to elucidate cellular mechanisms that would otherwise be impossible to visualize. Combined with molecular biology techniques, they give engineers invaluable tools in understanding the chemical pathways involved in mechanotransduction. Herein we briefly review the current knowledge on mechanical signal transduction in mammalian cells, focusing on the application of novel optical techniques in the ongoing research.

Animals↗
PubMed1999

Integrin alpha4beta1/VCAM-1 pathway mediates primary adhesion of RAW117 lymphoma cells to hepatic sinusoidal endothelial cells under flow.

Adhesion and stabilization of circulating tumor cells to endothelial cells in target blood vessels play an important role in the complex process of metastasis. We examined the cell surface receptors involved in the liver-metastatic adhesive interactions of murine RAW117 large-cell lymphoma cells to unstimulated hepatic sinusoidal endothelial cells (HSE) under physiological flow conditions. Flow cytometric analysis indicated that VCAM-1, ICAM-1 and PECAM-1 are constitutively expressed on the surfaces of both HSE and RAW117 cells. However, monoclonal antibody (mAb) blockade studies showed that ICAM-1 and PECAM-1 affected neither the attachment nor the stabilization step of the adhesion of RAW117 cells to HSE cell monolayers under flow. In contrast, RAW117 cells required a significantly lower shear stress to establish adhesion to HSE cells when VCAM-1 receptors on HSE cells were blocked with mAb. Furthermore, the presence of the anti-VCAM-1 mAb significantly decreased the extent of adhesion compared to that of the control, without affecting adherent cell stabilization times. Blocking the alpha4 integrin subunits present mainly on RAW117 cells produced similar results to those previously observed with anti-VCAM-1 mAb. Although constitutively present mainly on the surfaces of RAW117 cells, MAdCAM-1 and beta7 integrin subunit do not appear to play a role in either the arrest or stabilization of RAW117 cells on HSE cell monolayers. However, blocking the beta1 integrin subunit on the RAW117-H10 cells reduced adhesion to the same extent as anti-alpha4 and anti-VCAM-1 treatments. These observations suggest that an interaction of integrin alpha4/beta1 on RAW117 cells with liver endothelial VCAM-1 occurs during the early stages of the adhesion process and may be important in liver metastasis.

Animals↗
PubMed1998

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.

Aorta, Abdominal↗
PubMed1998

Shear-dependent rolling on von Willebrand factor of mammalian cells expressing the platelet glycoprotein Ib-IX-V complex.

Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.

Animals↗
PubMed1998

Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.

BACKGROUND: After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. METHODS AND RESULTS: Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. CONCLUSIONS: The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

Abciximab↗
PubMed1998

Biomechanics of cell interactions in shear fields.

Cellular interactions play a key role in diverse biological processes within the cardiovascular system. Targeting of leukocytes to sites of inflammation is viewed as a multistage process of sequential involvement of distinct adhesion molecules on the leukocyte and endothelial cell (EC) surface that is dictated by the local fluid dynamic environment. For neutrophils, the initial contact and rolling along the vessel wall are mediated primarily by selecting. Subsequent firm adhesion requires activation of neutrophil P, integrins and binding to their ligand ICAM-1 on the EC surface. The final step of this cascade of events includes neutrophil transmigration to extravascular tissue space. The neutrophil model of emigration in inflammation has been extended and refined to account for monocyte and T cell interactions with ECs. Platelet adhesion to thrombogenic surfaces (i.e. immobilized von Willebrand factor) under flow follows the general principles of leukocyte extravasation. More specifically, platelet glycoprotein (GP) Ib alpha appears to mediate an initial selectin-like tethering platelet-vWf interaction, followed by alpha II beta beta 3 integrin activation and firm adhesion. Some of the signaling mechanisms associated with cellular interactions in inflammatory and thrombotic processes are discussed. These basic principles are also discussed in the context of tissue engineering research.

Biomechanical Phenomena↗
PubMed1998

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.

Antibodies, Monoclonal↗
PubMed1998

Fibrillin containing elastic microfibrils support platelet adhesion under dynamic shear conditions.

The vascular subendothelium contains macromolecular structures called microfibrils. Type VI collagen is one protein found in microfibrils that supports platelet adhesion and aggregation and we have previously evaluated the roles of platelet receptors and vWf involved in these processes under physiological shear conditions. Here we investigate the ability of fibrillin containing elastic microfibrils to support mural thrombus formation. Our results show that elastic microfibril surfaces support platelet adhesion under low shear conditions at a level similar to collagen VI tetramers. However, the degree of aggregation on the elastic microfibril surface is much higher. Both adhesion and aggregation were shown to be mediated by the GPIIb-IIIa platelet receptor. Elastic microfibrils do not support the formation of mural thrombi under high shear conditions. These results suggest roles for both collagen VI and fibrillin containing elastic microfibrils in modulating the platelet response to blood vessel injury.

Actin Cytoskeleton↗
PubMed1997

Molecular mechanisms of monocyte adhesion to interleukin-1beta-stimulated endothelial cells under physiologic flow conditions.

This study identifies multiple pathways used by monocytes to adhere to 4-hour interleukin-1beta stimulated human umbilical vein endothelial cells under flow conditions. Physiologic shear stresses were simulated in a flow chamber with parallel plate geometry; quantitation of primary adhesion, secondary adhesion, and transmigration was performed using phase contrast videomicroscopy. Neuraminidase treatment of monocytes reduced primary interaction by 50%, whereas blocking L-selectin or very late antigen-4 showed significant but smaller effects (approximately 30% inhibition). However, a combined treatment against all three pathways was able to reduce interaction by 80%. Blocking beta2 and alpha4 integrin pathways together inhibited secondary/firm adhesion by 75%. Only 40% of firmly adherent monocytes transmigrated across the endothelial monolayer with significantly increased transmigration times when both beta2 and alpha4 integrins were blocked. These results demonstrate that monocytes can use multiple receptors to interact with endothelial cells at both primary and secondary adhesion stages, and that these pathways have to be blocked simultaneously for maximum inhibition.

Cell Adhesion↗
PubMed1997

Neutrophil CD18-dependent arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow can be activated through L-selectin.

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.

Adult↗