Biomedical subjects

Michael B Lawrence

Publications and source records attributed to Michael B Lawrence.

11 recordsLinked to original sources

PubMed2007

Microparticle adhesive dynamics and rolling mediated by selectin-specific antibodies under flow.

In vitro studies were performed to characterize the relative performance of candidate receptors to target microparticles to inflammatory markers on vascular endothelium. To model the interactions of drug-bearing microparticles or imaging contrast agents with the vasculature, 6 micron polystyrene particles bearing antibodies, peptides, or carbohydrates were perfused over immobilized E- or P-selectin in a flow chamber. Microparticles conjugated with HuEP5C7.g2 (HuEP), a monoclonal antibody (mAb) specific to E- and P-selectin, supported leukocyte-like rolling and transient adhesion at venular shear rates. In contrast, microparticles conjugated with a higher affinity mAb specific for P-selectin (G1) were unable to form bonds at venular flow rates. When both HuEP and G1 were conjugated to the microparticle, HuEP supported binding to P-selectin in flow which allowed G1 to form bonds leading to stable adhesion. While the microparticle attachment and rolling performance was not as stable as that mediated by the natural ligands P-selectin Glycoprotein Ligand-1 or sialyl Lewis(x), HuEP performed significantly better than any previously characterized mAb in terms of mediating microparticle binding under flow conditions. HuEP may be a viable alternative to natural ligands to selectins for targeting particles to inflamed endothelium.

Animals↗

PubMed2006

Bioactive hydrogel substrates: probing leukocyte receptor-ligand interactions in parallel plate flow chamber studies.

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLe(X)) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine-glycine-aspartic acid-serine (RGDS) and leucine-aspartic acid-valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.

Biocompatible Materials↗

PubMed2005

Measuring molecular elasticity by atomic force microscope cantilever fluctuations.

In single-molecule mechanics experiments the molecular elasticity is usually measured from the deformation in response to a controlled applied force, e.g., via an atomic force microscope cantilever. We have tested the validity of an alternative method based on a recently developed theory. The concept is to measure the change in thermal fluctuations of the cantilever tip with and without its coupling to a rigid surface via the molecule. The new method was demonstrated by its application to the elasticity measurements of L- and P-selectin complexed with P-selectin glycoprotein ligand-1 or their respective antibodies, which showed values comparable to those measured from the slope of the force-extension curve. L- and P-selectin were found to behave as nearly linear springs capable of sustaining large forces and strains without sudden unfolding. The measured spring constants of approximately 4 and approximately 1 pN/nm for L- and P-selectin, respectively, suggest that a physiological force of approximately 100 pN would result in an approximately 200% strain for the respective selectins.

Calibration↗

PubMed2005

PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow.

P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLe(x))-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLe(x) or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLe(x) and PSGL-1 microspheres adhere to 4 h of IL-1beta-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLe(x) microspheres despite the fact that the sLe(x) microspheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLe(x) microspheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes.

Cell Adhesion↗

PubMed2004

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin.

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.

Binding Sites↗

PubMed2004

The molecular mechanics of P- and L-selectin lectin domains binding to PSGL-1.

A laser trap was used to compare the load-dependent binding kinetics between truncated P- and L-selectin to their natural ligand, P-selectin glycoprotein ligand-1 (PSGL-1) over the predicted physiological range of loading rates. Human PSGL-1 was covalently coupled to polystyrene beads. Chimeric selectins were adsorbed to nitrocellulose-coated glass beads on a coverslip. A PSGL-1 bead was held in a laser trap and touched to a vertical surface of the glass bead, allowing a bond to form between selectin and ligand. The surface was moved away from the microsphere, applying load at a constant rate until bond rupture. Rupture force for both selectins increased with loading rate, but significant differences in rupture force between P- and L-selectin were observed only above 460 pN/s. These data are best represented as two energy barriers to unbinding, with the transition from the low to high loading rate regime at 260-290 pN/s. The data also allow the first estimate of a two-dimensional specific on-rate for binding of these two selectins to their natural ligand (1.7 microm2/s). These data suggest that P- and L-selectin lectin domains have very similar kinetics under physiological conditions.

Binding Sites↗

PubMed2004

Sonorheometry: a noncontact method for the dynamic assessment of thrombosis.

Inappropriate blood coagulation plays a central role in the onset of myocardial infarction, stroke, pulmonary embolism, and other thrombotic disorders. The ability to screen for an increased propensity to clot could prevent the onset of such events by appropriately identifying those at risk and enabling prophylactic treatment. Similarly, the ability to characterize the mechanical properties of clots in vivo might improve patient outcomes by better informing treatment strategies. We have developed a technique called sonorheometry. Unlike existing methods, sonorheometry is able to assess mechanical properties of coagulation with minimal disturbance to the delicate structure of a forming thrombus. Sonorheometry uses acoustic radiation force to produce small, localized displacements within the sample. Time delay estimation is performed on returned ultrasound echoes to determine sample deformation. Mechanical modeling and parametric fitting to experimental data yield maps of mechanical properties. Sonorheometry is well suited to both in vitro and in vivo applications. A control experiment was performed to verify that sonorheometry provides mechanical characterization in agreement with that from a conventional rheometer. We also examined thrombosis in blood samples taken from four subjects. This data suggests that sonorheometry may offer a novel and valuable method for assessing the thrombogenicity of blood samples.

Blood↗

PubMed2003

Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan.

Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.

Animals↗

PubMed2003

Activation of A2A adenosine receptors inhibits expression of alpha 4/beta 1 integrin (very late antigen-4) on stimulated human neutrophils.

The alpha 4/beta 1 integrin very late antigen-4 (CD49d/CD29) is up-regulated on circulating neutrophils of septic patients. Although no individual agent mimics this effect of sepsis, we now report that following priming of human neutrophils with lipopolysaccharide or tumor necrosis factor alpha (TNF-alpha), addition of formyl-Met-Leu-Phe (fMLP) results in a "stimulated", sepsis-like, four- to fivefold rise in CD49d expression. TNF/fMLP stimulation also produced a similar increase in CD49d-mediated adhesion of neutrophils to a vascular cell adhesion molecule-1 (VCAM-1)-coated surface. Adenosine is a naturally occurring, anti-inflammatory mediator released from injured or inflamed tissues. We observed that stimulated neutrophil CD49d expression was decreased by activation of A(2A) adenosine receptors (A(2A)AR) with the selective agonist 4-[3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylicacid methyl ester (ATL146e; EC(50)=6.4 nM). ATL146e (100 nM) also reduced the fraction of stimulated neutrophils that adhered to VCAM-1 from 38 +/- 6% to 27 +/- 5%. Inhibition of CD49d expression was equally inhibited by ATL146e, added before or after TNF priming, and was reversed by incubation with the A(2A)AR-selective antagonist 4-[2-[7-amino-2-(2-furyl) (1, 2, 4)triazolo(2,3-a) (1, 3, 5)triazin-5-yl-amino]ethyl]-phenol (ZM241385; 100 nM). A suboptimal ATL146e concentration (1 nM) combined with the type IV phosphodiesterase inhibitor rolipram (100 nM) synergistically decreased stimulated CD49d expression by >50%. The cyclic adenosine monophosphate (cAMP)-dependent kinase [protein kinase A (PKA)] inhibitor H-89 (10 microM) reversed the effect of ATL146e on stimulated CD49d expression. Other means of increasing cAMP in neutrophils also decreased stimulated CD49d expression. We conclude that adenosine binding to A(2A)AR counteracts stimulation of neutrophil CD49d integrin expression and neutrophil binding to VCAM-1 via a cAMP/PKA-mediated pathway.

Adenosine↗

PubMed2002

Comparison of PSGL-1 microbead and neutrophil rolling: microvillus elongation stabilizes P-selectin bond clusters.

A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophil's surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21 microm at 0.4 dyn/cm2, and increased to 1.58 microm at 2 dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152 pN/microm at 0.4 and 2.0 dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.

Antibodies, Monoclonal↗

PubMed2002

Viscosity-independent velocity of neutrophils rolling on p-selectin in vitro or in vivo.

OBJECTIVE: To determine whether selectin-mediated leukocyte-rolling velocity in inflamed venules in vivo is determined by wall shear rate (WSR) or by wall shear stress (WSS). METHODS: WSS was manipulated independently of WSR by altering the viscosity of blood plasma in mice with an isovolemic exchange of blood for low- or high-viscosity dextran solutions. Rolling of neutrophils or beads coated with P-selectin glycoprotein ligand-1 (PSGL-1) was reconstituted on P-selectin immobilized on the wall of a parallel plate flow chamber at two different viscosities of the perfusion medium. RESULTS: Leukocytes in vivo showed no increase in rolling velocity when shear stress was doubled by doubling viscosity. Neutrophils in the parallel-plate flow chamber in vitro showed the same dependence on WSR as leukocytes in vivo, but bead-rolling velocities correlated best with WSS. Rolling leukocytes, but not beads, deformed significantly in shear flow, and deformation correlated better with WSS. CONCLUSION: These data suggest leukocyte deformation during rolling offsets increased bond breakage at higher shear stress. The stable rolling velocity allows sufficient surveillance of the endothelial surface, even in venules with high WSS. doi:10.1038/sj.mm.7800165

Animals↗