Timothy M. Wick

Activation of Vascular Endothelial Cell Adhesion Molecule Expression by Sickle Blood Cells

Microvascular complications in sickle cell disease occur as a result of obstruction of small vessels by deoxygenated sickle cells. Cerebrovascular complications are also common and result from obstruction of large blood vessels by thrombosis with changes in vessels that have some similarity to those found in arteriosclerotic vascular disease. Endothelial damage and activation from sickle cell-endothelial interactions may contribute to both. We find that endothelial cells have increased expression of VCAM-1, E-selectin, and ICAM-1 when exposed to sickle blood cells. The concentration-dependent, sickle-induced, adhesion molecule expression is significantly greater than that promoted by normal cells. The time course of Cell Adhesion Molecule (CAM) expression is similar to that induced by TNF-α. and IL1. Studies after white cell enrichment and reduction suggest leukocytes are the primary mediators. CAM expression by endothelial cells appears stimulated by soluble factors. Antibody inhibition studies support TNF-α and IL-1, produced by sickle leukocytes, as the primary soluble factors responsible for the observed CAM expression. Both the induction of endothelial CAM expression and subsequent endothelial adherence of sickle erythrocytes may play significant roles in the pathophysiology of sickle-related complications, and reduction in CAM expression may provide a new approach to treatment.

Hemodynamic modulation of monocytic cell adherence to vascular endothelium

Hemodynamic shear stress is hypothesized to contribute to the localization of atherosclerotic plaques to certain arterial sites. Monocyte recruitment to these sites is an early event in atherogenesis. To determine the possible mechanisms by which shear stress modulates monocyte adhesionin vivo, studies of human monocytic cell adherence to endothelium were conducted under different shear conditions in a parallel-plate flow chamber. The number of monocytes capable of developing firm adhesive contacts with endothelium decreased as shear stress-induced drag forces increased over the range of 0.5 to 30 dynes/cm2. The number of adherent monocytic cells at a given shear stress was highly dependent on the activation state of the endothelium. To test the direct effect of shear stress on endothelial cell adhesivity, endothelial cells were presheared for 2 to 6 hr at 2, 10, or 30 dynes/cm2, and monocytic cell adherence was quantified at 1 dyne/cm2. Adherence increased 330% or 370% when endothelial cells were presheared for 2 hr at 2 or 10 dynes/cm2, respectively, as compared to unsheared endothelium. In contrast, when endothelial cells were presheared at 30 dynes/cm2, monocytic cell adherence at 1 dyne/cm2 was not significantly different from unsheared controls. Increased monocytic cell adherence to presheared endothelium was via a vasuclar cell adhesion molecule 1 (VCAM-1)α4β1 mechanism, and enzyme-linked immunosorbent assay studies showed that preshearing at 2 dynes/cm2 for 2 hr increased endothelial VCAM-1 expression by 38%. These data demonstrated that low levels of shear stress induce enthelial VCAM-1 expression and increase monocytic cell adherence via a VCAM-1/α4β1 mechanism. Thus, shear stress can modulate monocyte adherence to vascular endothelium through drag forces that affect the establishment and maintenance of adhesive bonds and by directly modulating the expression of endothelial VCAM-1. This dual effect of shear stress produces the most favorable conditions for adhesion at low-shear regions, where drag forces are low and induction of VCAM-1 is likely. The preferential adherence of monocytes to these regions may contribute to the localization of atherosclerotic plaques to low-shear regions of the arterial circulationin vivo.

Inhibition of plasma‐mediated adherence of sickle erythrocytes to microvascular endothelium by conformationally constrained RGD‐containing peptides

Adherence of sickle erythrocytes to vascular endothelium likely initiates or participates in microvascular occlusion, leading to ischemic tissue and organ damage characteristic of sickle‐cell pain episodes. In vitro, sickle‐cell adherence to endothelium involves adhesive plasma proteins and integrin and nonintegrin receptors on sickle cells and endothelial cells. The involvement of arginine‐glycine‐aspartic acid (RGD) sequences in adhesive plasma proteins and integrin receptors suggests that RGD‐containing peptides may inhibit sickle‐cell/endothelial‐cell adherence. In the present study, inhibition of plasma‐mediated sickle‐erythrocyte adherence to endothelium using conformationally constrained RGD‐containing peptides was quantified in vitro under continuous flow at a shear stress of 1.0 dyn/cm2. Two conformationally constrained RGD peptides were investigated: 6Z (which has high affinity for α5β1, αvβ3, and αIIIbβ3 integrin receptors), and TP9201 (which preferentially binds to αIIbβ3). Peptide 6Z at 50 μM inhibited plasma‐mediated sickle‐cell adherence to microvascular endothelium 70% when incubated with sickle red cells, and 63% when incubated with endothelium. Under similar conditions, peptide TP9201 inhibited plasma‐mediated sickle‐cell adherence up to 85% at concentrations from 250 to 500 μM TP9201. The inhibition of plasma‐mediated adherence by conformationally constrained RGD peptides, but not by linear or circular constructs, suggests that the tertiary structure of the peptide containing the binding sequence is important. Inhibition of plasma‐mediated sickle‐cell adhesion with these peptides in vitro suggests that such conformationally constrained RGD peptides could provide therapeutic interventions in the course of the disease by inhibiting receptor‐ligand interactions.

Prolonged contact wild blood alters surgical gown permeability

INTRODUCTION Surgical gowns are designed to prevent or minimize transmission of blood and pathogens between patients and hospital personnel. During prolonged procedures, a gown will probably be presented with repeated challenges of blood and other liquids. These multiple insults may alter the fabrics permeability to subsequent blood contact. METHODS In this study, a pressing-leaning simulator was used to quantify changes in fabric permeability to blood after surgical gowns were prewetted with anticoagulated or coagulating blood. RESULTS Of the five commercially available gowns tested, contact with blood for 1 hr before application of an external pressure increased permeability for two gowns, decreased permeability for two gowns, and did not alter the permeability of one gown (as compared with test conditions in which the fabrics were not prewetted with blood). These data indicate that at least in some cases prolonged contact with blood increases the amount of blood penetration on application of an external pressure, such as may occur during a pressing or leaning motion. CONCLUSION Because increased fabric permeability results in an increased risk of skin contact with liquid-borne pathogens for gown users, a major criterion in the design and selection of a gown should be its ability to resist blood penetration for prolonged periods.

Histamine increases sickle erythrocyte adherence to endothelium.

Complications of sickle cell anaemia include vascular occlusion triggered by the adherence of sickle erythrocytes to endothelium in the postcapillary venules. Adherence can be promoted by inflammatory mediators that induce endothelial cell adhesion molecule expression and arrest flowing erythrocytes. The present study characterised the effect of histamine stimulation on the kinetics of sickle cell adherence to large vessel and microvascular endothelium under physiological flow. Increased sickle cell adherence was observed within minutes of endothelial activation by histamine and reached a maximum value within 30 min. At steady state, sickle cell adherence to histamine‐stimulated endothelium was 47 ± 4 adherent cells/mm2, 2·6‐fold higher than sickle cell adherence to unstimulated endothelial cells. Histamine‐induced sickle cell adherence occurred rapidly and transiently. Studies using histamine receptor agonists and antagonists suggest that histamine‐induced sickle cell adhesion depends on simultaneous stimulation of the H2 and H4 histamine receptors and endothelial P‐selectin expression. These data show that histamine release may promote sickle cell adherence and vaso‐occlusion. In vivo histamine release should be studied to determine its role in sickle complications and whether blocking of specific histamine receptors may prevent clinical complications or adverse effects from histamine release stimulated by opiate analgesic treatment.

Cytokine Upregulation of CD36, But Not ICAM-1, Increases Plasmodium Falciparum-Infected Erythrocyte Adherence to Microvascular Endothelial Cells under Shear Conditions

Cytoadherence of parasitized red blood cells (PRBC) to microvascular endothelial cells (MEC) contributes to the pathogenesis of human cerebral malaria (MacPherson et al., 1985). We studied cytoadherence of two strains (HB3 and FC27) of Plasmodium falciparum PRBC to human MEC cultures under shear conditions to elucidate the pathways of adherence to MEC (Wick and Louis, 1991). Using antiICAM-1 and anti-CD36 antibodies, we found that HB3 PRBC bound exclusively to CD36 and ICAM-1 receptors. FC27 PRBC bound to MEC via CD36 (and at least one other pathway) but not to ICAM-1. Stimulation of MEC with interferon gamma (IFNy), which selectively increases CD36 expression, elevated HB3 and FC27 PRBC adherence 64% and 71%, respectively. Conversely, MEC stimulation with tumor necrosis factor (TNF), which selectively upregulates ICAM-1, did not increase HB3 PRBC adherence, in contrast to previous data using large vessel endothelium. Downregulation of CD36 and ICAM-1 expression by phorbol 12, 113-dibutyrate completely abolished HB3 PRBC adherence. These data suggest that cytoadherence to microvascular EC is distinct from adherence to large vessel EC (Berendt et aL, 1989). PRBC adherence to MEC is strain specific, regulatable by cytokines which increase CD36 expression, and abrogated by pharmacologic modulation of MEC receptors. These data have defined the relative contributions of both CD36 and ICAM-1 to PRBC binding to microvascular endothelium and have provided evidence for the presence of an additional adhesion mechanism. antibody therapy of pharmacological manipulation of endothelial cell receptor expression may reduce PBRC adherence to MEC and ameliorate the sequestration associated with human cerebral malaria.

Thrombospondin from Activated Platelets Promotes Sickle Erythrocyte Adherence to Human Microvascular Endothelial Cells Via CD36 and Integrin Receptors

Adherence of erythrocytes to vascular endothelium likely contributes to the pathophysiology of episodic microvascular occlusion in patients with sickle cell disease (Wick, et al., 1987). In addition, coagulation activation has been reported in sickle patients during complications such as pain episodes (Francis and Johnson, 1991). Since platelets contain adhesion proteins (e.g. von Willebrand factor, thrombospondin and fibrinogen), we investigated whether factors released from activated platelets from sickle patients promote adherence of sickle erythrocytes to human microvascular endothelial cells (MEC) under flow conditions.