Red Cell Adhesion Research Articles

Microvascular Rheology and Hemodynamics

The goal of elucidating the biophysical and physiological basis of pressure-flow relations in the microcirculation has been a recurring theme since the first observations of capillary blood flow in living tissues. At the birth of the Microcirculatory Society, seminal observations on the heterogeneous distribution of blood cells in the microvasculature and the rheological properties of blood in small bore tubes raised many questions on the viscous properties of blood flow in the microcirculation that captured the attention of the Society's membership. It is now recognized that blood viscosity in small bore tubes may fall dramatically as shear rates are increased, and increase (dramatically with elevations in hematocrit. These relationships are strongly affected by blood cell deformability and concentration, red cell aggregation, and white cell interactions with the red cells anti endothelium. Increasing strength of red cell aggregation may result in sequestration of clumps of red cells with either reductions or increases in microvascular hematocrit dependent upon network topography. During red cell aggregation, resistance to flow may thus decrease with hematocrit reduction or increase due to redistribution of red cells. Blood cell adhesion to the microvessel wall may initiate flow reductions, as, for example, in the case of red cell adhesion to the endothelium in sickle cell disease, or leukocyte adhesion in inflammation. The endothelial glycocalyx has been shown to result from a balance of the biosynthesis of new glycans, and the enzymatic or shear-dependent alterations in its composition. Flow-dependent reductions in the endothelial surface layer may thus affect the resistance to flow and/or the adhesion of red cells and/or leukocytes to the endothelium. Thus, future studies aimed at the molecular rheology of the endothelial surface layer may provide new insights into determinants of the resistance to flow.

Effects of Single Nucleotide Polymorphisms of the β2 Adrenergic Receptor and of Adenylate Cyclase on Sickle Red Cell Adhesion to Laminin.

Single nucleotide polymorphisms (SNPs) of the β2 adrenergic receptor (β2AR) gene (ADRB2) on chromosome 5 have been implicated in clinical variability of several cardiopulmonary disorders. Also, Hoppe et al. [2004] have recently found evidence that polymorphisms of ADRB2 correlate with the incidence of stroke in sickle cell disease (SCD). Stimulation of sickle red cell (SS RBC) adrenergic receptors by epinephrine, as well as stimulation of adenylate cyclase by forskolin, activates the B-CAM/LU laminin receptor [Hines et al. 2003]. There is also evidence that activation of B-CAM/LU on SS RBCs occurs in vivo [Zen et al. 2004]. However, SS RBCs from different individuals show variable baseline adhesion to laminin, as well as variable responsiveness to stimulation by epinephrine. We have found that butoxamine, which specifically inhibits the β2AR, blocked epinephrine-induced SS RBC adhesion by 91%, thus implicating β2ARs in activation of adhesion. Thus, we explored whether SNPs of the genes encoding β2AR (the mediator of epinephrine effects) and adenylate cyclase (a downstream effector of β2AR) affect the degree of baseline adhesion to laminin, as well as the degree to which RBCs upregulate adhesion in response to epinephrine. All samples studied were obtained from non-transfused Hb SS patients in steady state. SS RBCs from 14 of 20 patients studied showed increased adhesion in response to epinephrine, with a mean fold increase of 1.98±0.9. These results are similar to those previously described by Hines. Four SNPs of ADRB2 were examined by direct sequencing; two of these reside in the leader sequence, while the other two encode amino acid substitutions. Four intronic SNPs in ADCY6 were studied by Taqman assays. We found that the arg16gly substitution in β2AR is associated with elevation of baseline SS RBC adhesion measured at 1 dyne/cm2: arg/arg=122, arg/gly=149, gly/gly=262 (mean adherent cells/mm2, n=3, 11, 6 respectively, p=0.03 for gly/gly vs arg/arg + arg/gly). However, the arg16gly substitution was not found to be associated with a statistically significant difference either in the maximal adhesion observed after stimulation by epinephrine: arg/arg=116, arg/gly=263, gly/gly=286 (mean adherent cells/mm2), or in the fold increase observed after stimulation. In addition, two linked SNPs (HCV1244841 and RS3730070, r2=0.8) of ADCY6, which is encoded by chromosome 12, also had a statistically significant effect on baseline but not on epinephrine-stimulated SS RBC adhesion to laminin. For HCV1244841, mean baseline adhesion to laminin at 1 dyne/cm2 was 118 and 303 cells/mm2 for AA+AG and GG, respectively (n=13 and 6, p=0.007), while for RS3730070 mean baseline adhesion was 130 and 327 cells/mm2 for CC+CG and GG, respectively (n=15 and 5, p=0.01). However, these ADCY6 SNPs also showed no statistically significant effect on epinephrine-stimulated adhesion. Overall, both the β2AR and ADCY6 polymorphisms appeared to significantly affect baseline adhesion but not epinephrine-stimulated adhesion in this study. We conclude that β2AR and adenylate cyclase polymorphisms affect red cell adhesive function. We theorize that signaling pathway polymorphisms, by affecting baseline activation of SS RBC adhesion, may influence the severity of SCD.

Epinephrine-Induced Sickle Red Cell Adhesion and Vaso-Occlusion In Vivo Is Inhibited by the β-Adrenoceptor Blocker Propranolol.

Painful vaso-occlusive episodes in SCD are commonly associated with infection and other less definable stressors. Since epinephrine activates sickle red cell (SS RBC) adhesion in vitro, we studied the physiologic effect of adrenoceptor signaling activation by epinephrine on SS RBC adhesion to endothelium in vivo. We also investigated whether β-adrenoceptor blockade by propranolol would reduce adhesion and vaso-occlusion. Boluses of washed fluorescently labeled human SS RBCs, treated with epinephrine or vehicle in vitro, were infused into anesthetized athymic nude mice with window chambers implanted into their dorsal skin. Intravital microscopy of contralateral subdermal microvasculature was then performed to observe the dynamic interactions between flowing human SS RBCs and non-activated endothelium. Epinephrine induced human SS RBC adhesion, with frequent postcapillary obstruction. In contrast, neither sham-treated SS RBCs nor epinephrine-treated normal RBCs adhered appreciably to endothelium. Blood flow rates in venules of mice infused with epinephrine-treated SS RBCs was dramatically decreased, with fluxes of 19667±9048 and 6622±1494 circulating RBC/min/μm2 for sham-treated and epinephrine-treated cells, respectively (p=0.0074). SS RBC trapping in lung, spleen and kidney was assessed by fluorescence microscopy of frozen tissue sections collected 30 minutes post injection. Sham-treated SS RBCs were trapped to some degree in the lungs and spleen but only minimally in the kidney. However, epinephrine treatment markedly increased SS RBC trapping in all organs. To quantitate cell survival, sham-treated and epinephrine-treated SS RBCs labeled with different fluorescent dyes were co-infused into the same mouse. Blood samples were collected at intervals after infusion and analyzed by flow cytometry. Ten minutes after infusion, the percentage of circulating sham-treated SS RBCs was 3-fold higher than for epinephrine-treated cells, thus showing an inverse relationship between the percentage of circulating SS RBCs and the degree to which these cells were trapped in the organs studied. Finally, to determine whether propranolol can block epinephrine-induced SS RBC adhesion, SS RBCs were pretreated with propranolol, followed by treatment with epinephrine, then washed before infusion. Propranolol significantly inhibited the effect of epinephrine on SS RBC adhesion, resulting in markedly decreased obstruction of postcapillary vessels. Propranolol improved epinephrine-treated SS RBC circulation, with fluxes of 18809±7868, 3560±1443 and 16722±4985 circulating RBCs/min/μm2 for propranolol-treated, epinephrine-treated, and propranolol+epinephrine-treated cells, respectively (p<0.0001, propranolol-treated vs epinephrine-treated; p<0.0001, epinephrine-treated vs epinephrine+propranolol-treated). Ten minutes after infusion, the percentage of propranolol+epinephrine-treated SS RBCs in the circulation was similar to the percentage of propranolol-treated SS RBCs. Intravenous propranolol administration also blocked epinephrine-treated SS RBC adhesion, so that epinephrine-treated SS RBCs showed no increased adhesion in animals who received IV propranolol. We have thus demonstrated that (1) the stress hormone epinephrine can induce vaso-occlusion in vivo via activation of SS RBC adhesion, and (2) propranolol can inhibit epinephrine-induced SS RBC adhesion and prevent vaso-occlusion in this setting. Thus, we theorize that β-blockers may be useful in preventing or treating vaso-occlusion in SCD.

Effect of fibrinogen on leukocyte margination and adhesion in postcapillary venules.

To quantitatively evaluate the role of fibrinogen (Fb) as a determinant of leukocyte (WBC) margination in postcapillary venules in light of its ability to induce red blood cell (RBC) aggregation with reductions in shear rate (gamma) and increase adhesiveness of WBCs to endothelium (EC). Red cell aggregation (RCA), WBC margination (flux at the EC), rolling velocity, and adhesion to the EC were measured in rat mesenteric postcapillary venules upon reducing gamma, prior to and following systemic infusion of Fb. Proximal occlusion of feeding microvessels with a blunted probe facilitated reductions in gamma from 600 to 50 s(-1). An index of aggregation (G) was derived from light-scattering properties of RBCs, where G was proportional to the number of RBCs per aggregate. WBC margination was measured as the percentage of total luminal WBC flux that rolled on the EC, F*(WBC). For normal levels of Fb (0.07 g%), reductions in gamma resulted in a 4-fold rise in F*(WBC) and no change in G as gamma was reduced to 50 s(-1). Infusion of Fb to achieve a plasma concentration to 0.7 g% caused a modest 20% increase in G and a 2.5-fold increase in F*(WBC) at gamma = 50 s(-1). WBC-EC adhesion appeared to increase significantly, but much less than with infusion of high molecular weight dextran (Dx). With Dx, G increased 3-fold, with reductions in gamma, but F*(WBC) increased only half the amount incurred with Fb at low shear. The greater margination in the presence of Fb results from RBC rouleaux that promote radial migration of WBCs. In contrast, clumps of RBCs resulting from high molecular weight Dx entrain WBCs within plasma gaps along the vessel centerline. In the presence of Fb, margination of WBCs increases dramatically at low shear due to rouleaux formation, which enhances radial migration of WBCs. This effect is much greater than with Dx because disruption of the much weaker Fb induced rouleaux precludes reductions in H(MICRO), whereas clumping aggregates induced by Dx form plasma gaps. Thus, modest levels of RCA caused by increased Fb may greatly enhance margination and with an enhancement of adhesiveness synergistically promote firm WBC-EC adhesion in the low flow state.

In Vivo Studies of Sickle Red Blood Cells

The defining clinical feature of sickle cell anemia is periodic occurrence of painful vasoocclusive crisis. Factors that promote trapping and sickling of red cells in the microcirculation are likely to trigger vasoocclusion. The marked red cell heterogeneity in sickle blood and abnormal adhesion of sickle red cells to vascular endothelium would be major disruptive influences. Using ex vivo and in vivo models, the authors show how to dissect the relative contribution of heterogeneous sickle red cell classes to adhesive and obstructive events. These studies revealed that (1) both rheological abnormalities and adhesion of sickle red cells contribute to their abnormal hemodynamic behavior, (2) venules are the sites of sickle cell adhesion, and (3) sickle red cell deformability plays an important role in adhesive and obstructive events. Preferential adhesion of deformable sickle red cells in postcapillary venules followed by selective trapping of dense sickle red cells could result in vasoocclusion. An updated version of this 2-step model is presented. The multifactorial nature of sickle red cell adhesion needs to be considered in designing antiadhesive therapy in vivo.

Sickle red cell microrheology and sickle blood rheology.

The hallmark of the phenotypic expression of sickle cell disease is the remarkable degree of heterogeneity in the clinical manifestations. They vary latitudinally among patients and longitudinally in the same patient. The pathogenesis of sickle cell anemia centers on the sequence of events that occur between polymerization of deoxy hemoglobin S and increased red cell destruction, vasoocclusion, and end organ damage. Cellular dehydration, changes in sickle red blood cell rheology, adhesion of sickle red cells to vascular endothelium, inflammatory response, and tissue injury are some of the factors that contribute to hemolytic anemia, vasoocclusion, and eventual multiorgan damage. The focus of this review is on the rheology of sickle blood and microrheology of sickle RBC. Determinants of sickle RBC rheology and the factors that modulate its severity are discussed.

Pathophysiology of stroke in sickle cell disease.

Stroke affects both motor and cognitive function in patients with sickle cell disease (SCD). Symptomatic stroke is associated with intimal disease of the large cerebral arteries. Silent stroke, defined as cerebral infarction in the absence of overt clinical neurologic symptoms, is often due to microinfarcts suggestive of microvascular disease. While the natural history of stroke in SCD is well described, the pathophysiology remains poorly understood and probably varies with the site of vascular injury. Increased red cell adhesion, oxidative injury of the vessel wall, inflammation, abnormal vasomotor tone regulation, and increased activity of the coagulation system all may contribute to cerebral vasopathology in SCD.

Pathophysiology of Stroke in Sickle Cell Disease

Stroke affects both motor and cognitive function in patients with sickle cell disease (SCD). Symptomatic stroke is associated with intimal disease of the large cerebral arteries. Silent stroke, defined as cerebral infarction in the absence of overt clinical neurologic symptoms, is often due to microinfarcts suggestive of microvascular disease. While the natural history of stroke in SCD is well described, the pathophysiology remains poorly understood and probably varies with the site of vascular injury. Increased red cell adhesion, oxidative injury of the vessel wall, inflammation, abnormal vasomotor tone regulation, and increased activity of the coagulation system all may contribute to cerebral vasopathology in SCD.

Alpha Thalassemia is associated with decreased risk of abnormal transcranial Doppler ultrasonography in children with sickle cell anemia.

Cerebrovascular complications of sickle cell disease (SCD) are common, but the risk factors remain unclear. The multicenter Stroke Prevention Trial in Sickle Cell Anemia (STOP) provided an opportunity to examine alpha thalassemia-2 as a modifying risk factor, using abnormal transcranial Doppler ultrasonography (TCD) as a surrogate marker for cerebrovascular disease. The authors hypothesized that children with abnormal TCD are less likely to have alpha thalassemia-2, and an increased hemoglobin level accounts for this protective effect. A retrospective study was conducted of children with SCD who had both alpha gene and TCD data from STOP: 128 with TCD of at least 200 cm/s (abnormal TCD) and 172 with TCD less than 170 cm/s (normal TCD). Alpha thalassemia-2 was more frequent in the normal TCD group compared with the abnormal TCD group. The odds ratio for normal TCD and alpha thalassemia-2 was 4.1. Adjusting for either hemoglobin level or red cell size (mean corpuscular volume) reduced the odds ratio only slightly. Age, normal TCD, and alpha thalassemia-2 had significant statistical interaction, so that alpha thalassemia-2 was not related to TCD for age 10 years or older. The frequency of alpha thalassemia-2 was significantly higher in children with normal TCD. Speculation on mechanisms of effect includes improved erythrocyte deformability, reduced red cell adhesion, and reduced nitric oxide scavenging in alpha thalassemia-2. The association of alpha thalassemia-2 and normal TCD adds to the evidence on the protective effects of alpha thalassemia-2 in SCD and highlights the contribution of epistatic factors.

Musculoskeletal manifestations of hemoglobinopathies.

Patients with sickle cell disease often seek treatment for rapid periorbital swelling due to infarction of the orbital bones. Because of resulting orbital compression syndrome, treatment with corticosteroids and antibiotics is advisable. If spinal tuberculosis occurs in patients with sickle cell anemia, radiologic signs will be a combination of the two conditions. The diagnosis of juvenile rheumatoid arthritis is usually delayed in patients with sickle cell disease. Sulphasalazine is the disease-modifying drug of choice for treating juvenile rheumatoid arthritis, because it also reduces the adhesiveness of sickled red cells. TNF-alpha inhibitors may also be useful for treating these patients. A volumetric method to determine the size and special distribution of the necrotic lesions of the femoral head has been developed using magnetic resonance imaging scans. With this method it will be easier to determine which early lesions require core decompression, or which ones should be treated conservatively. Osteomyelitis can be differentiated from bone infarction with the use of segmental radionuclide bone-marrow and bone scans. Reduction in frequency of painful crises can be achieved by increasing fetal hemoglobin with the use of hydroxyurea. The treatment of the actual pain requires decisions about the analgesics that are used as well as the route of their administration. Ketorolac monotherapy is likely to fail in the presence of an initial high pain score or with involvement of four or more pain sites.

HODGKIN'S DISEASE IN A CHILD WITH SICKLE CELL DISEASE TREATED WITH HYDROXYUREA

Hydroxyurea (HU) is an oral drug that ameliorates the clinical course of sickle cell anemia by ¯ increasing the levels of fetal hemoglobin and decreasing the adhesion of red cells to endothelium. Although HU has minimal short-term toxicity, few data are available about the long-term safety and the potential risk for carcinogenesis or leukemogenesis. An 8-year-old child with sickle cell / g 0- thalassemia who received HU treatment for painful crises is described. Six months after the initiation of the HU treatment he developed Hodgkin's disease, lymphocyte predominance subtype. Chemotherapy induced a complete remission. After discontinuation of chemotherapy the painful crises recurred and bone marrow transplantation was decided at the age of 12 years. Two years after the bone marrow transplantation, the child is in complete remission without painful crises. Although the authors suggest that the development of Hodgkin's disease is a coexisting event, questions arise about the safety of HU treatment in childhood.

Exciting New Treatment Approaches for Pathyphysiologic Mechanisms of Sickle Cell Disease

During the past several decades, our understanding of the complex pathophysiology of vasoocclusion associated with sickle cell disease has improved greatly. Interaction of genes, hemoglobin molecules, red cell membrane and metabolic changes, cell-cell interactions and cell-plasma interactions, red cell adhesion to vascular endothelium, activation of coagulation, and vascular reactivity play a role in vasoocclusion. Penicillin prophylaxis of pneumococcal infections and appropriate use of blood transfusions and other supportive measures improved survival of sickle cell patients. Hydroxyurea made a major impact on sickle cell therapy when it was shown to decrease acute painful episodes, acute chest syndrome, and the need for blood transfusion in adults. Significant experience in the use of hydroxyurea has been accumulated in older children. The benefits and risks of hydroxyurea for younger children and long-term risks in all patients will be evaluated in future investigations. Other promising therapies include butyrate compounds, clotrimazole, magnesium supplementation, poloxamer 188, antiadhesion agents, anticoagulant approaches, and nitric oxide. Hemopoietic transplantation remains the only curative therapy. However, several transgenic mouse models are available for studies of gene therapy or other treatment approaches on biochemical, cellular, and pathologic effects of mutant genes.

EXCITING NEW TREATMENT APPROACHES FOR PATHYPHYSIOLOGIC MECHANISMS OF SICKLE CELL DISEASE

During the past several decades, our understanding of the complex pathophysiology of vasoocclusion associated with sickle cell disease has improved greatly. Interaction of genes, hemoglobin molecules, red cell membrane and metabolic changes, cell-cell interactions and cell-plasma interactions, red cell adhesion to vascular endothelium, activation of coagulation, and vascular reactivity play a role in vasoocclusion.Penicillin prophylaxis of pneumococcal infections and appropriate use of blood transfusions and other supportive measures improved survival of sickle cell patients. Hydroxyurea made a major impact on sickle cell therapy when it was shown to decrease acute painful episodes, acute chest syndrome, and the need for blood transfusion in adults. Significant experience in the use of hydroxyurea has been accumulated in older children. The benefits and risks of hydroxyurea for younger children and long-term risks in all patients will be evaluated in future investigations. Other promising therapies include butyrate compounds, clotrimazole, magnesium supplementation, poloxamer 188, antiadhesion agents, anticoagulant approaches, and nitric oxide. Hemopoietic transplantation remains the only curative therapy. However, several transgenic mouse models are available for studies of gene therapy or other treatment approaches on biochemical, cellular, and pathologic effects of mutant genes.

The role of cytokines in sickle cell disease.

Sickle cell disease (SCD) is characterized by chronic hemolysis, frequent infections, and recurrent occlusions of microcirculation, which cause painful crises and result in chronic organ damage and failure. Occlusions of the microcirculation and infections are important factors that stimulate the production of cytokines and acute-phase proteins. Cytokines seem to be involved with several possible mechanisms in the pathogenesis of vasoocclusive phenomena in SCD: vascular endothelial activation, induction of red-cell adhesiveness to vascular endothelium, induction of neutrophil adhesiveness to endothelium, development of vascular intimal hyperplasia, platelet activation, endothelin-1 production, and dysregulation of endothelial apoptosis. Cytokines are also thought to be involved in the regulation of hemopoiesis, the inhibition of immune functions, and the development of growth deficits. Investigation of cytokines in SCD patients will elucidate the pathogenesis of the disease and its complications and may help in assessing disease severity and prognosis.

Particulate air pollution and the blood

BACKGROUNDParticulate air pollution has been associated with excess deaths from, and increases in hospital admissions for, cardiovascular disease among older people. A study was undertaken to determine whether this may...

Critical Factors in Basal Cell Adhesion Molecule/Lutheran-mediated Adhesion to Laminin

Basal cell adhesion molecule (B-CAM) and Lutheran (LU) are two spliceoforms of a single immunoglobulin superfamily protein containing five Ig domains and comprise the sickle (SS) red cell receptor for laminin. We have now analyzed laminin binding to murine erythroleukemia cells transfected with various human B-CAM/LU constructs. B-CAM and LU bound equally well to laminin, indicating that the longer cytoplasmic tail of LU is not required for binding. However, binding of soluble laminin did require the presence of the membrane-proximal fifth immunoglobulin superfamily (IgSF) domain of LU, while deletion of IgSF domains 1, 2, 3, or 4 individually or together did not abrogate laminin binding. Under flow conditions, MEL cells expressing B-CAM, LU, and LU lacking domains 1, 2, 3, or 4 adhered to immobilized laminin with critical shear stresses over 10 dynes/cm2. However, MEL cells expressing LU lacking domain 5 bound to laminin poorly (critical shear stress = 2.3 dynes/cm2). Moreover, expression of only IgSF domain 5 of LU was sufficient to mediate MEL cell adhesion to immobilized laminin (critical shear stress >10 dynes/cm2). Finally, Scatchard analysis showed that SS red cells had an average of 67% more B-CAM/LU than normal red cells, and low density red cells from sickle cell disease patients expressed 40-55% more B-CAM/LU than high density SS red cells. B-CAM/LU copy number thus may also play a role in the abnormal adhesion of SS red cells to laminin.

Disturbance of plasma and platelet thrombospondin levels in sickle cell disease.

Thrombospondin (TSP), a large protein found in platelet alpha-granules (as TSP-1), mediates adhesion of sickle reticulocytes to cultured vascular endothelium. To further explore the physiologic relevance of this observation, we have measured plasma TSP levels and platelet TSP-1 content in subjects with sickle cell disease. Plasma TSP levels were similar for normal controls (mean 491 ng/ml, range 331-723) and steady-state HbSS patients (mean 536, range 333-1107) and were significantly (P = 0.012) but variably elevated for HbSS patients presenting with acute painful crisis (mean 868, range 442-2780). Some of these elevated plasma TSP levels reached those previously observed to support maximal red cell adhesion to endothelium in vitro. Compared to normals, both steady-state and in-crisis HbSS patients had significantly (P < 0.001) depressed platelet TSP-1 content (82.6 +/- 11.9, 47.1 +/- 16.0 and 45.9 +/- 20.7 ng/l0(6) platelets, respectively, mean +/- SD). HbSC disease patients, all examined during steady state, had low-normal plasma levels of TSP and either normal or depressed platelet TSP-1 content. Serial observations on three sickle cell anemia subjects indicated a probable relationship between platelet TSP-1 release, elevated plasma TSP levels, and acute vaso-occlusive episodes. These results suggest a state of ongoing release and depletion of TSP-1 from activated platelets in patients with sickle cell disease.

The pathophysiology of vascular obstruction in the sickle syndromes

Vasocclusive events in the sickle-cell syndromes have multiple determinants: first and foremost is the capacity of red cells to undergo intracellular polymerization of deoxy HbS. However, the impact of the sicklable red cell is not limited to mechanical obstruction of the microcirculation, but also results in other and sometimes unexpected consequences. For example, red-cell destruction leads to large numbers of young red cells with enhanced vascular adhesion and increased K:Cl cotransport expression, in addition to an elevated percent of erythrocytic HbE These pleiotropic effects, that is, multiple phenotypic effects from a single gene, can be further modulated by the action of epistatic effects, that is, the action of other genes besides β s The interaction of epistatic and pleiotropic effects leads to the interindividual phenotypic variations characteristic of sickle-cell disease. Further understanding of pleiotropic effects (i.e. mechanism of red-cell adhesion, production of vasoactive substances by damaged endothelium, etc.), will uncover new epistatic effects. At the end, we will be able to define not only the genotype, but also the phenotypic severity. This review covers the present knowledge of the red-cell and non-red-cell determinants of vasocclusion, and proposes models to explain the acute painful crises that commonly afflict these patients.

In vivo demonstration of red cell-endothelial interaction, sickling and altered microvascular response to oxygen in the sickle transgenic mouse.

Intravascular sickling, red cell-endothelium interaction, and altered microvascular responses have been suggested to contribute to the pathophysiology of human sickle cell disease, but have never been demonstrated under in vivo flow. To address this issue, we have examined a transgenic mouse line, alphaHbetaSbetaS-Antilles [betaMDD] which has a combined high (78%) expression of beta S and beta S-Antilles globins. In vivo microcirculatory studies using the cremaster muscle preparation showed adhesion of red cells, restricted to postcapillary venules, in transgenic mice but not in control mice. Electron microscopy revealed distinct contacts between the red cell membrane and the endothelium surface. Some red cells exhibiting sickling were regularly observed in the venular flow. Infusion of transgenic mouse red cells into the ex vivo mesocecum vasculature also showed adhesion of mouse red cells exclusively in venules. Under resting conditions (pO2, 15-20 mmHg), there were no differences in the cremaster microvascular diameters of control and transgenic mice; however, transgenic mice showed a drastic reduction in microvascular red cell velocities (Vrbc) with maximal Vrbc decrease (> 60%) occurring in venules, the sites of red cell adhesion and sickling. Local, transient hyperoxia (pO2, 150 mmHg) resulted in striking differences between control and transgenic mice. In controls, oxygen caused a 69% arteriolar constriction, accompanied by 75% reduction in Vrbc. In contrast, in transgenic mice, hyperoxia resulted in only 8% decrease in the arteriolar diameter and in 68% increase in VrBC; the latter is probably due to an improved flow behavior of red cells as a consequence of unsickling. In summary, the high expression of human sickle hemoglobin in the mouse results not only in intravascular sickling but also red cell-endothelium interaction. The altered microvascular response to oxygen could be secondary to blood rheological changes, although possible intrinsic differences in the endothelial cell/vascular smooth muscle function in the transgenic mouse may also contribute. These sickle transgenic mice could serve as a useful model to investigate vasoocclusive mechanisms, as well as to test potential therapies.

Plasmodium falciparum: Cytoadherence of malaria-infected erythrocytes to human brain capillary and umbilical vein endothelial cells—A comparative study of adhesive ligands

The cytoadherence of Plasmodium falciparum-infected erythrocytes (FCR-3 line) to human brain capillary endothelial cells (HBEC), C32 amelanotic melanoma cells, and human umbilical vein endothelial cells (HUVEC) was studied. The adhesion of infected red cells was HBEC > amelanotic melanoma > HUVEC. The presence or absence of the adhesive ligands ICAM-1 (CD54 or intercellular adhesion molecule 1), ICAM-2, and CD36 (=glycoprotein IV) was determined for each of these cells by indirect immunofluorescence using the monoclonal antibodies RR1/1, 6D5, and OKM 5/OKM 8, respectively. It appeared that a major ligand for the FCR-3 line of P. falciparum with amelanotic melanoma cells and HBECs was CD36. Binding to HUVECs was very low, presumably due to their lack of expression of CD36. HBECs, because of their ease of in vitro propagation, long-term maintenance of cytoadherent properties, and their high degree of adhesiveness, will be useful for in vitro studies of adherence.