Sickle Cell Adhesion Research Articles

Cell Membrane Sulfatide Promotes Sickle Cell Adhesion to Endothelium.

Sickle cell disease (SCD) affects millions of people worldwide, and is associated with significant morbidity and mortality. Although the clinical manifestations of the disease are very complex, much of the cause can be ascribed to occlusion of small vessels by the sickle red blood cells (RBCs). More than 30% of all deaths in SCD are due to the vasoocclusion, which results in ischemia, multiorgan failure and strokes. The proximate cause for vasoocclusion appears to be an increased adhesiveness of sickle cells to the vessel wall, and we postulate that the exposure of sulfatide on sickle cells accounts for their adhesive phenotype. Sulfatide binds with high affinity to many of the adhesion proteins known to be involved in cell adhesion to subendothelium and endothelium, including von Willebrand factor (VWF), thrombospondin (TSP), laminin and P-selectin. We therefore compared the expression and distribution of sulfatide in sickle cells to that in normal RBCs. When examined by flow cytometry using a previously described Alexa fluor-conjugated single-chain variable fragment (scFv) antibody, PA38, we found that sickle cells displayed more sulfatide on surface than normal RBCs (mean fluorescence 1.6±0.5 Vs. 0.9±0.3, p<0.05, n=6). When we examined sulfatide distribution by confocal microscopy using the labeled PA38, we found it to label more intensely in sickle cells than the normal RBCs and to be distributed heterogeneously, with areas of intense staining. The heterogeneous distribution suggested that the sulfatide might exist within membrane-microdomains/lipid rafts. We tested this possibility by sucrose density centrifugation of detergent lysates (1% Triton X-100) of erythrocyte ghosts from sickle and normal cells and found that sulfatide was distributed in raft fractions, as defined by being in the fractions containing the raft marker flotillin-1. Consistent with an important role for sulfatide in sickle vaso-occlusion, we found that both normal and sickle RBCs attached under flow to the surface of histamine-activated human umbilical vein endothelial cells (HUVEC). The sickle RBCs adhered more avidly, as they were able to rest higher shear stresses (1.86 and 2.5 dyne/cm2) than the normal RBCs before detaching Greater than 50% of the initial adhesion was inhibited by treatment with the anti-sulfatide scFv, PA38. We obtained similar results in terms of the greater shear resistance of sickle cells and the ability of PA38 to inhibit adhesion when we compared the adhesion of sickle and normal RBCs to surfaces coated with the adhesive ligands such as VWF (the ultra-large form) and laminin. Thus, our study elucidates an important role of red cell membrane sulfatide in sickle cell adhesion to the endothelium and to adhesive ligands, and suggests that this mechanism is important pathophysiologically in the development of sickle vaso-occlusion. Sulfatide distribution into lipid rafts may allow the formation of adhesive patches that facilitate adhesion.

Epinephrine-induced activation of LW-mediated sickle cell adhesion and vaso-occlusion in vivo

Sickle red cell (SS RBC) adhesion is believed to contribute to the process of vaso-occlusion in sickle cell disease (SCD). We previously found that the LW RBC adhesion receptor can be activated by epinephrine to mediate SS RBC adhesion to endothelial alphavbeta3 integrin. To determine the contribution of LW activation to vaso-occlusive events in vivo, we investigated whether in vitro treatment of SS RBCs by epinephrine resulted in vaso-occlusion in intact microvasculature after RBC infusion into nude mice. Epinephrine enhanced human SS but not normal RBC adhesion to murine endothelial cells in vitro and to endothelium in vivo, promoting vaso-occlusion and RBC organ sequestration. Murine sickle RBCs also responded to epinephrine with increased adhesion to postcapillary endothelium in nude mice. Epinephrine-induced SS RBC adhesion, vaso-occlusion, and RBC organ trapping could be prevented by the beta-adrenergic receptor (beta-AR) antagonist, propranolol. Infusion of soluble recombinant LW also significantly reduced adhesion and vaso-occlusion. In addition, epinephrine-treated SS RBCs induced activation of murine leukocyte adhesion to endothelium as well. We conclude that LW activation by epinephrine via beta-AR stimulation can promote both SS RBC and leukocyte adhesion as well as vaso-occlusion, suggesting that both epinephrine and LW play potentially pathophysiological roles in SCD.

Lactadherin mediates sickle cell adhesion to vascular endothelial cells in flowing blood

Increased exposure of sickle red blood cells to phosphatidylserine promotes its adhesion to the endothelium. A monoclonal antibody to lactadherin, a phosphatidylserine binding protein, inhibits sickle cell adhesion to histamine-stimulated endothelial cells in flowing blood. Added lactadherin enhances the adhesion via the integrin alphaVbeta3. These results indicate that lactadherin can mediate phosphatidylserine-expressing sickle cell adhesion to the endothelium.

Small-molecule cyclic αVβ3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.

Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell α4β1integrin: role in adhesion of sickle red blood cells to endothelial cells

AbstractThe Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin α5 chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin α4β1, the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin α4β1 expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin α4β1 under flow conditions. Antibody-mediated activation of integrin α4β1 induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)–Fc proteins. This novel interaction between integrin α4β1 in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

A Monoclonal Antibody to Lactadherin Inhibits Sickle Red Blood Cell Adhesion to Vascular Endothelial Cells in a Plasma Milieu.

Adherence of red blood cells to the endothelium initiates vaso-occlusion in sickle cell anemia. The increased adhesiveness of sickle erythrocytes is accompanied by several changes in the lipids of the erythrocyte membrane, including increased expression of phosphatidylserine (PS). One important PS-binding protein is lactadherin (also known as milk fat globule-EGF factor 8), a 45-kDa glycoprotein containing an Arg-Gly-Asp (RGD) sequence. It is secreted by macrophages and is present in normal plasma. Lactadherin promotes phagocytosis of PS-expressing apoptotic lymphocytes and sickle red blood cells by anchoring them to integrins on macrophages. Here, we investigated the role of endogenous lactadherin in adhesion of sickle erythrocytes to the endothelium. We developed a murine monoclonal antibody to human lactadherin, called L688, and investigated its effect on the adhesion of sickle red blood cells to histamine-stimulated human umbilical vein endothelial cells under hydrodynamic flow. In three experiments using washed erythrocytes resuspended in autologous plasma from three different patients with sickle cell anemia, L688 (20 μg/ml) inhibited adhesion by 24–30% (p<0.01). Further evidence for an important role for lactadherin in sickle erythrocyte adhesion to endothelial cells was provided by the observation that exogenous lactadherin enhanced adhesion in a concentration-dependent manner. Lactadherin-mediated adhesion was also inhibited by monoclonal antibody abciximab, (c7E3, 10 μg/ml) which targets the β3 integrin subunit common to both αIIbβ3 and αVβ3. Control antibodies had no effect. Finally, the lactadherin-dependent adhesion of sickle erythrocytes to activated endothelium was inhibited by PS vesicles but not by phosphatidylcholine vesicles, confirming an important role for PS in sickle cell adhesion. Consistent with this, normal erythrocytes can be induced to adhere to stimulated HUVEC in a lactadherin-dependent manner by treatment with N-ethylmaleimide (10 mM) and calcium ionophore A23187 (4 μM) — treatment that exposes PS on the outer leaflet of the red cell membrane. Together, these results indicate that lactadherin mediates sickle cell adhesion to the endothelium by bridging PS on the erythrocytes with αVβ3 integrin on the endothelium. We propose that anemia in sickle cell disease is at least partially due to phagocytosis of lactadherin-coated sickle erythrocytes in the spleen, liver, and lymph nodes. Those erythrocytes that are not ingested immediately by macrophages will become more adhesive for endothelium. Thus, lactadherin appears to be involved both in sickle cell clearance from the circulation and in adhesion to endothelium.

Mechanism of Soluble Laminin-Mediated Enhancement of Sickle Red Cell Adhesion to Endothelial Cells.

We have previously shown that erythrocytes from patients homozygous for hemoglobin S (SS RBC) adhere to immobilized laminins −10,11 (α5 LAM) with high affinity through the B-CAM/LU receptor. SS RBC also avidly bind soluble LAM via B-CAM/LU. In addition, the interaction between endothelial cell (EC) integrin αVβ3 and erythrocyte ICAM4 is a significant contributor to the adhesion of SS RBC to EC. We have recently shown that soluble LAM, either purified or in plasma, enhances SS RBC adhesion to EC. We have now studied the mechanism by which soluble LAM enhances RBC adhesion to EC and the possible relationship between LAM-enhanced and ICAM-4 mediated adhesion of SS RBC. We tested the adhesion of SS RBC to EC under various potentially adhesion-blocking conditions to determine possible modes of interaction between SS RBC, LAM, SS RBC pre-incubated in LAM (SS RBC+LAM), and EC. We first studied whether blocking RBC-bound LAM with an anti-LAM antibody (Ab) could reduce the adhesion-enhancing effects of LAM. SS RBC were pre-incubated in 10 μg/mL soluble LAM for 1 hr at 37°C. After LAM incubation, SS RBC+LAM were washed and re-incubated for 1 hr in 4C7 mAb, an anti-LAM Ab, followed by washing. Adhesion of SS RBC+LAM incubated in 4C7 was compared to adhesion of SS RBC+LAM incubated with an irrelevant Ab and SS RBC not first incubated in LAM. SS RBC+LAM showed on average more than a two-fold increase in adhesion compared to SS RBC not incubated with LAM, at shear stresses of both 2 and 5 dynes/cm2 (n=3). In contrast, SS RBC+LAM incubated in Ab 4C7 showed < a 50% increase in adhesion compared to SS RBC not pre-incubated in LAM. However, due to variability in adhesion among patient samples, these differences were not significant. In order to determine the EC structure to which SS RBC+LAM might bind, we then incubated EC for 1 hr in an IgM Ab to dystroglycan, a putative LAM receptor on EC. SS RBC+LAM were tested for adhesion to untreated EC, EC treated with anti-dystroglycan, and EC treated with a control IgM. Treatment of EC with anti-dystroglycan decreased the adhesion of SS RBC+LAM by 39.1% and 50.1% at shear stresses of 2 and 5 dynes/cm2, respectively, while EC treated with control IgM showed insignificant decreases in adhesion of 3.3% and 6.6% at shear stresses of 2 and 5 dynes/cm2, respectively (n=3; p=.0013 at 2 dynes/cm2; p=.0175 at 5 dynes/cm2). These results suggest that enhancement of SS RBC adhesion in the presence of LAM is due to LAM interaction with an EC receptor, possibly dystroglycan. We then explored whether the adhesion enhancing effects of LAM could still be observed when the interaction between erythrocyte ICAM4 and EC αVβ3 was blocked. EC were pre-treated for 1 hr with 25 μg/mL of soluble recombinant (sr) ICAM4, in order to block αvβ3 binding sites. We found that pre-incubation of SS RBC in 10 μg/mL LAM failed to enhance RBC adhesion to sr-ICAM4 treated EC (p=.0276 at 2 dynes/cm2, n=3). Similar results were obtained with the 7E3 mAb directed against αVβ3. This suggests that blocking the interaction between ICAM4 and αVβ3 abolishes adhesion despite the presence of RBC-bound LAM. Therefore, we conclude that soluble LAM enhances SS RBC adhesion to EC, probably by providing secondary adhesive interactions that can be abrogated by anti-LAM as well as anti-dystroglycan antibodies. However, the contribution of LAM to SS RBC adhesion was negligible in the absence of interaction between ICAM4 and αVβ3. Thus, the mechanism by which LAM enhances SS RBC adhesion to EC appears to be a complex process, dependent on both the binding of RBC-bound LAM to EC as well as ICAM4 interaction with EC αVβ3.

Increased Red Blood Cell Adhesion Is Associated with Priapism in Sickle Cell Disease.

The enhanced adhesion of sickle RBCs likely plays a role in the pathogenesis of vaso-occlusive complications in patients with sickle cell disease (SCD). In preliminary studies, we have previously found that RBCs from patients with overt stroke associated with large vessel disease have an abnormally enhanced adhesive phenotype. Priapism, another common vaso-occlusive complication that occurs in approximately 30% of males with SCD, has been associated with the development of stroke as well as pulmonary hypertension. The exact pathophysiology of priapism in the context of SCD is not completely understood. Recently, a potential mechanism of hemolysis-associated NO resistance, endothelial dysfunction and end-organ vasculopathy, including priapism, has been described. In this study, we hypothesized that increased RBC adhesion will also be linked to the development of priapism in patients with SCD. After obtaining informed consent, citrated blood for RBC adhesion analysis was obtained from greater than 3 year old males with homozygous HbSS disease during routine clinic visits (steady state) to the Wisconsin Sickle Cell Comprehensive Center. Female patients, those less than 3 years of age, or those with a history of stroke or recent blood transfusion were excluded. Washed RBCs were perfused through flow chambers previously coated with thrombospondin (TSP) (2μg/cm2) at a wall shear stress of 1 dyne/cm2 that mimics the forces in post-capillary venules and optimizes sickle RBC adhesion. After rinsing, adherent RBCs per unit area were counted in four random fields in each of two duplicate wells by direct microscopic visualization. Mean adhesion levels were compared between patients with a history of priapism and those patients without a history of priapism using an independent samples t-test. We studied 27 male patients who met the above inclusion criteria. The priapism group contained 8 patients and the control group 19 patients. The priapism group was older (18.7 vs. 12.9 years). The two groups had comparable laboratory data (Hb, retic, LDH), except for a trend toward a higher total bilirubin in the priapism group (6.4 vs. 3.1 μg/dL), which may be a marker of increased hemolysis. The level of adhesion to TSP for RBCs from patients with priapism was significantly higher compared to the patients without priapism (1336 ± 65 vs. 672 ± 93 RBCs/mm2; mean ± SEM; p= <.0001). Of the 8 patients with priapism, 2 were taking hydroxyurea (HU) at the time of adhesion analysis. Because HU has been shown to decrease sickle cell adhesion, we also analyzed our data excluding all patients taking HU at the time of the adhesion assay. After excluding patients on HU, we still found increased levels of adhesion for RBCs from patients with priapism compared to RBCs from patients without priapism (p= <.0001). In summary, sickle RBCs from patients who develop priapism have increased adhesion to TSP when compared to patients who have not developed priapism. Enhanced RBC adhesion may be an important marker of male patients at increased risk for priapism and perhaps for other severe vaso-occlusive complications associated with SCD.

Vasoocclusion in Sickle Cell Anemia

After reviewing a peripheral blood smear from a patient with sickle cell anemia, one might predict that the clinical consequences of sickle hemoglobin would result simply from vascular obstruction by misshapen red cells. In fact, the pathophysiology of sickle cell disease is far more complex. Besides red cell adhesion to vascular endothelium, it includes the participation of leukocytes, blood coagulation proteins, and platelets. See page 1626 More than 25 years ago, Hebbel et al observed that sickle red cells adhere to cultured endothelial cells regardless of their state of oxygenation.1 It was found subsequently that sickle red cells, and in particular sickle reticulocytes, express a number of adhesion receptors including the integrin α4β1, the immunoglobulin receptor family member ICAM-4, basal cell adhesion molecule/Lutheran (BCAM/Lu), and CD36 that are normally present on red cell progenitors in the bone marrow, but disappear as the progenitors mature into circulating red cells.2,3 Their presence on sickle red cells may be a consequence of the accelerated rate of red cell production in sickle cell anemia and the absence of a functioning spleen. But why these proteins are more prevalent on sickle red cells than on red …

Zinc Status But Not Iron Stores Are Inversely Associated With Soluble Vascular Cell Adhesion Molecule‐1 (sVCAM‐1) Levels in Children With Sickle Cell Disease (SCD)

VCAM-1, a protein expressed on endothelial cells, increases the risk of vasooclusion in SCD patients through adhesion of sickle red blood cells. Suboptimal zinc status and iron overload due to blood transfusion have been implicated in disease severity through oxidative stress. However, the influence of these factors on VCAM-1 has not been previously investigated. We measured sVCAM-1, zinc, hemoglobin [Hb], hematocrit [Hct], and serum ferritin in 76 SCD children and 96 non-SCD children, mean age 7.73 y and 11.24 y, respectively. Although mean zinc levels of both groups were within the normal range (≈14.5 μmol/l), 14.5 % of SCD and 11% of non-SCD children had levels below normal (10.7 μmol/L). As expected, mean Hb and Hct of SCD children were below normal and serum ferritin levels were 2–3 fold higher than those usually seen in healthy children. Mean sVCAMs1 levels of SCD children (837 μg/l) were higher than those of controls (627 μg/l) (p <0.0001). Differences persisted after taking into account age, Hb phenotypes, and inflammation (α1-acid glycoprotein >1g/l and C-reactive protein >10 mg/l). sVCAM-1 negatively correlated with serum (r=−0.444) and red blood cells zinc (r=−0.242, p <0.05), but not acute phase proteins. Hb and Hct, but not serum ferritin levels tended to inversely correlate with sVCAM-1 in children with SS and Sβthal Hb phenotypes (r≤−0.3364, p >0.05). Data suggest that zinc, but not body high iron stores may modulate SCD clinical status through VCAM-1 expression, and zinc supplementation may be beneficial in these patients. Funds: Pediatrics

Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants

In sickle cell anemia (SCA), inflammatory (i.e., intravascular sickling and transient vasoocclusive) events result in chronic endothelial activation. In addition to sickling behavior, sickle (SS) red blood cells exhibit abnormal interaction with the vascular endothelium, which is considered to have an important role in initiation of vasoocclusion. Upregulation of endothelial adhesion molecules caused by oxidants (and cytokines) may lead to increased SS red cell adhesion. We hypothesize that endothelial activation is indispensable in SS red cell adhesion to the endothelium and that antioxidants will have an inhibitory effect on this interaction. We examined the effect of selected antioxidants in ex vivo mesocecum vasculature, a well-established model that allows measurement of hemodynamic parameters and, by intravital microscopy, can allow quantification of adhesion. We tested antioxidant enzymes (SOD and catalase) and an intravascular SOD mimetic, polynitroxyl albumin (PNA), in the presence of platelet-activating factor (PAF); the latter causes endothelial oxidant generation and endothelial activation, which characterize SCA. In ex vivo preparations, PAF not only induced marked endothelial oxidant generation, it also enhanced SS red cell adhesion, resulting in frequent blockage of small-diameter venules. The adhesion, inversely related to venular diameter, and vasoocclusion were markedly inhibited by antioxidants, resulting in improved hemodynamics. PNA, the most effective antioxidant, also abolished SS red cell adhesion in non-PAF-activated preparations. Thus SS red cell adhesion and related vasoocclusion may be ameliorated by antioxidant therapy with a stable and long-acting molecule (e.g., PNA).

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/mm(2), 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 H(2) and H(4) 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.

The Effect of Sickle Red Blood Cell Adhesion on Gene Expression in a PAF-Pre-Treated Microcirculatory Bed.

The expression profile of a number of genes in the rat mesocecum linked to sickle (SS) red blood cell (RBC) adhesion was examined by microarray analysis. The abnormal adherence of SS RBC to the vascular endothelium has been proposed to play an important role in vaso-occlusion in a double capacity; first, by actual physical blockage of the post capillary venules and, second, by inducing the expression of pleiotropic genes that could lead to further modification of the SS phenotype. The goals of these experiments were to identify these pleiotropic genes expressed upon adhesion of SS RBC, to understand how they contribute to the SS phenotype and, to identify potential targets for therapy. The rat mesocecum preparation was prepared as described (Kaul et al, Blood 95:368, 2000) and platelet activating factor (PAF) was used to induce endothelial adhesion receptors to accentuate human SS RBC binding. Rat cecum/ mesocecum RNA was prepared by Qiagen RNeasy mini-kit and fluorescent labeled cDNA hybridized to 32K oligonucleotide microarrays followed by analysis using Gene Pix Pro 4.1. The effect of SS RBC perfusion on PAF-treated tissues vs untreated tissues was compared to PAF-only tissues vs untreated tissues using hierarchical clustering. SS RBC adhesion was analyzed as a function of venular diameter and was significantly different (p<0.00001) in the PAF treated tissues as revealed by a significantly higher Y-intercept (work quoted above). Of the 533 transcripts that were up-regulated and 353 transcripts that were down-regulated more than 2 fold, 68 were up-regulated and 32 were down-regulated more than 4-fold; of this subset, 91 transcripts were compared by Cluster analysis. Many of the up-regulated genes detected in the PAF treated SS RBC adherent tissues were associated with inflammation such as phospholipase A2, group IIA; glutathione peroxidase 2; sialyltransferase 4C; and integrin beta 4. Interestingly, a number of proteasome subunits and ubiquitin D were also highly up-regulated; these genes were only present in the SS RBC perfused PAF treated mesocecum and could then be considered potential targets for specific therapies e.g. proteasome inhibitors. Recent studies also indicate that the ubiquitin system controls NF-kappaB pathways that, in turn, control integrin expression that can increase cell adhesion. The genes that were down-regulated included apolipoprotein B; cytochrome P450 Cyp4b1; cyclin G associated kinase; and metallothionein 2 (MT2). The down-regulation of MT2 is interesting because it is known to be highly regulated in response to the plasma zinc concentration; due to the chronic inflammatory/oxidative condition present in SS disease, zinc may be depleted and feedback negatively on MT2 synthesis. SS RBC adhesion however, not just PAF treatment, is necessary for MT2 downregulation and may, paradoxically, increase zinc availability since MT expression and zinc depletion are inversely correlated. Overall, the results suggest that SS RBC adhesion regulates a number of mesocecum genes involved in the inflammatory response, regulation of oxidative damage, increased intracellular protein degradation, decreased vesicular transport and regulation of zinc ion concentration. These pleiotropic genes are candidates for epistatic (modifier) genes if found to be polymorphic in different individuals and ethnicities. Enhancing or interfering with these specific genes or metabolic pathways may open up new therapeutic strategies for SS disease.

Transfusion and Iron Overload in Thalassemia and Sickle Cell Disease: Impact of Infusion Chelation Therapy on Quality of Life.

Introduction: Blood transfusions are required as supportive care in thalassaemia and sickle cell patients. Regular blood transfusions may result in iron overload (IO) and, left untreated, in morbidity and earlier morality. Current infusion chelation therapy (ICT) requires 8–12 hour infusions, 5–7 days per week, potentially limiting quality of life (QoL) and inhibiting adherence in patients already limited by thalassaemia and sickle cell disease.Methods: To assess the impact of ICT on the QoL of patients with thalassemia and sickle cell disease and adherence, a literature review in Medline (539 abstracts; 130 articles), analysis of IO patient transcripts (4 thalassaemia, 1 sickle cell disease) and five IO clinical expert interviews were conducted.Results: Few published studies (11) used validated QoL instruments and even fewer assessed IO in thalassaemia (4) and sickle cell patients (7). No IO-specific QoL instruments were found. ICT in sickle cell patients may cause additional needle-site pain/discomfort, and may impact on evening social life and sleep, recreational activities, as well as emotional and physical well-being, occupational capacity and feelings of independence. ICT in thalassaemia patients may impact on emotional wellbeing such as self-esteem and worry about the future, family relationships and development of intimate relationships. Published articles and patient and clinician input suggested that QoL impact inhibited prescription of and adherence to ICT and therefore there is a need for easier, efficacious and safe oral chelation therapy (OCT).Discussion: Limited empirical studies assessed ICT's impact on QoL in patients with thalassaemia or sickle cell disease, though results indicated that ICT's QoL impact is significant and prescription and adherence rates would likely improve with OCT. We recommend further qualitative and empirical studies to assess the impact of ICT and OCT in IO patients with thalassaemia and sickle cell patients, using validated instruments.

Lactadherin Enhances the Adhesion of Sickle Cells to Vascular Endothelial Cells.

Adherence of red blood cells to the endothelium initiates vaso-occlusion in sickle cell anemia. The increased adhesiveness of sickle erythrocytes is accompanied by several changes in the lipids of the erythrocyte membrane, including increased expression of phosphatidylserine (PS). One important PS-binding protein is lactadherin (also known as milk fat globule-EGF factor 8), a 45-kDa glycoprotein containing an Arg-Gly-Asp (RGD) sequence. Lactadherin is secreted by many cell types, including macrophages, and is known to have a role in the clearance of apoptotic lymphocytes by binding PS on the cell surface and thus presenting a binding site for macrophage integrins, including αvβ3. We recently showed that lactadherin, added exogenously to both normal and sickle erythrocytes, enhances phagocytosis of these cells by macrophages. Here, we investigated the effect of bovine lactadherin on the adhesion of sickle erythrocytes to the endothelium in flowing blood. Lactadherin (100 nM) promoted the adhesion of sickle erythrocytes to histamine-stimulated human umbilical vein endothelial cells (HUVECs) at shear stresses from 2.5 to 10 dyne/cm2. Adhesion was inhibited significantly by abciximab (10μ g/ml), an antibody against αvβ3 (P<0.05). Normal erythrocytes were induced to adhere to stimulated HUVEC in a lactadherin-dependent manner by treatment with 10 mM N-ethylmaleamide and 4 μ M calcium ionophore A23187—treatment that exposes phosphatidylserine on the erythrocyte surface. These results indicate that lactadherin mediates sickle cell adhesion to the endothelium by bridging PS on erythrocytes to αvβ3 integrin on the endothelium. We propose that sickle erythrocytes acquire lactadherin as they traverse macrophage-rich zones such as spleen, liver, and lymph nodes. Those that are not ingested immediately will become more adhesive for endothelium. Thus, lactadherin appears to be involved in both hemolysis and vaso-occlusion in sickle cell anemia.

Superoxide Inhibits ADAMTS-13 Activity and Regulates Sickle Cell Adhesion.

The major clinical manifestations of sickle cell anemia are due to a vaso-occlusive process that leads to severe pain crises, tissue infarction, and in some instances, death. The pathophysiology of vaso-occlusion is extremely complex, the proximate cause appearing to be an increase in the adhesive interaction between sickle erythrocytes and vascular endothelium. There is ample evidence that it is the highest molecular weight forms of VWF, the ultra-large multimers (ULVWF), expressed on endothelium support sickle erythrocyte adhesion most potently. We postulate that variation in the activity of the plasma metalloprotease, ADAMTS-13, which processes ULVWF to the VWF form normally found in plasma will be an important determinant of the propensity for individuals with sickle cell anemia to develop vaso-occlusive crises. It has also been postulated that increased production of reactive oxygen species (ROS) also contributes to the pathology of sickle cell disease. ROS have been implicated in diverse cellular processes, acting through oxidation of cysteine residues. ADAMTS-13 is a cysteine-rich protein; we therefore hypothesized that its function could be affected by ROS. We measured ADAMTS-13 activity in the plasmas of sickle cell disease patients, both in the “steady state” and at various times during vaso-occlusive crises. We used three assays: a static assay that measures the ability of patient plasma to reduce the size of endothelial-derived ULVWF, a static assay that assesses the ability of patient plasma to cleave a recombinant VWF A2 fragment, and an assay performed under flow that evaluates the ability of patient plasma to cleave ULVWF attached to the surfaces of histamine-stimulated human umbilical vein endothelial cells (HUVECs). Five of nine patients with sickle cell anemia had significantly lower ADAMTS-13 activity than measured in normal controls during the chronic phase of the illness, some with activity measuring below 50% of normal levels. We also evaluated activity in two patients during acute vaso-occlusive crisis. Both patients had levels on the day of hospitalization that were indistinguishable from the levels found in patients with thrombotic thrombocytopenic purpura. Recovery of activity correlated with clinical recovery, to the point that in both cases activity was normal upon hospital discharge. We then compared superoxide generation facilitated by sickle erythrocytes with that generated by normal erythrocytes and found, as others have a significantly higher generation by sickle erythrocytes. We also found that superoxide (produced using a hypoxanthine-xanthine oxidase system) significantly decreased the activity of both plasma and recombinant ADAMTS-13 in vitro. Superoxide was also able to stimulate release of ULVWF and cell-membrane expression from cultured HUVEC. These results suggest that the enhanced production of superoxide in patients with sickle cell disease plays an important role in promoting erythrocyte adhesion to the endothelium by both stimulating release of ULVWF and inhibiting its cleavage by ADAMTS-13.

Hydroxyurea Therapy Reduces the Gene Expression of Adhesion Molecules VLA-4 and CD36 in Sickle Erythrocytes, with a Concomitant Decrease in Adhesion to Fibronectin.

Adhesive interactions between sickle red cells, leukocytes and the endothelium are thought to initiate vaso-oclusive events in the microcirculation. An increased expression of adhesion molecules such as CD 36 (thrombospondin receptor) and VLA-4 integrin (α4β1, CD49d/CD29) has been observed on the surface of sickle reticulocytes and the increased adhesive properties of sickle erythrocytes are well documented. Gene expression of adhesion molecules and their modulation by drugs such as hydroxyurea (HU) have yet to be fully explored. We compared the expression of the CD36 gene and the VLA-4 integrin subunit genes, CD29 and CD49d, in the reticulocytes of steady-state SCD patients and patients on HU therapy (20–30 mg/Kg/day) using Real Time PCR and also compared the basal adhesion of erythrocytes from these subjects using static adhesion assays. Basal adhesion of sickle erythrocytes (2x108 cells/ml) to fibronectin (20mg/ml) - coated plates was significantly higher than normal erythrocyte adhesion (15.4 ± 2.8% compared to 5.9 ± 1.1%, n=9, p=0.008). In contrast, the adhesion of erythrocytes from SS patients on HU therapy to fibronectin was significantly lower than that of SS erythrocytes of patients not on HU (5.8 ± 0.7%, n=9, p=0.006). Pre-treatment of cells, in vitro, with varying concentrations of HU (0.1 → 250μM, 30 minutes, 37oC), had no significant effect on either normal or sickle cell erythrocyte adhesion under our experimental conditions, suggesting that HU may not reduce sickle cell adhesion by altering adhesion molecule function due to affinity changes. We next used real time PCR to investigate the expression of the mRNA of the CD36 gene and the genes for the VLA-4 subunits, CD49d and CD29, in the reticulocytes of steady-state SCD patients and patients on HU therapy. CD 36 gene expression was reduced by 62.5% ± 10.4% in the reticulocytes of patients on HU compared to the reticulocytes of SCD patients not on HU (n=8, p < 0.001). The β1 (CD29) integrin subunit expression was reduced by 87.9 ± 8.7% (n=8, p < 0.001) in patients on HU compared to SS reticulocytes; likewise α4 integrin (CD49d), expression was reduced by 50.0% ± 2.6% (n=8, p <0.001). In addition, reticulocyte mRNA was collected from one steady-state SCD patient before and 3 months after initiation of HU therapy and decreases of 99.5%, 98.0% and 98.0% were observed in the expressions of CD36, CD29 and CD49d mRNA, respectively, in the reticulocytes of this patient following HU therapy. We speculate that the decrease in adhesive properties observed in the erythrocytes of sickle cell patients during HU therapy may be the consequence of a down-regulation of the gene expressions of adhesion molecules and, consequently, protein expression by sickle cells rather than as a result of alterations in adhesion molecule function.

Erythrocyte adhesion is modified by alterations in cellular tonicity and volume

We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.

Protein Kinase A-dependent Phosphorylation of Lutheran/Basal Cell Adhesion Molecule Glycoprotein Regulates Cell Adhesion to Laminin α5

Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha5 in sickle cell disease.

L-Glutamine therapy reduces endothelial adhesion of sickle red blood cells to human umbilical vein endothelial cells

BackgroundWe have previously demonstrated that therapy with orally administered L-glutamine improves nicotinamide adenosine dinucleotide (NAD) redox potential of sickle red blood cells (RBC). On further analysis of L-glutamine therapy for sickle cell anemia patients, the effect of L-glutamine on adhesion of sickle RBC to human umbilical vein endothelial cells (HUVEC) was examined.MethodsThe first part of the experiment was conducted with the blood samples of the 5 adult sickle cell anemia patients who had been on L-glutamine therapy for at least 4 weeks on a dosage of 30 grams per day compared to those of patient control group. In the second part of the experiment 6 patients with sickle cell anemia were studied longitudinally. Five of these patients were treated with oral L-glutamine 30 grams daily and one was observed without treatment as the control. t-test and paired t-test were used for determination of statistical significance in cross-sectional and longitudinal studies respectively.ResultsIn the first study, the mean adhesion to endothelial cells with the autologous plasma incubated cells were 0.97 ± 0.45 for the treated group and 1.91 ± 0.53 for the nontreated group (p < 0.02). Similarly with lipopolysaccharide (LPS) incubated cells the mean adhesion to endothelial cells were 1.39 ± 0.33 for the treated group and 2.80 ± 0.47 for the untreated group (p < 0.001). With the longitudinal experiment, mean decrease in the adhesion to endothelial cells was 1.13 ± 0.21 (p < 0.001) for the 5 treated patients whereas the control patient had slight increase in the adhesion to endothelial cells.ConclusionIn these studies, oral L-glutamine administration consistently resulted in improvement of sickle RBC adhesion to HUVEC. These data suggest positive physiological effects of L-glutamine in sickle cell disease.