Vaso-occlusion In Sickle Cell Disease Research Articles

Effect of sodium butyrate on lung vascular TNFSF15 (TL1A) expression: Differential expression patterns in pulmonary artery and microvascular endothelial cells

Vascular endothelial growth inhibitor TNFSF15 ( TL1A), a ligand for TNFRSF25 ( DR3) and decoy receptor TNFRSF6B ( DcR3), is expressed in human pulmonary arterial (HPAEC) and lung microvascular (HMVEC) endothelial cells where it might modulate inflammation and sickle vasculopathy. Pulmonary disease, endothelial abnormalities and inflammation are prominent features of sickle cell disease (SCD). Butyrate has opposing effects on endogenous TNFSF15 expression in pulmonary endothelium, acting as an inhibitor in HPAEC and an inducer in HMVEC. Similar effects were observed with a known cytokine TNF-α in these two cell types. Furthermore the TNFSF15 promoter utilized different combinations of cis-elements for its expression in these two cell types. AP1-like and G-rich sequence elements were critical for promoter activity in large vessel HPAEC while AP1-like and NF-κB consensus sequence elements were required in small vessel HMVEC. The requirement of an NF-κB sequence element by the TNFSF15 promoter in HMVEC but not in HPAEC supported the notion that HMVEC might be a target of inflammation and vasoocclusion in SCD. The dual effects of butyrate-dependant TNFSF15 regulation in lung endothelium may help in identify inflammatory pathways and understand the role of HMVEC in pathogenesis of vasoocclusion in SCD.

Three-Dimensional Endothelium-Lined Microfabricated Channels as in Vitro assays for Sickle Cell Vaso-Occlusion

Vaso-occlusion in sickle cell disease is fundamentally biophysical in nature, involving a complex set of cellular interactions. Originally attributed solely to the entrapment of abnormally rigid sickle red cells (RBCs) in the microcirculation, this process is now known to involve the decreased deformability of white blood cells (WBCs) and increased endothelial adhesion to different cell types (sickle RBCs, reticulocytes, WBCs, platelets). These biophysical interactions, which are also mediated biochemically by a variety of soluble factors (coagulation proteins, inflammatory mediators, reactive oxygen species, free hemoglobin, etc.), then ultimately lead to microvascular obstruction. However, in vitro experimental approaches that measure the vaso-occlusive properties of sickle blood cells have been unable to separate the contributions of decreased cell deformability and cell-cell adhesion in a single assay. Historically, cell deformability is measured using techniques such as micropipette aspiration, micropore filtration, and ektacytometry, whereas adhesive interactions between the endothelium, blood cells, and soluble factors are assessed using endothelial-lined flow chamber assays. No existing technique effectively evaluates both cell deformability and cell adhesion simultaneously, which is required to comprehensively study sickle cell vaso-occlusion.In the current study, we present an “endothelialized” microfluidic system that simultaneously integrates cell deformability and cell adhesion under physiologic microvascular flow conditions to investigate the underlying biophysical mechanisms of sickle cell vaso-occlusion. Briefly, a layer of human umbilical vein endothelial cells (HUVECs) was cultured along the inner walls of microfabricated microchannels made of the biocompatible and optically transparent polymer polydimethylsiloxane, or PDMS (Figure). Standard lab-on-chip photolithography techniques were utilized to design and create the microchannels, which geometrically emulate a branching microvasculature network with the smallest lumen size approximately 15 μm in diameter. Then, the inner lining of the microchannels were coated with fibronectin and seeded with HUVECs, which grew to a confluent monolayer within 4–5 days and encompassed the entire inner surface in all three dimensions.Whole blood or cell suspensions can then be perfused into the system, and cells can be visualized under flow using standard immunofluorescence microscopy techniques, which allows for identification of specific cellular subpopulations. Pressure and flow rate can be tightly controlled, and cell transit time, time to cell aggregation or obstruction of the system can be recorded using automated image processing software. The effect of different cell types and biologic modifiers (i.e. cytokines, coagulation factors, etc) on in vitro vaso-occlusion can be systematically analyzed and quantified. Furthermore, adhesion-blocking antibodies and drugs that increase cell deformability can be used to quantify the vaso-occlusive effect of adhesion versus cell rigidity. As this system physically constrains cells three-dimensionally in lumens the size of the human microvasculature, physical cellular interactions that are likely to occur in vivo and the endothelial cell response to these processes can also be directly evaluated with live cell fluorescence, immunofluorescence or western blotting.Overall, this system will provide a quantitative and controlled approach to investigate the underlying complex biophysical processes that govern sickle cell vaso-occlusion. In addition, this platform will also serve as a useful complimentary technology to in vivo experiments using sickle cell mouse models. [Display omitted]

The Hydroxyurea Effects on Plasma Levels and Surface Expression of Adhesion Molecules ICAM-1 VCAM-1, E-SELECTIN and on Plasma Levels of Protein C and Protein S in Patients with Sickle Cell Disease and β-Thalassemia Intermedia

Abstract The pathophysiological mechanism of vaso-occlusion in Sickle Cell disease (SCA) is complex, involving adhesive processes between sickle red blood, leukocyte, and the activated endothelium. Reduced activity of the protein C and protein S may contribute to vaso-occlusion. We investigated the effect of Hydroxyurea (HU) on surface expression of the circulating endothelial cells (CEC) ICAM-1 (CD54), VCAM-1 (CD106), E-SELECTIN (CD62E), on plasma levels of soluble sICAM-1, sVCAM-1, sESELECTIN, and on plasma levels of protein C and protein S in patients with SCA and β-Thalassemia Intermedia (TI) Methods : We studied 10 patients with SCA disease, 15 patients with TI, and 12 healthy controls (HC). Flow cytometric analysis was performed with a FC-500. For surface expression on CEC of CD54, CD106, CD62E whole blood (EDTA) is incubated with a panel of MoAbs. Activated CEC were defined as CD45 negative, CD54, CD106, CD62E positive. Plasma was separated from blood of patients and controls, and sICAM-1, sVCAM-1, sE-SELECTIN levels were quantified using specific kit Flowcytomix human adhesion 6plex (Bender MedSystem) according to the manufacturer’s instructions. Results : The percentage (%) of positive CEC expressing markers of endothelial-cell activation were significantly higher in patients with SCA and TI no taking HU, comparated with healthy controls, so as plasma levels (ng/ml) of sICAM-1, sVCAM-1, sE-SELECTIN. SCA patients: CD54+ = 10,9±6,5 % vs 1,4±0,7 % respectively, P= 0,001; CD106+ = 19,7±18,5 % vs 2,1±1,1 % P= 0,03; CD62E+ = 21,2± 20,8 % vs 3,7±1,6 % P= 0,02; sICAM-1=1263,12±445,48 (ng/ml) vs 102,24±14,50 (ng/ml), P=0,03; sVCAM= 1978,67± 985,08 (ng/ml) vs 300,81±70,35 (ng/ml) P=0,01; sESELECTIN= 935,04±313,54 (ng/ml) vs 352,59±50,32 (ng/ml) P= 0,03; TI patients: CD54+ = 12,1±7,1 % vs 1,4±0,7 % P=0,003; CD106+ = 3,68± 2,0 % vs 2,1±1,1 % P=0,42; CD62E+ = 7,08± 9,1 %vs 3,7±1,6 % P=0,87; sICAM-1 = 1911,16±509,90 (ng/ml) vs 102,24±14,50 (ng/ml) respectively, P=0,05; sVCAM = 1067,61±755,37 (ng/ml) vs 300,81±70,35 (ng/ml) P=0,03; sE-SELECTIN = 746,92± 236,47 (ng/ml) vs 352,59±50,32 (ng/ml) P=0,05.The expression of adhesion molecules on CEC and plasma levels of soluble sICAM-1, sVCAM-1, sE-SELECTIN were significantly reduced during HU therapy. SCA patients: CD54+ = 5,01± 4,5 % P=0,01; CD106+ = 9,9± 6,7 % P=0,26; CD62E+ = 11,6± 8,4 % P=0,38;sICAM-1 = 511,81±180,95 (ng/ml) P=0,01; sVCAM = 870,41±419,04 (ng/ml) P=0,02; sE-SELECTIN = 587,49± 364,16 (ng/ml) P=0,03. TI patients: CD54+ = 2,0±2,5% P=0,003; CD106+ = 2,1±0,9 % P=0,42; CD62E+ = 1,8±1,0 % P=0,87;sICAM-1 = 1294,18±350,69 (ng/ml) P=0,05; sVCAM = 1067,61±755,32(ng/ml) P=0,03; sE-SELECTIN = 746,92± 236,47 (ng/ml) P=0,05. The plasma levels of protein C and protein S were increased in SCA and TI patients on HU therapy comparated to those no taking HU; however, this difference was not significant. (SCA patients: Protein C = 106,76±15,79 % vs 77,59±24,73 P=0,14; Protein S = 79,070±1,38 % vs 65,96±6,36 P=0,49; TI patients: Protein C = 83,46±24,99 % vs 64,65±26,67 % P=0,23; Protein S = 80,50±0,77 % vs 74,33±2,36 % P=0,12). Conclusion : Our results confirm that the vascular endothelium is activated in patients with SCA and TI disease, as evidenced from the higher expression of adhesion molecules CD54, CD106, CD62E on the surface of CEC and from the higher levels of soluble sICAM-1, sVCAM-1, sE-SELECTIN, reflecting an increased capacity for the adhesion of sickle erythrocytes and leucocytes to the endothelium. HU therapy reduce the expression on the endothelial cell surface, so as plasma levels, of adhesion molecules, reducing the activation state of the endothelium. Furthermore, HU therapy increase plasma levels of anticoagulants protein C and protein S, reducing hypercoagulable state, which means smaller endothelial damages and vaso-occlusive-events.

Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium

AbstractInfusion of epinephrine-activated human sickle erythrocytes (SS RBCs) into nude mice promotes both SS RBC and murine leukocyte adhesion to vascular endothelium in vivo. We hypothesized that interaction of epinephrine-stimulated SS RBCs with leukocytes leads to activation of leukocytes, which then adhere to endothelial cells (ECs). In exploring the underlying molecular mechanisms, we have found that coincubation in vitro of epinephrine-treated SS RBCs with human peripheral blood mononuclear cells (PBMCs) results in robust adhesion of PBMCs to ECs. Sham-treated SS RBCs had a similar but less pronounced effect, whereas neither sham- nor epinephrine-treated normal RBCs activated PBMC adhesion. PBMC activation was induced via at least 2 RBC adhesion receptors, LW and CD44. In response to SS RBCs, leukocyte CD44 and β2 integrins mediated PBMC adhesion to ECs, a process that involved endothelial E-selectin and fibronectin. SS RBCs activated adhesion of both PBMC populations, lymphocytes and monocytes. Thus, our findings reveal a novel mechanism that may contribute to the pathogenesis of vaso-occlusion in sickle cell disease, in which SS RBCs act via LW and CD44 to stimulate leukocyte adhesion to endothelium, and suggest that RBC LW and CD44 may serve as potential targets for antiadhesive therapy designed to prevent vaso-occlusion.

Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation

Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.

Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease

The Duffy glycoprotein (Fy) on red blood cells (RBCs) has been hypothesized to promote clearance of inflammatory cytokines, which may play a role in the pathogenesis of vasoocclusion in sickle cell disease (SCD). Persons with the African-type Fy(a-b-) phenotype--whose RBCs lack expression of Duffy--may less efficiently clear inflammatory cytokines. Therefore, the Duffy-negative genotype may be associated with more severe disease among patients with SCD. Genotyping was performed on blood samples from 249 adult patients with HbSS at the Duffy gene (FY) locus GATA site (rs2814778) that determines RBC expression of Duffy antigens. Patients with discordant genotype and phenotype data were excluded (n = 12). Differences in demographic, clinical and laboratory findings, end-organ damage, and overall disease severity were compared between FY+ and FY- patients. Of the 237 patients studied, 174 (73%) were FY-. FY+ patients had a higher mean white blood cell (WBC) count (13.2 x 10(9) +/- 4.1 x 10(9)/L vs. 11.8 x 10(9) +/- 3.3 x 10(9)/L; p = 0.03) and higher rates of treatment with hydroxyurea (72% vs. 49%; p = 0.002). In contrast, FY- status was strongly associated with chronic organ damage (85% of FY- patients vs. 65% of FY+ patients; p = 0.018) and proteinuria (32% vs. 12%; p = 0.02). These associations remained, even after controlling for the effects of age and sex. Duffy genotype may be a potential biomarker for the development of end-organ damage in SCD, particularly kidney dysfunction. The association of both WBC counts and hydroxyurea use with Duffy expression provides another avenue for investigation of the biologic role of this protein.

Arginine Therapy Does Not Benefit Children with Sickle Cell Anemia — Results of the CSCC Clinical Trial Consortium Multi-Institutional Study.

Nitric oxide (NO) is an important inflammatory mediator produced from arginine. NO has a multitude of functions which could impact favorably on vaso-occlusion in sickle cell disease (SCD) including improvement in endothelial dysfunction, increased blood flow and a decrease in antioxidant stress. SCD patients have low NO and arginine levels. In preliminary animal studies using the sickle mouse model, arginine supplementation decreased the mean corpuscular hemoglobin concentration and the percentage of dense cells via a reduction in Gardos channel activity1. In humans with SCD, oral arginine increased NO levels in patients with vaso-occlusive crisis but not when patients were at well. In an attempt to determine if daily oral arginine would result in an increase in NO and other beneficial effects, the CSCC launched a multi-institution, blinded, phase II study of arginine in SCD. Children and adults were treated for three months with one of two doses of arginine (.05g/kg/day or .1 g/kg/day) or placebo. The pediatric and adult cohorts were analyzed separately. For this phase II study, the following laboratory values were chosen as surrogates for clinical benefit: NOx, RBC Gardos channel activity, RBC density, sVCAM, nitrotyrosine, ektacytometry, endothelin-1, and fetal hemoglobin. To determine the impact of arginine on these laboratory measures, patients had 3 baseline assessments in the 4 weeks prior to starting therapy with arginine. An average of these 3 measurements was used to determine change from baseline during therapy with arginine. Laboratory outcomes after one month and three months of therapy with arginine were compared with the average baseline measurement. This report summarizes the results of the pediatric cohort of patients. Fifty-one children were enrolled in the study and 48 completed at least one month of study drug making them evaluable. Arginine levels did not change significantly during treatment in any of the arms. The average percent change from baseline to 3 months on study drug being +18% for placebo, +35% for low dose arginine, and +19% for high dose arginine). None of the three primary outcome variables (NO, Gardos channel activity, RBC density) changed significantly with arginine therapy after one month or after 3 months of arginine. After 3 months of study drug, the average percentage change from baseline in NO was −33% for low dose arginine, +4% for high dose arginine, and +8% for placebo. Gardos channel activity changes were −1% for low dose, +3% for high dose, and +9% for placebo. RBC density changes were +10% for low dose, +11% for high dose, and −6% for placebo. Similarly, none of the other outcome variables changed with treatment at either one or 3 months. There were no significant differences in adverse events between the three treatment groups. Our data indicate that oral arginine therapy does not seem to provide clinical benefit for pediatric patients with SCD based on assessment of these selected laboratory outcomes. It is possible that the doses of arginine used in this study were not sufficient to produce significant changes in the laboratory measures chosen. It is also possible that children, due to less severe arginine deficiency, did not respond as well as adults with SCD.Supported by NHLBI U54HL070587.

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.

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.

Comment on “A Novel Role of Hypoxia-Inducible Factor in Cobalt Chloride- and Hypoxia-Mediated Expression of IL-8 Chemokine in Human Endothelial Cells”

Tissue hypoxemia is common in several pathological diseases, including vaso-occlusion in sickle cell disease and myocardial infarction. One finds increased presence of leukocytes during lung injury and at sites of inflammation in vascular endothelium. In this study, we used human pulmonary microvascular endothelial cells and human dermal microvascular endothelial immortalized cell line to delineate the cellular signaling mechanism of hypoxia- and CoCl2 (a mimetic of hypoxia)-induced IL-8 expression, and the latter’s role in chemotaxis of polmorphonuclear neutrophils. We show that hypoxia- and CoCl2-induced IL-8 mRNA and protein expression involved activation of PI3K/Akt and p38 MAPK, but not MEK kinase. Analysis of some transcription factors associated with IL-8 promoter revealed that hypoxia and CoCl2 increased DNA-binding activity of hypoxia-inducible factor-1α (HIF-1α), NF-κB, and AP-1. In addition, we show that hypoxia- and CoCl2-induced IL-8 expression requires activation of HIF as demonstrated by the following: 1) EMSA; 2) transfection studies with IL-8 promoter reporter constructs with mutation in HIF-1α binding site; 3) attenuation of IL-8 expression by both HIF-1α small interfering RNA and R59949; 4) augmentation of IL-8 expression by either transfection with HIF-prolyl hydroxylase-2 small interfering RNA or overexpression of HIF-1α; and 5) chromatin immunoprecipitation analysis. Moreover, conditioned medium from hypoxia-treated endothelial cells augmented chemotaxis of neutrophils, due to release of IL-8. These data indicate that hypoxia-induced signaling in vascular endothelium for transcriptional activation of IL-8 involves PI3K/Akt, p38 MAPK, and HIF-1α. Pharmacological agents, which inhibit HIF-1α, may possibly ameliorate inflammation associated with hypoxia in pathological diseases.

The Impact of Local Air Quality on the Number of Hospital Admissions with Acute Pain in Sickle Cell Disease within an Urban Environment.

The clinical severity of sickle cell disease (SCD) is dependent on genetic and environmental variables. The impact of environmental factors on disease is a major public health issue and air pollution has been consistently correlated with poor health outcomes. Environmental factors in SCD have been poorly studied. We have retrospectively studied the numbers of daily admissions with vaso-occlusive sickle cell pain to King's College Hospital, London, in relation to local daily air quality measurements. We analysed 1047 patient episodes over 1400 days (1st January 1998 to 31st October 2001). Statistical time series analysis was performed using cross-correlation function (CCF), where the observations of one series are correlated with the observations of another series at various lags and leads, values >0.05 being significant. This showed a significant association between increased numbers of admissions and low levels of nitric oxide (NO) (CCF=0.063), low levels of carbon monoxide (CO) (CCF=0.064) and high levels of ozone (O3) (CCF=0.067). There was no association with sulphur dioxide (SO2), nitric dioxide (NO2) or dust. The significant results were further examined using quartile analysis. This confirmed that increased numbers of hospital admissions were associated with high levels of O3 (oneway ANOVA p=0.039) and low levels of CO (oneway ANOVA p=0.042). Low NO levels were also associated with increased admissions, not however reaching statistical significance on quartile analysis (oneway ANOVA p=0.158). O3 levels show marked seasonal variation, with high levels occurring in summer months in the UK. We have previously shown a trend towards increased admissions in the summer months with acute sickle related pain, whereas other groups, which are primarily based in rural tropical climates, found increased admissions in cold, rainy months. The adverse effect of high O3 levels may explain this difference. There is no direct evidence to explain the mechanism by which high O3 levels leads to vaso-occlusion in SCD but high levels of O3 have been linked to reduced respiratory function, which may in turn precipitate vaso-occlusion. Independent studies have shown high CO levels are linked to increased respiratory and cardiovascular admissions. Paradoxically we found that high CO levels were linked to decreased admission numbers and may be protective against acute pain in SCD. CO may confer benefit by forming carboxyhaemoglobin that cannot polymerise resulting in decreased sickling. Previous studies have shown prolonged red cell survival in vivo following administration of CO to patients with SCD. Our study also suggests higher levels of atmospheric NO are linked to fewer admissions. NO is known to be central in the pathophysiology of vaso-occlusion and sickle cell patients are thought to have functional deficiency of NO. Many groups have reported inhaled NO as beneficial in the treatment of sickle pain. Our study suggests air quality has a significant effect on acute pain in SCD and that patients should be counselled accordingly. Based on these findings it would be appropriate to warn patients that high O3 levels might precipitate complications of SCD. The potential beneficial effect of CO and NO is intriguing and requires further investigation.

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.

A Novel Role of Hypoxia-Inducible Factor in Cobalt Chloride- and Hypoxia-Mediated Expression of IL-8 Chemokine in Human Endothelial Cells

Tissue hypoxemia is common in several pathological diseases, including vaso-occlusion in sickle cell disease and myocardial infarction. One finds increased presence of leukocytes during lung injury and at sites of inflammation in vascular endothelium. In this study, we used human pulmonary microvascular endothelial cells and human dermal microvascular endothelial immortalized cell line to delineate the cellular signaling mechanism of hypoxia- and CoCl2 (a mimetic of hypoxia)-induced IL-8 expression, and the latter's role in chemotaxis of polmorphonuclear neutrophils. We show that hypoxia- and CoCl2-induced IL-8 mRNA and protein expression involved activation of PI3K/Akt and p38 MAPK, but not MEK kinase. Analysis of some transcription factors associated with IL-8 promoter revealed that hypoxia and CoCl2 increased DNA-binding activity of hypoxia-inducible factor-1alpha (HIF-1alpha), NF-kappaB, and AP-1. In addition, we show that hypoxia- and CoCl2-induced IL-8 expression requires activation of HIF as demonstrated by the following: 1) EMSA; 2) transfection studies with IL-8 promoter reporter constructs with mutation in HIF-1alpha binding site; 3) attenuation of IL-8 expression by both HIF-1alpha small interfering RNA and R59949; 4) augmentation of IL-8 expression by either transfection with HIF-prolyl hydroxylase-2 small interfering RNA or overexpression of HIF-1alpha; and 5) chromatin immunoprecipitation analysis. Moreover, conditioned medium from hypoxia-treated endothelial cells augmented chemotaxis of neutrophils, due to release of IL-8. These data indicate that hypoxia-induced signaling in vascular endothelium for transcriptional activation of IL-8 involves PI3K/Akt, p38 MAPK, and HIF-1alpha. Pharmacological agents, which inhibit HIF-1alpha, may possibly ameliorate inflammation associated with hypoxia in pathological diseases.

Oxidative Stress and Vaso-Occlusion in Sickle Cell Disease: Role of Activated Leukocytes and Redox Active Iron.

Sickle cell disease (SCD) is a disease of oxidative stress. We and others have demonstrated increased oxidative stress, inflammation, endothelial cell activation and white blood cell counts in human patients and transgenic murine models of SCD. Leukocytosis in SCD is associated with increases in the incidence of pain crisis, acute chest syndrome, stroke and mortality. We hypothesize that reactive oxygen species (ROS) derived from leukocytes and excess redox active iron promote vascular inflammation and vaso-occlusion. Leukocytes were activated in S+S-Antilles sickle mice compared to normal C57BL/6 control mice as measured by the percentage of leukocytes expressing CD11b on their surface in ambient air (25.4% vs. 19.3%, p<0.05) and after exposure of mice to hypoxia-reoxygenation (31.7% vs. 23.0%, p<0.05). In addition, resting leukocytes from S+S-Antilles mice produce 1.8-fold more H2O2 than normal mice (p<0.05) as measured by Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) fluorescence. These leukocyte oxidants are especially toxic in the presence of excess redox active iron. Histopathology of the lungs and livers of 10 week old S+S-Antilles and BERK sickle mice showed red blood cell (RBC) congestion compared to normal. In addition, the sickle livers had multiple areas of infarction and inflammatory leukocyte infiltration. The heme contents of S+S-Antilles sickle lungs and livers were increased by 37- and 4.9-fold, respectively, compared to normals (p<0.05 for both organs). Furthermore, there was significantly more chelatable iron that is potentially redox active as measured by Ferene-S in sickle lungs (21.0-fold, p<0.05) and livers (2.4-fold, p<0.05) compared to normals. Thus, these data demonstrate there is an explosive pro-oxidative environment in sickle mice. These excess oxidants lead to NF-kB activation, VCAM-1 and ICAM-1 expression, and increased oxidative injury, as seen histopathologically by nitro-tyrosine and dihydroethidium staining in organs. Hypoxia-reoxygenation, which induces RBC sickling and enhances ROS production in sickle mice, causes an increase in leukocyte rolling (4.4-fold, S+S-Antilles vs. normal, p<0.05) and adhesion (6.5-fold, p<0.05). Hypoxia-reoxygenation induces transient vaso-occlusion in 12% and 24% of the subcutaneous venules of S+S-Antilles and BERK mice respectively. No vessels become static in normal mice (p<0.05 sickle vs. normal). Hypoxia-reoxygenation-induced vaso-occlusion can be inhibited by antibodies to P-selectin, VCAM-1 or ICAM-1. Furthermore, scavenging ROS with the SOD and catalase mimetic, polynitroxyl albumin or the iron chelator Trimidox, inhibited hypoxia-reoxygenation-induced vaso-occlusion (p<0.05). We conclude that oxidative stress derived from activated leukocytes and excess redox active iron plays a critical role in promoting vaso-occlusion and organ injury in SCD. We speculate that iron chelators, leukocyte adhesion molecule blockade and anti-oxidants will modulate vaso-occlusion in patients with SCD.

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.

Haemostasis Alterations in Sickle Cell Disease.

Sickle cell disease (SCD) is considered by many workers to be a hypercoagulable state. The vaso-occlusion in sickle cell disease is complex, involving adhesive processes between sickle red blood cells, leukocytes, and the activated endothelium. Loss of normal membrane phospholipids asymetry with the appearance of phosphatidylserine on the erythrocyte surface has been documented as promoting an exacerbation of the anaemia and an activation of coagulation. The aim of this study was to monitor changes in coagulation and platelet activation (monitored by measuring the levels of procoagulant phospholipids microparticles - PPM) in SCD (both in steady state and crisis). Coagulation screening tests (PT, APPT), Protein S (PS), D-Dimer (D-Di) and PPM were measured in plasma samples obtained from 18 patients with SCD (8 in crisis state and 10 in steady state) and in twenty one normal volunteers as control. All blood samples were collected and the plasma prepared in the same hospital using the same conditions. All plasma samples were stored at -80°C until assayed. PT, aPTT, PS and D-dimers were measured using reagents from Diagnostica Stago, Asniéres, France. PPM was monitored with a novel factor Xa-based coagulation assay. In this assay shortened clotting times are associated with increased levels of PPM and thus platelet activation.APTT Sec.PT %PPM SecPS %D-Di μg/lControls34.6 (29.4–39.6)93.1 (79–109)57.5 (51.1–74.9)88.8 (62–112)0.22 (0.22–0.45)Sickle cell – in crisis37.3 (33.4–47.4)71.1 (56.5–86.5)44.6 (34.0–60.0)48.9 (25.0–89.5)1.6 (0.6–2.6)Sickle cell – steady state.38.0 (33.0–47.5)75.0 (61.0–93.0)52.2 (42.5–62.8)84.7 (76.5–103.0)0.7 (0.2–1.0)In sickle cell crisis a significant increase in both coagulation and platelet activation, as monitored by PPM, was found (all at p < 0.001). The decrease in % PT and PS levels confirms the findings of others. In steady state only DDi levels are significantly higher than controls. Further studies are needed to explain these findings. Although this was a small pilot study and further studies are needed it is interesting to speculate on the possible usefulness of the PPM assay in monitoring SCD crisis.

Bases moléculaires et physiopathologiques des maladies de l'hémoglobine

Among haemoglobin diseases, two types are particularly important to consider: the structural abnormalities and the synthesis deficiencies. In the first type, abnormal haemoglobin (Hb) may be silent or functionally altered. Because it causes sickle cell disease, HbS is the most significant. Synthesis deficiencies are encountered among the very heterogeneous group of thalassaemias. Haemoglobin diseases represent an important public health problem in endemic countries, and in other parts of the world because of immigration. The pathophysiological mechanisms differ. Sickle cell disease is mostly a rheological disease and, more than anaemia, vaso-occlusion is its major feature. Even though it results from a single mutation, it is very variable in its clinical presentation, from very severe cases early in life, to a well tolerated condition. Anaemia is the hallmark of thalassaemias. Multiple mutations are involved, but the severity of the disease is only partially related to the nature of the mutation. In the two types, modulating genes are involved. Recent insights have shown the involvement and the modulating role of the vascular endothelium, more particularly in the pathophysiology of vaso-occlusion in sickle cell disease. Other factors are progressively unveiled, that modify the evolution of thalassaemia.

Blood mononuclear cells and platelets have abnormal fatty acid composition in homozygous sickle cell disease

Leukocyte adhesion to vascular endothelium contributes to vaso-occlusion and widespread organ damage in sickle cell disease (SCD). Previously, we found high expression of the adhesion molecules alphaMbeta2 integrin and L-selectin in HbSS individuals with severe disease. Since membrane n-6 and n-3 polyunsaturated fatty acids modulate cell adhesion, inflammation, aggregation and vascular tone, we investigated the fatty acid composition of mononuclear cells (MNC) and platelets of HbSS patients in steady state (n=28) and racially matched, healthy HbAA controls with similar age and sex distribution living in the same environment (n=13). MNC phospholipids of the patients had lower levels of docosahexaenoic acid (DHA, p<0.01) and increased arachidonic acid (AA, p<0.005) relative to HbAA controls. Similarly, platelets from HbSS patients had less eicosapentaenoic acid (EPA, p<0.05) and more AA (p<0.05) in choline phosphoglycerides (CPG), with reduced DHA (p<0.05) in ethanolamine phosphoglycerides. Platelet CPG had lower DHA levels in SCD patients with complications compared to those without (p<0.05). Reduced cell content of EPA and DHA relative to AA favours the production of aggregatory and proinflammatory eicosanoids that activate leukocytes and platelets. This facilitates inflammation, leukocyte adhesion, platelet aggregation and vaso-occlusion in SCD.

Polynitroxyl albumin inhibits inflammation and vasoocclusion in transgenic sickle mice

Individuals with sickle-cell disease (SCD) and transgenic sickle mice expressing human betaS globin exhibit enhanced reactive oxygen species (ROS) production, vascular inflammation, and episodic vasoocclusion. We hypothesize that reduction of ROS will reduce endothelial-cell activation and adhesion-molecule expression, thereby inhibiting vasoocclusion. To test this hypothesis, we measured endothelial-cell activation, adhesion-molecule expression, and vasoocclusion in sickle mice after administering i.v. polynitroxyl albumin (PNA), a superoxide dismutase and catalase mimetic. Untreated sickle mice, compared with normal mice, showed increased activation of nuclear factor-kappaB (NF-kappaB), an oxidant-sensitive transcription factor, in their lungs, livers, and skin. NF-kappaB activation was increased further in the livers and skin of sickle but not normal mice after hypoxia-reoxygenation. IV administration of PNA inhibited NF-kappaB activation by 60% (P < .01) in the lungs and by 33% (P < .05) in the livers of sickle mice after hypoxia-reoxygenation. PNA also reduced the expression of vascular cell-adhesion molecule-1 (VCAM-1) by 57% in lung (P < .05) and by 33% in liver (P < .05) and reduced the expression of intercellular-adhesion molecule-1 (ICAM-1) by 40% in lung (P < .05) and by 53% in liver (P < .05). PNA inhibited a hypoxia-reoxygenation-induced increase in leukocyte rolling (P < .01) and adhesion (P < .05) in venules of the dorsal skin. Most importantly, PNA completely inhibited hypoxia-reoxygenation-induced vasoocclusion (P < .001). Control albumin had no effect on NF-kappaB, VCAM-1, ICAM-1, rolling, adhesion, or vasoocclusion. We speculate that therapies to reduce oxidative stress will inhibit inflammation and vasoocclusion in SCD.

Differential effects of hydroxyurea and zileuton on interleukin-13 secretion by activated murine spleen cells: Implication on the expression of vascular cell adhesion molecule-1 and vasoocclusion in sickle cell anemia

Background Interleukin-13 (IL-13), a TH2 cytokine, upregulates the expression of vascular cell adhesion molecule-1 on endothelial cells, a factor involved in vasoocclusion in sickle cell disease (SCD). Hydroxyurea improves clinical status of SCD patients in part by induction of fetal hemoglobin. Its effect on IL-13 secretion has not been investigated. Objective To determine whether hydroxyurea and zileuton, a hydroxyurea derivative with antiinflammatory properties, affect IL-13 secretion. Methods We measured IL-13 in the supernatant of murine spleen cells incubated without and with hydroxyurea, zileuton (10 μg/ml), concanavalin A (2.5 μg/ml), and anti-CD3 (50 ng/ml) ( n = 8). Results Hydroxyurea and zileuton do not affect baseline IL-13 secretion. Unexpectedly, hydroxyurea increases IL-13 levels above baseline (120%, 216.5%, [ p < 0.05] after 24 h and 48 h, respectively) in lymphocytes activated by anti-CD3, while zileuton reduces them by 59%–78% ( p < 0.005). In lymphocytes activated by concanavalin A, hydroxyurea and zileuton reduce IL-13 secretion by 24–36% and 50–87%, respectively ( p < 0.05). Hydroxyurea, but not zileuton, significantly inhibits spleen cell proliferative responses to mitogens ( p < 0.005). Conclusion Data suggest that hydroxyurea up-regulates IL-13 secretion in anti-CD3-activated lymphocytes through gene activation but not by altered cell proliferation. Increased IL-13 secretion may contribute to unresponsiveness of certain SCD patients to hydroxyurea. The potential benefit of zileuton in the management of vasoocclusion is discussed.