Adhesion Of Sickle Erythrocytes Research Articles

TSH Receptor Reduces Hemoglobin S Polymerization and Increases Deformability and Adhesion of Sickle Erythrocytes.

SCD is a hereditary disorder caused by genetic mutation in the beta-globin gene, resulting in abnormal hemoglobin, HbS that forms sickle-shaped erythrocytes under hypoxia. Patients with SCD have endocrine disorders and it was described that 7% of these patients have clinical hypothyroidism. Recent studies have shown that mature erythrocytes possess TSH receptors. Thus, we aimed to assess the effects of TSH on SCD erythrocytes. The experiments were conducted using different concentrations of TSH (1, 2, 3, and 5 mIU/L). In HbS polymerization assay, erythrocytes were exposed to TSH in hypoxia to induce polymerization, and measurements were taken for 30 minutes. The deformability assay was made using Sephacryl-S 500 columns to separate deformable from nondeformable cells. Static adhesion test utilized thrombospondin to assess erythrocyte adhesion in the presence of TSH. TSH at all contractions were able to reduce polymerization of HbS and increase deformability. The static adhesion of erythrocytes at the lowest concentrations of 1 and 2 mIU/L were increased, but at higher contractions of 3 and 5 mIU/L, static adhesion was not modulated. The results suggest that TSH has potential involvement in the pathophysiology of sickle cell disease by inhibiting HbS polymerization, positively modulating deformability and impacting static adhesion to thrombospondin.

Radchip: A Laminin-Lined Red Blood Cell (RBC) Adhesion Device for Functional Characterization and Clinical Evaluation of Sickle RBCs at Steady-State

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the beta-globin gene producing an abnormal hemoglobin (HbS) which, under deoxygenation, polymerizes and causes severe distortion of the red blood cell (RBC). RBCs may be dehydrated, poorly deformable and highly adhesive to the endothelium, with abnormal adhesion being of primary interest to this study. Recently, several new disease modifying drugs have been FDA approved, with more in development. These novel agents target specific aspects of SCD pathophysiology, including adhesion. Changes induced by such therapies are not always detectable by common conventional lab instruments. LORRCA (oxygen-gradient ektacytometry) is the gold-standard for deformability measurement; it has been well described both analytically and clinically in SCD. Adhesion and deformability have a dynamic, yet complementary relationship that we explore here. While these may behave in an interdependent manner, each determinant must be considered independently. There is a growing demand for innovative tools to evaluate rheological properties under physiological conditions, particularly microfluidic devices. However, many current devices are limited in their clinical utility due to, choice of ligand, complexity of design, or reliance on endothelial cell culture (typically HUVEC cells) which are difficult to use, vary with passage number, and do not represent adult SCD endothelium. We developed a Red blood cell Adhesion Chip (RAdChip) to model RBC to endothelial adhesion using laminin, a physiologically relevant ligand, to easily, effectively, and reproducibly measure RBC adhesion. Here, we describe our analytic and clinical validation of our device. Peripheral blood samples were collected in EDTA from individuals with SCD and healthy controls under an IRB approved protocol. RBCs were extracted via centrifugation, washed and resuspended in PBS to a 0.5% HCT. Microfluidic devices were fabricated using polydimethylsiloxane (PDMS). A 5ug/ml solution of recombinant laminin was applied to each channel, then blocked with 1% bovine serum albumin (BSA). Resuspended RBCs were introduced to the device through tubing connected to the inlet port. Harvard Apparatus syringe pumps were used to perfuse samples and wash channels at ~1dyne/cm 2. Adhesion was visualized with a Keyence inverted bright-field microscope at 20x (Itasca, IL). Adhered cells were counted within a 1.8mm 2 region of interest (Roi) using the iCLOTS automated cell quantification software (Fay et al, Nat comm accepted 7/2023). We measured adhesion index via RAdChip for common SCD genotypes: HbSS/SB0: (median (x͂) =756; range (r): 154-1889; n=26) HbSC/SB+ : (x͂ =299; r=: 114-731; n=11 ) HbAA: (x͂) =13; r= 5-90; n=7). RBC adhesion values were independent of age (p=0.65) and gender (p=0.63). An increase of 0.1 unit in EImin and EImax decreases RBC adhesion by 214 and 268 cells/mm 2, respectively. A 1mmHg increase in PoS increased adhesion by 19.5 cells/mm 2. sRBC adhesion had a significant positive association with vaso-occlusive events (VOE) (p=0.005), absolute neutrophil count (ANC) (p=0.005), and EImax (p<0.001). Notably, analytical validation of RAdChip produced a mean CV of 0.13 ± 0.94. RAdChip adds a key component of functional RBC evaluation that conventional lab equipment does not measure. We have analytically validated our device, demonstrating an excellent coefficient of variability. 44 patient samples, each examined in triplicate produced a mean CV of 0.13. We have demonstrated clinical validity of our device by establishing correlations with known markers of severity and pain event frequency. RBC adhesion values increase as the number of VOE events increase, reaffirming the belief that adhesion is a principal element in the multistep VOE cascade. In like manner, improvements in deformability are associated with decreased RBC adhesion, strengthening the interdependent nature of VOE molecular events. Recognizing the dynamic nature of SCD, it is necessary to explore sickle erythrocyte adhesion longitudinally to investigate changes before, during, and after VOE occurrences. Continued and increased integration of adhesion measurements with RAdChip and other rheology devices into routine SCD care may be helpful in assessing disease severity, identifying need for additional disease modifying agents, and assist with second line agent selection and monitoring.

Membrane bending and sphingomyelinase-associated, sulfatide-dependent hypoxic adhesion of sickle mature erythrocytes.

Abnormal erythrocyte adhesion owing to polymerization of sickle hemoglobin is central to the pathophysiology of sickle cell disease (SCD). Mature erythrocytes constitute >80% of all erythrocytes in SCD; however, the relative contributions of erythrocytes to acute and chronic vasculopathy in SCD are not well understood. Here, we showed that bending stress exerted on the erythrocyte plasma membrane by polymerization of sickle hemoglobin under hypoxia, enhances sulfatide-mediated abnormal mature erythrocyte adhesion. We hypothesized that sphingomyelinase (SMase) activity, which is upregulated by accumulated bending energy, leads to elevated membrane sulfatide availability, and thus, hypoxic mature erythrocyte adhesion. We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856± 481 vs 78± 23, mean± SEM), whereas sickle reticulocyte (early erythrocyte) adhesion, high to begin with, does not change (1281± 299 vs 1258± 328, mean± SEM). We showed that greater mean accumulated bending energy of adhered mature erythrocytes was associated with higher acid SMase activity and increased mature erythrocyte adhesion (P= .022, for acid SMase activity and P= .002 for the increase in mature erythrocyte adhesion with hypoxia, N= 5). In addition, hypoxia results in sulfatide exposure of the erythrocyte membrane, and an increase in SMase, whereas anti-sulfatide inhibits enhanced adhesion of erythrocytes. These results suggest that the lipid components of the plasma membrane contribute to SCD complications. Therefore, sulfatide and the components of its upregulation pathway, particularly SMase, should be further explored as potential therapeutic targets for inhibiting sickle erythrocyte adhesion.

The 'scintilla' starting vaso-occlusion in sickle cell disease.

RBCEV SS RBC SS Reduced adhesion to endothelial cells# RBCEV AA RBC AA ADAMTS-13 is a metalloproteinase that cleaves von vWF and ultra large vWF. The importance of ADAMTS-13 is well known for conditions where vaso-occlusion occurs in microvessels, such as in thrombotic thrombocytopenic purpura (TTP). Similarly to hemopexin, the demonstration of a direct involvement of ADAMTS-13 in the adhesion process of SCD is relevant for potential therapeutical development. In fact, An Ran et al. showed that in the presence of hemopexin and ADAMTS-13, adhesion of sickle erythrocytes is returned to the level seen when normal erythrocytes are tested. The finding of an involvement of vWF in sickle cell adhesion suggests a role for anticoagulant proteins in SCD. This is also in keeping with similar findings of reduced naturally occurring anticoagulant (Protein C and Protein S) and vaso-occlusion in SCD.2, 10 In our opinion, this study has a strong scientific value, since it has been conducted in both in vitro and in vivo studies, in a mouse model with SCD. It will now be important to validate these findings in human trials, possibly in a large cohort of patients with SCD. If these findings were to be further corroborated, RBCEVs could potentially be used as a new parameter for monitoring SCD patients in a steady state and during SCD crises. RBCEV trials may possibly even predict SCD crises. Finally, the link between RBCEV, hemopexin and ADAMTS-13 offer an excellent target for the development of future treatment strategies for SCD.

Membrane Bending Enhances Sulfatide Mediated Sickle Mature Erythrocyte Adhesion

Introduction: Abnormal red blood cell (RBC) adhesion due to polymerization of sickle hemoglobin (HbS) is central to the pathophysiology of SCD. Early erythrocytes (reticulocytes), expanded in number due to sustained stress erythropoiesis stimulated by anemia and hypoxia, are more adhesive to vascular endothelial proteins and surfaces than are mature erythrocytes. However, mature erythrocytes typically constitute >80% of all RBCs in SCD, and the relative contribution made by erythrocytes vs. reticulocytes to acute and chronic vasculopathy is not well understood. We hypothesized that sphingomyelinase (SMase) activity that is upregulated by accumulated bending energy leads to elevated membrane sulfatide availability and thus hypoxic mature erythrocyte adhesion under hypoxia. Methods: We probed the adhesion of RBCs obtained from SCD patients (#NCT02824471) stained with acridine orange under various physiological conditions in microchannels that were coated with laminin. Then we analyzed the morphology of single sickle reticulocytes and mature erythrocytes to estimate the membrane bending energy. Results and Discussion: We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856±481 vs. 78±23, mean±SEM), while sickle reticulocyte adhesion, high to begin with, does not change (1281 ±299 vs. 1258±328, mean±SEM). Furthermore, we show that among the adherent RBCs, mature erythrocytes have greater elongation and accumulated membrane bending energy compared to reticulocytes under hypoxia (Figure 1A). Accumulated membrane bending is known to elicit SMase, and laminin establishes highly specific bonds with sulfatides which are downstream of the SMase. These results suggest that SMase is likely elicited particularly in sickle mature erythrocytes, which become highly adhesive with deoxygenation. This is likely due to loss of membrane surface area with RBC maturation, which may be excessive in SCD due to membrane vesiculation and microparticle release. To elucidate the roles of SMase and sulfatide in the adhesion of mature erythrocytes, we repeated the adhesion experiments with SMase and anti-sulfatide incubation. We found that SMase increases the hypoxia-enhanced adhesion of mature erythrocytes and the anti-sulfatide antibody inhibits the enhanced adhesion of mature erythrocytes seen under hypoxia (Figure 1B). Collectively, these data demonstrate the effect of accumulated membrane damage on the abnormal adhesion of sickle mature erythrocytes. Conclusions: Patient-to-patient variability in hypoxia enhanced mature erythrocyte adhesion suggests a role in modulating clinical comorbidities and or genetic polymorphisms relevant to red cell health and hypoxic regulation. These results also implicate the lipid components of the plasma membrane in the pathophysiology of RBC dysfunction in SCD. Therefore, sulfatide and the components of its pathway, such as SMase, could be explored as potential therapeutic targets to inhibit sickle erythrocyte adhesion in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

SummaryIn this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.

Role of Band 3 Tyrosine Phosphorylation in Causing the Vaso-Occlusive Events in Sickle Cell Disease

Background: Sickle cell disease (SCD) derives from a mutation in sickle hemoglobin (HbS) that leads to vaso-occlusive events that eventually cause organ failure. Based on observations from multiple labs, we hypothesize that the events linking HbS denaturation to vaso-occlusion are: HbS denaturation→oxidative stress→inhibition of erythrocyte tyrosine phosphatases→enhanced tyrosine phosphorylation of band 3→dissociation of the spectrin/actin cytoskeleton from the membrane→release of membrane-derived microparticles (MPs) and discharge of cell-free HbS→induction of microthromboses and activation of the vascular endothelium→vascular occlusion. In the studies below, we document each step of this pathway and demonstrate that inhibitors of Band 3 tyrosine phosphorylation block all downstream events. Methods: Band 3 tyrosine phosphorylation, plasma MP counts, plasma cell-free Hb concentration, sickle cell deformability, sickle cell flow through micro-capillaries, and adhesion of sickle erythrocytes to activated endothelial cells are quantitated using fresh sickle blood both before and after incubation with inhibitors of Band 3 tyrosine phosphorylation. Findings: Imatinib and other Syk kinase inhibitors block Band 3 tyrosine phosphorylation, suppress release of membrane MPs, reduce discharge of cell-free HbS, improve sickle cell deformability, enhance flow of sickle cells through microcapillaries, and reduce adherence of sickle cells to heme-activated endothelium. Interpretation: Inhibition of Band 3 Tyrosine phosphorylation restores membrane integrity and thereby suppresses the downstream sequelae that lead to vaso-occlusive events in SCD. Inhibitors of Band 3 tyrosine phosphorylation may therefore constitute a novel effective therapy for SCD. Funding Statement: NIH grants R01GM24417-40 (P.S.L.), 1RF1 NS110049-01 (J.W.), R01HL133574 (U.A.G. and J.A.L.), T32HL134622 (R.A.), and NSF #1552782 (U.A.G.). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All sickle cell blood samples were obtained following informed consent using procedures approved by the local IRBs.

Consistent Compliance with Hydroxyurea and Hematology Measures during L-Glutamine Therapy for Sickle Cell Anemia

Background. Oxidative stress contributes to the pathophysiology of sickle cell anemia (SCA). Oral l-glutamine increases the proportion of reduced NAD in sickle erythrocytes,1 which may decrease oxidative stress and SCA complications.2 Recently, a multicenter, randomized, placebo-controlled, double-blind, phase 3 trial of l-glutamine in SCA reported that the l-glutamine group had fewer pain crises and hospitalizations over 48 weeks of treatment than the placebo arm.3 There was a consistent treatment effect of l-glutamine in subjects who used and did not use hydroxyurea. The purpose of the present analysis was to determine if there was consistent use of hydroxyurea in both arms of the trial and if l-glutamine therapy led to a reduction in hemolysis and an amelioration of anemia.Methods. Because increase in mean corpuscular volume (MCV) is a sensitive indicator of hydroxyurea therapy,4 we used MCV as a surrogate marker of hydroxyurea compliance. We analyzed MCV at 4-week intervals according to hydroxyurea therapy separately in the l-glutamine and placebo arms. We also analyzed hemoglobin concentration and reticulocyte count at 12-week intervals according to experimental treatment with l-glutamine versus placebo as stratified by hydroxyurea use. We imputed missing variables by carrying the last measured value forward. We used the student t-test to compare linear variables, Pearson chi square to compare categorical variables and repeated measures analysis of variance to analyze trends over time.Results. The patients receiving l-glutamine and placebo were well matched (Table 1). During the 48-week trial, the MCV was consistently greater in patients using hydroxyurea than those not using hydroxyurea in both the l-glutamine arm (Figure 1) and the placebo arm (Figure 2). Furthermore, there was no significant change over 48 weeks in the MCV in the patients using hydroxyurea in either arm (P >0.7). In the hydroxyurea group, there were no significant differences according to experimental therapy with l-glutamine versus placebo in hemoglobin concentration or reticulocyte count at 12 weekly intervals. In the group not using hydroxyurea, the hemoglobin concentration was significantly higher at baseline in patients given l-glutamine compared to those given placebo, but this observation did not persist at the subsequent determinations; reticulocyte counts did not differ at any time point (Table 2). We performed many multivariate and repeated measures analyses of hemoglobin concentration and reticulocyte count that adjusted for age, gender and blood transfusions, but did not observe evidence that l-glutamine therapy was associated with an increase in hemoglobin concentration and/or a decline in reticulocyte count.Discussion. Based on the MCV, patients who were assigned to hydroxurea complied with hydroxyurea therapy at baseline and throughout the study; this supports the concept that the beneficial clinical effects of l-glutamine therapy for SCA are additive to those of hydroxyurea. Therapy with l-glutamine did not reduce hemolysis or improve anemia, which parallels observations with therapy with an antibody against the adhesion molecule, P-selectin.5 The mechanism whereby l-glutamine leads to reduced vaso-occlusive complications in SCA requires further investigation. Glutamine levels are lower in sickle erythrocytes compared to erythrocytes from healthy volunteers.6 L-Glutamine reduces intercellular adhesion molecule expression in endothelial cells,7 decreases adhesion of sickle erythrocytes to endothelial cells,8 attenuates the expression of endothelial adhesion molecules and T cell migration markers when the intestines are injured, 9,10 and inhibits IL-6 and TNF-α production in LPS-stimuated monocytes.11 Future research will need to assess the effect of l-glutamine on redox potential, adhesion molecule expression, and inflammatory marker expression in erythrocytes, other circulating blood cells and the vascular endothelium.ReferencesAm J Hematol. 1998;58(2):117-121.IUBMB Life. 2012;64(1):72-80.N Engl J Med. 2018;379(3):226-235.Medicine (Baltimore). 1996;75(6):300-326.N Engl J Med. 2017;376(5):429-439.Blood. 2008;111(1):402-410.J Reprod Med. 2006;51(3):193-198.BMC Blood Disord. 2005;5:4.Eur J Pharmacol. 2010;643(2-3):304-315.Mediators Inflamm. 2014;2014:837107.Cytokine. 2013;62(1):52-57. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging

AbstractLutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C–derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid–binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor.

Adhesion of erythrocytes to endothelial cells lining the vascular wall can cause vaso-occlusive events that impair blood flow which in turn may result in ischemia and tissue damage. Adhesion of erythrocytes to vascular endothelial cells has been described in multiple hemolytic disorders, especially in sickle cell disease, but the adhesion of normal erythrocytes to endothelial cells has hardly been described. It was shown that calcium-loaded erythrocytes can adhere to endothelial cells. Because sickle erythrocyte adhesion to ECs can be enhanced by ultra-large von Willebrand factor multimers, we investigated whether calcium loading of erythrocytes could promote binding to endothelial cells via ultra-large von Willebrand factor multimers. We used (immunofluorescent) live-cell imaging of washed erythrocytes perfused over primary endothelial cells at venular flow rate. Using this approach, we show that calcium-loaded erythrocytes strongly adhere to histamine-stimulated primary human endothelial cells. This adhesion is mediated by ultra-large von Willebrand factor multimers. Von Willebrand factor knockdown or ADAMTS13 cleavage abolished the binding of erythrocytes to activated endothelial cells under flow. Platelet depletion did not interfere with erythrocyte binding to von Willebrand factor. Our results reveal platelet-independent adhesion of calcium-loaded erythrocytes to endothelium-derived von Willebrand factor. Erythrocyte adhesion to von Willebrand factor may be particularly relevant for venous thrombosis, which is characterized by the formation of erythrocyte-rich thrombi.

Effect of pentoxifylline on adherence of sickle erythrocytes to vascular endothelial cells

Effect of pentoxifylline on adherence of sickle erythrocytes to vascular endothelial cells

Evaluation of Purified Poloxamer-188 (vepoloxamer) on Sickle Red Blood Cell (RBC) Membrane Function Properties

Background: Sickle erythrocyte adhesion and membrane fragility contribute to vaso-occlusion and downstream tissue and organ ischemia in sickle cell disease (SCD). Vepoloxamer is an amphipathic triblock copolymer with multi-mechanistic properties believed to improve these pathophysiologic consequences, by sealing damaged cell membranes and inhibiting hydrophobic cellular adhesive interactions. Vepoloxamer reduced both acute vaso-occlusive crisis duration and total opioid analgesic requirements in previous clinical studies. A phase 3 clinical trial of vepoloxamer in acute vaso-occlusive crises is ongoing. Currently, there are no standardized clinical assays to assess membrane properties such as adhesion and fragility that vepoloxamer is believed to target. We evaluated if and to what extent vepoloxamer affected adhesion, thrombosis, and membrane fragility in individual patient samples in our standardized microfluidic flow-based whole blood assays.Methods: Blood was obtained from pediatric homozygous sickle cell patients at steady state (n=12). Patients were first characterized by measuring the extent of whole bloodadhesion to vascular cell adhesion molecule (VCAM-1) in a standardized microfluidic flow-based assay. Dynamic adhesive interactions (rolling or sliding) of whole blood and isolated leukocytes to P-selectin were assessed by measuring absolute rolling cell number, cell rolling flux (number of rolling cells per total number of flowing cells), and cell rolling velocity at 0.3dyn/cm2. Detachment assays were performed by exposing adherent cells to vepoloxamer (10 mg/mL) under physiological flow conditions. Erythrocyte membrane fragility was evaluated based on mechanical stress-induced hemolysis at 3 min (Hem3min, %).Results: Vepoloxamer reduced whole blood adhesion to VCAM-1 at 0.1 mg/mL by 18% (p = 0.0015), at 1.0 mg/mL by 69% and at 10.0 mg/mL by 79% (in both cases, p < 0.001), and to HUVECs by >50% 0.1mg/mL (p=0.003, n=10). Vepoloxamer (10mg/mL) did not significantly affect cell-rolling flux in dynamic adhesion assays to P-selectin (p = 0.51 and 0.94, isolated leukocytes and whole blood, respectively). Vepoloxamer reversed established cell adhesion by > 30% in 3 or 10 patient samples tested, although the total average difference in percent detachment did not reach statistical significance (16.3%vs 29.6%, p=0.42, n=10). Finally, vepoloxamer reduced shear-induced hemolysis compared to untreated blood samples (35.2 % vs. 44.6% control, p =0.033).Conclusion: Vepoloxamer can significantly reduce firm cell adhesion to both VCAM-1 and HUVECS under physiologic flow conditions. Shear-induced hemolysis is also reduced in a statistically significant manner, consistent with the proposed membrane protecting effects of vepoloxamer. Interestingly, P-selectin mediated rolling adhesion was not significantly affected. This may suggest that vepoloxamer may have distinct inhibitory effects to VCAM-1 vs. P-selectin. The current findings suggest that combinatory assessment of the response of adhesive properties and membrane fragility to vepoloxamer treatment may facilitate selection of patients most likely to benefit from vepoloxamer therapy. The microfluidic flow-based platform presented in this study may have the potential to predict and monitor individual patient response to anti-adhesive therapies such as vepoloxamer. There are few reliable biomarkers that objectively predict and assess vaso-occlusive potential, thus further clinical study of this microfluidic flow-based platform are warranted. DisclosuresWhite:Functional Fluidics: Employment, Equity Ownership. Hines:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment, Equity Ownership. Benjamin:Mast Therapeutics: Consultancy. Gao:Functional Fluidics: Employment, Equity Ownership. Emanuele:Mast Therapeutics: Employment.

Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay.

Sickle cell disease (SCD) is characterized by microvascular occlusion mediated by adhesive interactions of sickle erythrocytes (SSRBCs) to the endothelium. Most in vitro flow adhesion assays measure SSRBC adhesion during continuous flow, although in vivo SSRBC adhesive interactions occur during pulsatile flow. Using a well-plate microfluidic flow adhesion system, we demonstrate that isolated SSRBCs adhere to vascular cell adhesion molecule (VCAM-1) at greater levels during pulsatile versus continuous flow. A significant increase in adhesive interactions was observed between all pulse frequencies 1 Hz to 2 Hz (60–120 beats/min) when compared to non-pulsatile flow. Adhesion of isolated SSRBCs and whole blood during pulsatile flow was unaffected by protein kinase A (PKA) inhibition, and exposure of SSRBCs to pulsatile flow did not affect the intrinsic adhesive properties of SSRBCs. The cell type responsible for increased adhesion of whole blood varied from patient to patient. We conclude that low flow periods of the pulse cycle allow more adhesive interactions between sickle erythrocytes and VCAM-1, and sickle erythrocyte adhesion in the context of whole blood may better reflect physiologic cellular interactions. The microfluidic flow adhesion bioassay used in this study may have applications for clinical assessment of sickle erythrocyte adhesion during pulsatile flow.

MEK Inhibitors, Novel Anti-Adhesive Molecules, Reverse Sickle Red Blood Cell Adhesion and Vaso-Occlusion in Vivo

In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by obstruction of the vessels due in large part to sickle erythrocyte (SSRBC) adherence to both the endothelium and leukocytes. Despite the contribution of SSRBC adhesiveness to vaso-occlusion, we lack treatments that specifically target intracellular signaling mechanisms critical for SSRBC cell-cell interactions. We have previously shown that the mitogen-activated protein (MAP) kinase ERK1/2 is aberrantly activated in SSRBCs. ERK1/2 signaling activation is critical for SSRBC interactions with the vascular endothelium and leukocytes. We have now evaluated the anti-adhesive properties of RDEA119, an inhibitor of MEK1/2, the kinase that activates the MAP kinase ERK1/2, in in vitro adhesion assays and in vivo in both nude mouse and humanized models of vaso-occlusion analyzed by intravital microscopy. We used RDEA119 because this dryg has previously been developed for human use. We found that RDEA119 caused a dose-dependent inhibition of human SSRBC adhesion to cultured TNFα-activated human micro-vascular endothelial cells (HMVECs) in vitro with a half-maximum inhibitory concentration (IC50) &lt; 2 nmol/L. This suggests that RDEA119 would be effective at very low doses in reducing SSRBC adhesion and vaso-occlusion in vivo. To determine if RDEA119 prevents vaso-occlusion in nude mice, animals were injected with TNFα followed by infusion of RDEA119 (or placebo control), and then human SSRBCs. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants immediately after human SSRBC infusion showed persistent human SSRBC adhesion to both small and large vessels, with mean diameter of 29±3.5 μm, and vaso-occlusion in placebo-treated nude mice for at least 2 h, promoting blood stasis. However, treatment of nude mice with either 0.2 mg/kg or 2 mg/kg RDEA119 markedly reduced human SSRBC adhesion and mitigated vaso-occlusion. While human SSRBC adhered to 68.5±4.6% of total vessels recorded in placebo-treated animals, SSRBCs occupied only 33 ± 6.6% (p&lt;0.0001) and 25 ± 3.7% (p&lt;0.0001) of vessels in 0.2 and 2 mg/kg RDEA119-treated nude mice, respectively. As a result, normal blood flow was restored in 95±3.5% and 76±6.2% of vessels in 0.2 and 2 mg/kg RDEA119-treated animals, respectively, compared to 43±5.8% of vessels with normal blood flow in placebo-treated mice (p&lt;0.001). Nude mice treated with 0.2 mg/kg RDEA119 also exhibited reduced human SSRBC trapping in the lungs, liver and spleen (p&lt;0.0001), and an increased number of human SSRBCs remaining in circulation. RDEA119 was also administered to nude mice after onset of vaso-occlusion for a more clinically relevant outcome. Infusion of a single dose of 1 mg/kg RDEA119 to nude mice reduced human SSRBC adhesion and vaso-occlusion within 5 min of RDEA119 administration, and adhesion progressively decreased thereafter compared to SSRBC adhesion and vaso-occlusion in these same vessel segments prior to drug administration (p&lt;0.0001). RDEA119 led to a 72% reduction in the percentage of vessels occupied by adherent human SSRBCs (p&lt;0.001) and improvement in blood flow in 89±2.4% of vessels (p&lt;0.001). Similar therapeutic benefits were obtained when RDEA119 was administered after the inflammatory trigger of TNFα to induce vaso-occlusion in transgenic sickle mice. RDEA119 resulted in a significant decrease in adherent sickle cells in enflamed vessels of sickle mice treated with only one dose of RDEA119 at 0.1, 0.05 or 0.025 mg/kg (p&lt;0.001). RDEA119 at 0.1, 0.05 or 0.025 mg/kg also significantly reduced adhesion of leukocytes to the vascular endothelium compared with placebo-treated sickle mice (p&lt;0.001). The effect of RDEA119 on both sickle RBCs and leukocyte adhesion was rapid, because a reduction in adhesion of the cells was already detectable within the first 15 minutes after drug infusion, and adhesion further decreased thereafter. In contrast, adherence of leukocytes increased progressively over time in placebo-treated sickle mice. Treatment of sickle mice with RDEA119 led to improved microcirculatory blood flow in enflamed vessels visible through the dorsal skin-fold window chamber and improved survival. These results suggest that RDEA119 may represent a valuable novel therapeutic intervention for acute pain crises that should be further evaluated in humans with SCD. Disclosures No relevant conflicts of interest to declare.

Heme-Induced Neutrophil Extracellular Traps (NETs) Formation Contributes To Sickle Cell Disease Pathogenesis

Sickle cell disease (SCD) is characterized by chronic inflammation, continuous oxidative stress and severe hemolysis. Activation of endothelial cells, adhesion of sickle erythrocytes and polymorphonuclear neutrophils (PMNs) have been reported to mediate SCD vaso-occlusion. PMNs from SCD patients produce significantly higher basal levels of reactive oxygen species (ROS) and contain less intracellular ROS scavengers than normal individuals. The formation of neutrophil extracellular traps (NETs), which requires the upregulation of intracellular ROS, has been described as a critical innate mechanism to contain infectious insults and to participate in thromboinflammatory injury. In addition, histones and granular proteins released during NET formation can directly damage lung microvascular endothelial cells. Based on these results, we explored the possibility that neutrophils contribute to SCD pathophysiology through NETs. To investigate the presence of NETs in vivo, we used a humanized mouse model of SCD that expresses human sickle hemoglobin exclusively and shares many features of human SCD. Immunofluorescence images of lung samples revealed a significantly higher number of NET DNA fibers in SCD mice (22.6 ± 2.8 NETs/field of view) compared to control mice (5.96 ± 0.86 NETs/field of view, p<0.0001) following proinflammatory cytokine (TNF-α) challenge. We confirmed that these DNA fibers were not resulting from section artifacts or remnants of apoptotic or necrotic cells by co-staining these fibers with antibodies against citrullinated histone H3 and neutrophil elastase (NE), both of which have been identified as markers of NETs. Citrullinated histone H3+ NE+ NETs were identified in the lungs of SCD mice and were found within blood vessels. Concurrently, the soluble NET component plasma DNA was significantly higher in the peripheral blood of SCD mice compared to control mice (∼524 versus ∼48 ng/ml, p<0.001). Further, plasma nucleosomes were detectable only in SCD mice (∼0.11 U/ml). We also found that SCD mice experienced body temperature decline (a reduction of 5.3 ± 1.0°C) following TNF-α challenge whereas the body temperature of control mice was unaffected. A significant positive correlation was found between numbers of NETs and reduction of body temperature in these mice (r=0.72, p<0.0001). DNase I treatment reduced NETs by 38% in SCD mice (12.2 ± 0.9 versus 7.5 ± 0.7 NETs/field of view, for vehicle- and DNase I-treated, respectively, p<0.001), protected these mice from NET-associated hypothermia and significantly prolonged their survival (p<0.05) compared to vehicle-treated SCD mice. As a result of chronic hemolysis, SCD is associated with increased plasma heme. Like SCD patients, SCD mice have significantly higher plasma heme concentration compared to control mice (50.8 ± 5.2 versus 23.8 ± 2.8 μM, p<0.001). We hypothesized that heme is the plasma factor that stimulates PMNs to produce NETs in SCD mice. In vitro study showed that heme activated PMNs to produce citrullinated histone H3+ NE+ NETs in a ROS- and heme-iron dependent manner. Heme injection significantly increased NET formation in control mice (4.5 ± 0.7 versus 9.6 ± 1.7 NETs/field of view for vehicle- and heme-treated, respectively, p<0.01) and accounted for the acute body temperature decline in these mice (3.1 ± 0.8°C, p<0.01). On the other hand, hemopexin administration, which decreased total plasma heme concentration in SCD mice, reduced NET formation by 41% (11.6 ± 0.8 versus 6.9 ± 0.7 NETs/field of view, for vehicle- and hemopexin-treated, respectively, p<0.0001) and protected treated mice from NET-associated hypothermia. Our study thus demonstrates that NETs may contribute to the pathogenesis of SCD and that heme released during hemolysis promotes NET generation in vivo. Targeting heme-mediated NET formation may represent a useful strategy in managing SCD and other pathological states involving severe hemolysis. Disclosures:No relevant conflicts of interest to declare.

Thrombospondin‐1 and L‐selectin are associated with silent cerebral infarct in children with sickle cell anaemia

Thrombospondin‐1 and <scp>L</scp>‐selectin are associated with silent cerebral infarct in children with sickle cell anaemia

&amp;lt;i&amp;gt;ARFGEF2&amp;lt;/i&amp;gt; Knockdown Enhances TNF-&amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt; Induced Endothelial Expression of the Cell Adhesion Molecules &amp;lt;i&amp;gt;VCAM1&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;ICAM1&amp;lt;/i&amp;gt;

Sickle cell anemia (SCA) is an autosomal-recessive hemoglobinopathy with a highly variable phenotype. Multiple clinical complications are characteristic of SCA including inflammatory and oxidant damage to both small and large blood vessels, hemolysis, vasoocclusion, and premature mortality. The overall severity of SCA is affected by multiple genetic modifier loci, including ARFGEF2, a gene known to modify TNF-α receptor release from human endothelial cells. In this report, we examine the effect of siRNA mediated knockdown of ARFGEF2 inhuman pulmonary artery endothelial cells and report that TNF-α induced expression of ICAM1 and VCAM1, both important mediators of endo-thelial-leukocyte adhesion, is significantly enhanced after ARFGEF2 knockdown. Levels of ICAM-1 protein are also increased in TNF-α treated endothelial cells after ARFGEF2 knockdown; the increased ICAM-1 appears to be localized in the cytoplasm. IL-1β stimulation of endothelial cells without ARFGEF2 produced enhanced ICAM1 expression only. Additionally, ARFGEF2 knockdown distinctly altered endothelial cell morphology. Large-vessel pathology in SCA is believed to begin with endothelial activation by inflammatory cytokines and adhesion of sickle erythrocytes and leukocytes, leading to a progressive vasculopathy characterized by smooth muscle cell migration and proliferation. Understanding how variability in the function of ARFGEF2 alters the response of pulmonary vasculature to TNF-α might suggest new targets for SCA treatment.

Monocyte Chemotactic Protein-1 Is Associated with Microvascular Abnormalities and Serum Ferritin Concentrations in Sickle Cell Disease Patients

Abstract 3255 Introduction:Microvascular abnormalities underlie much of the morbidity observed in sickle cell disease (SCD). Adherence of sickle erythrocytes and leukocytes to the microvascular endothelium leads to vaso-occlusion and end organ ischemia/reperfusion injury. Monocyte chemotactic protein-1 (MCP-1) is a chemokine involved in the recruitment of monocytes to sites of endothelial activation or injury and thus contributes to the pathophysiology of vascular disease. In SCD patients, circulating MCP-1 concentrations are elevated during both steady state and vaso-occlusive crisis. However, the relationship between MCP-1 and microvascular abnormalities in SCD patients has not been assessed. In the present study, we measured a panel of circulating cytokines including serum MCP-1 and hematological biomarkers including serum ferritin in SCD patients who were being monitored by computer-assisted intravital microscopy (CAIM) to assess severity of microvasculopathy. Methods:The study included 31 steady state SCD patients (22 females, 9 males) ranging in age from 4–61 years. MCP-1 concentrations were measured by Luminex multiple analyte profiling. Ferritin concentrations were measured by chemiluminometric immunossay. The conjunctival microvasculature was assessed using CAIM. A severity index on a scale of 0–15 was calculated as described previously (Cheung et al, Am J Hematol 85:899, 2010) to quantify the degree of microvasculopathy observed among the patients. Results:The mean ± SD MCP-1 concentration was 445 ± 253 pg/ml (range: 122–1180 pg/ml), mean ± SD ferritin concentration was 1675 ± 1883 ng/ml (range 18–5825 ng/ml), and mean ± SD severity index was 5.0 ± 2.5 (range: 0–9). By multiple regression analysis, with controlling for age and gender, MCP-1 was directly correlated with severity index (p = 0.046), and serum ferritin was directly correlated with MCP-1 (p = 0.019). Conclusions:Monocyte recruitment to the site of endothelial injury involves chemotactic signaling mediated in part by MCP-1, levels of which rise in SCD. Our results indicate that MCP-1 is associated with and may directly contribute to the microvasculopathy observed in SCD patients. Furthermore, we find that MCP-1 levels are correlated with ferritin concentrations. Ferritin reflects body iron stores, and an increase of iron stores in SCD resulting from chronic blood transfusion as well as increased iron absorption associated with hemolysis may thus lead to monocyte/macrophage recruitment to the vascular endothelium and contribute to vasculopathy. Measurements of MCP-1 and ferritin may also serve as surrogate biomarkers of microvasculopathy severity and inflammation. Disclosures:Green:Emisphere - Consultancy : Consultancy; Teva Pharmaceuticals - expert testimony: Consultancy.

Application of Microfluidic Technology to Evaluate the Influence of Pulsatile Blood Flow On Adhesion of Pediatric Sickle Erytrhocytes

AbstractAbstract 3238 Introduction:Sickle cell disease (SCD) is characterized by microvascular occlusion mediated in part by adhesion of sickle erythrocytes (SS RBCs) to the vasculature. Advanced flow adhesion (FA) technology facilitates SS RBC adhesion studies in conditions that simulate in vivo microvascular physiology. Most currently available FA systems measure SS RBC adhesion in non-pulsatile flow conditions, versus pulsatile blood flow conditions generated by the cardiac cycle. The influence of pulsatile blood flow on SS RBC adhesion may be particularly important in pediatric SS RBC adhesion, as children have a broad range of heart rates. This study compares SS RBC adhesion in non-pulsatile and pulsatile flow conditions, utilizing a commercially available, microfluidic FA system. Methods:Peripheral blood was obtained from patients with homozygous SCD (n=7) in steady state (5–18 years) from the Sickle Cell Center at the Children’s Hospital of Michigan. FA assays were performed in non-pulsatile and pulsatile flow conditions, at a shear stress of 1.0 dyne/cm2, and increasing shear stress up to 20 dyne/cm2to assess avidity. A programmable control unit regulated pulse frequency, shear stress, and temperature. Adhesion was measured to immobilized human laminin and vascular cell adhesion molecule-1 (VCAM-1). A static adhesion assay was used to assess initrinsic adhesive properties of SS RBCs independent of flow dynamics. Results:Standard assays were performed with 30 mL of isolated SS RBCs (1× 107 cells/mL), and SS RBC adhesion was comparable to levels previously reported in parallel plate flow adhesion assays. FA assays showed that adhesion to both laminin and VCAM-1 was significantly increased in the context of pulsatile blood flow (1.67Hz) compared to non-pulsatile blood flow by 8-fold and 56-fold, respectively. The relationship of SS RBC adhesion to increasing pulse frequencies was variable from patient to patient, although adhesion to both laminin and VCAM-1 was uniformly greater in all pulse frequencies tested (1.0, 1.5, 1.67, and 2.0 Hz) compared to non-pulsatile blood flow. When avidity of adhesion was tested 78% of SS RBCs remained adhered to VCAM-1 at the maximum 20dyne/cm2 shear stress, whereas 6% of SS RBCs remained adhered to laminin at a shear stress of 20 dynes/cm2. Pulsatile adhesion to VCAM-1 and laminin was unaffected by protein kinase A (PKA) inhibition, although adhesion to laminin decreased by 31% in one of three patients. To determine if increased adhesion under pulse-flow conditions was due to increased contact time with the immobilized substrate versus a change in the SS RBC’s intrinsic adhesive state, we measured SS RBC adhesion in a static adhesion assay following exposure to pulsatile versus non-pulsatile conditions. There was no significant difference in static adhesion to VCAM-1, however adhesion of pulse-exposed SS RBCs to laminin was more variable. Static adhesion of pulse-exposed SS RBCs to laminin was reduced by 60% in the presence of a PKA inhibitor. Conclusions:Our data demonstrate the application of a commercially available microfluidic flow adhesion assay system for efficient assessment of SS RBC adhesive properties. In the future, such advances may allow SS RBC adhesive properties to be evaluated clinically as a predictive tool for future vaso-occlusive events, and to predict individual patient response to anti-adhesive therapy. The small volume of blood required makes this system particularly attractive for studying pediatric samples. Additionally, our data demonstrate that adhesion to both an endothelial cell substrate (VCAM-1) and a subendothelial matrix substrate (laminin) is significantly influenced by the presence of pulsatile blood flow. Although PKA may play a minor role in pulsatile adhesion to laminin, increased contact time with immobilized laminin and VCAM-1 may be a greater contributor to increased adhesion under pulsatile conditions versus non-pulsatile conditions. Pediatric SS RBCs adhered to VCAM at higher levels and with more avidity compared to laminin. The pulsatile flow conditions described in this study more closely approximate in vivo microvascular conditions compared to non-pulsatile conditions commonly used to study SS RBC adhesion. Based on these differences in adhesion under pulsatile versus non-pulsatile flow, incorporating pulsatile flow in future adhesion studies may be more representative of in vivo conditions. Disclosures:No relevant conflicts of interest to declare.

RNA Aptamer Therapy for Vaso-Occlusion in Sickle Cell Disease

Patients with sickle cell disease (SCD) often suffer painful vaso-occlusive episodes caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium. To investigate inhibition of SS-RBC adhesion as a possible treatment for vaso-occlusion, 2 adhesion molecules, α(v)β(3) and P-selectin, were targeted by high-affinity RNA aptamers. An in vitro flow chamber assay was used to test the antiadhesion activity of α(v)β(3) aptamer clone 17.16. Human SS-RBC were passed across a confluent monolayer of thrombin-stimulated human umbilical vein endothelial cells (HUVEC) at a constant rate. α(v)β(3) aptamer reduced SS-RBC adhesion to activated endothelial cells to the level seen with untreated HUVEC. An aptamer reactive with complement component 8 was used as a negative control and exerted no inhibition, confirming the specificity of α(v)β(3) aptamer (P=0.04). At 2 dyn/cm(2) shear stress, 30 nM α(v)β(3) aptamer showed maximal effect in decreasing SS-RBC adhesion to HUVEC. The antiadhesive activity of the P-selectin aptamer clone PF377 was also tested using HUVEC pretreated with IL-13 to upregulate expression of P-selectin as seen in activated endothelial cells. At 1 dyn/cm(2) shear stress, 60 nM of P-selectin aptamer had antiadhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin. A negative control did not prevent adhesion (P=0.05). These data show the potential utility of aptamers to block endothelial adhesion molecules to prevent or treat vaso-occlusion in SCD.