Pathology Of Sickle Cell Disease Research Articles

The Physical Foundation of Vasoocclusion in Sickle Cell Disease

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.

Erythrocyte plasma membrane–bound ERK1/2 activation promotes ICAM-4–mediated sickle red cell adhesion to endothelium

AbstractThe core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. β2-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72syk-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4–mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and β-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.

Pathology of sickle cell disease

Sickle cell disease (SCD) is a hereditary chronic hemolytic anemia with numerous clinical consequences. Intravascular sickling of red blood cells leads to multiorgan dysfunction. Although the pathophysiology of SCD has been well studied, there remains a lack of effective treatment. Refinements in overall care have improved quality of life; however, premature death is still not uncommon. SCD usually presents in childhood and is common in areas where malaria is (or was) common. The association with malaria is apparently of benefit to the individual because these individuals tend to contract a milder form of the disease. This review highlights the spectrum of pathology seen in people with SCD, with an emphasis on the pathogenesis of sudden death.

The rate of hemolysis in sickle cell disease correlates with the quantity of active von Willebrand factor in the plasma

AbstractVaso-occlusion, hemolysis, and oxidative stress are hallmarks of sickle cell disease (SCD). This pathology is accompanied by systemic endothelial activation, rendering the endothelium more adhesive for blood cells, including sickle erythrocytes. Activated endothelial cells display or secrete several adhesive molecules, including von Willebrand factor (VWF). We assessed several VWF parameters in SCD patients at baseline: multimer pattern, antigen concentration (VWF:Ag), activation factor (VWF:AF), and total active VWF (VWF:TA). VWF:AF was determined using a llama nanobody (AU/VWFa-11) that detects a platelet-binding conformation of the A1 domain; VWF:TA was calculated by multiplying VWF:Ag by VWF:AF. SCD plasma contained elevated VWF:Ag and ultralarge VWF multimers. VWF:TA, a measure of total VWF reactivity, correlated closely with hemolysis, as determined by serum lactate dehydrogenase. ADAMTS13 activity and antigen were normal in all patients. These findings suggest an important role for hyperreactive VWF in SCD pathology and connect SCD to other microangiopathies, particularly thrombotic thrombocytopenic purpura.

Lack of Clustering of SCD Complications Into Two Distinct Subphenotypes In An Adult Patient Population.

AbstractAbstract 1643The complex pathophysiologic mechanisms that contribute to disease pathology in sickle cell disease (SCD) include microvascular occlusion secondary to deoxy-Hb S polymerization, interaction of sickle RBCs with vascular endothelium and other blood cells, hemolysis with resultant nitric oxide (NO) scavenging, endothelial activation with inflammation, and activation of coagulation. It has been recently hypothesized that there may be two distinct sub-phenotypes in SCD: one where hemolysis and NO depletion predominates (hemolysis/endothelial dysfunction) and the other where vaso-occlusion and increased whole blood viscosity plays a more prominent role. The clinical complications of SCD thus cluster into one of the two subphenotypes: pulmonary hypertension, priapism, leg ulcers and stroke appear to be more commonly associated with the hemolysis/NO depletion/endothelial dysfunction subphenotype, whereas frequent pain episodes, acute chest syndrome, osteonecrosis and retinopathy tend to be more common in the viscosity/vaso-occlusion group. We had previously analyzed the records of 124 patients with Hb SS or S-b° thalassemia followed at the Medical College of Georgia Adult Sickle Cell Clinic to validate the clustering of disease complications into the aforementioned two sub-phenotypes and found no significant associations between phenotypes within each sub-group, as well as between phenotypes across the groups (all p values >0.1). The following criteria were used to define complications:Vaso-occlusive episode≥ 1 major pain episode in the past 2 yearsPulmonary hypertensionTR jet velocity ≥ 2.5 m/secNephropathyCR ≥ 1.5 mg/dl, proteinuria > 300 mg/24 h, or ESRD on dialysisOsteonecrosisMR documentation, hx core decompression or total hip replacementGallstonesUltrasound documentation or hx cholecystectomyAcute Chest SyndromeDocumentation in medical recordPriapismDocumentation in medical record or patient history [Display omitted] We have now extended our analyses to 203 patients with Hb SS or S-b° thalassemia (ages 12–60; 101 males, 102 females). We performed principal component analysis on the data from 203 patients. A total of 10 sub-phenotype variables were used in the analysis. The variables are VOE, ACS, retinopathy, gallstones, AVN, stroke, nephropathy, pulmonary hypertension, leg ulcers, and priapism. The individual factor map based on the first 2 principal components is shown below. Each dot represents a patient in the figure. If the hypothesis of two groups of sub-phenotypes were correct, we would see two clusters of patients. However, from this figure, there is no clear clustering of patients. [Display omitted] We also plotted the sub-phenotype variables factor map based on the results from the principal components analysis. As shown in the following figure, the sub-phenotypes are not clustered into two groups as predicted by the hypothesis. In particular, we have retinopathy in one extreme and gallstones in the other. They are not clustered in one group.Our results indicate that the complications of sickle cell disease do not cluster into two distinct subphenotypes as previously hypothesized. While this hypothesis may provide a useful conceptual framework in deconstructing and understanding various pathophysiologic mechanisms operative in sickle cell disease, such clear cut distinction is not applicable in clinical practice. Disclosures:No relevant conflicts of interest to declare.

Induced expression of bone morphogenetic protein-6 and Smads signaling in human monocytes derived dendritic cells during sickle-cell pathology with orthopedic complications

BMP-SMAD (bone morphogenetic protein) signaling pathways in association with APT play paramount roles in osteoblastic differentiation, bone formation and embryonic development of human and animals. However, the implications of potent components (BMP6, Smad1, Smad2 and APT) of this pathway in SCD (sickle cell disease) pathology with orthopedic complications (Ortho + SS) are poorly elucidated and substantially unknown. Here, we address the role of BMP6, Smad1, Smad2 and APT mRNA and protein expression in hMDDCs obtained from Ortho + SS patients, employing RT-PCR, qRT-PCR and immunoblotting. Interestingly, we observed that SCD pathology exhibited significantly up-regulated expression of those signaling components at the level of mRNA and protein. Furthermore, exogenous BMP6 induced apoptosis was observed to be significantly associated in Ortho + SS complication and markedly increased the percentage of cells undergoing apoptosis as compared to healthy group. Interestingly, the non-stimulated cells have shown higher apoptotic nuclei percentage than the stimulated cells in pathological condition. Thus, expression of BMP-SMAD signaling components augments apoptosis and up regulates the transcription of these genes and it suggests that induction is due to transcriptional regulation. Taken together, our findings provide evidence that BMP-SMAD signaling components along with APT were over expressed, mediates apoptosis and may play an important role in the SCD pathology with orthopedic complications.

Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

P02-305 Medicating malingerers: A case study of feigned sickle cell disease

Speculation exists that bone pain crises among sickle cell disease (SCD) patients are not adequately treated. We counter this assertion with the case of a 17 year-old African American woman receiving narcotics while malingering SCD.There are various complications of SCD with the most common resulting from ischemia of the bone marrow. While depression, anxiety, and post-traumatic stress disorder have been described, psychiatric complications are not well documented in SCD.A 17 year old African American female entered the emergency room (ER) with right tibia and fibula fractures. She admitted to having SCD with bone pain crises previously treated with Ketorolac and Meperidine.On this occasion, radiological studies did not confirm sickle cell changes. A subsequent immunoglobulin electrophoresis came back hemoglobin AA. After the patient received notification she did not have SCD, she attempted to leave against medical advice. Her fractures were treated and she was discharged.DSM IV defines malingering as “the intentional production of false or grossly exaggerated physical or psychological symptoms”. One previous report of malingering sickle cell crises exists.1 This case demonstrates that the motivation to gain narcotic analgesics is a determining factor for malingering vis-à-vis factitious disorder, in which the primary goal is to assume the sick role.1Clinicians may assume the validity of a self-reported SCD history. In this case, ER physicians prescribed narcotics without objective evidence of SCD pathology. We suggest verification of SCD diagnosis in order to prevent unnecessary prescription of narcotics.

HuGTPCH Expressing S+S-Antilles Mice Have Reduced Gardos Channel Activity, Endothelin-1 Expression, and Endothelin Receptor A and B Expression, While Total Biopterin and Percent Tetrahydrobiopterin Are Increased

An imbalance in the ratio between nitric oxide synthase (NOS) and its essential co-factor, tetrahydrobiopterin (BH4), leads to a loss of NO generating capacity and a shift to superoxide production, resulting in a vicious cycle in which BH4 is oxidized and more superoxide is generated. Low NO and hypoxia have been shown to regulate endothelin-1 (ET), an endogenous vasoconstrictor and regulator of vascular tone. There is evidence that BH4 levels can be regulated by ET that is reported to be abnormally high in sickle cell disease (SCD). We have demonstrated that NOS levels are elevated while arginine levels are decreased in sickle transgenic mice. We hypothesized that increasing BH4 bioavailability, even in the absence of arginine supplementation, would ameliorate stresses resulting from NOS dysfunction and would mimic some aspects of arginine supplementation and ameliorate some of the complications of SCD. We studied a sickle transgenic mouse model that was generated by crossing a transgenic mouse expressing human GTP-cyclohydrolase I (huGTPCH), the rate-limiting enzyme in BH4 production, with S+S-Antilles sickle transgenic mice that we have used to study the effect of arginine supplementation on Gardos channel activity and ET expression. We demonstrate that BH4 levels are increased in sickle transgenic mice expressing huGTPCH, and that this is correlated with reduction of Gardos channel activity and ET and ET receptor expression. We measured Gardos channel activity in freshly isolated red blood cells from S+S-Antilles mice and found that Gardos channel activity was decreased in huGTPCH expressing S+SAntilles mice by 63% (from 4.35±0.43 FU to 2.63±0.65 FU in huGTPCH expressors, p<0.01, t-test) which is slightly larger than the decrease previously reported with arginine supplementation (58%) of S+S-Antilles mice without huGTPCH expression. Using RT-PCR we demonstrated that ET and ET-receptor A and B expression were reduced in lung tissue, a major producer of ET, versus that observed in S+S-Antilles mice without the huGTPCH transgene (from 1.84-fold±0.02 to 1.23±0.02, 1.41-fold±0.02 to 0.41±0.02, and from 1.38-fold±0.02 to 0.67±0.02, p<0.05, 0.02, 0.03, respectively). S+S-Antilles mice have an increase of ET and ET-receptor A and B expression versus C57BL of 1.84-fold, 1.41-fold, and 1.38-fold, respectively. We measured total biopterin and percent BH4 in heart and liver of S+S-Antilles mice with and without huGTPCH. In heart, S+SAntilles-huGTPCH mice versus S+S-Antilles had increased percent BH4 (to 0.75±0.02 from 0.46±0.02 pmol/mg protein, p<0.001) and total biopterin was increased 3-fold. In liver, S+S-Antilles-huGTPCH mice versus S+S-Antilles had increased percent BH4 (to 0.30±0.02 from 0.19±0.07 pmol/mg protein, p<0.05) and total biopterin was increased 2.5-fold. In conclusion, BH4 dysregulation is observed in sickle transgenic mice and expression of huGTPCH increased total biopterin and percent BH4 in our sickle mouse model. ET has a profound effect on vascular tone and the reduction of ET mRNA and Gardos channel activity in huGTPCH expressing mice supports the conclusion that the altered BH4 levels observed are physiologically relevant. Thus our observations suggest that BH4 supplementation may ameliorate some of the pathology of SCD.

Gene Replacement Therapy for Sickle Cell Disease and Other Blood Disorders

Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing anti-sickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (betaS) with a normal copy of the gene (betaA). We recently derived embryonic stem (ES) cells from a novel knockin mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. Animals derived after gene replacement produced high levels of normal human hemoglobin (HbA), and the pathology associated with SCD was corrected. These experiments provided a foundation for similar studies in which our group collaborated with Rudolf Jaenisch's laboratory to correct SCD by gene replacement in iPS (induced pluripotent stem) cells derived by direct reprogramming of sickle skin fibroblasts. Corrected iPS cells were differentiated into hematopoeitic progenitors that were transplanted into irradiated sickle recipients. The transplanted animals produced high levels of normal human HbA, and the pathology of SCD was corrected. These proof-of-principle studies provide a foundation for the development of gene replacement therapy for human patients with SCD and other blood disorders.

The % Tetrahydrobiopterin (BH4) and Serine 1177 Phosphorylation Is Reduced in Sickle Transgenic Mice.

Tetrahydrobiopterin (BH4) is an essential co-factor of nitric oxide synthase (NOS), the enzyme that generates nitric oxide (NO) from arginine and oxygen. NO plays an essential role in maintaining vascular tone and endothelial function, reducing levels of adhesion molecules and platelet aggregation, and reducing inflammation; all of which are critical in sickle cell disease (SCD). In addition to BH4, arginine, calmodulin, intracellular Ca++ levels, hsp90, phosphorylation at serine 1177 and threonine 495, and monomer/dimer equilibrium have all been shown to modulate NO and superoxide production by eNOS. Arginine levels are decreased in plasma in SCD. We have demonstrated that plasma arginine is reduced in sickle transgenic mice and that arginine supplementation partially restores both red cell and vascular function. Channon et al have demonstrated that, in atherosclerosis and diabetes, enhanced superoxide production is associated with BH4 depletion; superoxide can interfere with both NO production and, by reacting with NO, with bioavailability. We hypothesized that a similar depletion of BH4 occurs in SCD and that treatment protective of tissue BH4 levels may interrupt the cycle of polymerization, vaso-occlusion and inflammation that is the origin of pathology in SCD. We report here depletion of BH4 in tissue and plasma of several lines of sickle transgenic mice and the effect of hypoxia on % BH4 and serine 1177 phosphorylation in NY1DD mice, a mild sickle transgenic mouse that becomes more severe when exposed to hypoxia. Only BH4 is active as a NOS cofactor, the oxidized forms BH2 and biopterin (B) are inactive. Results are presented as %BH4 (BH4/(BH4+BH2+B)). We find that %BH4 is the same in C57BL and NY1DD mice under room air in plasma and brain. After exposure to 4 days of hypoxia (8% O2), % BH4 is reduced from 75.7%±3.8% to 53.7%±3.8% in plasma, p<0.004 and from 82.0%±3.7% to 68.7%±3.7% in brain, p<0.006. At the same time total biopterin (BT= BH4+BH2+B) increased in plasma, but remained constant in brain. We also measured eNOS phosphorylated at serine 1177 in brain in NY1DD mice. We find that the % phosphorylation decreases from 45.0%±7.9% to 28.3%±7.3%, p<0.007 after 4 days of hypoxia, but that at the same time total eNOS and total nNOS nearly doubled. BERK mice express exclusively HbS and, under normoxia, we found decreased %BH4 in brain in BERK mice (58%±3.3% vs 76.7%±3.0% for C57, p<0.003) with constant BT. In red cells of BERK mice, the %BH4 is about the same as C57BL (81.7% vs 76.7%, respectively), but BT doubled from 124 to 257, p>0.01. In conclusion, increased total expression of eNOS and nNOS and decreased %BH4 with decreased % phosphorylation at serine 1177 of eNOS suggest that NO production in brain in NY1DD mice after hypoxia may be severely compromised. Percent BH4 is also reduced in brain of BERK mice under normoxia, which suggests that BH4 is reduced in more severe mice under room air. Plasma and red cell %BH4 and BT do not have a simple relation to tissue levels, but do respond to pathology and may be the only readily measurable variable in human studies. The multiple pathways for disregulation of NOS in SCD involve not only NO scavenging by plasma free Hb and arginine deficiency, but also reduced bioavailability of BH4.

Inhaled Carbon Monoxide: An Anti-Inflammatory Modulator in Transgenic Sickle Mice.

Inflammation plays a prominent role in vaso-occlusion and organ pathology in sickle cell disease (SCD). We have shown that heme oxygenase-1 (HO-1) plays a critical role in metabolizing the excess heme generated in this hemolytic disease, thereby modulating hypoxia/reoxygenation-induced vaso-occlusion in murine models of SCD. The products of HO-1 activity, carbon monoxide (CO), Fe2+ (ultimately incorporated into ferritin), and biliverdin/bilirubin have demonstrable anti-oxidant and anti-inflammatory effects. We have observed that brief (1 h/d × 3 d) inhalation of CO in SCD mice modulates vaso-occlusion (JCI , 116: 808–816; 2006). We hypothesize that prolonged treatment with inhaled CO will significantly decrease the inflammatory phenotype of SCD mice. Starting at three weeks of age, C57BL-6J and heterozygous BERK mice (mβ/hβS) were treated with inhaled CO at 0, 25, or 250 ppm (1 h/d × 3 d/wk × 10 wks). Over the duration of the ten week study no animals died as result of the CO treatments. Upon completion of the ten week treatment period animals were euthanized, blood was removed by cardiac puncture, and organs were harvested and homogenized. Carboxyhemoglobin levels immediately after 1h of CO treatment, (0, 25, and 250 ppm) were <1%,<1%, and 12%, respectively, but decreased to normal 24 h later. Untreated heterozygous BERK mice had a higher mean white blood cell count (WBC)(14,400/μL) than C57BL-6J mice (9,469/ μL, p<0.005). Treatment for 10 weeks with either 25 or 250 ppm CO significantly decreased heterozygous BERK WBC (8827 and 9222/μl, p<0.02), but had no effect on C57BL-6J WBC. The decrement in heterozygous BERK WBC was primarily due to a significant decrease in neutrophils (p<0.05). There was no significant difference in either the mean hematocrits or reticulocyte counts in CO-treated mice. Since redox-active iron (Fe2+) promotes oxidative stress and inflammation, we measured redox-active iron with Ferene-S in liver homogenates. In untreated heterozygous BERK mice, redox-active iron was two-fold greater than C57BL-6J mice (p<0.05) and treatment with 25 or 250 ppm CO significantly (p<0.01, p<0.02 respectively) decreased the redox-active iron (Fe2+) to levels comparable to untreated C57BL-6J. Since there is red cell congestion in heterozygous BERK livers, it was not surprising that the heme-content of untreated heterozygous BERK mice was three-fold (p<0.05) higher than untreated C57BL-6J. Treatment with 25 or 250 ppm CO significantly (p<0.05) decreased the heme-content of the livers to levels comparable to untreated C57BL-6J. The cytoprotective proteins HO-1 and ferritin heavy-chain were significantly increased in the livers of untreated heterozygous BERK mice compared to C57BL-6J (p<0.02). However, CO treatments had no significant effects on expression of these proteins on Western blots. We conclude that inhaled CO treatments decrease WBC, neutrophils, as well as liver redox-active iron and heme content in heterozygous BERK mice. Further studies are evaluating the effects of CO on inflammation, vaso-occlusion, and organ pathology in the homozygous BERK mouse model. We speculate that inhaled CO treatments may be a potential therapy for patients with SCD by acting as a modulator of oxidative stress and inflammation.

Fiber Depolymerization

Depolymerization is, by definition, a crucial process in the reversible assembly of various biopolymers. It may also be an important factor in the pathology of sickle cell disease. If sickle hemoglobin fibers fail to depolymerize fully during passage through the lungs then they will reintroduce aggregates into the systemic circulation and eliminate or shorten the protective delay (nucleation) time for the subsequent growth of fibers. We study how depolymerization depends on the rates of end- and side-depolymerization, k end and k side, which are, respectively, the rates at which fiber length is lost at each end and the rate at which new breaks appear per unit fiber length. We present both an analytic mean field theory and supporting simulations showing that the characteristic fiber depolymerization time τ = 1 / k end k side depends on both rates, but not on the fiber length L, in a large intermediate regime 1 ≪ k side L 2/ k end ≪ ( L/ d) 2, with d the fiber diameter. We present new experimental data which confirms that both mechanisms are important and shows how the rate of side depolymerization depends strongly on the concentration of CO, acting as a proxy for oxygen. Our theory remains rather general and could be applied to the depolymerization of an entire class of linear aggregates, not just sickle hemoglobin fibers.

Peptides based on αV-binding domains of erythrocyte ICAM-4 inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation

Growing evidence shows that adhesion molecules on sickle erythrocytes interact with vascular endothelium leading to vaso-occlusion. Erythrocyte intercellular adhesion molecule-4 (ICAM-4) binds alphaV-integrins, including alphaVbeta3 on endothelial cells. To explore the contribution of ICAM-4 to vascular pathology of sickle cell disease, we tested the effects of synthetic peptides, V(16)PFWVRMS (FWV) and T(91)RWATSRI (ATSR), based on alphaV-binding domains of ICAM-4 and capable of inhibiting ICAM-4 and alphaV-binding in vitro. For these studies, we utilized an established ex vivo microvascular model system that enables intravital microscopy and quantitation of adhesion under shear flow. In this model, the use of platelet-activating factor, which causes endothelial oxidant generation and endothelial activation, mimicked physiological states known to occur in sickle cell disease. Infusion of sickle erythrocytes into platelet-activating factor-treated ex vivo rat mesocecum vasculature produced pronounced adhesion of erythrocytes; small-diameter venules were sites of maximal adhesion and frequent blockage. Both FWV and ATSR peptides markedly decreased adhesion, and no vessel blockage was observed with either of the peptides, resulting in improved hemodynamics. ATSR also inhibited adhesion in unactivated microvasculature. Although infused fluoresceinated ATSR colocalized with vascular endothelium, pretreatment with function-blocking antibody to alphaVbeta3-integrin markedly inhibited this interaction. Our data strengthen the thesis that ICAM-4 on sickle erythrocytes binds endothelium via alphaVbeta3 and that this interaction contributes to vaso-occlusion. Thus peptides or small molecule mimetics of ICAM-4 may have therapeutic potential.

Aggregates of Reticulocytes, Monocytes and Platelets Exist in Whole Blood of Sickle Cell Patients: Roles for Plasma Fibronectin, α4 Integrins and PSGL-1.

Leukocyte and platelets are understudied contributors to the overall pathology of sickle cell disease (SCD). Elevated leukocyte counts are common in these patients and correlate inversely with patient lifespan and overall disease severity. For example, a drop in neutrophil count typically predicts a patient's response to hydroxyurea, while increased monocyte counts correlate directly with increased reporting of pain crises. Moreover, both RBCs and WBCs have been detected as components in vaso-occlusive blockages in mouse models, where adhesive RBCs appear to interact directly with WBCs at the vaso-occlusive site. Platelets are activated in SCD and are thought to promote the hypercoagulability in these patients. Despite the potential contribution of all blood cells to the pathology of sickle cell disease, neither a mechanism of adhesion between the WBC and RBC nor a role for soluble matrix proteins in this interaction has been elucidated in humans. To detect potential adhesive interactions between the blood cells in SCD, we collected whole blood into anticoagulants that spare divalent cations (PPACK or factor Xa inhibitor) and assayed for heterotypic cell associations by two and three color flow cytometry. Our results indicate that RBCs, WBCs and platelets exist in heterotypic, multi-cellular aggregates in blood from SCD patients but not unaffected (AA) individuals. By detecting monocyte specific markers, we determined that the primary WBC component of these aggregates was the monocyte, and the primary RBC was the young SS “stress” reticulocyte. Using both in vitro RBC/monocyte adhesion studies and whole blood samples, we demonstrate that α4-containing integrins on both SS RBCs and WBCs mediate this interaction by interacting directly with endogenous plasma fibronectin. Furthermore, we show that the α4 integrin on SS RBCs binds to the RGDS site in fibronectin, whereas the α4 integrin on monocytes binds to the CS-1 site in the molecule, suggesting a novel mechanism of interaction between SS RBCs and monocytes via a fibronectin bridge. Antibodies against the CS-1 binding site in fibronectin substantially disrupt the monocyte/RBC interaction in whole blood, further underscoring the role of fibronectin as a linker between the two cell types. However, platelet incorporation in the aggregate was insensitive to inhibition of the α4 integrin, but was sensitive to inhibition of PSGL-1, suggesting that platelet inclusion likely occurs via a P-selectin/PSGL-1-mediated interaction between the platelet and the monocyte. Interestingly, similar aggregates were also detected in two patients with chronic hemolysis and brisk reticulocytosis, potentially extending the relevance of such aggregates beyond SCD. Taken together our results suggest a new adhesive paradigm for SS RBCs and monocytes as central components of heterotypic blood cell aggregates that include platelets and that are present in whole blood of patients with SCD. Our data therefore illustrate a potentially pathological interaction of all major blood cell types in SCD patients that may impact vaso-occlusion and contribute to other erythrocyte disorders.

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

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

Elevated Plasma Arginase Levels in Hemoglobinopathies.

The decreased bioavailability of arginine (Arg) and the resulting lower nitric oxide (NO) production has been shown to be an important factor in the pathology of sickle cell disease (SCD) and thalassemia. Vascular alterations leading to pulmonary hypertension are important factors of heart failure and death in these hemoglobinopathies. Red blood cell (RBC) hemolysis in these patients will release arginase into the circulation and contribute to the reduction of plasma Arg levels, change the Arg-to-ornithine ratio, and increase other downstream amino acid metabolites. Such compounds, including proline and polyamines, may contribute to vascular and airway remodeling. To study the contribution of arginase released from RBC, we measured the arginase activity and arginase protein concentration in the plasma and RBC lysates of normal controls, SCD patients, and thalassemia patients.Arginase activity was determined by the conversion of 14C-labeled guanidine-L-arginine to 14C-labeled urea, then to 14CO2 by urease and trapped as Na214CO3 for scintillation counting. Arginase and hemoglobin concentration were measured by ELISA (BioVendor, Candler, NC and Bethyl Labs Inc., Montgomery, TX respectively). Mean cellular hemoglobin concentration was measured with a Coulter Counter (Beckman Coulter, Fullerton, CA) or Technicon H3 analyzer (Tarrytown, NY). RBC were separated by density gradient centrifugation with OptiPrep (Axis Shield, Dundee, UK). Arginase specific activity (SA) was calculated by dividing the activity by the concentration to yield mole of Arg metabolized per g of arginase per hour. We found a higher level of arginase in the plasma and RBC of SCD and thalassemia patients as compared to normal controls. These levels correlated with cell-free hemoglobin content (plasma) or reticulocyte count (RBC). The light density fraction (<1.077 g/ml) of RBC, enriched in reticulocytes, showed higher arginase levels confirming increased arginase levels in young RBC. Interestingly, as the plasma arginase protein and activity increased with cell-free hemoglobin, the SA decreased. Similarly in RBC lysates, the SA decreases with hemoglobin concentration. This suggests that a factor exists within the RBC that negatively modulates arginase activity. Due to the high SA of arginase at low hemoglobin concentrations, low levels of intravascular hemolysis can generate relatively high levels of Arg breakdown.We conclude that low levels of intravascular hemolysis of RBC with increased arginase content in SCD and thalassemia, play a role in the altered Arg metabolism and decreased NO production. While the reason for high levels of arginase in the RBC as well as complete clinical impact of elevated arginase activity in plasma remain to be determined, dysregulated Arg metabolism and a shift toward Arg catabolic products, may be associated with the development of pulmonary hypertension and mortality in SCD and thalassemia.

Polymorphisms of chemokine receptors and eNOS in Brazilian patients with sickle cell disease

Sickle cell disease (SCD) is an inherited disorder that presents extremely variable clinical manifestations. For the past few decades, it has been approached as an inflammatory disorder, and several researchers have tried to determine the factors involved in such characteristic. In order to contribute to the identification of the genetic differences underlying this phenotypic diversity in SCD, we proposed to study the distribution of polymorphic variants of the genes encoding the chemokine receptors CCR2 and CCR5, as well as three polymorphisms in the NOS3 gene, in Brazilian SCD patients. These genes are involved in the development of inflammatory immune reactions, a feature believed to be of extreme importance in SCD pathology. Our results indicate that the polymorphisms studied here are not directly associated with severe clinical manifestations in SCD patients. Nevertheless, we observed a tendency for the development of a severe clinical course in carriers of the variant alleles CCR2-64I and CCR5delta32 and in homozygotes for the -786C variant of the NOS3 gene. Further studies should be carried out in order to assess the role of such variants in the clinical picture of SCD.

The Effects of Erythrocyte Membranes on the Nucleation of Sickle Hemoglobin

Pathology in sickle cell disease begins with nucleation-dependent polymerization of deoxyhemoglobin S into stiff, rodlike fibers that deform and rigidify red cells. We have measured the effect of erythrocyte membranes on the rate of homogeneous nucleation in sickle hemoglobin, using preparations of open ghosts (OGs) with intact cytoskeletons from sickle (SS) and normal adult (AA) red cells. Nucleation rates were measured by inducing polymerization by laser photolysis of carboxy sickle hemoglobin and observing stochastic variation of replicate experiments of the time for the scattering signals to reach 10% of their respective maxima. By optical imaging of membrane fragments added to a hemoglobin solution we contrast the rate of nucleation immediately adjacent to membrane fragments with nucleation in a region of the same solution but devoid of membranes. From analysis of 29,272 kinetic curves obtained, we conclude that the effect of AA OGs is negligible (10% enhancement of nucleation rates ±20%), whereas SS OGs caused 80% enhancement (±20%). In red cells, where more membrane surface is available to Hb, this implies enhancement of nucleation by a factor of 6. These experiments represent a 10-fold improvement in precision over previous approaches and are the first direct, quantitative measure of the impact of erythrocyte membranes on the homogeneous nucleation process that is responsible for polymer initiation in sickle cell disease.

Di-Annexin, a Novel Tool To Identify and Protect Phosphatidylserine Exposing Cells.

Phosphatidylserine (PS) is found exclusively in the inner monolayer of plasma membranes. During programmed cell death this well-preserved asymmetry of phopholipid distribution is lost, and PS is exposed on the outer surface. PS exposure is also found in subpopulations of red blood cells (RBC) in disease states such as sickle cell disease (SCD). PS exposure leads to increased cell-cell interactions as well as recognition and removal by macrophages. PS serves as a binding site for factors involved in blood coagulation and provides a target for secretory phospholipase A2 (sPLA2) mediated membrane degradation. Measurement of PS-exposing cells (PSRBC) relies on the use of proteins that bind to PS and can be fluorescently labeled and visualized by flow-cytometry. The human protein annexin V (AV) has a high affinity for PS and has been shown to interfere with the physiologic processes induced by the exposure of PS. To improve detection of PS exposing cells, and develop a probe that may be used in vivo, we generated plasmids encoding a dimer of human AV (DAV), and expressed the protein in E.coli. We show that DAV has a 5–8 fold higher sensitivity than AV in binding to PSRBC, and effectively identifies PSRBC in SCD blood samples. Moreover, DAV protects PSRBC from sPLA2-induced hemolysis. While normal RBC show only 2% hemolysis after incubation with 100 ng/ml sPLA2 for one hour, sPLA2 induces more than 90% hemolysis in PSRBC. DAV, when added to PSRBC before incubation with sPLA2, protects more than 90% of the PSRBC from hemolysis. Our data show that DAV effectively competes with factor Va for PS binding and inhibits thrombin formation in the presence of the pro-thrombinase complex. At low concentrations, DAV was significantly more efficient in inhibiting thrombin formation than AV (P<0.001). The increased size of DAV gives the molecule a significantly increased survival time in the murine circulation compared to AV, and allows visualization of PS exposing membranes in vivo. Together this data shows that DAV is a superior probe to identify PS-exposing membranes, protects PSRBC from sPLA2-induced hemolysis, and interferes with cellular hemostasis. The use of DAV will allow more precise determination of PS exposure by flow-cytometry as compared to currently available commercial probes. Moreover, it will allow the measurement of PS exposing membranes in vivo, and may ultimately be proven to be useful as a therapeutic agent in SCD pathology by masking PS exposing cell membranes.