Sickle RBC Research Articles

Assessing the Clinical Outcomes of Voxelotor Treatment in Patients with Sickle Cell Disease

The results of Voxelotor treatment in Sickle Cell Disease, as assessed through clinical outcomes, demonstrated its efficacy in reducing hemolysis and improving hemoglobin levels. This effect can be attributed to Voxelotor's inhibition of HbS polymerization, thereby restoring the deformability and compliance of sickle RBCs. Our mathematical model provided mechanistic insights into how altered rheological properties of sickle RBCs impact their flow dynamics in microcirculation. Specifically, we observed that Voxelotor-induced improvements in RBC deformability and compliance lead to enhanced plasma film height in capillaries, promoting smoother blood flow and reducing the risk of vaso-occlusive events. We established a direct relationship between the molecular mechanism of Voxelotor action and its clinical outcomes. The mathematical model served as a bridge, elucidating how Voxelotor-induced changes in sickle RBC mechanics translate to improved microcirculatory flow and reduced vaso-occlusion. This holistic understanding not only validates the therapeutic efficacy of Voxelotor but also underscores the importance of considering biomechanical factors in evaluating treatment outcomes in SCD. Furthermore, this study highlighted the relevance of microfluidics based diagnostic tools in assessing the efficacy of Voxelotor treatment. By leveraging insights from our mathematical model, microfluidics devices can be designed to mimic physiological conditions and effectively evaluate the deformability of sickle RBCs before and after treatment. This integrated approach not only facilitates the development of personalized therapeutic interventions but also accelerates the translation of novel treatments, such as Voxelotor, from preclinical studies to clinical practice for the management of Sickle Cell Disease. Int. J. Appl. Sci. Biotechnol. Vol 12(1): 46-53.

Is Epigenetic Modulation to Induce Fetal Hemoglobin Translatable in Sickle Cell Mice?

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the β-globin subunit of hemoglobin, resulting in the formation of HbS. Under deoxygenation, HbS polymerizes leading to red blood cell sickling, increased hemolysis and multi-organ damage. During human fetal development, due to expression of the γ-globin gene, the predominant type of hemoglobin produced is fetal hemoglobin (HbF). However, after birth, this shifts to adult globin (HbA or HbS in SCD). This is driven by epigenetic repression of γ-globin due to an increased methylation of its gene promoters. DNA methyltransferase 1 (DNMT1) is an important enzyme for DNA methylation and is known to be pivotal in regulation of HbF expression. Reactivation of HbF provides a functional hemoglobin, and therefore has the potential to treat SCD symptoms ( Steinberg et al. JAMA 2003;289:1645-51). HbF induction is an accepted treatment strategy in SCD; even small increases result in decreased vaso-occlusive crises and mortality ( Kato et al. Nat Rev Dis Primers 2018;4:18010). Decitabine inhibits DNMT1 activity, thus promoting γ-globin expression and thereby increasing HbF levels. While inhibition of DNA methylation is known to cause HbF induction in humans and non-human primates, the effects observed in SCD mouse models are more ambiguous. This study investigated to which extent decitabine treatment could induce HbF expression in the Townes SCD model. In Townes mice, the murine globin genes have been substituted for their sickle human counterparts, HBA, HBB sickle and HBG1, and mimic many features of human SCD. Mice aged 4-5 weeks were dosed subcutaneously 3 times a week for 12 weeks with research-grade decitabine (0.6 mg/kg or 0.4 mg/kg) or vehicle. Blood samples were collected for analysis of HbF protein levels by HPLC (represented as % of total Hb) and F-cells by flow cytometry (represented as % of the total RBC population positive for HbF), complete blood counts, histology, and other markers of disease and organ damage. Results are presented as mean ± SD. Groups were compared using an ANCOVA model, adjusting for multiple comparisons (Dunnett) with p<0.05 considered statistically significant. For both treatment groups, HbF was significantly elevated compared to the control group, with the maximal response observed at 12 weeks (Figure 1). F-cells at the end of the study were increased in both 0.4 mg/kg (11.4±1.5%) and 0.6 mg/kg (11.8±1.4%) groups compared to vehicle (4.5±0.6%). The same was observed for protein levels: 0.4 mg/kg group (1.2±0.3%), 0.6 mg/kg (1.4±0.3%) compared to vehicle (0.4±0.1%). Interestingly, after 12 weeks RBC and reticulocyte counts were significantly decreased in treatment groups and mean corpuscular volume (MCV) was significantly increased. No changes on LDH, bilirubin and liver markers (ALP, ALT, AST) were observed at 6 or 12 weeks. The results showed that treatment groups had significantly higher HbF levels than the vehicle group. However, the level of induction was lower than observed for inhibition of DNA methylation in humans and primates. The decrease in RBCs and reticulocytes could be partially explained by a combination of HbF induction, reduction of sickle RBC, and inhibition of erythropoiesis. The lack of changes in disease markers suggests that the increase in HbF levels in Townes mice might not be sufficient to alleviate the disease. This observation is consistent with recently published data (Woodard et al. Dis Model Mech. 2022;15(6)) that indicates that Townes mice are less suitable for studying this mechanism of action since, even though they have all human globin genes and some proximal regulatory elements, they lack key cis-acting distant DNA elements that regulate the globin genes. However, in primates the globin structure and regulation mechanisms are similar to human (Hardison. Cold Spring Harb Perspect Med. 2012;2(12)). In conclusion, although the measured levels of HbF protein were no higher than 2%, we found that decitabine can induce HbF expression in the Townes mouse model of SCD. However, within the 12-weeks, there was no clear beneficial effect on the disease markers studied. The successful induction of HbF suggests that better outcomes might be achieved with further optimization of the dosing and duration of the study. This indicates that SCD mice may be useful for preclinical studies of HbF induction, but caution is needed when extrapolating results to humans.

S2741 Sickling Manifestations: A Challenging Case of Sickle Cell Hepatopathy

Introduction: Sickle cell hepatopathy (SCH) is an entity describing a pattern of liver injury seen in an estimated ∼10% of patients with sickle cell disease (SCD). It is more prevalent in the Sub-Saharan African region and South-East Asia. This entity is classified according to its clinical presentation into acute and chronic which differs in clinical diagnosis and management. Case Description/Methods: A 51-year-old African American male with SCD presented to our ER with complaint of joint pain and shortness of breath. Patient-reported progressive bilateral lower extremity arthralgia, sharp, 8/10 in severity for 1 week. Reported Dyspnea on exertion. Recurrent sickle cell crises were noted. The patient recently discontinued hydroxyurea because of non-healing cutaneous ulcerations. Vitals marked hypoxia at 87%, otherwise stable. Physical examination revealed icteric sclerae with bilateral lung crackles prominent at the bases. Labs showed hemoglobin at 3.0 g/dl, HB S 28.7% with reticulocyte count at 17.41%. Chemistry panel showed alkaline phosphatase (ALP) at 209 U/L, Total bilirubin at 7.0 mg/dl with direct bilirubin at 4.7 mg/dl. Gamma-glutamyl transpeptidase (GGT) was normal. Chest Xray showed bilateral multifocal infiltrates. Abdominal ultrasound was unremarkable. Computed tomography of the chest showed interspersed ground-glass opacities. Hepatology was consulted for assessment of elevated ALP and Total bilirubin. Clinical suspicion was (SCH). Patient was given multiple blood transfusions. The need for liver transplant (LT) was investigated and deemed not necessary. Pathology was reviewed. Discussion: (SCH) is included in the differential of liver injury, therefore, identifying this entity from other causes of liver disease is important. It is more prevalent in the US now. Hence, an urgent need to find new treatment options in addition to emergent blood transfusion and LT is required. Iron quantification and determining need for chelation therapy is crucial and affects long-term outcomes. LT in SCD patients is controversial due to high risk of post-transplant complications. More studies and international registries are needed to expand our knowledge of the benefits and risks of LT in patients with SCD. We anticipate that (SCH) will become a burden on the health care system in the aging population of SCD patients as they will require more blood transfusions. With a current national shortage supply, we fear future complications will include an increase in mortality rates within this patient population.Figure 1.: a (H&E stain 400x) showing sickled RBC in the sinusoids. b (H&E stain 40x) The lobules show marked sinusoidal dilatation and congestion with elongated RBCs, more pronounced in zone 3 with extravasated RBCs. c (Trichrome stain 200x) Trichrome stain shows diffuse sinusoidal fibrosis, portal, periportal with bridging.

Validation of BCL11A As Therapeutic Target in Sickle Cell Disease: Results from the Adult Cohort of a Pilot/Feasibility Gene Therapy Trial Inducing Sustained Expression of Fetal Hemoglobin Using Post-Transcriptional Gene Silencing

Validation of BCL11A As Therapeutic Target in Sickle Cell Disease: Results from the Adult Cohort of a Pilot/Feasibility Gene Therapy Trial Inducing Sustained Expression of Fetal Hemoglobin Using Post-Transcriptional Gene Silencing

Transfusion Medicine Equations Made Internet Accessible.

Multiple mathematical equations inform the practice of transfusion medicine. These equations apply to a wide range of topics: dosage of blood products, calculation of fluid volumes, and even specific treatment decisions (e.g. corrected count increment for determination of platelet refractoriness). The calculation of these equations can be complicated, prone to error, and time-consuming. A trusted source is needed to accurately perform these calculations 24 hours a day without error and without monetary cost. We sought to build internet-enabled calculators relevant to the practice of transfusion medicine. We partnered with MDCalc, an online host of medical calculators with 1 million monthly users in 196 countries, to design and host the calculators. The calculators guide users in the application of transfusion medicine equations by providing indications for use, inputs for the equations variables, error-checking, warnings for bad inputs, and interpretive guidance of the result. The following calculators were built: blood volume, corrected count increment (CCI), plasma dosage, cryoprecipitated antihemophilic factor dosage, approximate number of units for compatibility testing, maternal-fetal hemorrhage Rh(D) immune globulin dosage, intrauterine RBC transfusion dosage, neonatal polycythemia partial exchange, theoretical removal of a substance by plasmapheresis, sickle cell RBC exchange volume, peripheral blood stem cell collection, and a calculator relevant to donor lymphocyte infusion. Clinicians can now utilize this reputable and highly visible online source to access these common transfusion medicine equations at any time with an internet-enabled device (https://www.mdcalc.com/search?filter=transfusion+medicine).

Rheological Assessments of Sickle Cell Patients Post Allogeneic Hematopoietic Cell Transplant

Introduction: Mixed donor chimerism (MDC) occurs in nearly 35% of patients with sickle cell disease (SCD) post allogeneic hematopoietic cell transplant (alloHCT). Donor myeloid engraftment of >20% is considered necessary to support disease resolution. However, in a disease known for its clinical variability, we hypothesize that patients (pts) with a severe pre-HCT phenotype may have abnormal RBC rheology at a level of chimerism and %HbS believed to be consistent with a cure. RBC rheology is markedly abnormal in SCD; these abnormalities are associated with SCD related clinical complications. Even fully oxygenated, sickle RBC are less deformable than those of HbAA or HbAS individuals; upon deoxygenation, deformability further declines. Sickle RBC morphology reflects the tendency to polymerize, with characteristic sickle forms. Sickle RBC are more adherent to the endothelial cell lining of the vasculature than HbAA or HbAS RBCs. The goal of any cell-based therapy of curative intent should be normalize the red cell rheology. To determine if the rheology of blood from SCD pts post-HCT with varying degrees of donor chimerism fall into the HbSS range of values, we propose to functionally assess the peripheral blood from a series of 6 post alloHCT SCD patients using a battery of rheological tests measuring deformability, sickling, adherence, percent dense cells (%DRBC), and RBC morphology. Methods: Peripheral blood samples (EDTA) from 6 SCD pts post alloHCT with a matched sibling donor were collected and immediately analyzed using oxygenscan (Lorrca), artificial microvascular network (AMVN), microfluidic image acquisition, and an ADVIA hematology analyzer. The Lorrca with oxygenscan measures RBC deformability (elongation index EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which RBC deformability rapidly declines. RBC adhesiveness is measured by the difference in rate of perfusion of RBC through the AVMN coated with adhesive proteins (adherent AVMN) and a non-adherent, uncoated AVMN. %DRBC was measured by an ADVIA hematology analyzer. Microscopic RBC images were acquired and classified to determine the fraction of sickled RBCs in well-oxygenated samples. Additional clinical data including Hb profile, donor chimerism, and symptoms were obtained via chart review. Reference ranges were generated as described above using n=45 HbSS samples ages 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA. Results: In Figure 1, we show the measurements obtained by the Lorrca with oxygenscan on 5 pts post-alloHCT with a range of donor chimerism, as well as typical values for HbAA, HbAS, and HbSS patients. Patient 1 and 3 exhibit normal rheology; both were transplanted with a HbAA donor, with high chimerism and no detectable HbS. Patient 2 has 40% whole blood chimerism, well above the 20% threshold described with myeloid chimerism, and a HbS less than 50%, yet their plot resembles that of an untransplanted HbSS pt; the donor to Patient 2 exhibits the expected HbAA deformability. Patients 5 and 6 exhibit rheology in the HbAS range; they have intermediate chimerism from a HbAS donor. Clinical details and biomarker panel values for all 6 pts analyzed are in Table 1, as well as ranges of values for HbSS and HbAS subjects generated in the Sheehan and Shevkoplyas labs. Patient 2 is the only subject with values in the HbSS range, and the only subject with SCD symptoms. Conclusions: Our rheological tests identified a pt with values consistent with a HbSS pt of moderate severity or treated with hydroxyurea despite variable donor chimerism above the 20% threshold and HbS <50% thought to be sufficient for cure. The pt reported pain events beginning two years prior, indicating a clinical correlation supporting the validity of the rheological tests we propose to use to distinguish cure from persistent SCD. Current post-HCT evaluation depends on chimerism and hemoglobin profiles, and would not detect the significant functional abnormalities visible by Lorrca with oxygenscan biomarkers that indicate that Patient 2 is at risk for SCD related complications. Our results suggest that this functional analysis may help with management of post alloHCT SCD pts, and may be even more essential to assessing new gene-based therapy approaches to curing patients with SCD. Disclosures Rab: RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. Shevkoplyas:SSS: Research Funding; New Health Sciences: Consultancy.

The Gut Microbiome Regulates Psychological Stress-Induced Inflammation in Sickle Cell Disease

Psychological stress (PS) is reported to precipitate vaso-occlusive episides (VOE) in sickle cell disease (SCD), but the mechanisms by which it does, are largely unknown. We subjected humanized (Berkeley) SCD mice to restraint stress and analyzed VOE using intravital microscopy. We found that stress exacerbated VOE as shown by reduced venular blood flow rates (control: 494 ± 30 × 103 pL/s; stress: 380 ± 20 × 103 pL/s; n = 25 - 43; p = 0.009) and reduced survival time (median survival time in control and stress groups: 394 min and 175 min, respectively; p = 0.02, Log-rank test). As previous studies show that adherent polymorphonuclear neutrophils (PMNs), especially aged PMNs marked by low CD62L and high CXCR4 expression, contribute to VOEs, we evaluated the behavior of leukocytes in live mice and observed a robust PMN response manifested by significantly increased adhesion and interactions with sickle RBC in stressed SCD animals. Flow cytometry analysis revealed a striking increase in aged PMNs in stressed SCD mice (control: 0.7 ± 0.1 × 106/ml; stress: 2.9 ± 0.3 × 106/ml; n = 3 - 4; p = 0.001), suggesting that the aged PMN expansion induced by stress may lead to the exacerbation of VOE in stressed SCD mice. Since aged PMN numbers are regulated by microbiota under steady state, we investigated whether response to PS also required the presence of the microbiota. Depletion of microbiota with broad-spectrum antibiotics (ABX) in SCD mice significantly prevented stress-induced aged PMN expansion (control: 4.8 ± 1.2 × 106/ml; ABX: 1.2 ± 0.3× 106/ml; n = 4 - 5; p = 0.0006), which was further confirmed using C57BL/6 germ-free (GF) mice (control: 1.1 ± 0.3 × 106/ml; GF: 0.2 ± 0.1 × 106/ml; n = 4; p = 0.003). Interestingly, analysis of inflammatory cytokines revealed a microbiota-dependent augmentation of IL-17A in the stressed mice. To investigate the role of IL-17A in the stress response, we neutralized IL-17A using an antibody (Ab) in C57BL/6 mice and found that IL-17A inhibition significantly dampened the aged PMN expansion induced by stress (isotype control Ab: 0.6 ± 0.1 × 106/ml; anti-IL-17A: 0.1 ± 0.0 × 106/ml; n = 4 - 5; p = 0.0004), which was also confirmed using IL-17A-/- mice. Further analyses revealed that Th17 lymphocytes were the major IL-17A-producing cells contributing to the stress response. In addition, depletion of microbiota with ABX or neutralization of IL-17A significantly alleviated stress-exacerbated VOE as measured by reduced PMN activation, improved blood flow rate and prolonged SCD mouse survival. As segmented filamentous bacteria (SFB) commensals were previously found to induce a Th17 response, we next tested the hypothesis that SFB was the driving bacteria in the stress response. Indeed, we found that PS-induced aged PMN expansion was greater in SFB+ mice (from Taconic) than in SFB- mice (from Jackson lab) (Taconic: 0.64 ± 0.11× 106/ml; Jax: 0.13 ± 0.02 × 106/ml; n = 4 - 5; p = 0.005). Depletion of SFB using vancomycin, a drug that eliminates Gram+ bacteria (including SFB), significantly reduced stress-induced aged PMN expansion. Furthermore, colonization of GF mice with SFB augmented aged PMN expansion after stress compared to stress control GF mice (control GF: 0.08 ± 0.02 × 106/ml; GF + SFB: 0.34 ± 0.10× 106/ml; n = 4 - 5; p = 0.04), suggesting that SFB was the key bacteria promoting aged PMN expansion during stress. The stress mechanisms involved the hypothalamic-pituitary-adrenal (HPA) axis since the blockade of glucocorticoid (GC) production using metyrapone or following adrenalectomy significantly prevented aged PMN expansion in stressed mice (PBS: 0.5 ± 0.1 × 106/ml; metyrapone: 0.1 ± 0.1× 106/ml; n = 5; p = 0.02. sham: 1.2 ± 0.2 × 106/ml; adrenalectomy: 0.4 ± 0.2 × 106/ml; n = 4 - 5; p = 0.01). Moreover, PS and dexamethasone greatly increased the gut permeability. Blockade of GC synthesis using metyrapone significantly alleviated stress-induced exacerbation of VOE manifested by reduced inflammation, improved the blood flow rate and prolonged SCD mice survival (median survival time in control and metyrapone groups: 155 min and 200 min, respectively; p = 0.02, Log-rank test). Overall, our results suggest that stress activation of the HPA axis increases the intestinal permeability, thereby promoting exposure to commensals that stimulate Th17 cells to produce IL-17A, which enables the expansion of aged polymorphonuclear neutrophils driving vaso-occlusion in SCD. Disclosures Frenette: Albert Einstein College of Medicine, Inc: Patents & Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.

MetAP2 Inhibition Modifies Hemoglobin S (HbS) to Delay Polymerization and Improve Blood Flow in Sickle Cell Disease

Dilution of HbS with non-sickling hemoglobin or hemoglobin with increased oxygen affinity is clinically beneficial in sickle cell disease. Aldehydes, including 5-HMF, tucaresol or GBT440, modify the N-terminus of HbS by reversible covalent imine formation generating modified forms of HbS that resist polymerization under low oxygen concentrations. In contrast to reversible imine formation by aldehydes, we hypothesize that stable modification of HbS will result from N-terminal retention of the initiator methionine (iMet) and subsequent N-terminal acetylation of the iMet (acetyl-iMet). MetAP2 is the methionine aminopeptidase able to cleave iMet from Val1 on α-globin and βS-globin as the unfolded N-terminal peptides emerge from the ribosome. Enzyme kinetic studies with pure MetAP2 and N-terminal octapeptides showed that βS-globin peptide is a 5-fold better substrate than α-globin peptide. Lentiviral shRNA knock-down of MetAP2 in differentiating erythroid HUDEP cells in vitro confirmed that α-globin is more extensively modified than βS-globin, consistent with the enzyme kinetic data. Selective MetAP2 inhibitors used to treat cultured human erythroid cells (HUDEP and PBMC derived CD34+) and Townes SCD mice in vivo confirmed that both α-globin and βS-globin domains of HbS are extensively modified by N-terminal iMet and acetyl-iMet. N-terminal retention of iMet and subsequent acetylation creates a mixture of modified HbS tetramers with combined modifications on both globins. Cation exchange chromatography separated nine different modified HbS variants from unmodified HbS as identified by LCMS. Purified samples of HbS modified by N-terminal iMet and acetyl-iMet had increased oxygen affinity as measured by decreased P50. Modified HbS containing the acetyl-iMet-βS-globin were found to have delayed polymerization under complete hypoxia (sodium metabisulfite triggered hypoxia in 1.8 M phosphate). Two modified HbS variants were further purified for X-ray crystallography studies (βS-globin / iMet-α-globin and acetyl-iMet-βS-globin / iMet-α-globin). Oxyhemoglobin structures of both modified HbS variants were in the R2-state previously described in structures of aldehyde modified HbS. This R2-state stabilizes the oxygenated R-state of HbS from conversion to the deoxygenated T-state that initiates HbS polymerization in sickle RBC. Treatment by selective irreversible covalent or reversible MetAP2 inhibitors resulted in high levels of HbS modification (>75%) in cultured erythroid cells (HUDEP and CD34+ cells). Dose dependent modification of HbS was observed in Townes sickle cell mouse blood RBC in vivo with total modification of HbS approaching 50%. In whole blood ex vivo studies, modification of HbS decreased RBC sickling under hypoxia (4% O2) and significantly increased the affinity of RBC for oxygen (decreased P50). Blood samples from MetAP2 inhibitor treated mice were analyzed for single-cell O2 saturation of the RBC and for the fractional flow velocity drop in whole blood rheology under decreasing partial oxygen pressures. In blood from vehicle treated sickle mice, a low-saturation peak of deoxy-HbS was observed in 7.8% O2, in contrast to blood from MetAP2 inhibitor-treated mice where the low-saturation peak was only observed in 6.4% O2. Similarly, in an assay of O2 dependent blood flow rheology, the half-maximum fractional velocity drop occurred at 5% O2 in control blood decreasing to 2% O2 in MetAP2 inhibitor treated blood. Our studies show that MetAP2 inhibition results in retention of iMet on βS-globin and α-globin and allows further acetylation of the retained iMet to create a mixture of N-terminal modified HbS tetramers. These modified HbS variants resist polymerization and RBC sickling under conditions of low O2 by delaying HbS polymerization and increasing O2 affinity. Our data suggests that MetAP2 may warrant further study as a potential therapeutic target for sickle cell disease. Disclosures Demers: Sanofi: Employment. Sturtevant:Sanofi: Employment. Guertin:Sanofi: Employment. Gupta:Sanofi: Employment. Desai:Sanofi: Employment. Vieira:Sanofi: Employment. Hicks:Sanofi: Employment. Ismail:Sanofi: Employment. Safo:Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Wood:Sanofi: Consultancy, Research Funding. Higgins:Sanofi: Consultancy, Research Funding. Light:Sanofi: Employment.

Hemodynamics of Capillary Networks in Sickle Cell Disease

ObjectiveSickle cell disease (SCD) is a complex hereditary blood disorder that can significantly affect the rheological properties of patients. Researches on the change of the rheological properties of blood and hemodynamics in a single capillary vessel have been carried out in recent three decades. The target of this work is to explore the hemodynamic behavior of diseased capillary networks based on anatomical data, in order to provide advice on the generation and prevention of the vaso‐occlusive crisis of patients with sickle cell disease.MethodIn this work, we use numerical simulations to calculate the sickle RBC velocity, blood hematocrit and blood viscosity in capillary bifurcations and networks based on mesenteric anatomical data. Then, the model predictions are discussed with reported clinical manifestations and compared with normal networks.ResultsThe predictions from our simulations show agreement with clinical manifestations. Compared with the properties of normal capillary networks, the blood flow in diseased micro vessel networks has 10%–25% larger hematocrit, slower RBC velocity and larger blood viscosity, which can lead to the vaso‐occlusive crisis and the pain of patients with sickle cell disease.ConclusionsThis study helps us to understand the blood flow properties of capillary networks of patients with sickle cell disease, and may help us to prevent the vaso‐occlusive crisis. To improve our study, we can use capillary networks from the diseased part of sickle cell patients instead.Support or Funding InformationThis research is supported by Shenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Patrolling monocytes in sickle cell hemolytic conditions

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis associated with vascular injury and dysfunction. Painful vaso-occlusive crisis (VOC) involving increased attachment of sickle erythrocytes and activated leukocytes to damaged vascular endothelium is a hallmark of SCD. Patrolling monocytes, which normally scavenge damaged cells and debris from the vasculature, express higher levels of anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme with anti-cytotoxic and anti-inflammatory properties. Recent data show that patients with SCD have a novel subset of patrolling monocytes expressing very high levels of HO-1 (HO-1hi) which are decreased in numbers in patients who had a recent VOC episode. This patrolling monocyte subset was responsible for protection of endothelium against sickle RBC stasis in an experimental model. This raises the possibility that patrolling monocytes may also offer protection against vascular endothelium damage in hyperhemolytic conditions in SCD.

Adaptation of a microbead assay for the easy evaluation of traditional anti-sickling medicines: application to DREPANOSTAT and FACA

Context: Sickle cell disease is a common inherited blood disorder affecting millions of people worldwide. Due to lack of progress in drug discovery for a suitable treatment, sufferers often turn to traditional medicines that take advantage of the plant extracts activity used by traditional healers.Objective: This study optimizes an anti-sickling screening test to identify preparations capable of reverting sickle cells back to the morphology of normal red blood cells. We focused on the miniaturization and practicability of the assay, so that it can be adapted to the laboratory conditions commonly found in less developed countries.Materials and methods: We tested two traditional anti-sickling herbal medicines, FACA® and DREPANOSTAT®, composed of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Rutaceae) and Calotropis procera (Aiton) Dryand. (Apocynaceae) at screening concentrations of hydroethanol extracts from 0.2 to 1 mg/mL. Potential bioactive molecules present in the extracts were profiled using Ultra High Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UHPLC-HRMS/MS) method, identified through HRMS, MS/MS spectra and in silico fragmentation tools.Results: Hydroethanol extracts of FACA® and DREPANOSTAT® showed low anti-sickling activity, inhibiting less than 10% of the sickling process. The UHPLC-HRMS/MS profiles identified 28 compounds (18 in FACA® and 15 in DREPANOSTAT®, including common compounds) among which l-phenylalanine is already described as potential anti-sickling agent. When used as positive control, 7 mg/mL phenylalanine reduced the sickled RBC to 52%.Discussion and conclusions: This assay has been optimized for the easy screening of plant extracts or extracted compounds from bioassay guided fractionation, valuable to laboratories from less developed countries.

Protective Role of HO-1 Expressing CD16+ Patrolling Monocytes Against Hemolysis-Induced Endothelial Damage and Vaso-Occlusive Crisis in Sickle Cell Disease

Causing leukocyte activation and upregulation of adhesion molecules on endothelial cells. CD16+ monocytes, also known as endothelial patrolling monocytes, normally scavenge the damaged cells and debris from the vasculature. As compared to other monocyte subsets or immune cell types, the CD16+monocyte subset expresses higher levels of the anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme. Given the role of CD16+ monocytes as scavengers of debris on endothelial cells, we tested the hypothesis that this subset may protect SCD vasculature from the ongoing hemolytic insult through expression of high levels of HO-1. We found roughly 35% of circulating CD16+ monocytes from SCD patients expressed very high levels of HO-1 as compared to 5% in healthy controls. The HO-1hi SCD monocytes expressed significantly (30%) less TNF-a compared to HO-1lo monocytes following stimulation, consistent with anti-inflammatory effects of HO-1. We hypothesized that uptake of free hemoglobin/heme was responsible for high HO-1 expression levels in SCD CD16+ monocytes. To test this, healthy donors (HDs) or SCD patient monocytes were treated with different doses of free heme or hemolysed RBCs. We found dose-dependent HO-1 induction (five-fold at 20mM heme) in purified CD16- monocytes, but surprisingly none in CD16+ subset. However, upon co-culture with human umbilical vein endothelial cells (HUVEC), continuous or prior exposure to heme induced HO-1hi expression exclusively in CD16+ monocytes (5 fold in HD and further two fold in SCD compared to non-heme treated cocultures, p<0.001). Using imagining flow cytometric analysis, we found marked increase in uptake of heme-exposed endothelial cell-derived material by CD16+ monocytes (HD: 2% to 13% ± 3%; in SCD: 20% ± 3% to 30% ± 4%, p< 0.001) but none by CD16- monocytes. Our transwell studies demonstrated that cell-cell contact between CD16+ monocytes and heme-exposed HUVEC was required for HO-1hi expression. We found roughly 4-fold increase in expression of phosphatidylserine (PS, annexin V+), ICAM-1 and vCAM-1 on heme-treated HUVEC cells. Antibody blocking studies identified PS moieties as well as ICAM-1 as key molecules involved in monocyte-HUVEC interactions that mediated HO-1hi induction, suggesting that high levels of HO-1 expression in SCD CD16+ monocytes is in part the result of attachment to and engulfment of apoptotic, activated endothelial cells damaged by heme. SCD patients suffer from vaso-occlusive crisis (VOC), resulting from increased attachment of SCD RBCs to damaged and activated endothelium. We hypothesized that inadequate numbers or lower HO-1hi levels in CD16+ monocyte will predispose SCD patients to episodes of VOC due to decreased removal by CD16+ monocytes of damaged endothelial and sickle RBCs. Amongst SCD patients receiving chronic transfusions, we found a two-fold lower frequency of circulating CD16+ monocytes and half the numbers of CD16+HO-1hi monocytes in patients with a recent history of VOC episode as compared to those without VOC (p< 0.01); the former group also expressed higher levels of circulating sVCAM-1 (997 ± 210 vs 765 ± 236 ng/m, p=0.02), a marker of endothelial activation. To formally test the role of patrolling monocytes in endothelial damage induced by SCD RBCs and heme, we injected RBCs from Townes SCD mice alone or after 24hrs with heme into Nr4a1-knockout mice which have a selective loss of patrolling monocytes. Immunofluorescence analysis of liver vasculature showed a 3 fold increase in the activated endothelial marker, ICAM-1 within 24hrs following injection of sickle RBCs and two-fold increase in circulating sVCAM-1 levels in mice treated with sickle RBC plus heme (p <0.001). Transfer of HO-1+ patrolling monocytes (LY6Clo), but not a classical monocyte subset (LY6C+) reversed activated endothelial phenotype, indicating that patrolling monocytes can inhibit SCD-induced endothelial activation. Altogether, these data suggest that SCD patrolling monocytes remove hemolysis-damaged endothelial cells, resulting in HO-1 upregulation and dampening of vascular inflammation. Perturbations in CD16+ monocyte numbers resulting in lower local HO-1 levels can predispose SCD patients to VOC, thus identifying HO-1+ patrolling monocytes as key players in VOC pathophysiology and as therapeutic targets. DisclosuresNo relevant conflicts of interest to declare.

Anti-Thrombin III Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to the Endothelium in a Sickle Cell Mouse Model: Potential Therapeutic Application for Vaso-Occlusive Episodes

Painful vaso-occlusive episodes are a hallmark of sickle cell disease (SCD), an inherited blood disorder caused by a point mutation in the β-globin gene. Vaso-occlusive events account for the majority of SCD-related hospital admissions and contribute importantly to major organ damage and decreased survival. Therapies to prevent and treat acute vaso-occlusive episodes are limited and there is a need for new treatment options. Adhesive interactions between circulating sickle red blood cells (sRBC), leukocytes, and endothelial cells have been implicated as critical pathologic events for the development of vaso-occlusive episodes. These cell adhesive interactions further manifest with increased local and systemic inflammation and activated coagulation evidenced in patients and mouse models of SCD.Anti-thrombin III (ATIII) is an endogenous inhibitor of thrombin and other serine proteases in the coagulation pathway. Apart from the role in hemostasis, ATIII exerts strong anti-inflammatory properties. Plasma levels of ATIII and ATIII activity are decreased in SCD and further decline during vaso-occlusive crisis. These findings indicate that ATIII deficiency may contribute by multiple mechanisms to the pathophysiology of acute vaso-occlusion and that repletion therapy with ATIII may provide a novel, multi-targeted therapeutic approach to this difficult-to-treat complication.In this study, we examined whether ATIII supplementation is able to reduce adhesion of sRBC and leukocytes to vascular endothelial cells in the calvarial bone marrow microvasculature in a mouse knockout-transgenic mouse model of SCD (Ryan, 1997). Homozygous SCD mice were injected intravenously with 230 U/kg ATIII (Thrombate III; Grifols, USA) or vehicle (saline). After 48 hours, the mice were subjected to 3 hours of hypoxia (8% O2 in medical air) followed by 3 hours of reoxygenation at room air (21% O2). Intravital microscopy analysis of the bone marrow microcirculation was performed to monitor adhesive interactions between blood cells and vascular endothelium. Leukocytes were labeled in vivo with phycoerythrin-conjugated anti-CD45 antibody which was infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery to the recipient mice. Effects of ATIII on sRBC and leukocyte adhesive interactions were compared to vehicle. Administration of ATIII to SCD mice resulted in more than 5 fold decrease in sRBC adhesion (p<0.01) compared to vehicle. Next the effects of ATIII on leukocyte flow dynamics were analyzed quantitatively. Mice pretreated with ATIII, but not those treated with vehicle, showed two-fold decrease in leukocyte rolling flux (p<0.01) and a 3-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Furthermore, ATIII improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and calculated wall shear rates. The pretreatment with ATIII was associated with decreased plasma levels of IL-1β and a trend to decreased soluble levels of vascular cell adhesion molecule 1 and thrombin-antithrombin complexes.In conclusion, our study demonstrates significant anti-adhesive activities of ATIII, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in a mouse model of SCD. ATIII may represent a novel and effective therapeutic intervention in the treatment of acute vaso-occlusive crisis in patients with SCD. DisclosuresGutsaeva:Grifols: Research Funding.

Increased Oxidative Stress In Sickle Cell Disease Activates The Renin-Angiotensin-TGF-β Pathway To Mediate Sickle Nephropathy

Sickle nephropathy (SN) is a common cause of morbidity and mortality in sickle cell disease (SCD), begins with hyposthenuria in childhood, and progresses to albuminuria, focal sclerosing glomerulo-sclerosis (FSGS), glomerular hypofiltration, and end stage renal disease in 30-50% adults The precise molecular mechanisms underlying SN are largely unexplored, as SN has been presumed to result from sickling associated ischemia/necrosis. Herein, we explored mechanisms of sickle renal pathologies utilizing the Berkeley sickle mouse model (HbS mice). We show that HbS mice develop renal pathologies similar to human SN, with hyposthenuria, progressive albuminuria, FSGS and nephron loss. HbS mice 4-8 weeks of age have high GFR compared to WT mice, that rapidly declines to subnormal levels by 16-24 weeks of age. We next explored the role of increased oxidant stress in mediating SN. We recently showed that sickle RBC are likely major contributors of reactive oxygen species (ROS) in SCD, and these high levels of ROS in RBC are also generated enzymatically by NADPH oxidase (George, et al Blood 2013). We now show that SCD-associated ROS initiate pathologically significant processes, including increased conversion of oxidized angiotensinogen (ANG) to angiotensin II (AT), and secondary AT receptor 1 (AT1R)-mediated generation of TGFβ1 in the HbS kidneys, which then phosphorylates Smad 2/3. We tested if activated Renin-Angiotensin-system (RAS) -AT1R-mediated TGFβ1 signaling causes albuminuria and FSGS in HbS mice. We blocked the AT1R with losartan, or its ligand AT by an angiotensin converting enzyme inhibitor, Captopril, starting at an early age (4wk) for 6-12 months. This prevented albuminuria and FSGN development in HbS mice. However, sickle hyposthenuria was worsened with losartan, and was even more severe with captopril. These data suggest that excessive AT1R signaling causes sickle glomerulopathy, and AT1R promotes urine concentrating ability; however, the captopril effect suggests that AT binds another receptor to further mediate urine concentrating ability. Increased RAS signaling is known to mediate glomerulopathy in other diseases, but its role in urine concentration has not been described. AT can also bind AT2 receptor that has been identified as a renoprotective receptor. We therefore investigated the role of AT1R and AT2R in sickle glomerulopathy and hyposthenuria by transplanting bone marrow from HbS mice into WT mice, AT1R-/- mice (HbS/AT1R-/-) and AT2R-/- mice (HbS/AT2R-/-) and followed them for 6-12 months. Bone marrow from WT mice was concurrently transplanted into WT, AT1R and AT2R deficient mice as controls. HbS/WT mice developed similar SN as in HbS mice with progressive albuminuria and hyposthenuria, the former reversible with losartan and captopril, and the latter worsened by these drugs as described above. However, HbS/AT1R mice were protected from development of albuminuria and FSGS, had reduced active TGFβ1 and PSmad-2/3, unlike HbS/WT mice, but developed significant hyposthenuria, which was worse than HbS/WT mice, and reminiscent of the effect of losartan. The HbS/AT2R mice also developed significantly worse hyposthenuria than HbS/WT mice, and were additionally not protected from albuminuria. These data suggest both AT1R and AT2R mediate urine concentrating ability, an effect blocked more effectively by captopril than losartan. AT1R signaling is known to activate NADPH oxidase to generate ROS. Indeed, mice placed on Captopril and Losartan had reduced ROS in RBC and platelets (cell types known to express AT1R) and kidneys, and consequently reduced RAS activation (significantly less oxidized ANG and AT), breaking the ROS-RAS-AT1R feedback loop. Significantly higher RBC and platelet ROS, oxidized ANG, and AT levels were also confirmed in patients with SCD as compared to their unaffected sibling controls. In summary, our data show that SN occurs from two distinct mechanisms – a) glomerulopathy that results in albuminuria, glomerulosclerosis and renal failure, which occurs primarily from increased AT1R signaling, and b) a tubulopathy, that results in inability to concentrate urine, and is worsened by AT1R signaling blockade, and AT2R signaling protects tubules against worsening hyposthenuria. Targeted therapies that block AT1R signaling but increase AT2R signaling may improve both glomerular and tubular pathologies in SCD and can now be explored. Disclosures:No relevant conflicts of interest to declare.

How Does CD47-SIRPα ‘Don't Eat Me Signal' Physically Signal Self

Resident macrophages in spleen and liver are particularly adept at recognizing foreign pathogens through recognition of ‘non-self' proteins on the pathogen surface but also through the absence of ‘self' proteins that are highly displayed on circulating blood cells. Red blood cells display a ‘marker of self' protein CD47 which increases the in vivo half-life and decreases red-pulp splenic macrophage uptake of mouse RBC (Oldenborg et al, Science 2000) and also of particles displaying human-CD47 in recent studies by our group (Rodriguez et al, Science 2013). CD47 signals self through its counter receptor SIRPa, which is highly expressed on the surfaces of myeloid cells but also highly polymorphic. The CD47 protein functions in vitro as a marker of self toward human SIRPa on human macrophages and monocytes, inhibiting accumulation of myosin II motor protein to the phagocytic synapse (Tsai 2008). The work here aims to clarify when and how CD47-SIRPa inhibition physically signals ‘self' during macrophage phagocytosis uptake. While it is clear that CD47 reduces the number of uptake events, here we use time-lapse and confocal microscopy to examine the forces of distortion imparted by phagocytes on opsonized red blood cell targets during uptake. Glutaraldehyde-fixed RBC are also used as a model to assess the affects of cell rigidity in this self-recognition process, since rigidity is relevant to processes as diverse as RBC aging and sickle RBC to malaria but also because adhesion (by macrophages) is expected to activate the myosin-II contractility system and oppose CD47 signaling. Through blocking and pharmacological approaches, we parse the pathways between foreign, self, and rigidity sensing. Disclosures:No relevant conflicts of interest to declare.

Proteomic Analysis of ERK1/2-Mediated Human Sickle Red Blood Cell Membrane Protein Phosphorylation.

Abstract 2120In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human erythrocytes. ERK1/2 is involved in activation of ICAM-4-mediated sickle red blood cell (SSRBC) adhesion to the endothelium. However, other effects of the ERK1/2 activation in SSRBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are still unknown. To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2 activation, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Consistent with other RBC membrane phosphorylation studies, the phosphoproteins of SSRBC membrane ghosts with the highest number of uniquely phosphorylated peptides (>10), were ankyrin-1 of the ankyrin complex (n=33), spectrin β chain of the cytoskeleton network (n=15), and proteins of the junctional complex involved in binding integral membrane proteins to cytoskeletal proteins, including α- and β-adducins (n=22 and n=18, respectively), dematin (n=16) and protein 4.1 (n=17). In addition, several other phosphoproteins with >5 unique phosphorylated peptides, affecting RBC shape, flexibility, anion transport and protein trafficking, and adhesion, all of which contribute to the pathophysiology of SCD, were also observed. However, the MEK1/2 inhibitor U0126 was able to significantly down-regulate 37 unique RBC membrane phosphopeptides (from 21 unique phosphoproteins) in SSRBCs. We found that MEK1/2-dependent ERK1/2 activation in SSRBCs affected membrane-bound proteomes of both the junctional and ankyrin complexes, including dematin, α-adducin, β-adducin with phosphorylation of residues within the ERK1/2 consensus motif, and glycophorin A. MEK1/2/ERK1/2 signaling in SSRBCs induced changes within the actins/spectrins network as well by affecting phosphorylation of β-spectrins. Furthermore, the peptide metabotropic glutamate receptor 7 (mGlu7) also underwent serine phosphorylation at the ERK consensus motif. This could explain the rate of active glutamate transport in these cells. Significant changes only in membrane ghosts prepared from SSRBCs treated with U0126 or after addition of exogenous active ERK2 to these membrane ghosts, were also observed in the status of leucine-rich repeats and immunoglobulin-like domains protein 2, leucine-zipper-like transcriptional regulator 1, glucose transporter 1, and adenylyl cyclase-associated protein 1 (CAP1), which may potentially disturb degradation of misfolded glycoproteins and receptor ubiquitination, protein transcription, glucose transport and cAMP production, respectively. These data also suggest that a negative regulatory mechanism might exist in normal cells to prevent activation of ERK1/2-dependent phosphorylation of these membrane proteins. Among all these phosphorylated proteomes, glycophorin A was the most affected protein in SSRBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreased in both anion transport by band 3 and band 3 trafficking. The abundance of thirteen of the thirty-seven phosphopeptides was subsequently increased in normal RBCs co-incubated with recombinant ERK2, and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2. These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD. Disclosures:No relevant conflicts of interest to declare.

Elevated Reactive Oxygen Species Production In Sickle Erythrocytes Is Modulated by a Pathway Involving Endothelin-1, TGFβ1, PKC, and Rac GTPases

AbstractAbstract 1634Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease. One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle erythrocytes. Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. The potential role of enzymatic mechanisms in ROS production in RBCs has not been fully explored. One candidate for enzyme-mediated ROS production in SS RBCs is NADPH oxidase, which is activated by the small Rho GTPases Rac1 and Rac2 in a variety of cell types (Hordijk P.L. Circ. Res. 2006; 98:453-462).Using flow cytometry with 5-(and 6-)-chloromethyl-2‘,7‘-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe, we determined that ROS generation is elevated in HbSS RBCs by 150–250% relative to that in HbAA RBCs. The NADPH oxidase NOX subunit homologs NOX1 and NOX5 were expressed in erythrocytes, and treatment of SS RBCs with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) reduced ROS generation in a dose-dependent manner. Inhibition of PKC or Rac activity by the small molecule-inhibitors calphostin and NSC23766, respectively, also resulted in decreased ROS production in HbSS-RBCs in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production. This effect of PMA could be inhibited by parallel treatment with NSC23766, implicating a PKC-Rac axis in erythrocyte ROS production. Enzymatic ROS production in sickle RBCs was dependent on the availability of free intracellular calcium, since it was inhibited by BAPTA-AM, a cell-permeable calcium chelator. Moreover, we found that ROS generation in SS RBCs was modulated by humoral factors. Incubation of AA RBCs in blood type-matched SS patient plasma resulted in increased ROS generation, while the incubation of SS RBCs in AA plasma decreased ROS production. Immunoblotting of AA and SS RBC membranes with specific antibodies revealed receptors for the inflammatory signaling molecules TGFβ1 and Endothelin-1 (ET-1), both of which are present in elevated levels in the plasma of patients with SCD. Incubation of AA RBCs with TGFβ1 or ET-1 resulted in increased Rac activation and increased ROS production in these treated cells.Our results suggest that ROS production in sickle RBCs is mediated by NADPH oxidase through Ca2+-regulated PKC and Rac signals, which in turn are modulated by plasma TGFβ1 and ET-1 via their receptors. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte deformation and fragility in sickle cell disease. Erythrocyte ROS generation, RBC lysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive feedback loop to drive the pathophysiology of sickle cell disease. These data may offer new therapeutic targets to counteract inflammation and RBC fragility and deformation in sickle cell disease. Disclosures:No relevant conflicts of interest to declare.

Apoliprotein A-I Mimetic Peptide and Sickle Vasculopathy: Mouse Model Study of Acute Administration.

Abstract 1521Poster Board I-544Epidemiologic observations associate low levels of Apolipoprotein A-1 (ApoA-I) and low high density lipoprotein (HDL) with greater prevalence of pulmonary hypertension in sickle cell patients. Sickle cell mice with knockout of apoliproteins have greater pulmonary hypertension and endothelial dysfunction than sickle cell mice with wild-type levels of ApoA-I, and they have further worsening of the global dysregulation of the nitric oxide axis (NO) seen in sickle vasculopathy. Ou et al. (Circulation 2003; 107(18):2337-41) showed that chronic administration (3-4 weeks) of the ApoA-I mimetic L-4F improves vasodilation in hypercholesterolemic and sickle mice. Remaley et al. (Circulation 2006;114:II_23) showed atheroprotective effects after a single (1mg/mouse) intravenous administration of the ApoA-I mimetic peptide 5A as a result of increased reverse cholesterol transfer. These preclinical data provide a mechanistic basis for the effects of ApoA-I in sickle cell disease and suggest that ApoA-I mimetics deserve further study as a potential therapy for sickle cell disease. However, it is unknown whether acute administration of an ApoA-I mimetic has any efficacy for sickle cell disease, and whether all ApoA-I mimetic peptides will be helpful. We hypothesized that acute administration of ApoA-I mimetic could improve endothelial dysfunction in sickle cell mouse models. Bone marrow harvested from Berkeley sickle cell mouse donors and hemizygote non-sickling control mice were used to generate three groups of mice after myeloablative irradiation of wild-type C57BL6 recipient mice. Three million cells whole marrow was injected per mouse. Mice were fully engrafted by 3 months. Hemoglobin analysis showed one group produced only sickle RBC, another group only hemizygote RBC, and a third group were mixed chimeras with 30% sickle RBC and 70% hemizygote RBC (intended to model a sickle cell patient on chronic transfusion). Pulse-wave velocities, a measure of arterial stiffness, showed no improvement after IV injection of the ApoA-I mimetic peptide, 5A-POPC (170 mcl of 8.6 mg/ml). Aortic rings prepared from mice of all three groups showed blunted relaxation response to acetylcholine and sodium nitroprusside, as expected for abnormal nitric oxide bioavailability - these vasorelaxation responses were not significantly different in the absence vs. presence of 5A-POPC incubation in vitro. These results agree with our previous observation that sickle bone marrow transplanted into transgenic mice overexpressing ApoA-I had no difference in vasculopathy from sickle bone marrow transplanted into wild-type mice We conclude that acute exposure to this ApoA-I mimetic, either in vivo or in vitro, is not sufficient to relieve the vasculopathy of sickle cell mouse models. Higher doses or long term therapy may be necessary for benefit. An alternative explanation is that ApoA-I mimetic peptides differ in their benefit for sickle cell vasculopathy, and that the key property is the high level of oxidant scavenging by L-4F rather than cholesterol removal by 5A-POPC. Future studies can determine whether the cardiovascular benefits of ApoA-I mimetics correlate with their antioxidant function. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic and Genetic Discordance in Monozygotic Twins with Sickle Anemia and ð-Thalassemia.

Abstract 5084 IntroductionDiscordant monozygotic twins with sickle cell anemia (SCA) are rare. In the present study we describe current clinical and laboratory data in a set of 49 year old discordant monozygotic twins with coexistent SCA and α-/αα thalassemia. We compare the current findings to their previously described data (Hemoglobin 1991;15:247-56). MethodsEvaluations of painful episode frequency, episode severity, frequency of the acute chest syndrome, standard blood counts and chemistries, hemolysis, pulmonary hypertension, irreversibly sickled cells (ISC) and RBC rheology were obtained. DNA from peripheral blood was genotyped using Illumina 610K SNP (single nucleotide polymorphism) beadchip allowing analysis of SNP genotypes and DNA copy number variations (CNVs). ResultsTwin I is right handed and Twin II is left handed. Frequency of hospitalized painful episodes examined over 34 years were similar (approximately 1 episode per year) and were asynchronous. Prior to approximately 25-27 years of age, the painful episodes in Twin I were moderately severe with decreasing severity as ageing occurred. In contrast, Twin II, who also had moderately severe painful episodes during early years, showed marked worsening of severity as ageing occurred. Episodes of acute chest syndrome were asynchronous with similar frequency (Twin I had 2 episodes in 18 years; Twin II had 2 episodes in 21 years). Systemic blood pressures were similar. Osteonecrosis, leg ulcers, priapism, stroke were absent.Laboratory results (Current)*Twin ITwin IIHb(gm/dl)9.238.76Reticulocytes (%)9.111.4Fetal Hb (%)5.04.5Plasma Hb(uM) (Normal <2uM)3.727.79Total GPA(+)CD71, RBC-derived vesicles x 105/l blood2.986.32Total Bilirubin(mg/dl)5.356.83- Indirect Bilirubin (mg/dl)4.635.7RBC survival(51Cr T1/2 days)(Hemoglobin1991;15:247-56)1312.3Irreversibly sickled cells(%)24.417.5Plasma nitrite (nM)(Normal 114 ± 11)84.52100.04Tricuspid regurgitant velocity(m/sec)2.512.51Rheology- Extent of RBC aggregation (Normal 16.3 ± 0.34)9.8811.58- Strength of RBC aggregation (Normal 78 ± 3.6)49134- T1/2 time for RBC aggregation (Normal 2.8 ± 0.09)4.755.33- RBC deformabilityIdentical maximum deformation indexCT scanGallstones, Aortic aneurysmGallstonesLDH-T1(u/l)254248LDH-1(%)3435Blood group phenotype, Chance of monozygosity(%)99.4005399.005*Levels of Hb, Reticulocytes, Hb F, Total and Indirect bilirubin are the Means of 3 samplesConcordance of SNP genotypes confirmed that the twins are monozygotic. Analysis of the entire genome for CNVs revealed a number of candidate loci that differ. Most differences in CNVs occurred in Twin II. ConclusionsClinical and laboratory findings in these twins include differences and similarities. Clinical differences have been observed in handedness, asynchronism of painful episodes and differences in episode severity with ageing. Similarity was observed in the frequency of the painful episodes. Laboratory differences indicate that the degree of intravascular hemolysis is greater in Twin II than in Twin I, as shown by the differences in plasma Hb and RBC-derived vesicle levels (Brit J Haematol 2008;142:126-35). In contrast, the current pulmonary pressures and the current and previous rheologic studies are similar. Ageing contributes to the extent of intravascular hemolysis, as noted when the current findings are compared to those observed in the earlier study. Levels of ISC,which are related to the life span of sickle RBC (Am J Med 1978;64:253-7, Brit J Haemat 1969;17:527-533), appear to be inversely associated with indicators of intravascular hemolysis, such as plasma Hb and RBC-derived vesicles (Twin I, ISC 24.4%, Pl Hb 3.72, RBC-vesicles 2.98; Twin II, ISC 17.5%, Pl Hb 7.79, RBC-vesicles 6.32). The difference in CNVs between the twins, which is greater in Twin II, may explain the phenotypic discordance (Amer J Human Genetics 2008;82:763-71). The loci showing the differences will require further study. To what extent genetic differences in handedness in the twins are associated with the phenotypic differences have not been examined (Mol Psychiatry 2007;12:1129-39). DisclosuresNo relevant conflicts of interest to declare.

Single-Stranded Oligonucleotide Binding to P-Selectin Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to Endothelial Cells in Sickle Cell Model Mice: Novel Therapeutics for Vaso-Occlusive Episodes.

Abstract 1545Poster Board I-568To date, a variety of therapeutic approaches to sickle cell disease (SCD) have been explored, however, knowledge about the efficacy of anti-adhesive agents in this disorder is limited. In SCD, P-selectin expressed on endothelial cells plays a key role in leukocyte recruitment as well as in the adhesion of sickle red blood cells (sRBC) to the endothelium. The interaction of P-selectin and its ligands is thus likely to contribute to impairment of the microvascular flow and thereby to the development of painful vaso-occlusive episodes. Studies with anti-P-selectin antibody and P-selectin-deficient mice support the notion that P-selectin-directed interventions may be a potential approach to the treatment of vaso-occlusive episodes in SCD. Aptamers, short single-stranded oligonucleotides, have been developed for a wide range of therapeutic targets (Keefe & Schaub, 2008). Aptamers bind molecular targets with high affinity and specificity, do not elicit immune responses, and can be readily delivered to the vascular compartment intravenously or subcutaneously. Although anti-P-selectin aptamers have been shown to inhibit leukocyte rolling in vitro and to display efficacy in mouse models for inflammation, the anti-adhesion activity of anti-P-selectin aptamers has never been evaluated in SCD. The purpose of this study was to determine in vivo whether the anti-P-selectin aptamer ARC5690 can inhibit adhesion of sickle RBC and leukocytes to vascular endothelial cells in the bone marrow microvasculature of SCD model mice. Knockout-transgenic SCD mice generated by Ryan et al. (Ryan et al., 1997) were used in this study. SCD mice were injected with 20 mg/kg ARC5690, scrambled aptamer ARC5694 (negative control), or vehicle (saline). Anti-P-selectin monoclonal antibody was also used as a positive control. After 2.5 hours, the mice were subjected to 1 hour of hypoxia (12% O2 in air) followed by 1 hour of reoxygenation at room air. Intravital observations of the bone marrow microcirculation were performed to monitor adhesive interactions between blood cells and endothelial cells. Leukocytes were labeled in vivo with rat anti mouse CD45 antibody conjugated with PE which were infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery. Effects of ARC5690 on sRBC and leukocyte adhesive interactions were compared to those of ARC5694 and vehicle. Administration of the anti-P-selectin aptamer ARC5690 significantly reduced sRBC adhesion (p<0.05) in SCD mice compared to those treated with vehicle. The anti-adhesive activity of ARC5690 for sRBC was comparable to anti-P-selectin monoclonal antibody, whereas the scrambled aptamer did not show any anti-adhesive activity. We also studied the effects of the anti-P-selectin-specific ARC5690 on leukocyte flow dynamics. Mice pretreated with ARC5690, but not those treated with scrambled aptamer ARC5694, showed a greater than four-fold decrease in leukocyte rolling (p<0.001) and a six-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Again, the anti-adhesive activity of ARC5690 and its effect on leukocyte rolling and adhesion were similar to those of the anti-P-selectin antibody. Furthermore, ARC5690 improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and d wall shear rates. In conclusion, our study demonstrates significant anti-adhesive activities of ARC5690, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in SCD model mice. ARC5690 may represent a novel therapeutic strategy that can be used to treat vaso-occlusive episodes in SCD DisclosuresGutsaeva:Archemix Corporation: Research Funding. Parkerson:Archemix Corporation: Research Funding. Schaub:Archemix: Research Funding. Kurz:Archemix Corporation: Employment. Head:Archemix Corporation: Research Funding.