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Sickle cell disease (SCD)-associated pulmonary hypertension (PH) causes significant morbidity and mortality. Here, we defined the role of endothelial specific peroxisome proliferator-activated receptor γ (PPARγ) function and novel PPARγ/HUWE1/miR-98 signaling pathways in the pathogenesis of SCD-PH. PH and right ventricular hypertrophy (RVH) were increased in chimeric Townes humanized sickle cell (SS) mice with endothelial-targeted PPARγ knockout (SSePPARγKO) compared with chimeric littermate control (SSLitCon). Lung levels of PPARγ, HUWE1, and miR-98 were reduced in SSePPARγKO mice compared with SSLitCon mice, whereas SSePPARγKO lungs were characterized by increased levels of p65, ET-1, and VCAM1. Collectively, these findings indicate that loss of endothelial PPARγ is sufficient to increase ET-1 and VCAM1 that contribute to endothelial dysfunction and SCD-PH pathogenesis. Levels of HUWE1 and miR-98 were decreased, and p65 levels were increased in the lungs of SS mice in vivo and in hemin-treated human pulmonary artery endothelial cells (HPAECs) in vitro. Although silencing of p65 does not regulate HUWE1 levels, the loss of HUWE1 increased p65 levels in HPAECs. Overexpression of PPARγ attenuated hemin-induced reductions of HUWE1 and miR-98 and increases in p65 and endothelial dysfunction. Similarly, PPARγ activation attenuated baseline PH and RVH and increased HUWE1 and miR-98 in SS lungs. In vitro, hemin treatment reduced PPARγ, HUWE1, and miR-98 levels and increased p65 expression, HPAEC monocyte adhesion, and proliferation. These derangements were attenuated by pharmacological PPARγ activation. Targeting these signaling pathways can favorably modulate a spectrum of pathobiological responses in SCD-PH pathogenesis, highlighting novel therapeutic targets in SCD pulmonary vascular dysfunction and PH.

Objective— The adaptive response to vascular injury is the formation of functional collateral vessels to maintain organ integrity. Many of the clinical complications associated with sickle cell disease can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of collateral vessel formation after injury are largely unknown in sickle cell disease. Here, we characterize postischemic neovascularization in sickle cell disease and the role of neutrophils in the production of reactive oxygen species. Approach and Results— We induced hindlimb ischemia by ligation of the femoral artery in Townes SS (sickle cell) mice compared with AA (wild type) mice. Perfusion recovery, ascertained using LASER (light amplification by stimulated emission of radiation) Doppler perfusion imaging, showed significant diminution in collateral vessel formation in SS mice after hindlimb ischemia (76±13% AA versus 34±10% in SS by day 28; P P Conclusions— Our data suggest dysfunctional collateral vessel formation in SS mice after vascular injury and provide a mechanistic basis for the multiple vascular complications of sickle cell disease.

Background: Sickle hemoglobin (HbS) under conditions of deoxygenation polymerizes to cause sickling of red blood cells (RBCs) and other rheological abnormalities. Voxelotor has been previously shown in a preclinical model of sickle cell disease (SCD) to increase HbS affinity to oxygen, thus reducing its polymerization and sickling with subsequent increase in the half-life of RBCs. We hypothesized that given this mechanism of action, we would observe improvements in RBC physiology in patients receiving voxelotor. In the Phase 3 GBT HOPE trial, the use of voxelotor in patients with SCD caused a significant reduction in markers of hemolysis and anemia. Ektacytometry is considered the gold standard to study deformability of RBCs with membrane protein disorders. The deformability of RBCs can be assessed using a defined value of shear stress with an increasing osmotic gradient (osmoscan) as well as with a newer technology to subject these cells to gradual deoxygenation (oxygenscan). Both assays can be measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics, NL). In this pilot study, we analyzed samples from patients with SCD receiving voxelotor, before and 12 weeks after starting therapy to assess the benefits of voxelotor on RBC physiology. Methods: Our pilot study obtained whole blood from children ages 4-11 years with SCD, who were enrolled in the IRB approved GBT 440-007 clinical trial (NCT02850406; a study evaluating multiple doses of voxelotor at 1500 mg/day equivalent exposure to adults based on body weight) at Emory University/Children's Healthcare of Atlanta. All participants in this cohort continued their stable, optimal hydroxyurea dose during treatment with voxelotor. The below measurements were performed on the pre-dose and Week 12 visit samples. Deformability of RBCs was performed at a shear stress of 30 Pa and varying osmolality gradients (0-600 mOsm/Kg) for Osmoscan. Omin corresponds to the value of the hypotonic osmolality, where 50% of the cells hemolyze in an osmotic fragility assay and provides information on the initial surface area:volume ratio. Maximal deformability or Elongation Index (EImax) near isotonic osmolality informs us of the RBC cytoskeleton mechanics and Ohyper, the osmolality corresponding to 50% of the Elmax, provides information regarding the cytoplasmic viscosity. Oxygenscan was performed but under controlled deoxygenation using nitrogen. Point of Sickling (POS) is a point on the curve during deoxygenation when sickling begins, and EImin corresponds when sickle RBCs can least elongate. Oxygen dissociation curves were obtained using a HemOx Analyzer (TCS Scientific). Complete blood count parameters were determined on a clinical laboratory hematology analyzer (ADVIA, Siemens). Data was analyzed with Prism using a paired T-test. Results: Both pre-dose and Week 12 visit samples were available for 10 participants. Mean hemoglobin at baseline was 9.0 g/dL (7.6-10.0) and at 12 weeks, 10.3 g/dL (8.2-12.3). Six out of 10 participants had a hemoglobin response at Week 12 (defined as an increase in Hb from baseline by >1 g/dL), of which 5 had hemoglobin over 10 g/dL. Mean % change in percentage of reticulocytes was -17.0%. Significant improvement in EImax on osmoscan was noted at Week 12 (p=0.0147), suggesting RBCs were more deformable with improved cytoskeleton mechanics. In addition, oxygenscan curves shifted upwards towards normal with a significant increase in EImax (p=0.0347) and EImin (p=0.0079). These findings combined with a decrease in POS (p=0.0001) during deoxygenation suggests that at low oxygen tension, voxelotor treated RBCs were more deformable possibly from reduced HbS polymer inside these cells. Significant reductions in P50 (p=0.0011) and P20 (p=0.0001) with a left shift of the oxygen dissociation curve further demonstrates the effect of voxelotor on RBCs. Discussion: Voxelotor therapy in children with HbSS is associated with reductions in anemia and reticulocyte response, and recovery in RBC health as early as 12 weeks of treatment. Voxelotor's ability to inhibit HbS polymerization and RBC sickling is associated with specific modulation in red cell rheology at normoxic and deoxygenating conditions. Left shifted oxygen dissociation curves confirm voxelotor's ability to increase oxygen affinity. These findings suggest that voxelotor improves RBC deformability and anemia and delays the initiation of RBC sickling. Figure Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Baratz:Prolong Pharmacuticals: Honoraria; Global Blood Therapeutics: Research Funding. Pochron:Global Blood Therapeutics: Employment, Equity Ownership. Dixon:Global Blood Therapeutics: Employment, Equity Ownership. Tonda:Global Blood Therapeutics: Employment, Equity Ownership. Lehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Brown:Pfizer: Research Funding; Novartis, Inc: Research Funding; Imara, Inc: Consultancy, Research Funding; Global Blood Therapeutics, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Archer:AstraZeneca: Research Funding; Prolong Pharmaceuticals: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding.

Objectives: Despite the increased incidence and worse outcomes of cardiovascular diseases among African-Americans compared to the general population, the contribution of sickle cell trait (AS) to this racial disparity remains unknown. We aimed to delineate the impact of AS on the pathogenesis of cardiovascular diseases alone, and in the setting of other risk factors. Methods: We compared multiple cardiovascular endpoints between AS and wild-type genotype (AA) using the humanized Townes sickle cell mouse model. To determine whether AS modifies a pre-existing cardiovascular risk factor, diabetes was induced in a cohort of mice using streptozotocin (STZ). Hind limb ischemia model was employed to evaluate collateral vessel formation, and perfusion recovery was characterized with LASER Doppler perfusion imaging (LDPI). The running wheel assay assessed spontaneous motor function after ischemia. Angiotensin II (Ang II) and L-NAME were used to evaluate hypertensive response. Experimental stroke was induced by unilateral common carotid artery ligation and daily 15 min exposure to 7.5% oxygen for 3 days, and the infarct size was quantified as the ratio of the infarcted area to the total area of the uninjured contralateral hemisphere. PCSK9 gain-of-function mutation in the setting of Ang II and high-fat diet was used to evaluate atherosclerosis development. Results: 4 groups of mice were studied: AA, AS, AA-STZ and AS-STZ. The formation of collateral vessels was equivalent between the AA and AS (day 28, AA: 80 ± 6%, AS: 85 ± 5%, p=0.8), and in the setting of diabetes (39 ± 5% AA-STZ; 36 ± 4% AS-STZ). Systolic blood pressure at baseline (110 ± 4 mmHg vs 112 ± 5 mmHg), and in response to Ang II (148 ± 4 mmHg vs 142 ± 5 mmHg) and L-NAME (135 ± 3mmHg vs 138 ± 5 mmHg) were equivalent. Mortality from stroke was equivalent between AA and AS. After 1 month of PCKS9 gain-of-function mutation/high-fat diet, AA and AS demonstrated similar lipid profiles. Mortality and infarct volume after stroke induction were identical between AA and AS. Conclusions: All major indices of cardiovascular outcomes were similar between AA and AS, even in the presence of diabetes. These various experimental models suggest that the sickle trait likely does not contribute significantly to the poor cardiovascular outcomes seen in the African-American population.

Introduction: Stroke, a devastating complication in children with Sickle Cell Anemia (SCA), consists of silent cerebral infarct (SCI) and large overt stroke. The current management relies on blood transfusion without the use of thrombolytic agents. However, a recent study showed that co-existent SCA does not impact the safety of tissue plasminogen activator (tPA) treatment. This finding calls for systemic analysis of the effects of thrombolysis in experimental stroke. Hypothesis: We hypothesize that sickle mice are highly vulnerable to hypoxia/ischemia-induced stroke, but respond to tPA-thrombolytic therapy. Methods: Townes sickle mice (knock-in/out mice that express the human α, γ, and sickle-β hemoglobin genes) were subjected to Doppler measurement of the carotid artery and evaluated for their responses to repetitive-mild hypoxia-ischemia (rmHI) and transient hypoxia-ischemia (tHI)-induced stroke at 3 and 6 months of age, respectively. The effects of tPA treatment after tHI in sickle mice were also examined. Results: First, 3-month-old sickle mice of SS genotype with a higher resistive index (RI) in common carotid artery were also prone to rmHI-induced cerebral infarct and mortality. Second, 6-month-old SS mice developed elevated flow velocity and greater RI without stenosis of the carotid artery akin to those previously implicated in large overt stroke in SCA. Finally, 6-month-old SS mice endured 20-min, but manifested enhanced leukocyte and platelet adherence to cerebral blood vessel, as well as, extensive vascular perfusion deficits and fibrin deposition at 4 h post-injury, followed by greatly increased mortality than AA and AS mice at 24 h recovery (p8 for each group). Importantly, intravenous tPA treatment at 0.5 h post-tHI markedly improved vascular reperfusion, mitigated fibrin deposition, and cut the mortality of SS mice by nearly 60%. Conclusions: Humanized sickle mice develop hyper-coagulation and hypersensitivity to HI-induced stroke without large-vessel obstructive vasculopathy at up to 6 months of age. Elevated RI may be an early ultrasonic marker for sickle cell vasculopathy and the risk of SCI in SCA. Future studies are warranted to confirm the therapeutic benefits of thrombolytic stroke therapy in SCA.

Background and Purpose— The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. Methods— We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia–ischemia- and transient hypoxia–ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia–ischemia-injured sickle mice. Results— Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia–ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia–ischemia. Yet, post-transient hypoxia–ischemia treatment with tPA reduced thrombosis and mortality in SS mice. Conclusions— Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy.

Stroke is a dramatic complication of sickle cell disease (SCD), which is associated with cerebral vasculopathies including moya moya, intravascular thrombi, cerebral hyperemia, and increased vessel tortuosity. The spontaneous occurrence of these pathologies in the sickle cell mouse model has not been described. Here, we studied Townes humanized sickle cell and age-matched control mice that were 13 months old. We used in vivo two-photon microscopy to assess blood flow dynamics, vascular topology, and evidence of cerebral vasculopathy. Results showed that compared to controls, sickle cell mice had significantly higher red blood cell (RBC) velocity (0.73 mm/s vs. 0.55 mm/s, p = 0.013), capillary vessel diameter (4.84 µM vs. 4.50 µM, p = 0.014), and RBC volume flux (0.015 nL/s vs. 0.010 nL/s, p = 0.021). Also, sickle cell mice had significantly more tortuous capillary vessels ( p

Sickle cell nephropathy begins with hyperfiltration and microalbuminuria and may progress to renal failure. The aim of this study was to determine the effects of losartan on glomerular function and albumin excretion in sickle cell anemia (SCA). Individuals with SCA on hydroxyurea with persistent albuminuria were enrolled in a 1-year study of losartan. Glomerular filtration rate (GFR) measured by iohexol clearance, albumin excretion rate (AER), and fractional clearance of dextran were assessed at baseline, short-term (1-2month), and long-term (≥12month) intervals. Twelve subjects (6 microalbuminuria, 6 macroalbuminuria) completed short-term studies; 8 completed long-term studies. Baseline GFR was 112ml/min/1.73m2 (71-147ml/min/1.73m2). AER decreased significantly at the short-term (median decrease -134 mcg/min, p=0.0063). GFR was not significantly-different at short-term or long-term intervals. Dextran clearance improved for diameters smaller than albumin (

Sickle cell disease (SCD) produces many structural and functional abnormalities in the kidney, including glomerular abnormalities. Albuminuria is the most common manifestation of glomerular damage, with a prevalence between 26 and 68% in adult patients. The pathophysiology of albuminuria in SCD is likely multifactorial, with contributions from hyperfiltration, glomerular hypertension, ischemia-reperfusion injury, oxidative stress, decreased nitric oxide (NO) bioavailability, and endothelial dysfunction. Although its natural history in SCD remains inadequately defined, albuminuria is associated with increased echocardiography-derived tricuspid regurgitant jet velocity, systemic blood pressure, and hypertension, as well as history of stroke, suggesting a shared vasculopathic pathophysiology. While most patients with albuminuria are treated with angiotensin converting enzyme inhibitors/angiotensin receptor blockers, there are no published long-term data on the efficacy of these agents. With the improved patient survival following kidney transplantation, SCD patients with end-stage renal disease should be considered for this treatment modality. Given the high prevalence of albuminuria and its association with multiple SCD-related clinical complications, additional studies are needed to answer several clinically important questions in a bid to adequately elucidate its pathophysiology, natural history, and treatment.

Increased frequency and risk of infection is one of the well described complications of sickle cell anemia (SCA). Dietary supplementation in children with SCA and growth retardation improved growth and decreased incidence of infection. We investigated the impact of a high protein diet on weight gain, hematological profile, and immune cytokine levels in the Berkeley model of SCA, 16 of which were randomized to either regular mouse diet with 20% of calories from protein ( n = 8) or a test feed with 35% of calories from protein ( n = 8). Control mice (C57BL/6, n = 16) were correspondingly randomized, and were all feed ad libitum for three months with actual intake estimated by subtracting the weight of gnaw waste from that of the feed given. Blood was collected at sacrifice by cardiac puncture and plasma levels of T helper cell 1 (TH1) and TH2 associated cytokines were measured using a multiplex antibody immobilized bead assay. SCA mice receiving the 35% protein diet had modest improvements in weight, red blood cell count, and hemoglobin level, with a slight decrease in reticulocyte count compared with SCA mice on the regular mouse diet. Furthermore, they also had significantly higher plasma levels of cytokines tumor necrosis factor (TNF)-α ( P = 0.02), interferon (IFN)-γ ( P = 0.01), interleukin 10 (IL-10; P = 0.02), and IL-4 ( P = 0.02) compared with those that received the 20% protein diet. We conclude that providing additional protein calories to transgenic SCA mice increased the plasma levels of acute inflammatory cytokines associated with immune response to infection, which might partly explain decreased episodes of infection observed among supplemented children with SCA.

INTRODUCTION: Repeated bout of tissue ischemia is a hallmark of sickle cell disease (SCD). Neutrophils are critical first responder cells that initiate host response to sterile injury, including ischemia. Of the numerous neutrophils in circulation, a small proportion undergo physiologic changes (termed aging) which directs their homing to the bone marrow for clearance. How neutrophil homeostasis is regulated in sickle cell disease (SCD) to influence ischemic repair is unknown. HYPOTHESIS: We hypothesized that SCD may alter the homeostasis and function of aged neutrophil. METHODS: Aged neutrophils from the Townes humanized sickle cell (SS) mice were compared to aged neutrophils from MerTK deficient mice (MerKO), which age in the circulation owing to defective efferocytosis. Hind limb ischemia (HLI) was used as model of vascular injury. LASER Doppler perfusion imaging (LDPI) measured perfusion recovery after HLI. RESULTS: Compared to wild-type mice (7 ± 1.2%), the proportion of CXCR4HiCD62Llo aged neutrophils in circulation was markedly elevated in the SS (34 ± 4%) and MerKO mice (38 ± 4%). However, in contrast with aged neutrophils from MerKO mice, those from SS mice showed impaired phagocytosis, increased capacity to produce reactive oxygen species, and pronounced release of extracellular traps (NETs). In response to HLI, perfusion recovery in the MerKO and wild type mice were similar, but this was dramatically impaired in SS mice. Importantly, the phenotype of SS mice was reversed by treatment with the anti-oxidant N-acetyl cysteine (NAC), but NAC therapy had no impact on MerKO mice. CONCLUSIONS: Although SS and MerKO mice demonstrated elevated numbers of aged neutrophils, our data suggest that oxidant stress causes aged CXCR4HiCD62Llo neutrophils in SS to impede vascular repair after ischemia. This specialized subset of neutrophils may be targeted to improve vascular health in SCD. Disclosures No relevant conflicts of interest to declare.

The prevention and treatment of acute chest syndrome (ACS) is a major clinical concern in sickle cell disease (SCD). However, the mechanism underlying the pathogenesis of ACS remains elusive. We tested the hypothesis that the hemolysis byproduct hemin elicits events that induce ACS. Infusion of a low dose of hemin caused acute intravascular hemolysis and autoamplification of extracellular hemin in transgenic sickle mice, but not in sickle-trait littermates. The sickle mice developed multiple symptoms typical of ACS and succumbed rapidly. Pharmacologic inhibition of TLR4 and hemopexin replacement therapy prior to hemin infusion protected sickle mice from developing ACS. Replication of the ACS-like phenotype in nonsickle mice revealed that the mechanism of lung injury due to extracellular hemin is independent of SCD. Using genetic and bone marrow chimeric tools, we confirmed that TLR4 expressed in nonhematopoietic vascular tissues mediated this lethal type of acute lung injury. Respiratory failure was averted after the onset of ACS-like symptoms in sickle mice by treating them with recombinant hemopexin. Our results reveal a mechanism that helps to explain the pathogenesis of ACS, and we provide proof of principle for therapeutic strategies to prevent and treat this condition in mice.

The prevention of organ damage and early death in young adults is a major clinical concern in sickle cell disease (SCD). However, mechanisms that control adult progression of SCD during the transition from adolescence are poorly defined with no cognate prophylaxis. Here, we demonstrate in a longitudinal cohort of homozygous SCD (SS) mice a link between intravascular hemolysis, vascular inflammation, lung injury, and early death. Prophylactic Nrf2 activation in young SS mice stabilized intravascular hemolysis, reversed vascular inflammation, and attenuated lung edema in adulthood. Enhanced Nrf2 activation in endothelial cells in vitro concurred with the dramatic effect on vascular inflammation in the mice. BM chimeric SS mice lacking Nrf2 expression in nonhematopoietic tissues were created to dissect the role of nonerythroid Nrf2 in SCD progression. The SS chimeras developed severe intravascular hemolysis despite having erythroid Nrf2. In addition, they developed premature vascular inflammation and pulmonary edema and died younger than donor littermates with intact nonhematopoietic Nrf2. Our results reveal a dominant protective role for nonhematopoietic Nrf2 against tissue damage in both erythroid and nonerythroid tissues in SCD. Furthermore, we show that prophylactic augmentation of Nrf2-coordinated cytoprotection effectively impedes onset of the severe adult phenotype of SCD in mice.

BACKGROUND The pathogenesis of albuminuria in SCD remains incompletely understood. We evaluated the association of albuminuria with measures of endothelial function, and explored associations of both albuminuria and measures of endothelial function with selected biological variables (vascular endothelial growth factor [VEGF], endothelin-1 [ET-1], soluble fms-like tyrosine kinase-1 [sFLT-1], soluble vascular cell adhesion molecule-1 [soluble VCAM-1] and plasma hemoglobin). METHODS Spot urine measurements for albumin-creatinine ratio (UACR) and 24-hour urine protein were obtained. Endothelial function was assessed using brachial artery ultrasound with measurements of flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NTMD) and hyperemic velocity. RESULTS Twenty three subjects with varying degrees of albuminuria were evaluated. UACR was significantly correlated with FMD (ρ = -0.45, p = 0.031). In univariate analysis, UACR was correlated with VEGF (ρ = -0.49; 95% CI: -0.75 --0.1, p = 0.015), plasma hemoglobin (ρ = 0.50; 95% CI: 0.11-0.75, p = 0.013) and ET-1 (ρ = 0.40; 95% CI: -0.03-0.69, p = 0.06). Multivariable analysis showed significant associations of ET-1 (estimate: 455.1 [SE: 198.3], p = 0.02), VEGF (estimate: -1.1 [SE: 0.53], p = 0.04) and sFLT-1 (estimate: -1.14 [SE: 0.49], p = 0.02) with UACR. Only ET-1 (estimate: -8.03 [SE: 3.87], p = 0.04) was significantly associated with FMD in multivariable analyses. Finally, UACR was correlated with both 24-hour urine protein (ρ = 0.90, p < 0.0001) and urine aliquots for albumin-creatinine ratio obtained from the 24-hour urine collection (ρ = 0.97, p < 0.0001). CONCLUSION This study provides more definitive evidence for the association of albuminuria with endothelial dysfunction in SCD. Elevated circulating levels of ET-1 may contribute to SCD-related glomerulopathy by mediating endothelial dysfunction.

BACKGROUND: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease. STUDY DESIGN AND METHODS: Transgenic sickle mice, which express human α and βS globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffyb) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array. RESULTS: Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals. CONCLUSIONS: In sum, homozygous βS expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Purpose: Recent work indicates that transplanted neural stem cells (NSCs) can survive, migrate to the injury site, and facilitate recovery from traumatic brain injury (TBI). The present study manipulated timing and location of NSC transplants following controlled cortical impact injury (CCI) in mice to determine optimal transplant conditions. Methods: In Experiment 1 (timing), NSCs (E14.5 mouse) were injected into the host striatum, ipsilateral to the injury, at 2, 7, or 14 days. In Experiment 2 (location), NSCs or vehicle were injected into the mouse striatum (7 days post-CCI) either ipsilateral or contralateral to the injury and cognitive and motor abilities were assessed from weeks 1-8 post-transplant. Histological measures of NSC survival, migration, and differentiation were taken at 6 and 8 weeks post-transplant. Results: The results demonstrate that: (1) 2-7 days post-injury is the optimal time-range for delivering NSCs; (2) time of transplantation does not affect short-term phenotypic differentiation; (3) transplant location affects survival, migration, pheno- type, and functional efficacy; and (4) NSC-mediated functional recovery is not contingent upon NSC migration or phenotypic differentiation. Conclusions: These findings provide further support for the idea that mechanisms other than the replacement of damaged neu- rons or glia, such as NSC-induced increases in protective neurotrophic factors, may be responsible for the functional recovery observed in this model of TBI.

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPARγ ligands attenuate, whereas the loss of function mutations in PPARγ stimulate, endothelial dysfunction, suggesting that PPARγ may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPARγ in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPARγ gene to produce endothelial PPARγ null mice (ePPARγ−/−). When compared with littermate controls, ePPARγ−/−animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPARγ−/−mice. Similarly, intact aortic segments from ePPARγ−/−mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPARγ−/−aortas. Reduced nitric oxide production in ePPARγ−/−aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-κB in aortic homogenates. These findings demonstrate that endothelial PPARγ regulates vascular nitric oxide production and that the disruption of endothelial PPARγ contributes to endothelial dysfunction in vivo.

Stable mixed chimeric stem cell transplantation in hemoglobinopathies exploits shorter erythroid survival in hemolytic anemias, providing normal donor red blood cells with a competitive survival advantage. This study examined the level of stable mixed chimerism necessary for complete hematological cure of the thalassemic phenotype, using a nonmyeloablative busulfan chemotherapeutic preparation. Thalassemic mice transplanted from congenic wild-type donors developed partial mixed chimerism. Hematologic cure required >80% donor red blood cells and only >13% donor white blood cells. Murine and human transplant results were compared with a math model for survival advantage of donor peripheral blood cells produced by steady-state chimeric marrow.

The factors inducing normally innervated Schwann cells in peripheral nerve to divide are poorly understood. Transection of the fourth and fifth lumbar ventral roots (L4/5 ventral rhizotomy) of the rat is highly selective, sparing unmyelinated axons and myelinated sensory axons; Wallerian degeneration is restricted to myelinated efferent fibers. We found that L4/5 ventral rhizotomy prompted many normally innervated nonmyelinating (Remak) Schwann cells to enter cell cycle; myelinating Schwann cells of intact (sensory) axons did not. Three days after L4/5 ventral rhizotomy, [3H]thymidine incorporation into Remak Schwann cells increased 30-fold. Schwann cells of degenerating efferents and endoneurial cells also incorporated label. Increased [3H]thymidine incorporation persisted at least 10 d after ventral rhizotomy. Despite Remak Schwann cell proliferation, the morphology of unmyelinated nerve (Remak) bundles was static. Seven days after L5 ventral rhizotomy, Remak Schwann cells in the L5-predominant lateral plantar nerve increased slightly; endoneurial cells doubled. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive nuclei increased dramatically in peripheral nerve after L5 ventral rhizotomy; many of these were macrophage nuclei. In summary, we find that the degeneration of myelinated motor axons produced signals that were mitogenic for nonmyelinating Schwann cells with intact axons but not for myelinating Schwann cells with intact axons.

Studies demonstrating the versatility of neural progenitor cells (NPCs) have recently rekindled interest in neurotransplantation methods aimed at treating traumatic brain injury (TBI). However, few studies have evaluated the safety and functional efficacy of transplanted NPCs beyond a few months. The purpose of this study was to assess the long-term survival, migration, differentiation and functional significance of NPCs transplanted into a mouse model of TBI out to 1 year post-transplant. NPCs were derived from E14.5 mouse brains containing a transgene-expressing green fluorescent protein (GFP) and cultured as neurospheres in FGF2-containing medium. Neurospheres were injected into the ipsilateral striatum of adult C57BL/6 mice 1 week following unilateral cortical impact injury. Behavioral testing revealed significant improvements in motor abilities in NPC-treated mice as early as 1 week, and the recovery was sustained out to 1 year post-transplant. In addition, mice receiving NPC transplants showed significant improvement in spatial learning abilities at 3 months and 1 year, whereas an intermediate treatment effect on this behavioral parameter was detected at 1 month. At 14 months post-transplant, GFP(+) NPCs were observed throughout the injured hippocampus and adjacent cortical regions of transplanted brains. Immunohistochemical analysis revealed that the majority of transplanted cells co-labeled for NG2, an oligodendrocyte progenitor cell marker, but not for neuronal, astrocytic or microglial markers. In conclusion, transplanted NPCs survive in the host brain up to 14 months, migrate to the site of injury, enhance motor and cognitive recovery, and may play a role in trophic support following TBI.

The morbidity and mortality associated with sickle cell disease (SCD) is caused by hemolytic anemia, vaso-occlusion, and progressive multiorgan damage. Bone marrow transplantation (BMT) is currently the only curative therapy; however, toxic myeloablative preconditioning and barriers to allotransplantation limit this therapy to children with major SCD complications and HLA-matched donors. In trials of myeloablative BMT designed to yield total marrow replacement with donor stem cells, a subset of patients developed mixed chimerism. Importantly, these patients showed resolution of SCD complications. This implies that less toxic preparative regimens, purposefully yielding mixed chimerism after transplantation, may be sufficient to cure SCD without the risks of myeloablation. To rigorously test this hypothesis, we used a murine model for SCD to investigate whether nonmyeloablative preconditioning coupled with tolerance induction could intentionally create mixed chimerism and a clinical cure. We applied a well-tolerated, nonirradiation-based, allogeneic transplantation protocol using nonmyeloablative preconditioning (low-dose busulfan) and costimulation blockade (CTLA4-Ig and anti-CD40L) to produce mixed chimerism and transplantation tolerance to fully major histocompatibility complex–mismatched donor marrow. Chimeric mice were phenotypically cured of SCD and had normal RBC morphology and hematologic indices (hemoglobin, hematocrit, reticulocyte, and white blood cell counts) without evidence of graft versus host disease. Importantly, they also showed normalization of characteristic spleen and kidney pathology. These experiments demonstrate the ability to produce a phenotypic cure for murine SCD using a nonmyeloablative protocol with fully histocompatibility complex–mismatched donors. They suggest a future treatment strategy for human SCD patients that reduces the toxicity of conventional BMT and expands the use of allotransplantation to non–HLA-matched donors.

Previous reports have shown that a high protein diet improves weight gain and decreases expression of inflammatory markers in weanling Berkeley transgenic sickle cell mice. The effect of this diet on the underlying histopathology, however, has not been studied. Age-matched, male C57BL/6 controls (n = 24), Berkley sickle mice (n = 31) and Townes sickle mice (n = 14) were randomized in a terminal experiment at weaning to isoenergetic diets, with either normal (20%) or high (35%) amount of energy from protein, by replacing dextrin. Tissue sampling for blinded histologic study and scoring of changes at baseline and after 3 months of feedings showed progressive siderosis and infarcts in spleen, kidney, and liver in all sickle groups, and no significant changes in age- and sex-matched normal controls. High-protein (35%) fed Berkeley sickle mice had significantly fewer (p

Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis resulting in the release of hemin into the blood. Our preliminary microarray data indicate that PPARγ expression is significantly reduced in hemin-treated human pulmonary artery endothelial cells (HPAECs). To examine post-transcriptional mechanisms of PPARγ regulation in pulmonary hypertension in SCD (SCD-PH), Townes knockin SCD mice aged 12-weeks were gavaged daily with vehicle or rosiglitazone (RSG, 10 mg/kg/day) for 10 d. PPARγ was decreased and ET-1 expression was increased in sickle mice (SS) compared to littermate control mice (AA). Using an in vitro model that mimics acute extracellular hemin release, HPAECs were treated with DMSO or hemin (5 μM) for 72 h ± RSG (10 μM) for the final 24 h. Similarly, hemin reduced PPARγ and increased ET-1 expression in HPAECs. MicroRNA (miR-301b), which negatively regulates PPARγ, was increased in SCD mice and in hemin-treated HPAECs. In contrast, PPARγ activation attenuates increases in m...

Introduction: Endothelin-1 (ET1) is as a potent vasoconstrictor and mediates oxidative stress and inflammatory responses in various disease states. ET1 levels are elevated in individuals with physiologic measures of endothelial dysfunction. Further, urinary ET1 levels correlate with microalbuminuria in patients with sickle cell disease (SCD) suggesting ET1 mediates sickle cell nephropathy. We hypothesized that ET1 would be elevated in SCD patients with albuminuria and would correlate with endothelial dysfunction as measured by ultrasound imaging of the brachial artery. Methods: Subjects with sickle cell anemia (HbSS) were evaluated in non-crisis, “steady-states.” Individuals on hydroxyurea or renin-angiotensin-aldosterone system blocking agents (such as ACE-inhibitors) had to be on stable doses for at least 3 months. Spot urine samples were obtained for albumin-to-creatinine ratio (UACR) over 2-3 visits. Hematologic measures were obtained at baseline. At last visit, plasma ET1 levels were drawn, subjects provided 24hr urine collections, and endothelial function was assessed by brachial artery shear stress methods. We obtained ultrasound measures of flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NTMD), as indices of endothelium-dependent and endothelium-independent vasodilation, respectively. Hyperemic flow was quantified as the velocity-time integral (VTI), a reflection of small vessel endothelial function. ET1 levels were compared by categories of albuminuria (normal, micro- (>=30, =300 mg/g) by Kruskal-Wallis testing. We examined Spearman's correlations for ET1 and covariates of interest. Results: 24 subjects had measures of plasma ET1 available with mean age 43.0 (SD 11.9) years; 11 (45.8%) were male and 17 had albuminuria (9 micro-, 7 macro-). Other covariates are presented in Table 1. Median ET1 levels were significantly higher in micro- (0.69 pg/ml; IQR 0.62, 0.82) and macroalbuminuria (0.67 pg/ml; IQR 0.49, 1.02) subjects compared to normoalbuminuria (0.39 pg/ml; IQR 0.28, 0.58) subjects (p =0.03) (Fig 1). ET1 also correlated with UACR in continuous measures (ρ=0.44; 95% CI 0.05, 0.72; p =0.03). We saw no correlation between ET1 and hematologic indices or hemolysis markers including hemoglobin level, WBC and platelet counts, LDH, indirect bilirubin, or HbS or HbF content. In measures of endothelial dysfunction, ET1 had a negative correlation with FMD (ρ= -0.34, 95% CI -0.65, 0.08; p =0.1) that was not statistically significant. However, there was a strong negative correlation between ET1 and hyperemic VTI (ρ= -0.56, 95% CI -0.80, -0.18; p =0.008). Conclusions: In HbSS subjects, plasma ET1 levels associated with the presence of micro- or macroalbuminuria and also correlated with continuous measures of albuminuria. High ET1 levels correlated strongly with lower values of hyperemic VTI, indicating small vessel endothelial dysfunction. This latter finding may reflect glomerular capillary dysfunction leading to albuminuria. Our findings suggest ET1 plays a role in the pathogenesis of early kidney disease in HbSS as reflected by albuminuria and mediated by endothelial dysfunction. Table 1. Demographic and clinical data in 24 patients with HbSS. Normoalbuminuria (n=8) Microalbuminuria (n=9) Macroalbuminuria (n=7) Age (yrs) 38.8 (9.9) 42 (13.8) 49.1 (10.1) Male (N, %) 2 (25.0) 5 (55.6) 4 (57.1) SBP (mmHg) 107.5 (96, 117) 120 (117, 145) 138 (117, 151) DBP (mmHg) 57 (52, 65) 63 (56, 67) 81 (63, 83) Hemoglobin (g/dl) 9.3 (8.3, 10.0) 8.8 (7.5, 9.2) 8.8 (8, 9.5) WBC (109/L) 6.9 (5.6, 8.5) 7.0 (5.8, 10.6) 8.1 (4.8, 11.2) Platelet (109/L) 351 (306, 556) 288 (224, 374) 356 (253, 445) LDH (U/L) 736 (580, 1031) 1068 (771, 1389) 907 (759, 998) Indirect Bilirubin (g/dl) 2.1 (0.9, 4.8) 2.4 (1.2, 3.1) 2.3 (1.6, 4.1) Creatinine (mg/dl) 0.58 (0.55, 0.76) 0.68 (0.66, 0.98) 0.70 (0.54, 1.35) eGFR (ml/min/1.73m2) 121.9 (109.8, 153.7) 118.6 (78.4, 145.2) 127.4 (63.0, 136.8) 24hr CrCl (ml/min/1.73m2) 129 (89, 203) 101 (77, 116) 80 (53, 124) UACR (mg/g) 8.8 (5.1, 11.2) 95.8 (48.6, 142.7) 518.9 (398.6, 706.7) 24hr Urine Protein (mg) 93 (72.5, 106) 207 (140, 216) 910 (506, 1388) Disclosures Archer: Global Blood Therapeutics: Consultancy.

Introduction: Albuminuria is a common and sensitive marker of glomerular injury in SCD. We and others have previously observed the association of albuminuria with echocardiographically-derived tricuspid regurgitant jet velocity, systolic blood pressure, and history of stroke, suggesting a shared vasculopathic pathophysiology. However, the pathogenesis of albuminuria in SCD remains poorly defined. The purpose of this study was to evaluate the association of albuminuria with endothelial dysfunction, assessed non-invasively using ultrasound imaging of the brachial artery in SCD patients. In addition, we evaluated the association of albuminuria assessed by spot urine collection with 24-hour urine collections for protein. Methods: Spot urine measurements for microalbumin/creatinine ratio (UACR) were obtained during the "non-crisis," steady state over 2-3 visits, with a 24-hour urine collection for assessment of protein at the final visit. We obtained routine laboratory tests, including markers of hemolysis (lactate dehydrogenase, and total and indirect bilirubin) at the baseline visit. Endothelial function was assessed non-invasively at the final visit using a brachial artery shear stress method, with measurements of flow-mediated dilation (FMD), an index of endothelium-dependent vasodilation and nitroglycerin-mediated dilation (NTMD), a measure of endothelium-independent vasodilation. Patients on renin-angiotensin-aldosterone system (RAAS) blocking agents (ACE-inhibitors, angiotensin receptor blockers, direct renin inhibitors and mineralocorticoid receptor blockers), as well as those on hydroxyurea were on a stable dose for at least 3 months prior to enrollment. We evaluated associations between measures of endothelial function and other study covariates using Spearman's correlation coefficient for continuous covariates, and the Kruskal Wallis test for categorical covariates. Linear regression analysis was used to adjust for baseline arterial diameter. Reported p-values are for individual tests, unadjusted for multiple comparisons. Results: Spot urine samples were obtained in 23 subjects with sickle cell anemia (female = 13, male = 10), mean age, 43.3 ± 12.0 years, with normoalbuminuria (UACR < 30 mg/g creatinine) in 7 subjects, microalbuminuria (UACR = 30 - 299mg/g creatinine) in 9 subjects and macroalbuminuria (UACR ³300 mg/g creatinine) in 7 subjects. Albuminuria (assessed by spot microalbumin/creatinine ratio) was significantly correlated with FMD (ρ = -0.45, p = 0.031). However, albuminuria was no longer significantly associated with FMD when controlled for baseline arterial diameter (p = 0.09). Although FMD appeared to be lowest in patients with macroalbuminuria, no significant difference was observed when FMD was evaluated in the 3 albuminuria categories (p = 0.16). No significant correlations were observed between NTMD and albuminuria (ρ = 0.35, p = 0.20). Surprisingly, no significant correlations were observed between FMD and hemoglobin, fetal hemoglobin, lactate dehydrogenase or indirect bilirubin. We observed a strong correlation between albuminuria assessed by spot microalbumin-creatinine ratio and total urinary protein assessed by 24-hour urine collection (ρ = 0.90, p < 0.0001). Furthermore, albuminuria assessed by spot microalbumin-creatinine ratio was strongly correlated with urine aliquots for microalbumin-creatinine ratio obtained from the 24-hour urine collection (ρ = 0.97, p < 0.0001). Conclusion: In this exploratory study, we found that albuminuria is significantly correlated with FMD in patients with SCD, suggesting that endothelial dysfunction may contribute to the pathogenesis of this complication in SCD. We also find a very strong correlation between spot urine assessments for microalbumin/creatinine ratio and 24-hour urine collections for protein, providing validation for the use of spot urine assessments for the evaluation of albuminuria in patients with SCD. Disclosures Archer: Global Blood Therapeutics: Consultancy.

In the brains of aged humans and cases of Alzheimer disease, deposits of amyloid in senile plaques are located in proximity to nerve processes. The principal component of this extracellular amyloid is beta/A4, a peptide derived from a larger amyloid precursor protein (APP), which is actively expressed in brain and systemic organs. Mechanisms that result in the proteolysis of APP to form beta/A4, previously termed beta-amyloid protein, and the subsequent deposition of the peptide in brain are unknown. If beta/A4 in senile plaques is derived from neuronally synthesized APP and deposited at locations remote from sites of synthesis, then APP must be transported from neuronal cell bodies to distal nerve processes in proximity to deposits of amyloid. In this study, using several immunodetection methods, we demonstrate that APP is transported axonally in neurons of the rat peripheral nervous system. Moreover, our investigations show that APP is transported by means of the fast anterograde component. These findings are consistent with the hypothesis of a neuronal origin of beta/A4, in which amyloid is deposited in the brain parenchyma of aged individuals and cases of Alzheimer disease. In this setting, we suggest that APP is synthesized in neurons and delivered to dystrophic nerve endings, where subsequent alterations of local processing of APP result in deposits of brain amyloid.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates the cellular defense mechanism by mediating a coordinate induction of cytoprotective antioxidant responsive element-driven genes. Nrf2 agonists augment fetal hemoglobin expression in hematopoietic progenitors and have emerged as a new class of drugs for therapeutic induction of fetal hemoglobin in sickle cell anemia (SCA). However, the cytoprotective effect of Nrf2 on the pathobiology of SCA has not been previously defined. To investigate the role and mechanism of Nrf2 in SCA independent of globin gene modulation, we generated chimeric mice with disruption of Nrf2 in non-hematopoietic tissues. A total of twenty-six Nrf2-/- mice were transplanted with bone marrow harvested from the Berkeley sickle and hemizygous (Hemi) mice and the Townes sickle (SS) and control (AA) mice. All sickle/Nrf2-null chimeras (SSNHNrf2-/-; n=13) developed classical hematological and intravascular hemolysis features of SCA after eight weeks of transplantation. Despite the presence of erythrocyte Nrf2, SSNHNrf2-/- chimeras developed more severe intravascular hemolysis than SS wild-type (SSWT) donor-litter-mates. The concentration of plasma cell-free Hb (p Disclosures Archer: Global Blood Therapeutics: Consultancy.

Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life. We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry. In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of 30% calculated Hb target occupancy. Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD. | SS mice | RBC half life | Reticulocytes | Sickled RBCs | Hemoximetry | | -------------------------------------- | ------------- | ------------- | --------------- | ----------- | ----------- | | Chronic treatment, PO, BID, 10-12 days | (Days) | (%) | (% at 10 mm Hg) | p20 (mm Hg) | p50 (mm Hg) | | Vehicle-treated | 2.4 | 53 | 56 | 18 | 32 | | GBT440-treated (100mg/kg) | 3.8 | 32 | 19 | 4.5 | 21 | Table Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg: Global Blood Therapeutics: Employment, Equity Ownership. Zhou: Global Blood Therapeutics: Research Funding. Hutchaleelaha: Global Blood Therapeutics: Employment, Equity Ownership. Archer: Global Blood Therapeutics: Consultancy, Research Funding.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (γ-irradiation, depletion of the peripheral immune system) or administration of “mega” doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-γ-producing T cells re-emerged and skin grafts were rejected at ∼100 days. When applied to a murine β-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.

Acute chest syndrome (ACS) is a major cause of morbidity and mortality in sickle cell disease (SCD). The diagnosis, prevention and treatment of ACS pose major clinical concerns in SCD partly because the mechanism underlying the pathogenesis of this syndrome remains elusive. Our group first reported that excess intravascular hemin causes a lethal acute lung injury (ALI) in transgenic SCD mice reminiscent of ACS (Ghosh and Ofori-Acquah, Blood 116 Suppl 1:944, 2010). Subsequently, large-scale genomics studies by Bean et al., (Blood 120:3822-8, 2012) and Galarneau et al., (Blood, 122:434-42, 2013) have implicated hemin catabolism and inflammation in the pathogenesis of ACS. In addition, we have reported recently that raised plasma free hemin increases the odds of ACS in children with SCD (Adisa et al., Br J Haematol. 2013). Collectively, these studies support a new theme of ACS pathogenesis involving extracellular hemin. In the current study, we validated the respiratory dysfunction of this ACS model, tested the hypothesis that toll-like receptor 4 (TLR4) mediates the associated lung injury, and examined the efficacy of two strategies to treat the condition in mice. Arterial blood gas analysis of SS mice with the ACS-like disease confirmed severe hypoxemia (PaO2; 40.23 ±3.85 mmHg, SO2; 58.72±6.6%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 4836 Sickle cell disease (SCD) is a chronic hemolytic and inflammatory disorder characterized by repeated episodes of vaso-occlusion and hemolysis, resulting in oxidative stress and endothelial dysfunction. We have recently demonstrated that the heme scavenging capacity in SCD is severely impaired, highlighting the danger posed by excess heme in this disorder. Paradoxically, heme induces expression of several cyto-protective enzymes including the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in glutathione (GSH) synthesis, which is a crucial antioxidant in the lung. While the induction of cytoprotective enzymes is thought to attenuate the deleterious effects of heme in SCD the somatic origin of this protection has not previously been defined. Using transgenic mouse models we show for the first time that the level of GCLM in the sickle lung is markedly up-regulated due primarily to enhanced expression of the enzyme in the epithelium and blood mononuclear cells, but not in the endothelium. Based on these findings, we tested the hypothesis that leukocyte-derived GCLM was sufficient to protect the sickle lung from oxidative stress. Thus, bone marrow chimeric SCD mice with GCLM deficiency were generated by transplanting bone marrow from Berkeley SCD transgenic mice into GCLM null mice recipients. We confirmed that the chimeric GCLM-null-SCD mice had a SCD phenotype as determined by >95% engraftment of donor white blood cells, reticulocyte counts, urine osmolality and hemoglobin gel electrophoresis. Whole lung GCLM and total GSH levels in the chimeric mice were identical to the levels in the wild-type SCD mice. Moreover, lung function, as determined by oxygen saturation and breath rate, were identical in the two mouse strains. These results show that loss of GCLM expression in resident lung cells does not compromise production of GSH or the function of the lung in SCD. Disclosures: Ofori-Acquah: Emory University: Patents & Royalties.

Background: Patients with sickle cell disease have higher rates of red blood cell (RBC) alloimmunization following transfusion than any other patient population. However, it is unclear if they are inherently more immunizable, or if the high rates of RBC alloimmunization are simply due to antigenic differences between donors/recipients and heightened transfusion frequency. Utilizing a murine model, we have previously shown that rates of alloimmunization are influenced by the inflammatory status of the recipient at the time of the transfusion. Thus, we hypothesized that sickle patients may be more immunizable, due in part to the baseline inflammation associated with their disease. Herein, we present a reductionist murine sickle model of RBC alloimmunization, and investigate the hypothesis that mice with sickle cell disease are more likely to become alloimmunized following transfusion of RBCs containing a foreign antigen than sickle trait controls. Materials/Methods: Berkeley mice (with human α and βS-globin) were determined to be homozygous (sickle cell disease) or heterozygous (sickle cell trait) by cellulose acetate electrophoresis. We generated the HOD mouse, with RBC specific expression of the model humoral antigen hen egg lysozyme (HEL) fused to the model minor histocompatibility antigen ovalbumin (OVA), linked to the cell membrane by a human blood group antigen (Duffy). The inclusion of OVA in the HOD construct allows presentation of HOD peptides from H-2b MHC; thus, Berkeley mice are able to process and present the HOD antigen. 100 microliters of packed HOD RBCs were transfused IV into mice with sickle cell disease or sickle cell trait; alloimmunization to the HEL antigen was assessed by anti-HEL IgG ELISA 2 weeks following transfusion. Given that co-stimulatory molecule expression on antigen presenting cells in part determines the response of the CD4+ T cell to the antigen being presented, we examined baseline expression of B7-1, B7-2, Ox40L, CD70, 41BBL, CD30L, and CD40 on macrophages (F480 high) and dendritic cells (CD11c high) in sickle cell disease and sickle trait mice. Results: Data for a standard curve was fit to one phase exponential association, with a non-linear best fit regression analysis (R2 of 0.96). Utilizing the OD 415 value of the anti-HEL IgG ELISA, data was combined from 3 separate experiments (n=31 mice). An unpaired two-tailed t-test showed a mean of 6.25e-5, SD 5.87e-5 for sickle disease mice, and a mean of 4.14e-5, SD 2.84e-5 for sickle trait mice, p=0.217 (see figure). Baseline co-stimulatory molecule expression of all molecules examined on macrophages and dendritic cells was similar in both sickle cell disease and sickle trait mice. Figure Figure Conclusions: Generation of the HOD mouse, containing a model humoral antigen capable of being presented by the antigen presenting cells in the Berkeley mice, allowed these studies to be performed. These data rule out the hypothesis that Berkeley mice with sickle cell disease are substantially more immunizable to a single transfusion of RBCs containing the HOD foreign antigen than are those with sickle cell trait. However, there is a large standard deviation in alloantibody response between individual mice with sickle cell disease. The varied response may be explained by differing levels of illness in individual mice; ongoing studies are investigating such a potential correlation. Clinically, many patients with sickle cell disease are transfused in times of illness (i.e. acute chest syndrome), and the possibility that illness alters alloantibody response cannot be ruled out by these studies. Furthermore, although the model was designed to be as reductionist as possible, these studies are limited by the highly heterogeneous genetic background of the Berkeley mice (including FVB/N, 129, DBA/2, C57BL/6, and Black Swiss); this diverse background likely influences response to foreign antigen and makes identifying control groups difficult. Finally, although Berkeley mice with sickle cell disease are known to have baseline inflammation associated with their disease, this does not appear to influence co-stimulatory molecule expression on their antigen presenting cells.

In order to understand the mechanisms underlying the increased rates of stem cell transplant rejection in patients with sickle cell disease (SCD) we have developed a mouse model of transplant rejection utilizing the Berkeley SCD mouse. We find that Berkeley SCD mice exhibit a significantly higher rate of transplant rejection when transplanted with fully allogeneic bone marrow from an SJL donor after non-myeloablative conditioning and peri-transplant T cell costimulation blockade. Thus, while control C57BL/6 (BL/6) mice or hemizygous litter-mates exhibit 80–85% engraftment rates, only 20% of Berkeley SCD mice engraft with donor marrow. Transplant rejection in the SCD mice is mediated by CD8+ and/or NK1.1+ cells, as depletion with antibodies directed at either of these cell-surface molecules increases engraftment rates to 75–90%. To dissect the mechanistic underpinnings of these observations, we performed a multiparameter phenotypic and functional flow-cytometric analysis of leukocytes from the peripheral blood, lymph nodes and spleen from SCD mice, and compared them to the two non-SCD controls. We observed that SCD mice displayed striking phenotypic differences compared to normal mice, but that their hemizygous litter-mates were indistinguishable from the BL/6 controls, underscoring the sickle-specific nature of our observations. SCD mice showed minimal phenotypic variation in the peripheral blood, but the spleens and lymph nodes from SCD mice were highly abnormal. Sickle mice exhibited ∼10-fold more splenic NK and NK-T cells than either the BL/6 or hemizygous controls. These cells produced large quantities of both TNF-α and IFN-γ, potentially contributing to the inflammatory milleu that is associated with SCD. Furthermore, while absolute numbers of splenic T cells were not different in sickle mice compared to controls, their phenotype was skewed toward a highly activated state. Thus, while many (40%) splenic memory T cells from normal controls exhibited a Central Memory (Tcm) phenotype, there were fewer Tcm cells in SCD mice (6%), and a skewing of the memory repertoire towards a more activated Intermediate Memory phenotype (71% in SCD mice compared to 25% in controls). Finally, in ELISpot analysis, lymph node cells from naive sickle mice, having never been previously exposed to allo-antigen, were able to secrete IFN-γ when briefly co-cultured with donor SJL stimulators, a phenomenon not observed with either control BL/6 or hemizygous controls, again, pointing to the increased inflammatory milieu and potential for rejection in the sickle mice. In summary, we have observed evidence for a multi-faceted increase in immune activation in SCD mice, which correlates with their increased transplant rejection. These results point both toward possible therapeutic targets aimed at decreasing SCD-mediated transplant rejection and toward future studies in SCD patients, in order to determine whether similar mechanisms of immune activation also occur in the patient population.

Clinically, pulmonary hypertension is a major risk factor for mortality in adults with sickle cell disease. Contributing factors probably include red cell hemolysis and vaso-occlusive injury with their associated oxidative and inflammatory stimuli. Previously, we have described RBC hemolysis and endothelial oxidative stress in the Berkeley sickle mouse model and extend those studies in this work to investigate cardiovascular and endothelial dysfunction. Eight to ten month old homozygous and hemizygous Berkeley sickle mice and C57BL/6 control mice were used for all aspects of these experiments. In vivo measurements of mean arterial pressure and right ventricular pressures were conducted in fully anesthetized mice using a pressure transducer inserted in the carotid and right ventricle respectively. Following in vivo readings hearts were excised for measurement of ventricular mass. The ascending aorta was removed and cut into 5 mm rings for in vitro studies of agonist- induced contractility and relaxation. The mean arterial pressure of the hemizygous sickle mice (70.6 ± 3.4) was significantly lower than the control mice (86.0 ± 3.1) and the mean arterial pressure of homozygous sickle mice (59.0 ± 2.2 mmHg) was significantly lower than the hemizygous and control mice (p≤0.05 and p≤0.001, respectively). The right ventricular pressure showed a trend that approached significance (p= 0.08) such that pressures in homozygous mice were ≥ than those in hemizygous which were ≥ than those in control mice. Increased basal cardiac output was suggested by significant left ventricular hypertrophy. In vitro examination of potassium chloride activation of voltage gated calcium channels showed no significant difference in sensitivity or maximal contraction. Similarly, there was no difference in sensitivity to the α1 agonist, phenylephrine. However, both hemi- and homozygous mice showed a significant reduction in maximal force of contraction (normalized to cross sectional area when compared to controls. Maximal acetylcholine induced relaxation of aortic rings was significantly reduced (p≤0.05) in homozygous sickle mice compared to controls. The same effect was not seen with sodium nitroprusside induced relaxation indicating that the acetylcholine effect was not due to effects on the smooth muscle but was endothelium-dependent. The Berkeley mouse model shows cardiac hypertrophy consistent with the increased cardiac output associated with chronic anemia and a reduced basal mean arterial blood pressure similar to that seen in humans. 8–10 month old mice have increased right ventricular pressure and RV mass indicative of pulmonary hypertension. Further endothelial dysfunction is characterized by a reduction in the maximal relaxation elicited by acetylcholine. Therefore, the Berkeley mouse is a good model for investigating sickle related endothelial dysfunction.