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Sickle cell anemia (SCA), caused by a mutant β-globin gene, results in polymerization of the abnormal hemoglobin S and sickle shaped RBCs that cause vascular occlusions, chronic hemolytic anemia and cumulative organ damage. We have reported sickle nephropathy from elevated levels of angiotensin-II (AT), the bioactive peptide of the renin-angiotensin system (RAS), by signaling through the AT receptor-1 (AT1R), in both mice and humans with SCA, and that occurs in the absence of hypertension or hyper-reninemia [Roy et al, AJH, 2018]. Herein, we investigated the mechanisms underlying the hyperangiotensinemia in SCA. We found that AT levels are elevated due to higher oxidized state of its precursor, angiotensinogen (ATGN), which results from the elevated reactive oxygen species (ROS) in SCA. Oxidized ATGN is more rapidly converted to AT. Hence, the high ROS in SCA increases AT production. Blockade of AT-AT1R signaling in SCA mice, either globally (with captopril that reduces AT production, or losartan that blocks AT1R, or by constitutive genetic knockout of AT1R in SCA mice [SCA AT1R-/-]), or in erythroid cells by an erythroid-specific AT1R knockout (AT1Rf/f EpoR Cre+ termed eCre+) in SCA mice (SCA eCre+) resulted in significant reduction in RBC ROS (Figure 1a). These data show that AT-AT1R signaling in turn generates ROS in sickle erythroid cells, thus creating a positive feedback loop of ↑ROS -->↑RAS -->↑ROS, which causes, and perpetuates the hyperangiotensinemia seen in SCA. The AT-mediated ROS-RAS loop is driven largely by sickle erythropoiesis: SCA eCre+ mice have reduced RBC ROS, hence have reduced AT, and do not develop nephropathy. Surprisingly, while global AT1R deficiency in WT mice (WT AT1R-/-) had no RBC phenotype, SCA AT1R-/- mice developed profound anemia, suggesting AT signaling may be important for the stress erythropoiesis (Stress-E) state, present in SCA. AT is known for its role as a renal erythropoietin (Epo) secretagogue, but its role in Stress-E is unknown. Induction of Stress-E in WT AT1R-/- mice (with phenylhydrazine or daily phlebotomies) resulted in higher anemia in WT AT1R-/- mice than in WT AT1R+/+ mice, suggesting AT1R signaling is important for Stress-E, not baseline erythropoiesis (Base-E). However, WT mice with erythroid-specific deficiency of AT signaling (WT eCre+), when stressed, were able to maintain hemoglobin comparable to their controls (WT eCre-), with an exponential increase in Epo level. Epo levels were high in SCA mice to begin with but were insufficient to compensate with loss of AT signaling in SCA AT1R-/- mice, resulting in development of anemia. RNAseq analysis of sorted Stress-E nucleated erythroid precursors and enucleated erythrocytes in WT AT1R-/- mice showed a remarkable downregulation of the Hedgehog, BMP4, KIT and WNT Stress-E signaling pathways, when compared to WT (AT1R+/+) mice, further confirming that AT signaling is critical, and conceivably upstream to these established Stress-E pathways. However, AT-AT1R signaling in Stress-E states is a double-edged sword: on one hand it was essential to sustain erythropoiesis, while on the other hand it resulted in significant ROS production. This was seen in SCA, and in WT mice where Stress-E was associated with higher RBC ROS (Figure 1a). AT is known to activate NADPH oxidase (NOX), to generate ROS in other cell types. But pharmacological inhibition of NOX signaling, or genetic deficiency of Gp22phox (a common subunit of NOX isoforms) in SCA mice did not lower RBC ROS. However, AT-AT1R signaling in Stress-E increased the mitochondrial mass in erythroid precursors, and also resulted in retention of dysfunctional mitochondria with lower membrane potential in the enucleated erythrocytes, a source of high ROS (Figure 1b-c). Hence, AT signaling in Stress-E inhibited mitophagy, which is required for clearance of mitochondria after enucleation. The transcriptome profile corroborated these findings: With Stress-E, WT AT1R-/- mice had upregulation of genes involved in mitophagy, and genes involved in maintaining mitochondrial integrity/quality and cellular redox homeostasis under oxidative stress. Taken together, our results show that AT signaling plays a critical role in increasing erythroid cell mass during Stress-E, but also downregulates mitophagy, antioxidant genes, and results in increased retention of dysfunctional mitochondria, which are the source of high RBC ROS that mediates end-organ injury. Disclosures Kalfa: Forma Therapeutics, Inc: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Research Funding. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene, MICAL3 (microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3' untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3' UTR of ET-1 mRNA. Since miR-648 is located in a 5'-proximal intron of MICAL3, we examined which of three potential promoters was responsible for its expression. The MICAL3 distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.

Placental growth factor (PlGF) plays an important role in various pathological conditions and diseases such as inflammation, cancer, atherosclerosis and sickle cell disease (SCD). Abnormally high PlGF levels in SCD patients are associated with increased inflammation and pulmonary hypertension (PHT) and reactive airway disease; however, the transcriptional and post-transcriptional mechanisms regulating PlGF expression are not well defined. Herein, we show that treatment of human erythroid cells and colony forming units with erythropoietin (EPO) increased PlGF expression. Our studies showed EPO-mediated activation of HIF-1α led to subsequent binding of HIF-1α to hypoxia response elements (HREs) within the PlGF promoter, as demonstrated by luciferase transcription reporter assays and ChIP analysis of the endogenous gene. Additionally, we showed miR-214 post-transcriptionally regulated the expression of PlGF as demonstrated by luciferase reporter assays using wild-type (wt) and mutant PlGF-3′-UTR constructs. Furthermore, synthesis of miR-214, located in an intron of DNM3 (dynamin 3), was transcriptionally regulated by transcription factors, peroxisome proliferator-activated receptor-α (PPARα) and hypoxia-inducible factor-1α (HIF-1α). These results were corroborated in vivo wherein plasma from SCD patients and lung tissues from sickle mice showed an inverse correlation between PlGF and miR-214 levels. Finally, we observed that miR-214 expression could be induced by fenofibrate, a Food and Drug Administration (FDA) approved PPARα agonist, thus revealing a potential therapeutic approach for reduction in PlGF levels by increasing miR-214 transcription. This strategy has potential clinical implications for several pathological conditions including SCD.

Beta protein 1 (BP1), a human homeotic transcription factor, is expressed during hematopoeisis in the erythroid lineage. To determine the in vivo role of BP1 in erythropoiesis, we have undertaken two complementary approaches using enforced BP1 expression in both transgenic mice and embryonic stem (ES) cells. Despite repeated attempts, only one adult transgenic BP1 founder mouse among 121 mice was obtained. This mouse presumably survived due to transgene mosaicism because the transgene could not be transmitted. This mouse expressed BP1 and displayed splenomegaly, extramedullary erythropoiesis and severe amyloidosis A in the kidney, a phenotype compatible with thalassemia. Consistently, the presence of BP1 transgene in fetuses was associated with paleness and lethality. In ES cells, BP1 expression in primary differentiation appeared to antagonize adult beta-globin expression. In secondary differentiation, BP1 expression reduced significantly beta-globin gene expression in both primitive and definitive erythroid cells, whereas it impaired only the definitive erythroid cell differentiation. These studies showed that BP1 can negatively modulate adult beta-globin gene expression and definitive erythroid cell differentiation, and suggest that BP1 could play a role in thalassemia.

Background: Although increase prevalence of airway hyper-reactivity (AHR) has been detected in children, and even adults with sickle cell disease (SCD) using methacholine provocative challenge test (MCT), AHR was not been found to be associated with clinical asthma.Hence, the clinical implications of AHR in SCD are unknown. Furthermore, AHR studies in SCD patients using MCT lack comparisons with a control population. We recently showed that increased placenta growth factor (PlGF) levels in sickle mice causes AHR by upregulating leukotriene synthesis and inflammation. We therefore designed a prospective study to determine the prevalence of AHR in children and young adults with SCD, and in unaffected siblings living in the same household as a control population, and also determine the biochemical and clinical implications of AHR in SCD. Methods: A total of 67 patients with SCD and 14 unaffected siblings of the patients enrolled on to the study for determining baseline AHR (ClinicalTrials.gov Identifier: NCT00448370). Chief eligibility criteria included a diagnosis of SCD (HbSS, HbSC or HbS b-thalassemia) confirmed by hemoglobin electrophoresis or gene sequencing, ages 5.5 years to 30 years who were able to complete pulmonary function testing and were at baseline/steady state (no acute sickle events 3 weeks prior or acute chest syndrome (ACS) 4 weeks prior). Excluded were patients taking anti-leukotriene medications within 30 days prior, although patients with SCD who had a known history of asthma but not on leukotriene inhibitors were eligible. Patients with chronic transfusions, congenital lung or heart condition, or other medical condition that could affect the pulmonary function testing were also ineligible. Unaffected siblings of the 67 enrolled SCD patients living in the same household were offered enrollment for MCT and those consenting were enrolled. Results: 64 SCD patients and 14 siblings underwent a MCT; 3 of 67 SCD patients had FEV116mg/mL; borderline AHR=PC20 4-16mg/mL; mild AHR=PC20 1-3mg/mL and moderate-severe AHR=PC20 Conclusions: There was a higher prevalence of mild to severe AHR (51%) in patients with SCD, compared to a 14% prevalence of mild AHR in siblings living in the same household. The higher degree of AHR was associated with increased SCD morbidity - increased rates of admission from ACS and pain crisis, when compared to those with no AHR. AHR was associated with higher leukotrienes and a trend towards higher PlGF target gene induction. Together, our data suggests that AHR may be a biomarker for SCD morbidity, and may also be a therapeutic target, since blockade of PlGF or leukotriene synthesis in patients with severe AHR may reduce SCD morbidity. Disclosures No relevant conflicts of interest to declare.

Endothelin-1, a potent vasoconstrictor, plays an important role in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show that higher levels of placenta growth factor (PlGF), secreted by erythroid precursor cells, correlate with increased plasma levels of endothelin-1 (ET-1) and other functional markers of PH in SCD. PlGF-mediated ET-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively less is understood regarding how PlGF-mediated expression of HIF-1α and its downstream effector ET-1 are post-transcriptionally regulated. Herein, we show that PlGF treatment of endothelial cells resulted in reduced levels of miR-199a2, which targeted the 3′-UTR of HIF-1α mRNA and concomitantly led to augmented ET-1 expression. Plasma levels of miR-199a2 in SCD subjects were significantly lower with reciprocally high levels of plasma ET-1, unlike unaffected controls. This observation provided a molecular link between miR-199a2 and high levels of ET-1 in SCD. Furthermore, we show that miR-199a2 located in the DNM3os transcription unit was co-transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα). Binding of the latter to PPARα cis-elements in the promoter of DNM3os was demonstrated by promoter mutational analysis and ChIP. Additionally, we show that fenofibrate, a PPARα agonist, increased the expression of miR-199a2 and DNM3os; the former was responsible for reduced expression of HIF-1α and ET-1. In vivo studies of fenofibrate-fed Berkeley sickle mice resulted in increased levels of miR-199a2 and reduced levels of ET-1 in lung tissues. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-199a2 expression can ameliorate PH by reduction of ET-1 levels.

Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.

The β-hemoglobinopathies and thalassemias are serious genetic blood disorders affecting the β-globin chain of hemoglobin A (α(2)β(Α)(2)). Their clinical severity can be reduced by enhancing expression of fetal hemoglobin (γ-globin), producing HbF (α(2)γ(2,)). In studies reported here, γ-globin induction by 23 novel, structurally-unrelated compounds, which had been predicted through molecular modeling and in silico screening of a 13,000 chemical library, was evaluated in vitro in erythroid progenitors cultured from normal subjects and β-thalassemia patients, and in vivo in transgenic mice or anemic baboons. Four predicted candidates were found to have high potency, with 4- to 8-fold induction of HbF. Two of these compounds have pharmacokinetic profiles favorable for clinical application. These studies thus effectively identified high potency γ-globin inducing candidate therapeutics and validated the utility of in silico molecular modeling.

Airway hyper-responsiveness (AHR) affects 55-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the underlying mechanisms of this highly prevalent AHR in a hemoglobinopathy remain unknown. We hypothesized that Placenta Growth Factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates this AHR. We studied AHR in allergen-exposed PlGF-/- and SCD mice. Sickle mice genetically deficient in PlGF were not viable. PlGF augmented AHR, lung inflammation and blood/lung eosinophilia via IL-13 (a Th2-cytokine) and increased 5-lipoxygenase (a leukotriene synthetic enzyme) expression. AHR, or pulmonary inflammation and leukotriene levels were blunted in PlGF-/- mice or in PlGFWT mice treated with anti-PlGF-Ab, and was rescued by intratracheal administration of leukotrienes to PlGF-/- mice. Notably, Th2-mediated Stat-6 activation further increased PlGF expression from lung epithelium, eosinophils and macrophages, linking the PlGF-leukotriene pathway in a positive feedback loop. Th2 response is classically seen in asthma, and indeed, expression of PlGF and its downstream genes was increased in respiratory epithelial cells of asthma patients. Like patients with SCD, sickle hematopoietic chimeras developed increased AHR and had higher leukotriene levels; and both were abrogated by anti-PlGF-Ab or zileuton (5-lipoxygenase inhibitor) phenocopying the blunted AHR in PlGF-/- mice, and indicating that sickle erythroid-cells/RBC potentiate AHR. Overall, PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. Both zileuton, licensed for asthma and anti-PlGF-Ab, in clinical trials for cancer, could be promising therapies to reduce the pulmonary morbidity in SCD. Disclosures Jonckx: ThromboGenics: Employment.

Sickle cell disease (SCD) is associated with a high prevalence of airway hyperreactivity (AHR) and chronic inflammation at a steady state in the absence of infection or acute sickle events; SCD patients have also been reported to have increased circulating leukotrienes (LT) (Setty et al, 2002; Jennings et al, 2008): both the pro-inflammatory LT-B4 and cysteinyl leukotrienes (cys-LTs; LT-C4, -D4 and -E4), known to promote AHR. We sought to explore the mechanisms of increased AHR in SCD. Five-lipoxygenase (5LO) is a key enzyme in LT-B4 and cysLT synthesis, we have recently reported that expression of 5LO is significantly increased in blood mononuclear cells (MNCs) in patients with SCD (Patel et al 2009). Zileuton is a specific inhibitor of 5LO that decreases LT production, is licensed for asthma and is now available in BID dosing (Zyflo CR, Cornerstone Therapeutics), but has never been tested in patients with SCD. We performed a phase 1 clinical trial to determine the safety and pharmacodynamics (PK) of twice daily administration of ZL (Zyflo CR, the study drug was provided by Cornerstone Therapeutics) in patients with SCD after obtaining an IND from the FDA. The primary objective of this study was to determine a safe and tolerable dose of ZL using a 3x3 dose escalation design of twice daily ZL for 6 weeks, with monitoring of blood counts, serum and urine chemistries, echo, EKG and clinical events. Patients 7-30 years of age with SCD at steady state that were not on hydroxyurea or chronic transfusions were enrolled. A total of 14 subjects were consented and screened for the ZL trial; 4 failed screening and 11 subjects received the study drug; 2 subjects were withdrawn due to acute sickle events. The 1200 mg BID dose that is approved for asthma was well tolerated by 3 patients; the dose was then escalated to 3000 mg/day. We found that 1800 mg alternating with 1200 mg of ZL was a safe and clinically well tolerated dose in 6 patients. No clinically significant changes of any of the clinical/laboratory safety parameters were observed from baseline values that were attributable to ZL. PK, another primary objective of this study was performed using a D-optimal sampling strategy (day 1 through week 6) and data analyzed by nonlinear mixed effects modeling (NONMEM). ZL plasma concentrations were determined by validated LC-MS/MS assay. The Cmax of ZL were 2445 ± 795 ng/ml and 2510 ± 489 ng/ml at 2400 mg/d and 3000 mg/d and the minimum zileuton exposure as measured by predose troughs were 445.0 ± 137.7 ng/ml and 703.7 ± 172.2 ng/ml at 2400 mg/d and 3000 mg/d respectively. PK details will be presented. The secondary objective was to determine the pharmacodynamics (PD) of ZL in patients with SCD. PD end points were 1. CysLT levels before and after ZL administration for 6 weeks, 2. circulating levels of inflammatory biomarkers: sVCAM, sICAM, IL-6, IL-13 and macrophage chemoattractant protein-1 (MCP-1), 3. Methacholine challenge test (MCT) for AHR and 4. fetal hemoglobin. MCT was negative in 5 of 9 of patients; It is to be noted that exclusion of patients on hydroxyurea and chronic transfusions excluded the majority of patients with moderate to severe SCD that predominantly have AHR; 4 patients had a positive MCT, 2 of them became negative after receiving ZL while there was no change in the other 2 patients. LT and inflammatory markers are under analysis. There was no increase in HbF, F cells/F-retics in any of the patients, unlike results reported in vitro in erythroid cultures (Haynes et al, Blood 2004). Our study shows the safety and PK of ZL in SCD and that a higher dose of ZL (than used for asthma) was safely tolerated by SCD patients with good compliance. This phase I trial may form the basis of a phase II/III trial of zileuton in SCD. Clinicaltrial.gov # NCT01136941. Disclosures: No relevant conflicts of interest to declare.

Abstract 1078 Sickle cell disease (SCD), a chronic hemolytic anemia due to a mutant beta-globin, is associated with prominent inflammatory features with elevated levels of inflammation mediating molecules and an oxidative state, with elevated levels of reactive oxygen species (ROS). Additionally, elevated levels of adenosine (Ado) have recently been shown to play a role in exacerbating sickle cell pathophysiology. However, the etiology of increased Ado is unclear; and presumed to occur secondary to increased ATP release from cellular damage (Xu et al, Nature Medicine, 2010). Herein, we show that increased levels of Ado in the plasma of patients with SCD and the mouse model of SCD [Berkeley sickle (SS) mice] were accompanied by significantly reduced catalytic activity of the Ado degrading enzyme, adenosine deaminase (ADA) in sickle red blood cell (RBCs). ADA activity in Berkeley sickle (SS) RBCs was 246 ± 50 U/L as compared to 570 ± 36 U/L in normal mouse RBCs (derived from Berkeley AA/WT mice), as measured by a color-based enzymatic assay. Notably, despite lower enzyme activity of ADA, immunoblotting assays showed that the levels of ADA protein in sickle RBC were similar, or even slightly higher compared to that in wild type (WT) RBC, suggesting that ADA catalytic activity was impaired in sickle RBCs. In vitro plasma swap experiments, where WT RBC are incubated with sickle plasma and vice versa for an hour, revealed that sickle plasma induced a significant decrease in ADA activity in WT RBC, suggesting that plasma from sickle mice contains a soluble factor that is capable of impairing ADA enzyme activity. Interestingly enough, the decrease in ADA activity correlated inversely with the levels of intracellular RBC ROS in sickle mice. Next we directly assess in vivo the effect of induced oxidative stress on RBC ADA activity in sickle mice by injecting sickle mice with 35umoles/Kg body weight of hemin and saline respectively. Preliminary data suggests a decrease in RBC ADA activity in hemin–injected mice (294 ± 197 U/L) compared to the saline-injected group (346 ± 185.8 U/L), and this data was consistent with a concomitant increase in intracellular RBC and white blood cells ROS levels. We conclude that increase in oxidative stress levels mediates a post translational alteration of ADA catalytic activity and alters Ado metabolism in SCD. Disclosures: No relevant conflicts of interest to declare.