ALBERT

Description: Hydroxyurea and higher hemoglobin F improve the clinical course and survival in sickle cell disease, but their roles in protecting from pulmonary hypertension are not clear. We studied 399 children and adolescents with sickle cell disease at steady state; 38% were being treated with hydroxyurea. Patients on hydroxyurea had higher hemoglobin concentration and lower values for a hemolytic component derived from 4 markers of hemolysis (P ≤ .002) but no difference in tricuspid regurgitation velocity compared with those not receiving hydroxyurea; they also had higher hemoglobin F (P 〈 .001) and erythropoietin (P = .012) levels. Hemoglobin F correlated positively with erythropoietin even after adjustment for hemoglobin concentration (P 〈 .001). Greater hemoglobin F and erythropoietin each independently predicted higher regurgitation velocity in addition to the hemolytic component (P ≤ .023). In conclusion, increase in hemoglobin F in sickle cell disease may be associated with relatively lower tissue oxygen delivery as reflected in higher erythropoietin concentration. Greater levels of erythropoietin or hemoglobin F were independently associated with higher tricuspid regurgitation velocity after adjustment for degree of hemolysis, suggesting an independent relationship of hypoxia with higher systolic pulmonary artery pressure. The hemolysis-lowering and hemoglobin F–augmenting effects of hydroxyurea may exert countervailing influences on pulmonary blood pressure in sickle cell disease.

Description: Abstract 1897 Poster Board I-920 Background: Chuvash polycythemia is caused by homozygosity for the VHL598C〉T mutation, which leads to up-regulation of HIF-1a and HIF-2a in normoxia. As the result, circulating concentrations of erythropoietin are elevated. Chuvash polycythemia patients suffer from cardiovascular abnormalities that include pulmonary arterial hypertension, thrombosis and stroke. Phlebotomy is a common therapy for patients to decrease symptoms such as plethora and headache. However, the outcomes of phlebotomy have not been assessed for these patients. The objective of this analysis is to evaluate the effect of phlebotomy on hemoglobin concentration, serum concentrations of ferritin and erythropoietin, and echocardiographically-determined tricuspid regurgitation velocity, which reflects systolic pulmonary artery pressure. Methods: One hundred twenty patients homozygous for VHL598C〉T and 38 controls of comparable age and gender from Chuvash Republic of the Russian Federation were studied. Clinical and demographic characteristics were determined and echocardiography was performed. Serum ferritin and erythropoietin concentrations were measured by ELISA. Results: The median (interquartile) age for Chuvash polycythemia cases was 36 (22–48) years. They included 68 females (56%). Chuvash polycythemia patients had higher serum erythropoietin concentration (medians of 46 versus 8 mIU/ml, P = 0.0001) and lower serum ferritin concentration (medians of 12 versus 48 ng/ml, P = 0.0001) compared to controls. Tricuspid regurgitation velocity was higher in cases than controls (medians of 2.5 vs. 2.3 m/sec, P = 0.007). Among the cases, 87 (71%) had a history of phlebotomy and 54 of these had phlebotomy within the last year. Phlebotomy was associated with higher erythropoietin concentration (P = 0.033) and lower ferritin concentration (P = 0.024) but no significant difference in hemoglobin concentration (P = 0.9) (Table 1). After adjusting for the effect of age, phlebotomy was associated with significantly higher odds of tricuspid regurgitation velocity ≥2.5 (m/sec) (odds ratio: 3.3; 95% CI: 1.1–9.9). Conclusion: Patients with Chuvash polycythemia tend to mobilize iron stores and increase erythropoietin production to maintain a constant, elevated hemoglobin concentration despite phlebotomy therapy. In this process, estimated pulmonary systolic blood pressure appears to increase. Therefore, phlebotomy therapy might be a risk factor for pulmonary hypertension in the context of Chuvash polycythemia. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1765 Hypoxia may cause pulmonary and brain edema, pulmonary hypertension, aberrant metabolism and early mortality. To better understand pathological processes associated with hypoxia, we examined gene expression in Chuvash polycythemia blood mononuclear cells. Chuvash polycythemia is a congenital disorder of up-regulated hypoxic response at normoxia wherein VHLR200W homozygosity leads to elevated hypoxia inducible factor (HIF)-1 and HIF-2 levels, thromboses, pulmonary hypertension, lower systemic blood pressure (SBP) and increased mortality. VHLR200W homozygotes are often treated by phlebotomy resulting in iron deficiency, allowing us to evaluate an interaction of augmented hypoxia sensing with iron deficiency. Expression profiling of 8 VHLR200W homozygotes and 17 VHL wildtype individuals, matched for normal iron status as reflected in serum ferritin concentration, revealed altered regulation of 3069 genes at false discovery rate 1.5-fold change in expression level, mostly (74%) an increase. Seven showed a 〉2-fold increase: CA1 (carbonic anhydrase), SELENBP1 (selenium binding protein 1), IL1B (interleukin 1 beta), SLC4A1 (solute carrier family 4 member 1), HBB (hemoglobin beta), and AHSP (alpha hemoglobin stabilizing protein). Additional studies including 16 VHLR200W homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHLR200W for 51 of the 847 upregulated genes and suppressed the induction effect for 108 of the upregulated genes. Genes further upregulated by iron deficiency included CA1, CSDA (cold shock domain protein A), BCL2L1 (BCL-2 like 1), BPGM (2,3-bisphosphoglycerate mutase), DCAF12 (DDB1 and CUL4 associated factor 12), FECH (ferrochelatase), SELENBP1 and SLC4A1. Genes for which iron deficiency suppressed the induction included inflammatory and immune pathway genes such as CASP5, CXCL16, IFI30, IFI35, IRF5, LILRB1, NOD2, RELT, TCIRG1 and TNFAIP2. A number of the genes with altered regulation in VHLR200W homozygotes might modify risk of thrombosis (upregulated: F3, SERPINE1, SERPINB2, SERPING1, PLAUR, THBD; down regulated: SERBP1), elevated systolic pulmonary artery pressure (upregulated: HTR1B, THBS1; downregulated: S1PR1, STIM2), or benign hemangioma (downregulated: CCM2). However, expression of these genes tended not to be influenced by iron status. VEGF was induced in VHLR200W homozygotes and surprisingly this induction was suppressed by iron deficiency. Expression relationships suggested a broad effect of VHLR200W in reducing systolic blood pressure through inducing VEGF. We demonstrate that many genes have commensurate changes of their expression by both iron deficiency and VHLR200W associated normoxic up-regulation of HIFs, as expected. However, there are genes that are regulated asynchronously. Further research is needed to define the molecular bases of separate regulation of genes by HIFs and iron status and to define relative risks and benefits of therapeutic phlebotomy for polycythemia. The resulting elucidation of the genomic pathways affecting predisposition to thromboses, pulmonary hypertension, lower systolic blood pressure and the interaction of augmented hypoxia sensing with iron deficiency should have broad implications leading to a better understanding of the pathophysiology of many diseases and the development of targeted therapies. Disclosures: No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) is a characterized by hemolysis, vaso-occlusion and ischemia. Several previous studies pointed to a possibility that SCD patients might be protected from HIV-1 infection. These studies described low prevalence of anti-HIV-1 antibodies in SCD patients transfused with potentially HIV-1 infected blood;1 higher number of long-term non-progressors among HIV-1 infected SCD patients, 2 and a lower frequency of HIV diagnosis among SCD patients (odds ratio 0.33).3 This study aims to decipher a mechanism of HIV-1 restriction in PBMC from SCD patient infected with HIV-1 ex vivo. HIV-1 replication in SCD PBMC was inhibited at the level of reverse transcription and transcription implicating the involvement of post-entry and transcription restriction factors. SAM domain and HD domain-containing protein 1 (SAMHD1) restricts HIV-1 infection in in myeloid cells. 4,5 by reducing intracellular nucleotide pool and blocking reverse transcription. SAMHD1 phosphorylation on Thr-592 by CDK2 or CDK1 inactivates it and prevents HIV-1 inhibition. We showed that SAMHD1 phosphorylation was reduced in SCD PBMCs and in hemin-treated promonocytic THP-1 cells. Moreover, knock-down of SAMHD1 prevent hemin-mediated inhibition of HIV-1 in THP-1 cells. We also detected a reduction of CDK2 activity in SCD PBMCs and in hemin-treated THP-1 cells which can explain reduced SAMHD1 phosphorylation. Previously, we showed that CDK2 activity is inhibited when intracellular iron levels are depleted by iron chelators. We observed reduced intracellular labile iron levels and increased expression of iron export protein, ferroportin and HIF-1α in SCD PBMCs. Importantly, treatment of SCD PBMCs with hepcidin alleviated HIV-1 inhibition. Unaltered hepcidin levels in plasma of SCD patients suggest that ferroportin expression is sustained in SCD PBMC. Our study points out to ferroportin as upstream regulator of SAMHD1 and links a reduction in iron levels, inhibition of CDK2 activity and a decrease in SAMHD1 phosphorylation to the inhibition of HIV-1 infection in SCD. Acknowledgments. This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Literature 1. Castro O, Saxinger C, Barnes S, Alexander S, Flagg R, Frederick W. Prevalence of antibodies to human immunodeficiency virus and to human T cell leukemia virus type I in transfused sickle cell disease patients. J Infect Dis. 1990;162(3):743-745. 2. Bagasra O, Steiner RM, Ballas SK, et al. Viral burden and disease progression in HIV-1-infected patients with sickle cell anemia. Am J Hematol. 1998;59(3):199-207. 3. Nouraie M, Nekhai S, Gordeuk VR. Sickle cell disease is associated with decreased HIV but higher HBV and HCV comorbidities in US hospital discharge records: a cross-sectional study. Sex Transm Infect. 2012. 4. Hrecka K, Hao C, Gierszewska M, et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature. 2011;474(7353):658-661. 5. Laguette N, Sobhian B, Casartelli N, et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature. 2011;474(7353):654-657. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2869 Chuvash polycythemia (CP) is characterized by homozygosity for the R200W mutation in the von Hippel Lindau gene (VHL). This rare genetic disorder causes elevated levels of hypoxia inducible factor (HIF)-1 and HIF-2 that trigger constitutive hypoxia responses at normoxia. Hypoxia is a recognized cause of pulmonary hypertension. We recently reported that systolic pulmonary artery pressure (SPAP) estimated by echocardiography-determined tricuspid regurgitation velocity (TRV) was elevated in 120 CP patients compared to 31 Chuvash controls (P = 0.005), and that increasing age (P = 0.001), increasing systemic pulse pressure (P = 0.003) and lower serum ferritin concentration (P = 0.009) were independent predictors of higher estimated SPAP in CP patients [Sable et al., 2012]. In this study, we profiled gene expression for 16,642 genes in peripheral blood mononuclear cells (PBMCs) derived from a cohort of 43 CP patients, and measured the TRV for these individuals. Based on a prospectively chosen criterion of TRV ≥2.5 m/sec, 20 patients were classified as having elevated level of estimated SPAP and 23 were normal. Gene expression level between these two SPAP groups appeared to be homogenous. However, we identified 4777 genes at false discovery rate (FDR)

Description: Background Hypoxia and low iron induce hypoxia-induced factor 1(HIF-1) by stabilizing its alpha subunit and deregulate HIV-1 which transcription and several other steps of life cycle depend on cellular iron [1]. HIV-1 transcription is inhibited at low oxygen levels and reduced cellular iron through deregulation of CDK9/cyclin T1 and CDK2/cyclin E. Sickle cell disease has low odds of ratio for HIV-1 infection [2]. Sickle cell disease (SCD) leads to hemolytic anemia which results in local ischemia and release of heme. Induction of heme oxygenase-1 (HO-1) by hemin was shown to inhibit HIV-1 [1], although the mechanism of the inhibition was not clarified. Iron depletion by iron chelators or through the expression of ferroportin, an iron export protein, inhibits CDK2 and CDK9 activities and blocks HIV-1 transcription [1]. Because neither CDK2 nor CDK9 require iron for the enzymatic activity, we analyzed the expression of hypoxia and iron –dependent factors that may deregulate HIV-1 infection in SCD. Results Expression profiling followed by real-time PCR analysis showed induction of HO-1, p21, Erg-1, IKBα, HIF-1 and ferroportin mRNA and decrease of hepcidin mRNA in PBMCs from SCD patients. HIV-1 replication was reduced in SCD PBMCs comparing to normal controls, and also in THP1 cells treated with hemin. Subsequent treatment with hepcidin restored HIV-1 replication in SCD PBMC and in hemin-treated THP-1 cells, suggesting that ferroportin played a key role in the HIV-1 inhibition in these settings. Stable ferroportin knock down in THP-1 cells led to the inability of hemin to inhibit HIV-1, suggesting that ferroportin played a key role in the heme-meidated HIV-1 inhibition. Stable HIF-1a knockdown in promonocytic THP1 cells increased HIV replication suggesting that HIF1α is a restriction factor for HIV-1. Iron chelators induced the expression of IKBα, an inhibitor of NF-kB and also induced the expression of HIF-1 and p21. Iron chelators also inhibited enzymatic activity of CDK2 and shifted CDK9/cyclin T1 from the large to the small complex making it unavailable for HIV-1 Tat recruitment. Hemin treatment induced expression of HO-1, ferroportin, IkBα, HIF1α and p21 thus mimicking the effect of iron chelators. Conclusions Hemolytic conditions of sickle cell disease upregulate hypoxia and iron regulatory pathways leading to refraction of HIV-1. Targeting cellular iron, ferroportin and HO-1 may lead to novel anti-HIV-1 therapeutics. Acknowledgments This project was supported by NIH Research Grants 1SC1GM082325, 2G12RR003048, and P30HL107253. References 1. Nekhai S, Kumari N, Dhawan S: Role of cellular iron and oxygen in the regulation of HIV-1 infection. Future Virol 2013, 8(3):301-311. 2. Nouraie M, Nekhai S, Gordeuk VR: Sickle cell disease is associated with decreased HIV but higher HBV and HCV comorbidities in U.S. hospital discharge records: a cross-sectional study. Sex Transm Infect 2012, 88(7):528-533. Disclosures: No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) is associated with pleiotropic clinical outcomes, the severity of which exhibits remarkable inter-individual variability. Investigations of the pathophysiology of SCD have focused on the adverse effects of vaso-occlusion, chronic inflammation, and hemolysis. SCD is also characterized by up-regulation of the hypoxic response to chronic anemia. We postulated that the up-regulated hypoxic response in SCD contributes to altered gene expression that might impact pulmonary hypertension, a complication associated with early mortality. To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 sickle cell anemia untreated with hydroxyurea and 15 Chuvash polycythemia (CP) patients characterized by homozygous VHLR200W-induced constitutive up-regulation of hypoxia inducible factors in the absence of anemia or hypoxia. Gene expression of both cohorts was profiled on identical Affymetrix exon arrays. The degree and direction of differential gene expression were highly correlated between sickle cell anemia and CP (Spearman’s ρ = 0.73 between regression coefficients of differential gene expression), suggesting that 53% of expression variation in sickle cell anemia is related to hypoxic transcriptional responses. At 5% false discovery rate (FDR), 1040 genes exhibited a 〉1.15 fold change in both sickle cell anemia and VHLR200W homozygotes, among which 297 were up-regulated and 743 down-regulated. Hypoxia strongly induced inflammatory response pathways but suppressed T-cell activation in sickle cell anemia. MAPK8, encoding a mitogen-activated protein kinase important for stress-induced apoptosis, T-cell differentiation and inflammatory responses, was a hypoxia down-regulated gene and played a central role in hypoxic gene regulation in sickle cell anemia according to gene network analysis. To assess the genetic contribution to hypoxic transcriptional variation among SCD patients, we mapped expression quantitative trait loci (eQTL) for the 1,040 hypoxia response genes. Association mapping with a focus on local regulatory polymorphisms in 61 SCD patients identified eQTL for 103 of the hypoxia response genes at 5% FDR. We further tested the hypothesis that these hypoxic eQTL potentially underlie heterogeneity in risk of pulmonary hypertension in an additional SCD cohort (University of Illinois cohort). In this cohort, the A allele of an eQTL of MAPK8, rs10857560, was associated with pre-capillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency=0.66; OR=4.4, P=0.00037, n=238). This association was confirmed in another independent cohort (Walk-PHaSST cohort) (allele frequency=0.65; OR=7.2, P=0.0025, n=519). The homozygous AA genotype of rs10857560, which was associated with decreased MAPK8 expression, was present in all 14 identified pre-capillary pulmonary hypertension cases among the combined 757 SCD patients (P = 6 x 10-6 by the Fisher exact test). Our study demonstrates a prominent hypoxic transcription component in SCD that in part contributes to pre-capillary pulmonary hypertension. Disclosures: No relevant conflicts of interest to declare.