Pulmonary Hypertension In Sickle Cell Disease Research Articles

5612390 CORRELATION OF DOPPLER DEFINED ELEVATED PULMONARY PRESSURE WITH LABORATORY PARAMETERS IN ADULT PATIENTS WITH SICKLE CELL DISEASE - A RETROSPECTIVE STUDY

Background: Pulmonary hypertension (PH) is a relatively common complication and known cause of death in adult patients with sickle cell disease (SCD). Trans-thoracic Echocardiography is a non-invasive way to screen SCD patients for elevated pulmonary pressure. An estimation of the pulmonary artery systolic pressure can be made via assessment of the Tricuspid jet regurgitation velocity(TRV). TRV greater than or equal to 2.5 m/sec corresponds to an estimated Pulmonary Artery Systolic pressure of >35 mm Hg, which is approximately two standard deviations above normal 1.The prevalance of elevated pulmonary pressure in adults with SCD is 30-40%, while the prevalance of PH in SCD patients utilizing right heart catheterization (RHC) is between 6-10.5% 2. A major risk factor for PH in sickle cell disease is the severity of hemolytic anemia 3. The level of N terminal fragment of brain natriuretic peptide (NT ProBNP) correlates with the degree of pulmonary vascular resistance in SCD patients and may be an indirect reflection of the end organ damage 4. Aims: To analyse the prevalence of Doppler defined pulmonary hypertension in adult patients with Sickle cell Disease visiting our centre. To correlate the severity of hemolysis using laboratory parameters and NT-ProBNP with the severity of PH in these patients Methods: 92 patients with SCD underwent baseline clinical evaluation, laboratory testing, 2D echocardiography and NT-ProBNP analysis in our Hospital from November 2020 - December 2021. TRVmax of ≥2.5m/s, was used as a surrogate marker of Pulmonary Hypertension. The severity of PH was correlated with the degree of hemolysis and NT Pro BNP levels. Results: IBM SPSS Statistics 24.0 was used for Statistical Analysis. Doppler-defined pulmonary hypertension was found among 29 out of 92 patients denoting a prevalence of 31.5%. The prevalence among males was 20.9% and that among females was 40.8%. TRV max had significant positive correlation with NT-ProBNP(r=0.310,p<0.01) and with markers of hemolysis like LDH (r=0.322,p<0.01)and Reticulocyte count(r=0.212,p<0.05). Serum Ferritin(r=0.233,p<0.05),Hemoglobin S(%)(r=0.374,p<0.01) and Left Ventricular ejection fraction (LVEF) (r=0.352,p<0.01) also had significant correlation with TRV Max. Patients with high serum ferritin and HbS percent had higher TRV max while LVEF was low in those with high TRVmax. TRV Max also exhibited significant negative correlation with Hemoglobin (r=- 0.340, p<0.01). Summary: In our study, the prevalence of doppler - defined Pulmonary hypertension in adults with sickle cell disease was 31.5%. These patients had a positive correlation with markers of hemolysis and levels of NT-Pro BNP. Hence, we conclude, transthoracic echocardiography and NT proBNP can be used as a screening tool and surrogate test to Right heart catheterisation in order to diagnose PH in SCD patients in resource constrained settings.Both these tests have a high sensitivity in screening SCD patients with PH and having the advantage of being non-invasive procedure.

Increased Hemoglobin Affinity for Oxygen with GBT021601 Reduces Pulmonary Hypertension and Lung Vascular Inflammation in a Murine Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin gene coding sequence that results in the production of hemoglobin S (HbS). Under conditions of red blood cell (RBC) deoxygenation, HbS polymerizes and leads to RBC sickling, resulting in hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Despite improvements in treatment options, SCD remains a debilitating condition, and most patients experience progressive end-organ damage with associated complications and repeated hospitalizations. Pulmonary hypertension (PH), which is associated with increased mortality, is a common complication in patients with SCD, with nearly 10% of the SCD population reporting PH. PH in patients with SCD is multifactorial and may be caused by hemolysis, nitric oxide scavenging, and/or tissue hypoxia. Pharmacological increase of the concentration of oxygenated HbS (oxyHbS) in RBCs by voxelotor, a first-generation HbS polymerization inhibitor, reduces RBC sickling and preserves oxygen (O2) delivery by reducing anemia and improving blood rheology. Limited data are currently available on whether inhibiting HbS polymerization by increasing oxyHbS concentration may improve end-organ damage and associated complications, including PH. Objective: To examine the effects of GBT021601, a small-molecule, investigational, second-generation HbS polymerization inhibitor, on hemoglobin (Hb)-O2 affinity, PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Methods: Townes HbSS mice treated with GBT021601 (75 mg/kg orally once daily for 14 days) were exposed to acute hypoxia (8% O2) for 1 hour and reoxygenated for 72 hours. Untreated HbAA Townes mice were used as controls. Lung inflammation was assessed in terms of accumulation of inflammatory cells, activation of nuclear factor kappa B subunit 1, and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. To evaluate the impact of GBT021601 treatment on Hb-O2 affinity and anemia, p50 (partial O2 pressure at which Hb is 50% saturated with O2) and hematocrit levels were measured in SCD mice. In vivo pulmonary hemodynamic measurements included pulmonary artery pressure and right atrial pressure, whereas right ventricular hypertrophy was assessed using the Fulton index, which is the ratio of right ventricular weight to the weight of the left ventricle plus septum. Results: The total number of inflammatory cells and the specific numbers of lymphocytes, macrophages, and neutrophils were lower in untreated HbAA Townes mice and in HbSS Townes mice treated with GBT021601 compared with vehicle-dosed HbSS mice (Figure, panel A). Chronic treatment with GBT021601 increased Hb-O2 affinity, reducing the p50 in SCD mice from 32 mm Hg to 14 mm Hg. The treatment significantly reduced anemia, increasing hematocrit from 27.2% in the vehicle-dosed HbSS mice to 38.5% in the mice treated with GBT021601. Chronic GBT021601 treatment in HbSS mice also reduced pulmonary artery and right atrial pressure compared with those in untreated HbSS mice. Additionally, GBT021601 reduced right ventricular hypertrophy in HbSS mice, as evidenced by a reduction in the Fulton ratio (Figure, panel B). Conclusions: GBT021601 reduced PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Our findings suggest that reducing RBC sickling, anemia, and hemolysis by increasing HbS affinity for O2 plays a critical role in modulating the immune system in PH and lung vasculature. These findings highlight the potential therapeutic efficacy of GBT021601 based on its ability to increase HbS affinity for O2 and prevent HbS polymerization and RBC sickling to reduce PH in SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeted Proteomics of Pulmonary Hypertension in Sickle Cell Disease

BACKGROUND AND AIMHemolysis, inflammation and coagulopathy associated with sickle cell disease (SCD) can lead to pulmonary hypertension. More than 15% of adult patients with SCD are affected by pulmonary hypertension as measured by a tricuspid regurgitation velocity (TRV) ≥ 2.9 m/sec (Nouraie, M. et al., Validation of a composite vascular high-risk profile for adult patients with sickle cell disease. American Journal of Hematology, 94:E312-E314. 2019). Moreover, sickle cell disease patients with pulmonary hypertension have a much higher mortality risk than those without pulmonary hypertension. There is little knowledge on the proteomic profile of pulmonary hypertension in SCD. Our aim was to conduct a proof-of-concept study to explore targeted serum protein biomarkers that are differentially expressed in SCD patients with elevated tricuspid regurgitation velocities and explore their corresponding pathways. MATERIAL AND METHODSData from the Walk-PHaSST study was used for this study. The Walk-PHaSST was a multicenter study to assess the effect of sildenafil for SCD patients with pulmonary hypertension (Gladwin, M. T. et al., Risk factors for death in 632 patients with sickle cell disease in the United States and United Kingdom. PLoS One, 9:e99489. 2014). In the screening phase of the study, 720 patients with SCD were recruited. Study participants underwent clinical and lab examinations, as well as an echocardiography. We selected two groups of patients: one group of patients with a TRV≤2.6 m/sec and another with TRV≥2.9 m/sec (N =35 in each). The serum concentration of 92 protein biomarkers was measured using an OLINK cardiovascular panel. This panel assess a range of biological process including inflammatory response, angiogenesis, cell adhesion, coagulation, and response to hypoxia. T-tests were performed between the aforementioned groups for each of the serum biomarkers. The Hochberg method was used to calculate the false discovery rate. A volcano plot was created using the Bonferroni correction. Finally, we tested the correlation of the differentially expressed biomarkers with clinical variables measured in blood samples of the participants. These variables include white blood cell (WBC), neutrophil count, thrombospondin-1 (TSP as measure of coagulation), PIGF and VEGF (growth factors involved in angiogenesis), and NT-proBNP (elevated in pulmonary hypertension). RESULTSUsing a false discovery rate of 0.01, we discovered 14 significantly expressed proteins. Six of them passed a Bonferroni corrected overall critical p value < 0.00054 (Figure 1). These included T-cell surface glycoprotein (CD4), lymphotactin (XCL1), SLAM family member 7 (SLAMF7), galectin-9 (GAL9), TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), and tumor necrosis factor receptor superfamily member 11A (TNFRSF11A). We observed up to a 1.2-fold increase in these 6 protein biomarker levels in the high TRV groups.The correlogram (Figure 2) indicated that there is a slightly positive correlation between WBC and CD4, GAL9, TRAILR2, and TNFRSF11A (r > 0.20). For neutrophil count, we observed significantly negative correlation with selected markers including TNFRSF11A levels (r = -0.51), GAL9 (r = -0.35), XCL1 (r = -0.27), SLAMF7 (r = - 0.21), and CD4 (r < - 0.24). A significantly negative correlation between TSP and GAL9 levels (r = -0.31) was also observed. Finally, we observed a strong, positive correlation between all proteins and serum NT-proBNP levels (r > 0.44). CONCLUSIONCirculatory protein markers of immune response and coagulation are highly expressed in SCD patients with elevated TRV. This provides evidence that these protein biomarkers have potential to be utilized as prognostic markers for pulmonary hypertension in patients with SCD. [Display omitted] DisclosuresLittle: Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Patents & Royalties. Gibbs: Acceleron Pharma: Consultancy, Other: lecture fees; Actelion: Consultancy, Other: lecture fees; Aerovate: Consultancy, Other: lecture fees; Bayer: Consultancy, Other: lecture fees; Compexia: Consultancy, Other: lecture fees; Janssen: Consultancy, Other: lecture fees; MSD: Consultancy, Other: lecture fees ; Pfizer: Consultancy, Other: lecture fees; United Therapeutics: Consultancy, Other: lecture fees. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Nouraie: Phoenicia BioScience Inc.: Consultancy.

Pulmonary Hypertension in Sickle Cell Disease: The Role of Activated Blood Coagulation and Inflammation.

Introduction: Pulmonary hypertension (PHT) is a common complication of sickle cell disease (SCD). The pathogenesis of PHT in this setting is likely multifactorial. There is an abundance of evidence to suggest that the coagulation system is activated in patients with SCD. In addition, autopsy series have shown that an in situ thrombotic arteriopathy is seen in the majority of SCD patients with PHT. The purpose of this study is to determine whether markers of thrombin generation, platelet activation and inflammation are associated with PHT in SCD patients.Methods: This cross-sectional study was performed using a cohort of patients followed at the Sickle Cell Clinic at UNC-Chapel Hill. The pulmonary artery systolic pressure (PASP) was determined with a Doppler echocardiogram by applying the modified Bernoulli equation (PASP = 4V2 + right atrial pressure). Pulmonary hypertension was defined using an age-, sex- and BMI-adjusted reference range. Laboratory studies, including a complete blood count, soluble CD40 ligand [sCD40L] (a marker of inflammation and platelet activation) and markers of thrombin generation (thrombin-anti-thrombin complex [TAT], prothrombin fragment 1+2 [F1+2], and D-dimers) were obtained on all SCD subjects and on six healthy, race-matched control subjects without SCD. Wilcoxon Rank Sum Tests were used to compare median values in SCD patients with and without PHT, as well as between SCD patients and the control individuals.Results: Of the 76 subjects with SCD (SS = 56, SC = 9, Sβ0 = 4, Sβ+ = 7), PHT was found in 26 (34%). Compared with SCD patients without PHT, those with PHT had higher WBC counts (10.4 X 103/μL vs. 8.7 X 103/μL; p = 0.05). In addition, while PHT patients had consistently higher levels of such parameters as: D-dimers (1603 ng/mL [FEU] vs. 1233 ng/mL [FEU]; p = 0.3), TAT (7.05 μg/L vs. 5.7 μg/L; p = 0.8) and F1+2 (1.15 nmol/L vs. 0.9 nmol/L; p = 0.3), they failed to achieve statistical significance. On the other hand, the sCD40L level was lower (6.7 ng/mL vs. 7.6 ng/mL; p = 0.6) in the PHT group, although this difference was also not significant. In a second analysis, SCD patients had significantly higher levels of D-dimers (1251.5 ng/mL [FEU] vs. 318 ng/mL [FEU]; p = 0.02), sCD40L (7.3 ng/mL vs. 2.8 ng/mL; p = 0.0003), WBC count (9.2 X 103/μL vs. 7.1 X 103/μL; p = 0.02) and platelet count (399 X 103/μL vs. 251 X 103/μL; p = 0.002) when compared to control individuals. The TAT and F1+2 levels were also higher in SCD patients, although the differences were not significant.Conclusion: Compared to controls, SCD patients have increased thrombin generation, platelet activation and inflammation. In our cohort, SCD patients with PHT have higher levels of various markers of thrombin generation compared to patients without PHT. The lack of statistical significance is likely a result of the baseline elevation of these markers in SCD patients in steady state and as such, much higher increases in the levels of these markers and/or much larger sample sizes would be required to reveal a significant difference between both groups. This evidence of further activation of coagulation in SCD patients with PHT provides a rationale for clinical trials exploring the use of anticoagulation therapy in these patients.

Circular RNA Expression in the Lungs of Sickle Cell Disease Mice

Rationale and Objective Circular RNA (circRNA), a novel class of endogenous non-coding RNA, can regulate gene expression through binding to miRNAs (sponge effect). Pulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) that causes significant morbidity and mortality. The role of circRNAs in SCD-PH pathogenesis has not been defined. We sought to explore the expression profiles of circRNAs and mRNAs in lungs of sickle cell (SS) mice that spontaneously develop PH. Methods and Results Total RNAs isolated from the lungs of 5 SS male mice and 5 littermate control (AA) mice (15-17 weeks old) were subjected to circRNA expression profiling using the Arrystar™ mouse circRNA array V2 and mRNA expression profiling using Arraystar™ mouse array. For circRNA analysis, Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Volcano plot filtering was used to screen for differentially expressed (DE) circRNAs with statistical significance (Fold Change >1.5, p< 0.05). 21 circRNAs were upregulated (Up) and 127 circRNAs were downregulated (Dn) in lungs of SS mice compared to AA mice. For mRNA analysis, raw signal intensities were normalized in quantile method by GeneSpring GX software (Agilent Technologies). Volcano plot filtering was used to screen for DE mRNAs with statistical significance (Fold Change >2.0, p< 0.05). 1,753 mRNAs were upregulated and 1,455 mRNAs were downregulated in lungs of SS mice compared to AA mice. Database for Annotation, Visualization and Integrated Discovery bioinformatics analysis was used for Gene Ontology (GO) enrichment including biological process (BP), molecular function (MF), and cellular component (CC), and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database. GO analysis showed that the altered mRNAs were mainly distinct to negative (Up) or positive (Dn) regulation of biological processes in BP, ion binding (Up) or organic cyclic compound binding (Dn) in MF, and membrane-bounded organelle and vesicle (Up) and non-membrane-bounded organelle and intracellular organelle (Dn) in CC. Pathway analysis revealed that MAPK signaling pathway was significantly associated with upregulated mRNAs, whereas C-type lectin receptor signaling pathway was associated with downregulated mRNAs. circRNA-mRNA co-expression network analysis was evaluated by Coding-Non-coding gene Co-expression network analysis and generated by Cytoscape software (The Cytoscape Consortium). Conclusions These results identify new potential targets for therapeutic intervention in SCD and provide new insights into SCD pulmonary vascular dysfunction and PH pathogenesis.

Pathophysiology of Pulmonary Hypertension in Sickle Cell Disease

The pathophysiology of pulmonary hypertension (PH) in sickle cell disease (SCD) is multifactorial: hemolysis, hypercoagulability, hypoxemia, oxidative stress, platelet activation, increased adhesiveness, inflammatory cell activation and genetic susceptibility, all contributing in varying degrees to endothelial dysfunction. Intravascular hemolysis is the main pathological process contributing to vasculopathy by releasing toxic red blood cell products that impair endothelial function, cause hypercoagulable state and drive oxidative and inflammatory stress. Hemolysis induced nitric oxide imbalance is one the most important contributors to high pulmonary artery pressures seen in SCD. Multi-faceted, targeted interventions, before irreversible vasculopathy develops, will allow for improved patient outcomes and life expectancy, stressing the need for a better understanding of the multiple pathophysiological mechanisms involved in the development of PH before considering those patients for targeted therapies. Hemolysis is still considered as the main contributor of PH in SCD but the mechanisms by which it causes PH are still not completely known. This review precisely presents the various pathophysiological mechanisms and factors that have been proposed till date to help the reader get an overview.

Impact of Pulmonary Hypertension on in-hospital outcomes amongst Sickle Cell Disease hospitalizations

Introduction: Pulmonary hypertension (PH) is a common and severe complication of Sickle Cell Disease (SCD), and an independent risk factor for mortality. While there is a clear association between SCD and PH, the predictors of PH in SCD and the impact of PH on in-hospital outcomes of SCD hospitalizations remains unknown. In our study, we sought to assess the in-hospital prevalence, predictors, and the impact of PH in SCD hospitalizations. Methods: We used the 2016 and 2017 National Inpatient Sample (NIS) to identify all adult hospitalizations with a primary discharge diagnosis of SCD. The sample was then stratified based on the presence or absence of PH. We used the Pearson chi-square test and weighted Student's t-test to analyze categorical and continuous variables, respectively. Multivariate logistic regression analysis was performed to calculate the adjusted odds ratio for various clinical outcomes. SAS was used for the analysis, and the p-value was defined as &amp;lt;0.05. Results: We identified n=191,080 weighted hospitalizations for SCD, of which, 5.54% (n=10590) had concomitant PH. Female gender and comorbidities including hypertension, obesity, illicit drug use, hepatic cirrhosis, renal failure, prior venous-thromboembolism, valvular, and congenital heart disease were identified as significant predictors of PH in SCD. PH was associated with increased in-hospital mortality (1.04% vs 0.22%, AOR=2.14, 95% CI 1.15-3.98, p=0.0158). PH in SCD hospitalizations also increased the odds of - acute kidney injury (AKI), need for dialysis, acute respiratory failure (ARF), and need for mechanical ventilation for &amp;gt; 96 hours. The adjusted odds ratio for venous thromboembolism, shock-state, and the need for cardiac catheterization (both right and bilateral) were also higher in patients with PH. Overall hospitalization cost and length of stay increased (7.06±0.16 vs 1.82±0.02 days) in patients with SCD and PH (see table 1). Conclusion: In sickle cell disease hospitalizations, PH is independently associated with increased in-hospital morbidity and mortality, with an increased need for in-hospital catheterizations thereby, prolonging the length of stay and overall health care costs. Identifying and treating PH in the SCD population would improve in-hospital outcomes. Disclosures No relevant conflicts of interest to declare.

Echocardiographic evaluation of patients with sickle cell disease. A study based on Etendar Cohort

Abstract Background Echocardiography is the main non-invasive screening tool for pulmonary hypertension (PH) in Sickle Cell Disease (SCD), but relies mostly on the tricuspid regurgitation velocity (TRV) assessment, without distinction between pre and postcapillary patterns. Purpose Using Etendard Cohort data, the aim of this study was to refine echocardiographic assessment of PH in SCD patients. Methods The French Etendard Study is a prospective cohort initially designed to assess the prevalence of PH among 398 SCD participants. We analyzed echocardiography data of the 96 Etendard patients who underwent Right Heart Catheterization (RCH), because of a TRV≥2.5m/s, and compared them to invasive haemodynamic measurements. Based on RHC results, patients were classified as follows: Absence of pulmonary hypertension if mean pulmonary arterial pressure (mPAP) was &amp;lt;25mmHg. Precapillary PH if mPAP≥25mmHg and pulmonary-capillary wedge pressure (PCWP) ≤15mmHg. Postcapillary PH if mPAP≥25mmHg and PCWP &amp;gt;15mmHg. Results PH was found in 24/96 patients with 11 precapillary and 13 postcapillary. In addition to TRV, multivariate analysis identified indexed left atrial volume (LAVind) and lateral E' wave velocity (E'lat) as independent echocardiographic predictors of PH (OR=1.06 and 0.6 respectively, p&amp;lt;0.01). LAVind and E'lat showed good correlation with mPAP (R=0.51 and R=0.40 respectively and p&amp;lt;10–3 for both) and had good accuracy to predict PH with an optimal cut-off of 48ml/m2 for LAVind (AUC=0.78, Se=81% and Sp=75%) and 12cm/s for E'lat (AUC=0.8, Se=72% and Sp=79). Using the association of a TRV≥3m/s or a TRV [2.5; 2.9] m/s with LAVind&amp;gt;48ml/m2 and E'lat&amp;lt;12cm/s, we could predict PH with a PPV of 68% and NPV of 90%. Moreover, comparing echocardiography data of pre and post-capillary PH patients, we observed that Pulmonary acceleration time (PAcT) was different in the two groups (104±22ms vs. 160±21ms, p&amp;lt;0.001) with an excellent accuracy for the differentiation of both phenotypes (AUC=0.95, optimal cut-off=115ms, Se=100% and Sp=78%). PAcT correlated with pulmonary vascular resistance (PVR) assessed by RHC (R=0.34, p=0.001) but not with mPAP (R=0.1, p=0.2) and a PAcT&amp;gt;115ms ruled out pre-capillary PH with a NPV of 97%. Conclusion Echocardiography can accurately estimates the probability of PH in SCD patients with an integrated approach using TRV, LAVind and E'lat. In addition, among patients with suspected PH, measuring PAcT as a PVR surrogate allows a good differentiation between pre and post-capillary phenotypes. We propose a new echocardiographic algorithm for the diagnosis of PH in SCD that could be a true assistance in referring patients for RHC. TTE Algorithm for PH in SCD Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Funded by the French Ministry of Health and Assistance Publique–Hôpitaux de Paris

Integrative Analysis of Long Non‐Coding RNAs and mRNAs in Lungs of Sickle Cell Mice

RationalePulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) and causes significant morbidity and mortality. Emerging evidence indicates that long non‐coding RNAs (lncRNAs) play a pivotal role in biological processes such as, cellular proliferation, differentiation, and apoptosis. However, the role of lncRNA and mRNA interaction in SCD‐PH pathogenesis has not been defined. We sought to evaluate the expression profiles of lncRNAs and mRNAs in lungs of sickle cell (SS) mice that spontaneously develop PH.Methods and ResultsTotal RNAs were isolated from lungs of SS mice and littermate control (AA) mice at age 15–17 weeks and subjected to lncRNA and mRNA expression profiling using the Arrystar™ lncRNA array (Rockville, MD). Raw signal intensities were normalized in quantile method by GeneSpring GX software (Agilent Technologies, Santa Clara, CA). Volcano plot filtering was used to screen for differentially expressed (DE) lncRNAs and mRNAs with statistical significance (Fold Change &gt;2.0, p&lt; 0.05). To validate DE lncRNAs and mRNAs, 5 lncRNAs and 5 mRNAs were selected for quantitative real‐time PCR (qRT‐PCR). 2302 lncRNAs and 1753 mRNAs were upregulated (Up) and 2546 lncRNAs and 1455 mRNAs were downregulated (Dn) in lungs of SS mice compared to AA mice. Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics analysis was used for Gene Ontology (GO) enrichment including biological process (BP), molecular function (MF), and cellular component (CC), and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. GO analysis showed that the altered mRNAs were mainly distinct to negative (Up) or positive (Dn) regulation of biological processes in BP, ion binding (Up) or organic cyclic compound binding (Dn) in MF, and membrane‐bounded organelle and vesicle (Up) and non‐membrane‐bounded organelle and intracellular organelle (Dn) in CC. Pathway analysis revealed that malaria and MAPK signaling pathway were significantly associated with upregulated mRNAs, whereas C‐type lectin receptor signaling pathway, focal adhesion, and JAK‐STAT signaling pathway were related with downregulated mRNAs. LncRNA‐mRNA co‐expression network analysis was evaluated by Coding‐Non‐coding gene Co‐expression network (CNC network) analysis and generated by Cytoscape software (The Cytoscape Consortium, San Diego, CA).ConclusionsThese results identify potential new targets for therapeutic intervention in SCD and provide new insights into SCD pulmonary vascular dysfunction and PH pathogenesis.Support or Funding InformationNIH HL133053 (BYK)

Study of pulmonary hypertension as a complication of sickle cell disease

Background: Sickle cell anaemia is the most common inherited hematological abnormality across human race, particularly prevalent in pockets of central India and pulmonary hypertension (PH) supposedly worsens prognosis. Data from central India is lacking though. Present study aims to study incidence of pulmonary hypertension as a complication in patients of sickle cell disease.Methods: Patients aged more than 12 years diagnosed to have sickle cell anaemia on hemoglobin electrophoresis during the study period (total- 94; 54 SS and 40 AS) were enrolled as cases. Sixty four age/sex matched healthy close relatives of the cases with ‘AA’ Hb electrophoresis pattern doubled up as controls. Participants were thoroughly assessed with complete history, examination, haematological and biochemical tests, X-ray chest, ECG, pulmonary function tests and 2D-echocardiography and Doppler studies and data analysed.Results: Pulmonary hypertension was observed in a total of five SS cases (5/54, 9.26%). No participant from the AS group or the control group developed pulmonary hypertension. It was more common in females (3/23, 13%) than males (2/26, 7.7%). The mean age of the SS cases with PH (32±5.15 years) was found to be significantly higher than that of the SS cases without PH (24.2±6.21 years) (p&lt;0.01).Conclusions: The incidence of PH in sickle cell disease was relatively lower as compared to western countries and was found to affect females and older individuals more. Larger community-based studies are recommended for corroboration.

PULMONARY HYPERTENSION IN SICKLE CELL DISEASE: LOOKING BEYOND THE USUAL SUSPECT

SESSION TITLE: Monday Fellow Case Report Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM INTRODUCTION: Pulmonary hypertension (PH) is common in sickle cell disease (SCD) and is a major risk factor for death with 2-year mortality 40-50%. Autoimmune disease (AID) is an established cause of pulmonary hypertension. Patients with SCD present a defective alternate complement pathway that increases risk of infection and autoimmune diseases. However, coexisting AID and SCD is rarely reported, suggesting possible under-diagnosis due to overlapping symptoms. CASE PRESENTATION: 20 year old male with sickle cell S/O Arab presented with 2 days of hip pain, fever, dyspnea and reproducible, non-exertional chest pain. Admitted for management of acute chest syndrome. He was found to have nephrotic-range proteinuria (protein-creatinine ratio 3.65 mg/mg). Serology markers were positive for ANA titer 1:2560 (speckled pattern), positive RNP (> 8.0) and Smith antibodies (1.9). Echocardiogram revealed right ventricular systolic pressure (RVSP) of 75 mmHg. Patient was diagnosed with mixed connective tissue disease (MCTD) with systemic lupus erythematosus (SLE) predominant features. He underwent kidney biopsy which was consistent with focal segmental and global glomerulosclerosis (FSGS). Right heart catheterization (RHC) showed mean pulmonary artery pressure of 31 mmHg, pulmonary capillary wedge pressure of 5 mmHg, cardiac output/cardiac index 7.95L/min/4.69L/min/m2 respectively, pulmonary vascular resistance of 5.7 Wood units. Patient was initiated on hydroxychloroquine, losartan, and ambrisentan. DISCUSSION: Hemoglobin O Arab is an uncommon hemoglobinopathy due to substitution of lysine for glutamic acid at a position 121 of beta globin chain. Homozygotes are clinically asympatomatic. Compound heterozygous condition in combination with sickle hemoglobin results in Hb S/O Arab , which resembles homozygous sickle cell anemia and manifests with severe hemolytic anemia and recurrent painful crisis (Reference #1). Pulmonary hypertension in SCD is multifactorial, classified as Group V; pre-capillary PH due to hemolysis, impaired nitric oxide bioavailability, asplenia, chronic hypoxemia and thromboembolism; as well as post-capillary PH due to left ventricular dysfunction. Despite the prevalence and negative impact on mortality, there are few data to guide management of PH in SCD. A small number of clinical trials (Walk-PHaSST, ASSET-1 and ASSET-2 trials) failed to prove success with PAH specific therapies, thus it is not recommended for routine use in PH-SCD patients (Reference #2 and #3). Based on RHC data of our patient, he has PAH of combined etiology associated with SCD as well as MCTD with SLE features and therefore we chose to treat him with PAH-targeted therapy. CONCLUSIONS: This case demonstrates importance of increased awareness of coexisting etiologies for PAH leading to a rapid progression and increased morbidity. Reference #1: Hemoglobin S/OARAB: Thirteen New Cases and Review of the Literature. American Journal of Hematology 60:279–284 (1999) Reference #2: Machado RF, Martyr S, Kato GJ, et al. Sildenafil therapy in patients with sickle cell disease and pulmonary hypertension. Br J Haematol. 2005;130:445–453. Reference #3: Br J Haematol. 2010 May;149(3):426-35. https://doi.org/10.1111/j.1365-2141.2010.08097.x. Epub 2010 Feb 17. DISCLOSURES: No relevant relationships by Sandi Khin, source=Web Response No relevant relationships by Ruth Minkin, source=Web Response

Pulmonary Hypertension in Children with Sickle Cell Disease: a Review of the Current Literature

Pulmonary hypertension (PH) is a well-recognized complication of sickle cell disease (SCD) and is one of the strongest predictors of increased morbidity and mortality in adult patients. There is evidence that PH can develop in children with SCD and the clinical implications of this finding are an area of active research. We review the current literature examining the association of SCD and PH in childhood. The recent literature has focused on elucidating the multifactorial mechanisms for the development of PH in SCD with the goal of developing targeted therapies. In addition, there has been a focus on understanding the significance of echocardiographic evidence of PH in children with SCD, a finding that has recently been associated with adverse clinical factors. While still based on limited evidence, the increased understanding of the important prognostic implications of echocardiographic evidence of PH has led to the development of guidelines that recommend screening echocardiograms beginning in childhood in children with SCD. PH can develop in children with SCD and, while the exact clinical implications of this finding are still being elucidated, current guidelines and research are aimed at early identification and treatment to improve outcomes.

Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a leading cause of death in sickle cell disease (SCD) patients. Hemolysis and oxidative stress contribute to SCD-associated PH. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and, by interacting with its receptor CD47, limits vasodilation of distal pulmonary arteries ex vivo. We hypothesized that the TSP1-CD47 interaction may promote PH in SCD. We found that TSP1 and CD47 are upregulated in the lungs of Berkeley (BERK) sickling (Sickle) mice and patients with SCD-associated PH. We then generated chimeric animals by transplanting BERK bone marrow into C57BL/6J (n = 24) and CD47 knockout (CD47KO, n = 27) mice. Right ventricular (RV) pressure was lower in fully engrafted Sickle-to-CD47KO than Sickle-to-C57BL/6J chimeras, as shown by the reduced maximum RV pressure (P = 0.013) and mean pulmonary artery pressure (P = 0.020). The afterload of the sickle-to-CD47KO chimeras was also lower, as shown by the diminished pulmonary vascular resistance (P = 0.024) and RV effective arterial elastance (P = 0.052). On myography, aortic segments from Sickle-to-CD47KO chimeras showed improved relaxation to acetylcholine. We hypothesized that, in SCD, TSP1-CD47 signaling promotes PH, in part, by increasing reactive oxygen species (ROS) generation. In human pulmonary artery endothelial cells, treatment with TSP1 stimulated ROS generation, which was abrogated by CD47 blockade. Explanted lungs of CD47KO chimeras had less vascular congestion and a smaller oxidative footprint. Our results show that genetic absence of CD47 ameliorates SCD-associated PH, which may be due to decreased ROS levels. Modulation of TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH.

Abnormal Ventilation-Perfusion Scan Is Associated with Pulmonary Hypertension in Sickle Cell Adults.

Pulmonary hypertension (PH) in adults with sickle cell disease (SCD) is associated with early mortality. Chronic thromboembolic PH (CTEPH) is an important complication and contributor to PH in SCD but is likely underappreciated. Guidelines recommend ventilation-perfusion (V/Q) scintigraphy as the imaging modality of choice to exclude CTEPH. Data on V/Q scanning are limited in SCD. Our objective was to compare the performance of V/Q scanning with that of CT pulmonary angiography (CTPA) and to report clinical outcomes associated with abnormal V/Q findings. Methods: Laboratory data, echocardiography, 6-min-walk testing, V/Q scanning, CTPA, and right heart catheterization (RHC) were prospectively obtained. High-probability and intermediate-probability V/Q findings were considered to be abnormal. Included for analysis were 142 SCD adults (aged 40.1 ± 13.7 y, 83 women, 87% hemoglobin SS) in a stable state enrolled consecutively between March 13, 2002, and June 8, 2017. Results: V/Q results were abnormal in 65 of 142 patients (45.8%). CTPA was positive for pulmonary embolism in 16 of 60 (26.7%). RHC confirmed PH (mean pulmonary artery pressure ≥ 25 mmHg) in 46 of 64 (71.9%), of whom 34 (73.9%) had abnormal V/Q findings. Among those without PH by RHC (n = 18), 2 of 18 patients had abnormal V/Q findings. Thirty-three patients had a complete dataset (V/Q scanning, CTPA, and RHC); 29 of 33 had abnormal RHC findings, of whom 26 had abnormal V/Q findings, compared with 11 who had abnormal CTPA findings. There was greater concordance between V/Q findings and RHC (κ-value = 0.53; P < 0.001) than between CTPA and RHC (κ-value = 0.13; P = 0.065). The sensitivity and specificity for V/Q scanning was 89.7% and 75.0%, respectively, whereas CTPA had sensitivity of 37.3% and specificity of 100%. Abnormal V/Q finding swere associated with hemodynamic severity (mean pulmonary artery pressure, 35.2 ± 9.6 vs. 26.9 ± 10.5 mm Hg, P = 0.002; transpulmonary gradient, 21.5 ± 9.7 vs. 12.16 ± 11 mmHg, P = 0.005; and pulmonary vascular resistance, 226.5 ± 135 vs. 140.7 ± 123.7 dynes⋅s⋅cm-5, P = 0.013) and exercise capacity (6-min-walk distance, 382.8 ± 122.3 vs. 442.3 ± 110.6 m, P < 0.010). Thirty-four deaths were observed over 15 y. All-cause mortality was higher in the abnormal-V/Q group (21 [61.8%]) than in the normal-V/Q group (13 [38.2%]) (log-rank test, P = 0.006; hazard ratio, 2.54). Conclusion: V/Q scanning is superior to CTPA in detecting thrombotic events in SCD. Abnormal V/Q findings are associated with PH, worse hemodynamics, lower functional capacity, and higher mortality. Despite high sensitivity in detecting CTEPH, V/Q scanning is underutilized. We recommend the use of V/Q scanning in the evaluation of dyspnea in adult SCD patients given the important implications toward management.

The Long Non‐Coding RNA XIST Is Decreased in Lungs and Plasma Exosomes of Sickle Cell Mice and in Hemin‐Treated Human Pulmonary Artery Endothelial Cells

RationalePulmonaryhypertension (PH), a serious complication of sicklecell disease (SCD), causes significant morbidity andmortality. The release ofhemoglobin and free heme during hemolysis triggers endothelial dysfunction andcontributes to PH pathogenesis. Emerging evidence indicates that longnon‐coding RNAs (lncRNAs) play a pivotal role in cellular proliferation,differentiation, and apoptosis. Circulating exosomes, which are nano‐sized extracellular vesicles, contain lncRNAs and play an important role in intercellular communication. However, the role of lncRNAs released from lungs and plasmaexosomes in SCD‐PH pathogenesis has not been defined. We hypothesized that lncRNAs are differentially expressed in SSmouse lungs and plasma exosomes and in hemin‐treated human pulmonary artery endothelialcells (HPAECs).Methods and ResultslncRNAs were isolated from lungs and plasma exosomes of sicklecell (SS) mice and littermate control (AA) mice at age 15–17 weeks. Isolated plasma exosomes were characterized forlevels of exosomal markers (CD9, CD45, and CD63), following which exosomes andlung samples were subjected to determine theexpression profiles of 90 lncRNAs using the LncProfiler™ lncRNA qPCR array(System Bioscienes). 5 lncRNAs were increased and10 lncRNAs were decreased in the lungs and plasma exosomes of SS mice compared to AA mice. One of thelncRNAs found to be decreased in SS samples was X‐inactivespecific transcript (XIST). To further examinewhether hemin reduces lncRNA XIST expression, HPAECs were treated with DMSO or hemin (5 uM) for 72 hours. XIST expression was decreased in hemin‐treatedHPAECs. Hemin also stimulated dose (0–10 μM)− and time (0–72hours)‐dependent increases in HPAEC monocyteadhesion and proliferation (BrdU assays). Because PPAR gamma (PPARg) levels were reduced in the lungs of SS mice,and pharmacological PPARg activation attenuated SCD‐PH, we examined the impactof PPARg activation on XIST levels in SS mouse lungs and found that PPARgactivation attenuates reductions in XIST expression.ConclusionsTheseresults suggest that sickle cell disease can impact lncRNAs and these effectsmay be reversed by treatment with PPARg agonists. Therefore, characterizationof lncRNAs are impacted in SCD may provide new insights into SCD pulmonaryvascular dysfunction and PH pathogenesis and identify novel targets fortherapeutic intervention.Support or Funding InformationSupported by NIH HL102167 (RLS and CMH) and NIH HL133053‐01A1 (BYK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5′-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (αIIbβ3) integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

Sickle cell disease and H3Africa: enhancing genomic research on cardiovascular diseases in African patients.

SummaryBackgroundSickle cell disease (SCD) has a high prevalence in sub-Saharan Africa. There are several cardiovascular phenotypes in SCD that contribute to its morbidity and mortality.DiscussionSCD is characterised by marked clinical variability, with genetic factors playing key modulating roles. Studies in Tanzania and Cameroon have reported that singlenucleotide polymorphisms in BCL11A and HBS1L-MYB loci and co-inheritance of alpha-thalassaemia impact on foetal haemoglobin levels and clinical severity. The prevalence of overt stroke among SCD patients in Cameroon (6.7%) and Nigeria (8.7%) suggests a higher burden than in high-income countries. There is also some evidence of high burden of kidney disease and pulmonary hypertension in SCD; however, the burden and genetics of these cardiovascular conditions have seldom been investigated in Africa.ConclusionsSeveral H3Africa projects are focused on cardiovascular diseases and present major opportunities to build genome-based research on existing SCD platforms in Africa to transform the health outcomes of patients.

Peroxisome Proliferator-activated Receptor-α-mediated Transcription of miR-199a2 Attenuates Endothelin-1 Expression via Hypoxia-inducible Factor-1α

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.

Pulmonary hypertension in sickle cell disease: diagnosis and management.

The increased survival of patients with sickle cell disease (SCD) into adulthood is associated with an increased incidence of multiorgan dysfunction and a progressive systemic and pulmonary vasculopathy. The high prevalence of an elevated tricuspid regurgitant jet velocity and its association with an increased risk of death in adult patients is well established. However, there has been controversy regarding the prevalence of pulmonary hypertension (PH) and its association with mortality in SCD. Multiple recently published reports demonstrate that PH as diagnosed by right heart catheterization is common in adult SCD patients, with a prevalence of 6%-11%. Furthermore, PH is associated with an increased risk of death in SCD patients. In this chapter, we provide evidence for the high prevalence of PH in SCD and its association with mortality and make recommendations for its evaluation and management. Finally, we provide the rationale for screening for this life-threatening complication in adult patients with SCD.

Computed tomography correlates with cardiopulmonary hemodynamics in pulmonary hypertension in adults with sickle cell disease.

Our objective was to determine whether computed tomography angiography (CTA) measurements of pulmonary artery size can noninvasively assess hemodynamics and diagnose pulmonary hypertension (PH) secondary to sickle cell disease (SCD). Twenty SCD patients with confirmed PH were compared with 20 matched controls. Diameters of the pulmonary artery trunk and branches were measured with CTA and a semiautomatic segmentation algorithm. Measurements were normalized by body size and correlated (Spearman rank) with hemodynamic markers from right-heart catheterization. Receiver operating characteristic (ROC) curves were used to investigate the role of pulmonary artery sizes in diagnosing PH. Analysis of pulmonary artery sizes adjusted for body surface area (BSA) resulted in the most significant discrimination between subjects with PH secondary to SCD and controls (P < 0.001); PH was diagnosed accurately with an area under the ROC curve of 0.99. There was significant correlation between pulmonary artery sizes and body mass index (BMI) and BSA only in controls (r = 0.46-0.68, P < 0.04 for all). The most significant correlations with hemodynamic markers were found between BMI-adjusted pulmonary artery sizes and high systolic pulmonary arterial pressure, high pulmonary vascular resistance, high systemic vascular resistance, and low cardiac output (r = 0.47, 0.62, 0.61, and 0.66, respectively; P < 0.04 for all). BMI-adjusted CTA measures of the pulmonary artery relate to high pulmonary vascular resistance and reduced cardiac output in patients with SCD and PH. CTA with quantitative image analysis is a powerful noninvasive diagnostic tool for PH in SCD and shows promise as estimator of hemodynamic markers.