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Background: Many patients with heart failure with preserved ejection fraction have metabolic syndrome and develop exercise-induced pulmonary hypertension (EIPH). Increases in pulmonary vascular resistance in patients with heart failure with preserved ejection fraction portend a poor prognosis; this phenotype is referred to as combined precapillary and postcapillary pulmonary hypertension (CpcPH). Therapeutic trials for EIPH and CpcPH have been disappointing, suggesting the need for strategies that target upstream mechanisms of disease. This work reports novel rat EIPH models and mechanisms of pulmonary vascular dysfunction centered around the transcriptional repression of the soluble guanylate cyclase (sGC) enzyme in pulmonary artery (PA) smooth muscle cells. Methods: We used obese ZSF-1 leptin-receptor knockout rats (heart failure with preserved ejection fraction model), obese ZSF-1 rats treated with SU5416 to stimulate resting pulmonary hypertension (obese+sugen, CpcPH model), and lean ZSF-1 rats (controls). Right and left ventricular hemodynamics were evaluated using implanted catheters during treadmill exercise. PA function was evaluated with magnetic resonance imaging and myography. Overexpression of nuclear factor Y α subunit (NFYA), a transcriptional enhancer of sGC β1 subunit (sGCβ1), was performed by PA delivery of adeno-associated virus 6. Treatment groups received the SGLT2 inhibitor empagliflozin in drinking water. PA smooth muscle cells from rats and humans were cultured with palmitic acid, glucose, and insulin to induce metabolic stress. Results: Obese rats showed normal resting right ventricular systolic pressures, which significantly increased during exercise, modeling EIPH. Obese+sugen rats showed anatomic PA remodeling and developed elevated right ventricular systolic pressure at rest, which was exacerbated with exercise, modeling CpcPH. Myography and magnetic resonance imaging during dobutamine challenge revealed PA functional impairment of both obese groups. PAs of obese rats produced reactive oxygen species and decreased sGCβ1 expression. Mechanistically, cultured PA smooth muscle cells from obese rats and humans with diabetes or treated with palmitic acid, glucose, and insulin showed increased mitochondrial reactive oxygen species, which enhanced miR-193b–dependent RNA degradation of nuclear factor Y α subunit (NFYA), resulting in decreased sGCβ1-cGMP signaling. Forced NYFA expression by adeno-associated virus 6 delivery increased sGCβ1 levels and improved exercise pulmonary hypertension in obese+sugen rats. Treatment of obese+sugen rats with empagliflozin improved metabolic syndrome, reduced mitochondrial reactive oxygen species and miR-193b levels, restored NFYA/sGC activity, and prevented EIPH. Conclusions: In heart failure with preserved ejection fraction and CpcPH models, metabolic syndrome contributes to pulmonary vascular dysfunction and EIPH through enhanced reactive oxygen species and miR-193b expression, which downregulates NFYA-dependent sGCβ1 expression. Adeno-associated virus–mediated NFYA overexpression and SGLT2 inhibition restore NFYA-sGCβ1-cGMP signaling and ameliorate EIPH.

Background The severity of pulmonary hypertension (PH) is monitored by measuring pulmonary vascular resistance, which is a steady-state measurement and ignores the pulsatile load encountered by the right ventricle (RV). Pulmonary vascular impedance (PVZ) can depict both steady-state and pulsatile forces, and thus may better predict clinical outcomes. We sought to calculate PVZ in patients with PH associated with heart failure with preserved ejection fraction who were administered inhaled sodium nitrite to better understand the acute effects on afterload. Methods and Results Fourteen patients with PH associated with heart failure with preserved ejection fraction underwent right heart catherization and were administered inhaled sodium nitrite. A Fourier transform was used to calculate PVZ for both before and after nitrite for comparison. Inhaled sodium nitrite decreased characteristic impedance (inversely related to proximal pulmonary artery compliance) and total work performed by the RV. RV efficiency improved, defined by a reduction in the total work divided by cardiac output. There was a mild decrease in pulmonary steady-state resistance after the administration of inhaled sodium nitrite, but this effect was not significant. Conclusions PVZ analysis showed administration of inhaled sodium nitrite was associated with an improvement in pulmonary vascular compliance via a decrease in characteristic impedance, more so than pulmonary steady-state resistance. This effect was associated with improved RV efficiency and total work.

In the US, mortality in sickle cell disease (SCD) increases after age 18–20 years. Biomarkers of mortality risk can identify patients who need intensive follow-up and early or novel interventions. We prospectively enrolled 510 SCD patients aged 3–20 years into an observational study in 2006–2010 and followed 497 patients for a median of 88 months (range 1–105). We hypothesized that elevated pulmonary artery systolic pressure as reflected in tricuspid regurgitation velocity (TRV) would be associated with mortality. Estimated survival to 18 years was 99% and to 25 years, 94%. Causes of death were known in seven of 10 patients: stroke in four (hemorrhagic two, infarctive one, unspecified one), multiorgan failure one, parvovirus B19 infection one, sudden death one. Baseline TRV ≥2.7 m/second (>2 SD above the mean in age-matched and gender-matched non-SCD controls) was observed in 20.0% of patients who died vs 4.6% of those who survived (P = .012 by the log rank test for equality of survival). The baseline variable most strongly associated with an elevated TRV was a high hemolytic rate. Additional biomarkers associated with mortality were ferritin ≥2000 μg/L (observed in 60% of patients who died vs 7.8% of survivors, P < .001), forced expiratory volume in 1 minute to forced vital capacity ratio (FEV1/FVC)

Primary graft dysfunction (PGD) is directly related to ischemia-reperfusion (I/R) injury and a major obstacle in lung transplantation (LTx). Nitrite ([Formula: see text]), which is reduced in vivo to form nitric oxide (NO), has recently emerged as an intrinsic signaling molecule with a prominent role in cytoprotection against I/R injury. Using a murine model, we provide the evidence that nitrite mitigated I/R-induced injury by diminishing infiltration of immune cells in the alveolar space, reducing pulmonary edema, and improving pulmonary function. Ultrastructural studies support severe mitochondrial impairment in the lung undergoing I/R injury, which was significantly protected by nitrite treatment. Nitrite also abrogated the increased pulmonary vascular permeability caused by I/R. In vitro, hypoxia-reoxygenation (H/R) exacerbated cell death in lung epithelial and microvascular endothelial cells. This contributed to mitochondrial dysfunction as characterized by diminished complex I activity and mitochondrial membrane potential but increased mitochondrial reactive oxygen species (mtROS). Pretreatment of cells with nitrite robustly attenuated mtROS production through modulation of complex I activity. These findings illustrate a potential novel mechanism in which nitrite protects the lung against I/R injury by regulating mitochondrial bioenergetics and vascular permeability.

BACKGROUND RBC transfusion effectiveness varies due to donor, component, and recipient factors. Prior studies identified characteristics associated with variation in hemoglobin increments following transfusion. We extended these observations, examining donor genetic and nongenetic factors affecting transfusion effectiveness. METHODS This is a multicenter retrospective study of 46,705 patients and 102,043 evaluable RBC transfusions from 2013 to 2016 across 12 hospitals. Transfusion effectiveness was defined as hemoglobin, bilirubin, or creatinine increments following single RBC unit transfusion. Models incorporated a subset of donors with data on single nucleotide polymorphisms associated with osmotic and oxidative hemolysis in vitro. Mixed modeling accounting for repeated transfusion episodes identified predictors of transfusion effectiveness. RESULTS Blood donor (sex, Rh status, fingerstick hemoglobin, smoking), component (storage duration, γ irradiation, leukoreduction, apheresis collection, storage solution), and recipient (sex, BMI, race and ethnicity, age) characteristics were associated with hemoglobin and bilirubin, but not creatinine, increments following RBC transfusions. Increased storage duration was associated with increased bilirubin and decreased hemoglobin increments, suggestive of in vivo hemolysis following transfusion. Donor G6PD deficiency and polymorphisms in SEC14L4, HBA2, and MYO9B genes were associated with decreased hemoglobin increments. Donor G6PD deficiency and polymorphisms in SEC14L4 were associated with increased transfusion requirements in the subsequent 48 hours. CONCLUSION Donor genetic and other factors, such as RBC storage duration, affect transfusion effectiveness as defined by decreased hemoglobin or increased bilirubin increments. Addressing these factors will provide a precision medicine approach to improve patient outcomes, particularly for chronically transfused RBC recipients, who would most benefit from more effective transfusion products. FUNDING Funding was provided by HHSN 75N92019D00032, HHSN 75N92019D00034, 75N92019D00035, HHSN 75N92019D00036, and HHSN 75N92019D00037; R01HL126130; and the National Institute of Child Health and Human Development (NICHD).

To address the global burden of sickle cell disease and the need for novel therapies, the American Society of Hematology partnered with the US Food and Drug Administration to engage the work of 7 panels of clinicians, investigators, and patients to develop consensus recommendations for clinical trial end points. The panels conducted their work through literature reviews, assessment of available evidence, and expert judgment focusing on end points related to patient-reported outcome, pain (non–patient-reported outcomes), the brain, end-organ considerations, biomarkers, measurement of cure, and low-resource settings. This article presents the findings and recommendations of the end-organ considerations, measurement of cure, and low-resource settings panels as well as relevant findings and recommendations from the biomarkers panel.

The six-minute walk test (6MWT) has been used in patients with sickle cell disease (SCD), in conjunction with tricuspid regurgitant velocity (TRV) and plasma N-terminal pro-brain natriuretic peptide (NT-pro BNP), to assess risk of having pulmonary hypertension. Exercise-induced vital sign changes (VSCs) are predictors of clinical outcomes in other diseases. In this study, we assess the predictors and prognostic value of 6MWT VSC in adult SCD patients. Data from a multinational study of SCD patients (Treatment of Pulmonary Hypertension with Sildenafil: walk-PHaSST) were used to calculate the 6MWT VSC. Predictors of VSC were identified by a multivariable analysis, and a survival analysis was conducted by the Cox proportional hazard method. An increase in heart rate was observed in 90% of the 630 SCD adults, 77% of patients had an increase in systolic blood pressure (SBP), and 50% of patients had a decrease in oxygen saturation. TRV (odds ratio [OR] = 1.82, p = .020), absolute reticulocyte count (OR = 1.03, p

BackgroundThe COVID-19 pandemic continues to affect the United States and the world. Media reports have suggested that the wave of the alpha variant in the Spring of 2021 in the US caused more cases among younger patients and racial and ethnic subgroups.ApproachWe analyzed electronic health record data from a multihospital health system to test whether younger patients accounted for more cases and more severe disease, and whether racial disparities are widening. We compared demographics, patient characteristics, and hospitalization variables for patients admitted from November 2020 through January 2021 to those admitted in March and April 2021.ResultsWe analyzed data for 37, 502 unique inpatients and outpatients at 21 hospitals from November 1, 2020 to April 30, 2021. Compared to patients from November through January, those with positive tests in March and April were younger and less likely to die. Among patients under age 50, those with positive tests in March and April were three times as likely to be hospitalized and twice as likely to require ICU admission or mechanical ventilation. Individuals identified as Black represented a greater proportion of cases and hospitalizations in March and April as compared to November through January.ConclusionsWe found that relative COVID-19 hospitalization rates for younger individuals and individuals identified as Black were rising over time. These findings have important implications for ongoing public health measures to mitigate the impact of the pandemic.

Pulmonary arterial hypertension (PAH) leads to right ventricular cardiomyopathy and cardiac dysfunctions where in the clinical setting, cardiac arrest is the likely cause of death, in ~70% of PAH patients. We investigated the cardiac phenotype of PAH hearts and tested the hypothesis that the insulin-like hormone, Relaxin could prevent maladaptive cardiac remodeling and protect against cardiac dysfunctions in a PAH animal model. PAH was induced in rats with sugen (20 mg/kg), hypoxia then normoxia (3-weeks/each); relaxin (RLX = 0, 30 or 400 μg/kg/day, n ≥ 6/group) was delivered subcutaneously (6-weeks) with implanted osmotic mini-pumps. Right ventricle (RV) hemodynamics and Doppler-flow measurements were followed by cardiac isolation, optical mapping, and arrhythmia phenotype. Sugen-hypoxia (SuHx) treated rats developed PAH characterized by higher RV systolic pressures (50 ± 19 vs. 22 ± 5 mmHg), hypertrophy, reduced stroke volume, ventricular fibrillation (VF) (n = 6/11) and bradycardia/arrest (n = 5/11); both cardiac phenotypes were suppressed with dithiothreitol (DTT = 1 mM) (n = 0/2/group) or RLX (low or high dose, n = 0/6/group). PAH hearts developed increased fibrosis that was reversed by RLX-HD, but not RLX-LD. Relaxin decreased Nrf2 and glutathione transferases but not glutathione-reductase. High-dose RLX improved pulmonary arterial compliance (measured by Doppler flow), suppressed VF even after burst-pacing, n = 2/6). Relaxin suppressed VF and asystole through electrical remodeling and by reversing thiol oxidative stress. For the first time, we showed two cardiac phenotypes in PAH animals and their prevention by RLX. Relaxin may modulate maladaptive cardiac remodeling in PAH and protect against arrhythmia and cardiac arrest.

Background: Pulmonary complications of sickle cell disease (SCD) are diverse and encompass acute and chronic disease. The understanding of the natural history of pulmonary complications of SCD is limited, no specific therapies exist, and these complications are a primary cause of morbidity and mortality. Methods: We gathered a multidisciplinary group of pediatric and adult hematologists, pulmonologists, and emergency medicine physicians with expertise in SCD-related lung disease along with an SCD patient advocate for an American Thoracic Society–sponsored workshop to review the literature and identify key unanswered clinical and research questions. Participants were divided into four subcommittees on the basis of expertise: 1) acute chest syndrome, 2) lower airways disease and pulmonary function, 3) sleep-disordered breathing and hypoxia, and 4) pulmonary vascular complications of SCD. Before the workshop, a comprehensive literature review of each subtopic was conducted. Clinically important questions were developed after literature review and were finalized by group discussion and consensus. Results: Current knowledge is based on small, predominantly observational studies, few multicenter longitudinal studies, and even fewer high-quality interventional trials specifically targeting the pulmonary complications of SCD. Each subcommittee identified the three or four most important unanswered questions in their topic area for researchers to direct the next steps of clinical investigation. Conclusions: Important and clinically relevant questions regarding sickle cell lung disease remain unanswered. High-quality, multicenter, longitudinal studies and randomized clinical trials designed and implemented by teams of multidisciplinary clinician-investigators are needed to improve the care of individuals with SCD.

Cold storage of blood for 5 to 6 weeks has been shown to impair endothelial function after transfusion and has been associated with measures of end-organ dysfunction. Although the products of hemolysis, such as cell-free plasma hemoglobin, arginase, heme, and iron, in part mediate these effects, a complete analysis of transfused metabolites that may affect organ function has not been evaluated to date. Blood stored for either 5 or 42 days was collected from 18 healthy autologous volunteers, prior to and after autologous transfusion into the forearm circulation, followed by metabolomics analyses. Significant metabolic changes were observed in the plasma levels of hemolytic markers, oxidized purines, plasticizers, and oxidized lipids in recipients of blood stored for 42 days, compared with 5 days. Notably, transfusion of day 42 red blood cells (RBCs) increased circulating levels of plasticizers (diethylhexyl phthalate and derivatives) by up to 18-fold. Similarly, transfusion of day 42 blood significantly increased circulating levels of proinflammatory oxylipins, including prostaglandins, hydroxyeicosatrienoic acids (HETEs), and dihydroxyoctadecenoic acids. Oxylipins were the most significantly increasing metabolites (for 9-HETE: up to ∼41-fold, P = 3.7e-06) in day 42 supernatants. Measurements of arginine metabolism confirmed an increase in arginase activity at the expense of nitric oxide synthesis capacity in the bloodstream of recipients of day 42 blood, which correlated with measurements of hemodynamics. Metabolic changes in stored RBC supernatants impact the plasma metabolome of healthy transfusion recipients, with observed increases in plasticizers, as well as vasoactive, pro-oxidative, proinflammatory, and immunomodulatory metabolites after 42 days of storage.

BACKGROUND The major aims of the RBC-Omics study were to evaluate the genomic and metabolomic determinants of spontaneous and stress-induced hemolysis during RBC storage. This study was unique in scale and design to allow evaluation of RBC donations from a sufficient number of donors across the spectrum of race, ethnicity, sex, and donation intensity. Study procedures were carefully piloted, optimized, and controlled to enable high-quality data collection. METHODS The enrollment goal of 14,000 RBC donors across four centers, with characterization of RBC hemolysis across two testing laboratories, required rigorous piloting and optimization and establishment of a quality assurance (QA) and quality control (QC) program. Optimization of WBC elution from leukoreduction (LR) filters, development and validation of small-volume transfer bags, impact of manufacturing and sample-handling procedures on hemolysis parameters, and testing consistency across laboratories and technicians and over time were part of this quality assurance/quality control program. RESULTS LR filter elution procedures were optimized for obtaining DNA for analysis. Significant differences between standard and pediatric storage bags led to use of an alternative LR-RBC transfer bag. The impact of sample preparation and freezing methods on metabolomics analyses was evaluated. Proficiency testing monitored and documented testing consistency across laboratories and technicians. CONCLUSION Piloting and optimization, and establishment of a robust quality assurance/quality control program documented process consistency throughout the study and was essential in executing this large-scale multicenter study. This program supports the validity of the RBC-Omics study results and a sample repository that can be used in future studies.

Opioid overdose is the leading cause of death for Americans 25 to 64 years of age, and opioid use disorder affects >2 million Americans. The epidemiology of opioid-associated out-of-hospital cardiac arrest in the United States is changing rapidly, with exponential increases in death resulting from synthetic opioids and linear increases in heroin deaths more than offsetting modest reductions in deaths from prescription opioids. The pathophysiology of polysubstance toxidromes involving opioids, asphyxial death, and prolonged hypoxemia leading to global ischemia (cardiac arrest) differs from that of sudden cardiac arrest. People who use opioids may also develop bacteremia, central nervous system vasculitis and leukoencephalopathy, torsades de pointes, pulmonary vasculopathy, and pulmonary edema. Emergency management of opioid poisoning requires recognition by the lay public or emergency dispatchers, prompt emergency response, and effective ventilation coupled to compressions in the setting of opioid-associated out-of-hospital cardiac arrest. Effective ventilation is challenging to teach, whereas naloxone, an opioid antagonist, can be administered by emergency medical personnel, trained laypeople, and the general public with dispatcher instruction to prevent cardiac arrest. Opioid education and naloxone distributions programs have been developed to teach people who are likely to encounter a person with opioid poisoning how to administer naloxone, deliver high-quality compressions, and perform rescue breathing. Current American Heart Association recommendations call for laypeople and others who cannot reliably establish the presence of a pulse to initiate cardiopulmonary resuscitation in any individual who is unconscious and not breathing normally; if opioid overdose is suspected, naloxone should also be administered. Secondary prevention, including counseling, opioid overdose education with take-home naloxone, and medication for opioid use disorder, is important to prevent recurrent opioid overdose.

Introduction: Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children. Decreased nitric oxide bioavailability and responsiveness contribute to neurovascular disease. Cytochrome b5 reductase 3 (Cyb5R3) is a heme iron reductase that reduces oxidized soluble guanylate cyclase heme iron (Fe 3+ --> Fe 2+ ) to preserve nitric oxide signaling. A loss-of-function Cyb5R3 missense variant (T117S) occurs with high frequency (0.23 minor allele) in persons of African ancestry. Hypothesis: We hypothesized that impaired reductase function of T117S Cyb5R3 exacerbates brain damage after ischemic stroke in SCD. Methods: Bone marrow transplant was used to create male SCD mice with wild type (SS/WT) or T117S (SS/T117S) Cyb5R3. Blood was sampled before and after middle cerebral artery occlusion (55 minutes occlusion, 48 hours reperfusion). Infarct volume (IV) was determined by 2,3,5-triphenyltetrazolium chloride. Intravascular hemolysis and correlation (Pearson’s R) of hematology changes with IV were determined. Baseline Walk-PHaSST (NCT00492531) data were analyzed for stroke occurrence. Results: Brain IV (63 vs 27 cm 3 , P=0.003) and mortality (3/6 vs 0/8) were greater in SS/T117S vs SS/WT. Red blood cells, hemoglobin and hematocrit declined as IV increased. Plasma oxyhemoglobin increased in parallel with IV (r = 0.74, P=0.09). There were different signatures to hematologic changes that occurred with IV in SCD. Relative to wild type, T117S contracted the erythroid compartment (red blood cell: -13% vs 13%, P=0.003; hematocrit: -20% vs 1%, P=0.008; hemoglobin: -18% vs 2%, P=0.007). Mean platelet volume correlated with IV in SS/T117S (r = 0.87, P=0.06), while the inverse occurred in SS/WT (r = -0.63, P=0.09) Monocytes increased in parallel with IV in SS/T117S (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT (r = -0.77, P=0.04). WalkPHaSST participants with T117S Cyb5R3 self-reported more ischemic stroke (7.4% vs 5.1%) relative to wild type. Conclusion: Cyb5R3 is an important modifier of the evolution and outcome of ischemic brain injury in SCD and its hematologic consequences. Our findings indicate a bidirectional relationship between stroke and anemia in SCD that may axially turn on Cyb5R3 activity.

Background People living with human immunodeficiency virus (PLWH) are at risk of developing pulmonary hypertension (PH) and right ventricular (RV) dysfunction, but understanding of the relationship of RV function to afterload (RV-PA coupling) is limited. We evaluated the clinical and hemodynamic characteristics of human immunodeficiency virus (HIV)-associated PH. Methods We performed a retrospective review of patients with a diagnosis of HIV undergoing right heart catheterization (RHC) from 2000–2016 in a tertiary care center. Inclusion criteria were diagnosis of HIV, age ≥ 18 years and availability of RHC data. PH was classified as either pulmonary arterial hypertension (PAH; mean pulmonary arterial pressure [mPAP] ≥ 25mmHg with pulmonary artery wedge pressure [PAWP] ≤ 15mmHg) or pulmonary venous hypertension (PVH; mPAP ≥ 25mmHg with PAWP > 15). We collected demographics, CD4 cell count, HIV viral load, RHC and echocardiographic data. The single beat method was used to calculate RV-PA coupling from RHC. Results Sixty-two PLWH with a clinical likelihood for PH underwent RHC. Thirty-two (52%) met PH criteria (15 with PAH, 17 with PVH). Average time from diagnosis of HIV to diagnosis of PH was 11 years. Eleven of 15 individuals with PAH were on antiretroviral therapy (ART) while all 17 patients with PVH were on ART. Compared to PLWH without PH, those with PH had an increased likelihood of having a detectable HIV viral load and lower CD4 cell counts. PLWH with PAH or PVH had increased RV afterload with normal RV contractility, and preserved RV-PA coupling. Conclusion PLWH with PH (PAH or PVH) were more likely to have a detectable HIV viral load and lower CD4 count at the time of RHC. PLWH with PAH or PVH had increased RV afterload, normal RV contractility, with preserved RV-PA coupling suggestive of an early onset, mild, and compensated form of PH. These results should be confirmed in larger studies.

Introduction: Prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, especially among older adults. HFpEF is commonly associated with exercise intolerance and skeletal muscle weakening which likely contribute to functional decline. We studied the utility of nitrite (NO 2 ) supplementation with the aim to augment skeletal muscle mitochondrial respiration. Hypothesis: NO 2 administered via oral supplements would be associated with improved mitochondrial respiration. Whereas prior studies with other oral and inhaled nitrate and NO 2 therapy in HFpEF patients have been inconsistent, we used novel NO 2 capsules that we hypothesized would achieve higher serum levels of nitrite and nitrate, and greater cellular and clinical benefits. Methods: A randomized, placebo-controlled pilot trial to study the utility of oral sodium (Na) NO 2 over 4 weeks in an older population (≥70 years) was done. Participants received 20 or 40 mg Na-NO 2 supplements 3 times daily based on their hemodynamic responses. Pre- and post-NO 2 intervention participants underwent biopsies of the vastus lateralis muscle with associated assessments of mitochondrial respiration in permeabilized fibers and also completed cardiopulmonary exercise test. Results: 15 participants were randomized and 13 completed the interventions (n=6 on NO 2 , n=7 on placebo, 69% men, mean age 75.7 years, range 70-91). Post-intervention serum NO 2 level increased (+0.61±0.67 μmol on NO 2, +0.25±0.15 on placebo). Ex-vivo analysis of mitochondrial respiration showed increased O 2 consumption in muscle (+27.1±27.4 pmol/(s*mg) on NO 2 , -11.7±11.3 on placebo). Rated perceived exertion during steady-state walking decreased (-1.2±2.0 on NO 2, +0.2±1.6 on placebo). Peak oxygen uptake (VO 2 ) increased (+1.4±5.2 ml/kg/min on NO2, -3.4±5.2 on placebo). Conclusion: Our study demonstrated efficacy and potential clinical utility of oral NO 2 supplementation in older HFpEF patients with an internally consistent physiologic composite of improved skeletal muscle mitochondrial respiration in association with improved cardiorespiratory fitness and diminished perceived exertion during steady-state walking. Clinical implications are auspicious and further research is indicated.

Background Many Heart Failure with preserved Ejection Fraction (HFpEF) patients have metabolic syndrome and develop Exercise Induced Pulmonary Hypertension (EIPH). The pathogenesis of EIPH in HFpEF remains unclear as there is no rodent model. As the SGLT2 inhibitor Empagliflozin improves clinical outcome in patients with type 2 diabetes and cardiovascular risk, we tested its effect on EIPH in a novel rat model of HFpEF. Methods Obese ZSF1 (HFpEF model) with leptin receptor mutation have metabolic syndrome and received the VEGF-inhibitor SU5416 to stimulate PH (Obese + Sugen). Half also received Empagliflozin (0.2 mg/kg/day) in drinking water from 8 to 22 weeks old. Lean ZSF1 lacking the mutation served as controls. During treadmill exercise, right/left ventricle (RV/LV) hemodynamics were evaluated via catheters. Pulmonary artery vascular smooth muscle cells (PAVSMC) prepared from normal or diabetic patients were cultured in standard media, or with Palmitate acid, Glucose and Insulin (PGI) to induce metabolic stress. Flow cytometry was used to evaluate reactive oxygen species (ROS) in mitochondria (Mitosox) or cytoplasm (CellROX). Results Relative to Lean, Obese + Sugen had increased body weight and HgA1C (Fig. 1A). Relative to Lean and at rest, Obese + Sugen showed mildly elevated RVSP and LVEDP. After exercise, LVSP and LVEDP rose similarly in Lean and Obese + Sugen. However, after exercise, Obese + Sugen showed a markedly greater increase in RVSP and exercise intolerance consistent with EIPH (Fig. 1B). In MR imaging of PA, Lean showed dobutamine (5 μg/kg/min)-induced PA dilation, which was not seen in Obese + Sugen (Fig. 1C). Protein levels of sGCβ1 (key regulator of PA relaxation) and its transcription factor (NFYA) both were decreased in PA from Obese + Sugen relative to Lean (Fig. 1D). Obese + Sugen + SGLT2 inhibitor treated rats showed marked improvements metabolic syndrome (body weight, HgA1c), exercise induced increase in RVSP, PA response to dobutamine, and increased NFYA and sGCβ1 expression (Fig. 1A–D). We observed greater ROS-induced DNA damage (8-OHdG staining) (Fig. 1E) and mitochondrial complex I, III, and IV activity in Obese + Sugen PA that was normalized in Obese + Sugen + SGLT2 inhibitor (Fig. 1F), suggesting a role of ROS in EIPH. Control human PAVSMC treated with PGI media showed elevated cytoplasmic and mitochondrial ROS, associated with increased mitochondrial complex I, III, IV and V activity (Fig. 1F, G). PGI media also accelerated the degradation of NFYA RNA and protein level in a manner mimicked by H2O2, and prevented by catalase/SOD (Fig. 1H, I), suggesting PGI-induced ROS enhanced NFYA degradation. Diabetic human PAVSMCs cultured in normal media resembled PGI-treated normal cells with respect to sGCb1 and NFYA expression, and in response to catalase/SOD (Fig. 1H, I). Conclusions In this PH-HFpEF model, metabolic syndrome contributes to PA dysfunction and EIPH through mitochondrial dysfunction and enhanced ROS, which were improved by Empagliflozin treatment. Figure 1 Funding Acknowledgement Type of funding source: None

BackgroundHydroxychloroquine (HCQ) is widely used in the treatment of malaria, rheumatologic disease such as lupus, and most recently, COVID-19. These uses raise concerns about its safe use in the setting of glucose-6-phosphate dehydrogenase (G6PD) deficiency, especially as 11% of African American men carry the G6PD A- variant. However, limited data exist regarding the safety of HCQ in this population.Study design and methodsRecently, we created a novel "humanized" mouse model containing the G6PD deficiency A- variant (Val68Met) using CRISPR technology. We tested the effects of high-dose HCQ administration over 5 days on hemolysis in our novel G6PD A- mice. In addition to standard hematologic parameters including plasma hemoglobin, erythrocyte methemoglobin, and reticulocytes, hepatic and renal function were assessed after HCQ.ResultsResidual erythrocyte G6PD activity in G6PD A- mice was ~6% compared to wild-type (WT) littermates. Importantly, we found no evidence of clinically significant intravascular hemolysis, methemoglobinemia, or organ damage in response to high-dose HCQ.ConclusionsThough the effects of high doses over prolonged periods was not assessed, this study provides early, novel safety data of the use of HCQ in the setting of G6PD deficiency secondary to G6PD A-. In addition to novel safety data for HCQ, to our knowledge, we are the first to present the creation of a "humanized" murine model of G6PD deficiency.

The nitrate-nitrite-NO pathway regulates NO synthase–independent vasodilation and NO signaling. Ingestion of inorganic nitrite has vasodilatory and blood pressure–lowering effects. Preclinical studies in rodent models suggest there may be a benefit of nitrite in lowering serum triglyceride levels and improving the metabolic syndrome. In a phase 2 study, we evaluated the safety and efficacy of chronic oral nitrite therapy in patients with hypertension and the metabolic syndrome. Twenty adult subjects with stage 1 or 2 hypertension and the metabolic syndrome were enrolled in an open-label safety and efficacy study. The primary efficacy end point was blood pressure reduction; secondary end points included insulin-dependent glucose disposal and endothelial function measured by flow-mediated dilation of the brachial artery and intima-media diameter of the carotid artery. Chronic oral nitrite therapy (40 mg/3× daily) was well tolerated. Oral nitrite significantly lowered systolic, diastolic, and mean arterial pressures, but tolerance was observed after 10 to 12 weeks of therapy. There was significant improvement in the intima-media thickness of the carotid artery and trends toward improvements in flow-mediated dilation of the brachial artery and insulin sensitivity. Chronic oral nitrite therapy is safe in patients with hypertension and the metabolic syndrome. Despite an apparent lack of enzymatic tolerance to nitrite, we observed tolerance after 10 weeks of chronic therapy, which requires additional mechanistic studies and possible therapeutic dose titration in clinical trials. Nitrite may be a safe therapy to concominantly improve multiple features of the metabolic syndrome including hypertension, insulin resistance, and endothelial dysfunction. Registration— URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01681810.

Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice to assess the pathogenesis of pulmonary thrombosis following deionized water–induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbβ3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravascular ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet P2Y(12) purinergic receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to our knowledge to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles, and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis–triggered pulmonary thrombosis.

The coronavirus disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), marks a global event that will permanently reshape implementation of intensive care medicine. As of June 4, 2020, there are 6,606,455 reported cases of COVID-19 including 388,556 fatalities spanning 215 countries and territories, although epidemiological data remain incomplete. Early autopsy reports emphasize proximal airway and distal airspace involvement, including alveolar epithelial inflammation and capillary thickening.

Hemolysis is a hallmark of various inherited or acquired blood disorders including sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome. Epidemiological evidence suggests that in situ pulmonary thrombosis is a major pathological event contributing to cardiopulmonary morbidities affecting patients with hemolytic disorders. Intravascular hemolysis promotes the release of erythrocyte-derived damage associated molecular patterns (eDAMPs) molecules such as cell free hemoglobin (Hb), heme and adenosine diphosphate (ADP). Although these eDAMPS may activate platelets, promote endothelial dysfunction and drive sterile thrombo-inflammation, the pathogenesis of pulmonary thrombosis in hemolytic disorders and the role of platelets in this process remains unclear. Intravascular hemolysis in C57BL/6 (WT) mice was induced by intravenous (IV) administration of deionized water (H2O). Alternatively, eDAMPs or tissue derived DAMPs relevant to hemolysis or endothelial injury were administered IV to WT mice. The pulmonary microcirculation was visualized using quantitative intravital fluorescence lung microscopy (qFILM). Fluorochrome-conjugated anti-mouse CD49b Ab and dextran were administered IV for in vivo staining of circulating platelets and visualization of blood vessels, respectively. Time series of qFILM images were collected at baseline and post IV administration of agonists to assess pulmonary thrombosis. IV administration of H2O led to arrival of platelet-rich thrombi in the pulmonary arterioles that occluded the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries leading to their transient obstruction. DAMPs such as ADP, collagen, thrombin and tissue factor also triggered dose-dependent PT in mice. Identical to IV H2O, DAMPs triggered pulmonary thrombosis also involved entrapment of platelet-rich thrombi in the arteriolar bottle-necks, which led to ischemia in the pulmonary arteriole and the down-stream capillaries. Interestingly, pretreatment with IV heparin (300 U/kg) prevented TF-induced but not ADP-triggered pulmonary thrombosis in mice. In contrast, IV administration of αIIbβ3 receptor inhibitor eptifibatide (10 mg/kg) completely abrogated TF-, collagen and ADP-dependent PT. Our current findings provide the first real-time in vivo visual evidence establishing that intravascular hemolysis directly induces pulmonary thrombosis, which involves entrapment of platelet-rich thrombi in the pre-capillary pulmonary arterioles. Our results are the first to identify that platelet activation and procoagulant activity contribute to pathogenesis of hemolysis induced pulmonary thrombosis. Disclosures Ragni: OPKO: Research Funding; Bioverativ/Sanofi: Other: Advisory Board, Research Funding; Shire/Takeda: Other: Advisory Board, Research Funding; Sangamo: Research Funding; Spark Therapeutics: Other: Advisory Board, Research Funding; Alnylam/Sanofi: Other: Advisory Board, Research Funding; BioMarin: Other: Advisory Board, Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Gladwin:Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

Objective:Pulmonary hypertension (PH) due to left heart disease (group 2), especially in the setting of heart failure with preserved ejection fraction (HFpEF), is the most common cause of PH worldwide; however, at present, there is no proven effective therapy available for its treatment. PH-HFpEF is associated with insulin resistance and features of metabolic syndrome. The stable prostacyclin analog, treprostinil, is an effective and widely used Food and Drug Administration-approved drug for the treatment of pulmonary arterial hypertension. While the effect of treprostinil on metabolic syndrome is unknown, a recent study suggests that the prostacyclin analog beraprost can improve glucose intolerance and insulin sensitivity. We sought to evaluate the effectiveness of treprostinil in the treatment of metabolic syndrome-associated PH-HFpEF.Approach and Results:Treprostinil treatment was given to mice with mild metabolic syndrome-associated PH-HFpEF induced by high-fat diet and to SU5416/obese ZSF1 rats, a model created by the treatment of rats with a more profound metabolic syndrome due to double leptin receptor defect (obese ZSF1) with a vascular endothelial growth factor receptor blocker SU5416. In high-fat diet-exposed mice, chronic treatment with treprostinil reduced hyperglycemia and pulmonary hypertension. In SU5416/Obese ZSF1 rats, treprostinil improved hyperglycemia with similar efficacy to that of metformin (a first-line drug for type 2 diabetes mellitus); the glucose-lowering effect of treprostinil was further potentiated by the combined treatment with metformin. Early treatment with treprostinil in SU5416/Obese ZSF1 rats lowered pulmonary pressures, and a late treatment with treprostinil together with metformin improved pulmonary artery acceleration time to ejection time ratio and tricuspid annular plane systolic excursion with AMPK (AMP-activated protein kinase) activation in skeletal muscle and the right ventricle.Conclusions:Our data suggest a potential use of treprostinil as an early treatment for mild metabolic syndrome-associated PH-HFpEF and that combined treatment with treprostinil and metformin may improve hyperglycemia and cardiac function in a more severe disease.

Heart failure with preserved ejection fraction (HFpEF), a major public health problem that is rising in prevalence, is associated with high morbidity and mortality and is considered to be the greatest unmet need in cardiovascular medicine today because of a general lack of effective treatments. To address this challenging syndrome, the National Heart, Lung, and Blood Institute convened a working group made up of experts in HFpEF and novel research methodologies to discuss research gaps and to prioritize research directions over the next decade. Here, we summarize the discussion of the working group, followed by key recommendations for future research priorities. There was uniform recognition that HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach in both humans and animal models to improve understanding of mechanisms and treatment of HFpEF. It was recognized that advances in the understanding of basic mechanisms and the roles of inflammation, macrovascular and microvascular dysfunction, fibrosis, and tissue remodeling are needed and ideally would be obtained from (1) improved animal models, including large animal models, which incorporate the effects of aging and associated comorbid conditions; (2) repositories of deeply phenotyped physiological data and human tissue, made accessible to researchers to enhance collaboration and research advances; and (3) novel research methods that take advantage of computational advances and multiscale modeling for the analysis of complex, high-density data across multiple domains. The working group emphasized the need for interactions among basic, translational, clinical, and epidemiological scientists and across organ systems and cell types, leveraging different areas or research focus, and between research centers. A network of collaborative centers to accelerate basic, translational, and clinical research of pathobiological mechanisms and treatment strategies in HFpEF was discussed as an example of a strategy to advance research progress. This resource would facilitate comprehensive, deep phenotyping of a multicenter HFpEF patient cohort with standardized protocols and a robust biorepository. The research priorities outlined in this document are meant to stimulate scientific advances in HFpEF by providing a road map for future collaborative investigations among a diverse group of scientists across multiple domains.

Red blood cell (RBC) storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBC to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBC from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyse following oxidant stress. A subset of extreme hemolysers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBC from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBC stored in additive solution- 3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBC stored in additive solution-1. Merging metabolomics data with results from an independent genome-wide association study on the same cohort, we identified metabolic markers of hemolysis and glucose 6-phosphate dehydrogenasedeficiency, which were associated with extremes in oxidative hemolysis and dysregulation in nicotinamide adenine dinucleotide phosphate and glutathione- dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and glucose 6-phosphate dehydrogenase status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.

Background Biological and technical variability has been increasingly appreciated as a key factor impacting red blood cell (RBC) storability and, potentially, transfusion outcomes. Here, we performed metabolomics analyses to investigate the impact of factors other than storage duration on the metabolic phenotypes of stored RBC in a multicenter study. Study design and methods Within the framework of the REDS-III (Recipient Epidemiology and Donor Evaluation Study-III) RBC-Omics study, 13,403 donors were enrolled from four blood centers across the United States and tested for the propensity of their RBCs to hemolyze after 42 days of storage. Extreme hemolyzers were recalled and donated a second unit of blood. Units were stored for 10, 23, and 42 days prior to sample acquisition for metabolomics analyses. Results Unsupervised analyses of metabolomics data from 599 selected samples revealed a strong impact (14.2% of variance) of storage duration on metabolic phenotypes of RBCs. The blood center collecting and processing the units explained an additional 12.2% of the total variance, a difference primarily attributable to the storage additive (additive solution 1 vs. additive solution 3) used in the different hubs. Samples stored in mannitol-free/citrate-loaded AS-3 were characterized by elevated levels of high-energy compounds, improved glycolysis, and glutathione homeostasis. Increased methionine metabolism and activation of the transsulfuration pathway was noted in samples processed in the center using additive solution 1. Conclusion Blood processing impacts the metabolic heterogeneity of stored RBCs from the largest multicenter metabolomics study in transfusion medicine to date. Studies are needed to understand if these metabolic differences influenced by processing/storage strategies impact the effectiveness of transfusions clinically.

Identifying sickle cell disease patients at high risk of complications could lead to personalized treatment and better prognosis but despite many advances prediction of the clinical course of these patients remains elusive. We propose a system-type approach to discover profiles of multiple, common biomarkers that correlate with morbidity and mortality in sickle cell disease. We used cluster analysis to discover 17 signatures of 17 common circulating biomarkers in 2320 participants of the Cooperative Study of Sickle Cell Disease, and evaluated the association of these signatures with risk for stroke, pain, leg ulceration, acute chest syndrome, avascular necrosis, seizure, death, and trend of fetal hemoglobin and hemolysis using longitudinally collected data. The analysis shows that some of the signatures are associated with reduced risk for complications, while others are associated with increased risk for complications. We also show that these signatures repeat in two more contemporary studies of sickle cell disease and correlate with recently discovered biomarkers of pulmonary vascular disease. With replication and further study, these biomarker signatures could become an important and affordable precision medicine tool to aid treatment and management of the disease.

Pulmonary and systemic vasculopathies are significant risk factors for early morbidity and death in patients with sickle cell disease (SCD). An underlying mechanism of SCD vasculopathy is vascular smooth muscle (VSM) nitric oxide (NO) resistance, which is mediated by NO scavenging reactions with plasma hemoglobin (Hb) and reactive oxygen species that can oxidize soluble guanylyl cyclase (sGC), the NO receptor. Prior studies show that cytochrome b5 reductase 3 (CYB5R3), known as methemoglobin reductase in erythrocytes, functions in VSM as an sGC heme iron reductase critical for reducing and sensitizing sGC to NO and generating cyclic guanosine monophosphate for vasodilation. Therefore, we hypothesized that VSM CYB5R3 deficiency accelerates development of pulmonary hypertension (PH) in SCD. Bone marrow transplant was used to create SCD chimeric mice with background smooth muscle cell (SMC)–specific tamoxifen-inducible Cyb5r3 knockout (SMC R3 KO) and wild-type (WT) control. Three weeks after completing tamoxifen treatment, we observed 60% knockdown of pulmonary arterial SMC CYB5R3, 5 to 6 mm Hg elevated right-ventricular (RV) maximum systolic pressure (RVmaxSP) and biventricular hypertrophy in SS chimeras with SMC R3 KO (SS/R3(KD)) relative to WT (SS/R3(WT)). RV contractility, heart rate, hematological parameters, and cell-free Hb were similar between groups. When identically generated SS/R3 chimeras were studied 12 weeks after completing tamoxifen treatment, RVmaxSP in SS/R3(KD) had not increased further, but RV hypertrophy relative to SS/R3(WT) persisted. These are the first studies to establish involvement of SMC CYB5R3 in SCD-associated development of PH, which can exist in mice by 5 weeks of SMC CYB5R3 protein deficiency.

Purpose Patients with sickle cell disease (SCD) regularly experience abnormal sleep, characterized by frequent arousals and reduced total sleep time. However, obstructive sleep apnea syndrome (OSAS) is a common comorbidity of SCD, making it unclear whether the disease per se is impacting sleep, or sleep disruption is secondary to the presence of OSAS. Thus, we assessed sleep, independent of OSAS, using a mouse model of SCD. Methods Sleep was compared between 10-to-12-week-old Townes knockout-transgenic mice with the sickle cell phenotype SS (n = 6) and Townes mice with sickle cell trait AS (n = 6; control). The mice underwent chronic polysomnographic electrode implantation (4EEG/2EMG) to assess sleep architecture. Results The SS mice had significantly lower hemoglobin concentration compared to control AS mice (7.3 ± 1.3 vs. 12.9 ± 1.7 g/dL; p

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions which leads to red blood cell (RBC) distortion, calcium-influx mediated RBC dehydration, increased RBC adhesivity, reduced RBC deformability, increased RBC fragility, and hemolysis. These impairments in RBC structure and function result in multifaceted downstream pathology including inflammation, endothelial cell activation, platelet and leukocyte activation and adhesion, and thrombosis, all of which contribute vascular occlusion and substantial morbidity and mortality. Hemoglobin released upon RBC hemolysis scavenges nitric oxide (NO) and generates reactive oxygen species (ROS) and thereby decreases bioavailability of this important signaling molecule. As the endothelium-derived relaxing factor, NO acts as a vasodilator and also decreases platelet, leukocyte, and endothelial cell activation. Thus, low NO bioavailability contributes to pathology in sickle cell disease and its restoration could serve as an effective treatment. Despite its promise, clinical trials based on restoring NO bioavailability have so far been mainly disappointing. However, particular "NO donating" agents such as nitrite, which unlike some other NO donors can improve sickle RBC properties, may yet prove effective.

Patient case: An 18-year-old male patient with homozygous hemoglobin SS disease was evaluated for progressive dyspnea and elevated tricuspid regurgitant jet velocity (TRV) on echocardiography. The patient’s case is described in detail in Lancet.1 He had been treated with regular transfusions since childhood for stroke, had rare episodes of vaso-occlusive pain episodes, and did not take narcotic pain medications. He presented with progressive severe dyspnea on exertion and lower extremity edema. His laboratory tests were notable for a total hemoglobin level of 11.8 g/dL and hemoglobin S levels 140 mm Hg (4 times the TRV squared = 4V2). Additional images in Figure 1D show a dilated right ventricle and right atrium with a compressed left ventricle. The patient’s right heart catheterization revealed a pulmonary artery systolic pressure of 147 mm Hg and diastolic pressure of 49 mm Hg; note that the normal values are ∼25/10 mm Hg.

Sickle cell disease (SCD) patients have an increased risk of venous thromboembolism (VTE). Population-based studies demonstrated that VTE has a cumulative incidence rate of approximately 25% in adult SCD patients and is associated with higher risk of mortality. VTE in SCD is most commonly manifested as deep vein thrombosis (DVT) with associated pulmonary embolism (PE). Although autopsy studies have regularly discovered pulmonary thromboembolic lesions in SCD patients, the pathophysiology of VTE in SCD remains largely unknown mostly due to the lack of relevant animal VTE model. Understanding the mechanisms that promote VTE in SCD is imperative to identify its prevention and treatment measures. To elucidate the cellular, molecular and biophysical mechanisms of VTE in SCD we developed an innovative two stage intravital imaging analysis experimental model in SCD mice. DVT in SCD mice was induced by surgical ligation of femoral vein. Venous thrombus formation was visualized using intravital multi-photon-excitation (MPE) microscopy. Venous thrombus took the form of a large mass of elliptical shape which extended in the long-axis direction of the femoral vein. It was composed of fibrin, erythrocytes and sparse platelets. Over time, thrombus was infiltrated by migrating neutrophils. To trigger acute PE, femoral vein ligation was removed and the venous thrombus was observed to spontaneously detach and travel to the SCD mouse lung. This method allowed real time visualization of acute PE in vivo using MPE microscopy of intact lung in live breathing mice. Acute PE involved embolization of the pulmonary arterioles and the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries. The embolization of arteriolar circulation led to loss of blood flow in the arterioles and the down-stream capillaries. Herein we introduce an intravital microscopy approach to probe VTE in SCD live mouse. Our model has potential application in investigating the molecular determinants of VTE associated with SCD as well as evaluating efficacy of new antithrombotic drugs. Disclosures Sundd: Bayer: Research Funding; CSL Behring Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

To determine the relationship between the production of cell-free plasma hemoglobin and acute kidney injury in infants and children undergoing cardiopulmonary bypass for cardiac surgery.Prospective observational study.Twelve-bed cardiac ICU in a university-affiliated children's hospital.Children were prospectively enrolled during their preoperative outpatient appointment with the following criteria: greater than 1 month to less than 18 years old, procedures requiring cardiopulmonary bypass, no preexisting renal dysfunction.None.Plasma and urine were collected at baseline (in a subset), the beginning and end of cardiopulmonary bypass, and 2 hours and 24 hours after cardiopulmonary bypass in 60 subjects. Levels of plasma hemoglobin increased during cardiopulmonary bypass and were associated (p0.01) with cardiopulmonary bypass duration (R = 0.22), depletion of haptoglobin at end and 24 hours after cardiopulmonary bypass (R = 0.12 and 0.15, respectively), lactate dehydrogenase levels at end cardiopulmonary bypass (R = 0.27), and change in creatinine (R = 0.12). Forty-three percent of patients developed acute kidney injury. There was an association between plasma hemoglobin level and change in creatinine that varied by age (overall [R = 0.12; p0.01]; in age2 yr [R = 0.22; p0.01]; and in2 yr [R = 0.03; p = 0.42]). Change in plasma hemoglobin and male gender were found to be risk factors for acute kidney injury (odds ratio, 1.02 and 3.78, respectively; p0.05).Generation of plasma hemoglobin during cardiopulmonary bypass and male gender are associated with subsequent renal dysfunction in low-risk pediatric patients, especially in those older than 2 years. Further studies are needed to determine whether specific subgroups of pediatric patients undergoing cardiopulmonary bypass would benefit from potential treatments for hemolysis and plasma hemoglobin-associated renal dysfunction.

The Division of Lung Diseases of the NHLBI and the Cardiovascular Medical Education and Research Fund held a workshop to discuss how to leverage the anticipated scientific output from the recently launched "Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics" (PVDOMICS) program to develop newer approaches to pulmonary vascular disease. PVDOMICS is a collaborative, protocol-driven network to analyze all patient populations with pulmonary hypertension to define novel pulmonary vascular disease (PVD) phenotypes. Stakeholders, including basic, translational, and clinical investigators; clinicians; patient advocacy organizations; regulatory agencies; and pharmaceutical industry experts, joined to discuss the application of precision medicine to PVD clinical trials. Recommendations were generated for discussion of research priorities in line with NHLBI Strategic Vision Goals that include: (1) A national effort, involving all the stakeholders, should seek to coordinate biosamples and biodata from all funded programs to a web-based repository so that information can be shared and correlated with other research projects. Example programs sponsored by NHLBI include PVDOMICS, Pulmonary Hypertension Breakthrough Initiative, the National Biological Sample and Data Repository for PAH, and the National Precision Medicine Initiative. (2) A task force to develop a master clinical trials protocol for PVD to apply precision medicine principles to future clinical trials. Specific features include: (a) adoption of smaller clinical trials that incorporate biomarker-guided enrichment strategies, using adaptive and innovative statistical designs; and (b) development of newer endpoints that reflect well-defined and clinically meaningful changes. (3) Development of updated and systematic variables in imaging, hemodynamic, cellular, genomic, and metabolic tests that will help precisely identify individual and shared features of PVD and serve as the basis of novel phenotypes for therapeutic interventions.

Pulmonary hypertension (PH) associated with heart failure with preserved ejection fraction (PH-HFpEF; World Health Organization Group II) secondary to left ventricular (LV) diastolic dysfunction is the most frequent cause of PH. It is an increasingly recognized clinical complication of the metabolic syndrome. To date, no effective treatment has been identified, and no genetically modifiable mouse model is available for advancing our understanding for PH-HFpEF. To develop a mouse model of PH-HFpEF, we exposed 36 mouse strains to 20 weeks of high-fat diet (HFD), followed by systematic evaluation of right ventricular (RV) and LV pressure-volume analysis. The HFD induces obesity, glucose intolerance, insulin resistance, hyperlipidemia, as well as PH, in susceptible strains. We observed that certain mouse strains, such as AKR/J, NON/shiLtJ, and WSB/EiJ, developed hemodynamic signs of PH-HFpEF. Of the strains that develop PH-HFpEF, we selected AKR/J for further model validation, as it is known to be prone to HFD-induced metabolic syndrome and had low variability in hemodynamics. HFD-treated AKR/J mice demonstrate reproducibly higher RV systolic pressure compared with mice fed with regular diet, along with increased LV end-diastolic pressure, both RV and LV hypertrophy, glucose intolerance, and elevated HbA1c levels. Time course assessments showed that HFD significantly increased body weight, RV systolic pressure, LV end-diastolic pressure, biventricular hypertrophy, and HbA1c throughout the treatment period. Moreover, we also identified and validated 129S1/SvlmJ as a resistant mouse strain to HFD-induced PH-HFpEF. These studies validate an HFD/AKR/J mouse model of PH-HFpEF, which may offer a new avenue for testing potential mechanisms and treatments for this disease.

Introduction. Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children between 3 and 14 years old. A vast amount of clinical and experimental evidence has concluded that decreased nitric oxide (NO) bioavailability and/or NO responsiveness, as is seen in SCD, is a major contributing factor in the pathogenesis of neurovascular disease. NO responsiveness, which occurs via NO-induced activation of soluble guanylate cyclase (sGC), requires reduced heme iron (Fe2+) in the sGC active site. We recently identified cytochrome b5 reductase 3 (Cyb5R3) as an sGC heme iron reductase in vascular smooth muscle (VSM), where it reverses the oxidized heme iron of sGC (Fe3+ --> Fe2+) to preserve NO sensing/signaling under conditions of oxidative stress. In a mouse model of SCD we have shown that knockdown of Cyb5R3 in VSM accelerates the development of pulmonary hypertension and cardiac remodeling. A missense variant of Cyb5R3 (T117S) that results in loss-of-function methemoglobin reductase activity occurs at a high frequency in persons of African ancestry (0.23 minor allele frequency). Unpublished baseline data from the Walk-PHaSSt trial (NCT00492531) reveals that persons with SCD who carry the T117S variant are at increased risk of ischemic stroke; these individuals self-reported almost 50% more (74 vs 51 cases per 1000 individuals) ischemic stroke than those with wild-type (WT) Cyb5R3. Hypothesis. We hypothesized that impaired reductase function of Cyb5R3 T117S leads to sustained sGC heme oxidation, which drives cerebral vascular dysfunction and exacerbates brain damage after ischemic stroke in SCD. Methods. Bone marrow transplant was used to create SCD mice with global expression of WT or T117S Cyb5R3, hereafter referred to as SS/WT or SS/T117S, respectively. All mice were male, C57Bl/6 background, and >85% engrafted with SS Hb for 12 weeks. Ischemic stroke was induced using transient middle cerebral artery occlusion (MCAO: 55 min occlusion, 48 hr reperfusion), after which brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC,1%) to determine infarct volume. Blood was sampled before and after MCAO to assess effects of brain infarct on hematological parameters. Student's t-test was used for analysis of 2 groups and Pearson's R used for correlation analyses of brain infarct volume with hematology changes [(post-pre/pre) * 100]. Results. Global expression of T117S Cyb5R3 in SCD caused increased cerebral infarct volume (62.9 vs 26.7 cm3, P=0.003) and mortality (3/6 vs 0/6) relative to WT Cyb5R3. WT and T117S Cyb5R3 mice with SCD were similar in that both showed declining red blood cells (RBC), hemoglobin (Hgb) and hematocrit (Hct) as infarct volumes increased. In the SS/T117S group, the anemia was more severe in keeping with larger infarct volumes. There were different signatures to the hematologic changes that occurred with cerebral infarct in SCD. When compared to WT Cyb5R3, T117S caused the erythroid compartment to contract (RBC: -12.97% vs 13.41%, P=0.01; Hct: -19.75% vs 0.31%, P=0.025; Hgb: -17.93% vs 2.78%, P=0.017). In SS/WT mice platelet numbers increased more relative to SS/T117S (17.5 vs 9.7 * 103 cells/uL); and MPV, a measure of platelet activation, inversely correlated with brain infarct volume (r = -0.94, P=0.006), the opposite of what was seen in SS/T117S (r = 0.87, P=0.056). Monocytes seem to play an important role in the volume of brain infarct in SS/T117S as their numbers increased in parallel with infarct volume (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT mice (r = -0.75, P=0.14). Conclusion. These results indicate that Cyb5R3 is an important modifying factor in the evolution and outcome of ischemic brain injury in SCD. Our findings also raise questions on just how cerebral infarct modifies the anemia of SCD, as well as the role played by Cyb5R3 in the dynamics of that relationship. To what extent is the sGC-cGMP-PKG pathway involved at the cerebrovascular and erythropoietic levels? Does Cyb5R3 contribute resilience to ischemic stroke in SCD? The development and application of targeted therapies for effectively preventing and treating cerebrovascular disease in SCD rely on finding the answers to these questions. Disclosures No relevant conflicts of interest to declare.

The six-minute walk test (6MWT) is a well-established assessment of the cardiopulmonary function of sickle cell disease (SCD) patients. The test is used for people with SCD who are suspected of having hypoxia or an elevated estimated pulmonary arterial systolic pressure, which increases the risk for early death. Low 6MWT distances are associated with cardiopulmonary compromise. Six-minute walk distance examination has been proposed as a screening test, used in conjunction with plasma levels of brain natriuretic peptide and Doppler-echocardiography, through which to identify high-risk patients. Post-exercise tachycardia or lack of heart rate recovery following a 6MWT has been shown to be a predictor of pulmonary hypertension and mortality in people with pulmonary fibrosis. A rise in blood pressure after exercise is associated with impaired capacity for vasodilation in ischemic heart disease. The prognostic significance of patterns of vital sign change in adult SCD patients completing the 6MWT is currently unknown. In this study, we aimed to assess the distribution and predictors of vital sign change during 6MWT in adult SCD patients and test the association of these changes with patient survival. Data from a multinational observational study of SCD patients (Walk-PhassT), was used to calculate the change in vital signs (heart rate, O2 saturation, systolic blood pressure, diastolic blood pressure, pulse pressure) normalized for walk distance, after a 6MWT. Bivariate and LASSO regression analyses were performed to ascertain the significant predictors of change in each vital sign, in addition to Cox proportional hazard analysis to assess the impact of vital sign change and time to death. The median age of the 630 adult SCD patients was 37 years. 47% were male, 77% had the HbSS phenotype, and 22 (3.7% of 592 with follow-up data) died during a median time of follow up of 29 months. The most frequent changes in vital signs identified were increases in the heart rate (90%) followed by increases in systolic blood pressure (77%, Table 1b). Bivariate analysis revealed significant but weak positive correlations between tricuspid regurgitation velocity (TRV) and increases in both heart rate (r= 0.08; p These findings support links between changes of vital signs during the 6MWT and established markers of hemolysis and cardiovascular dysfunction in SCD patients. Evidence of a protective effect of increased systolic pressure is a novel finding. This might indicate that the ability to increase systolic pressure during submaximal exercise relates to cardiac output and conveys a physiological advantage for SCD patients. These findings could be used as the basis for future mechanistic studies of exercise effects on cardiovascular function in SCD patients. Disclosures Gordeuk: Imara: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; CSL Behring: Consultancy, Research Funding; Ironwood: Research Funding. Gibbs:Pfizer: Consultancy; United Therapeutics: Consultancy; MSD: Consultancy; GSK: Consultancy; Complexa: Consultancy; Bayer: Consultancy; Actelion: Consultancy; Acceleron: Consultancy. Little:BioChip Labs: Patents & Royalties: SCD Biochip (patent, no royalties); GBT: Membership on an entity's Board of Directors or advisory committees; NHLBI: Research Funding; Hemex Health, Inc.: Patents & Royalties: Microfluidic electropheresis (patent, no royalties); Bluebird Bio: Research Funding; GBT: Research Funding.