Sickle cell diseases are widespread in regions encompassing the Mediterranean, Middle East, sub-Saharan Africa, and specific parts of Asia, primarily due to the abnormal production of hemoglobin S. This genetic blood disorder stems from a mutation in the beta-globin gene, a crucial component of hemoglobin and the heme-containing protein found in red blood cells. Point mutations in the hemoglobin gene can be inherited as a heterozygous or homozygous pattern. These mutations disrupt the normal configuration of the protein, impeding its physiological function and altering the cell's shape, giving it a sickle-like appearance. The resulting sickle cells can lead to organ damage, intense physical discomfort, and anemia; in severe cases, the condition can be fatal. Early detection and effective treatment methods have the potential to progressively reduce the associated mortality rate over time. To diagnose sickle cell disease and its carrier states with unparalleled specificity, a variety of approaches have been developed. The most common method includes differential blood cell counts and their assessment, high-performance liquid chromatography (HPLC) and hemoglobin electrophoresis. Furthermore, innovative sensing technologies are currently under development, encompassing user-friendly, cost-effective and portable point-of-care devices that are capable of timely diagnosis at the genetic and molecular levels of these disorders. The review delves into a range of established and innovative strategies utilized in the detection of sickle cell disease, also underscoring the essential role played by diverse bio-sensing techniques in propelling the advancement of early diagnosis of SCD.

Invasive aspergillosis is characterized by lung hemorrhage and release of extracellular heme, which promotes fungal growth. Heme can also mediate tissue injury directly, and both fungal growth and lung injury may induce hemorrhage. To assimilate these interdependent processes, we hypothesized that, during aspergillosis, heme mediates direct lung injury independent of fungal growth, leading to worse infection outcomes, and the scavenger protein hemopexin mitigates these effects. Mice with neutropenic aspergillosis developed a time-dependent increase in lung extracellular heme and a corresponding hemopexin induction. Hemopexin deficiency resulted in markedly increased lung injury, fungal growth, and lung hemorrhage. Using a computational model of the interactions of Aspergillus, heme, and the host, we predicted a critical role for heme-mediated generation of neutrophil extracellular traps (NETs) in this infection. We tested this prediction using a fungal strain unable to grow at body temperature and found that extracellular heme and fungal exposure synergized to induce lung injury by promoting NET release, and disruption of NET was sufficient to attenuate lung injury and fungal burden. These data implicate heme-mediated NETosis in both lung injury and fungal growth during aspergillosis, resulting in a detrimental positive feedback cycle that can be interrupted by scavenging heme or disrupting NETs.

Acute kidney injury (AKI) is a common clinical complication in malaria, with AKI reported across all species that cause severe disease, including Plasmodium falciparum, Plasmodium knowlesi, and Plasmodium vivax. AKI during malaria varies based on host and parasite factors, including the growth potential of the parasite within host red blood cells, the extent of red blood cell lysis, and the capacity of the parasite to sequester within the microvasculature. In this review, we focus primarily on P. falciparum pathogenesis and the role of intravascular hemolysis in AKI through the depletion of endogenous hemoglobin and heme scavengers, resulting in oxidative stress and tissue injury. We discuss the etiology of blackwater fever as a hemolytic complication in severe malaria that has been rising in incidence. All patients with severe malaria should have a high index of suspicion for AKI, particularly when hemolytic features are present. Finally, we review potential interventions to mitigate the impact of hemolysis on kidney injury in severe malaria. Given the high burden of malaria in Africa, the incidence of AKI in severe malaria, and the number of malaria episodes over a person's lifetime, the cumulative impact of malaria-associated AKI on chronic kidney disease needs to be considered. Semin Nephrol 36:x-xx © 20XX Elsevier Inc. All rights reserved.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) is a novel mediator involved in abnormal angiogenesis. We aimed to investigate circulating LRG1 levels and their relationship with proangiogenic mediators in sickle cell disease (SCD).

A total of 50 patients with SCD, with 25 in steady-state condition (SCD-SS) and 25 in periods of painful vaso-occlusive crisis (SCD-VOC), and 25 healthy controls were included in the study. Demographical and clinical data were collected from hospital records. Serum LRG1, vascular endothelial growth factor A (VEGFA), and hypoxia-inducible factor 1-alpha (HIF1A) levels were measured by enzyme-linked immunosorbent assay (ELISA), and C-reactive protein (CRP) was measured by the nephelometric method. Routine biochemical parameters were assessed using an autoanalyzer. Multinomial logistic regression was used to analyze ELISA parameters, and receiver operating characteristic (ROC) curves were constructed to determine the optimal cut-off point for HIF1A to predict VOCs in SCD patients.

LRG1 and VEGFA levels were significantly higher in SCD patients than controls (p<0.001), with no difference between the SCD-SS and SCD-VOC groups. HIF1A, CRP, and lactate dehydrogenase levels differed significantly across all groups, being highest in the SCD-VOC group (p<0.001). After adjusting for age and sex, LRG1, HIF1A, and VEGFA remained elevated in the SCD groups. HIF1A correlated with CRP (r=0.351, p=0.024), but LRG1 showed no correlation with proangiogenic mediators in the SCD-VOC group. The area under the ROC curve was calculated as 0.694 (95% confidence interval: 0.542-0.845, p=0.021) and the optimal cut-off point was 494.5 pg/mL for HIF1A in predicting vaso-occlusive crises in patients with SCD.

Circulating LRG1 levels may reflect neutrophil activation and contribute to the cross-talk between proangiogenic mediators released in SCD.

Sickle cell disease (SCD) is a genetic disorder characterized by sickle red blood cells (RBCs). Sickle RBCs cause cerebral vasculopathies including vaso-occlusive events, leading to ischemia-reperfusion injury and hypoxic tissue environment. To date, the physiological blood flow velocities in cerebral vessels of preclinical SCD models has not been evaluated under hypoxic-reoxygenation. In our study, we used transcranial ultrasound techniques to measure abnormal blood flow velocities in the internal carotid (ICA) and middle cerebral arteries (MCA) of transgenic sickle cell mice (SS) challenged with hypoxia-reoxygenation. Our study showed that SS mice that underwent hypoxic stress exhibited lower relative mean velocities in the MCA compared to wildtype mice (AA) (0.67 ± 0.18 vs. 0.95 ± 0.15; p < 0.05). Comparison of the Lindegaard ratio between normoxia and hypoxia in SS mice suggested that the MCA underwent vasodilation (0.67 ± 0.18 vs. 0.95 ± 0.15; p < 0.05). Bilirubin, a potential biomarker for cerebral vasculopathies in SCD, was higher in SS than AA mice (0.56  ±   0.28 vs. 0.05  ±   0.07 mg/dL; p < 0.05). Correlation analyses revealed a significant association between bilirubin levels and blood velocities of MCA (r = -0.9377, p = 0.0002) and ICA (r = 0.8203, p = 0.0068), especially in hypoxic conditions of SS mice. We propose that the reactivity of cerebral vessels in SS mice is correlated with the elevated plasma bilirubin level.

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by sickle-shaped red blood cells (RBCs). Primary cilia are mechanosensory organelles and are projected in the lumen of blood vessels to detect blood flow. We previously reported that interaction between microvasculature endothelial cells and sickled RBCs resulted in altered blood flow that can elevate reactive oxygen species, leading to increased deciliation in SCD patients. However, the impact of deciliation mediated by sickled RBCs in the context of the ciliary protein profiles remains unclear. Here, we investigated cell-cilia stability under different physiological shear-stress magnitudes and examined cilia protein profiles in SCD, utilizing mouse models and human participants. Our results demonstrate that subjecting endothelial cilia to sickled RBCs at 5.0 dyn/cm2 led to significant deciliation events. The proteomic and bioinformatic analyses showed different ciliary protein profiles, distinct signaling pathways, and unique post-translational modification processes in the SCD mouse model. Consistent with the SCD mouse model results, our translational studies validated the enrichment of specific proteins, including Transferrin Receptor-1 (TfR1), Glyceraldehyde-3-Phosphate-Dehydrogenase (GAPDH), and ADP Ribosylation Factor Like GTPase-13B (ARL13B) in SCD patients. These findings underscore the clinical relevance of cilia in SCD and suggest that ciliary proteins are potential biomarkers for assessing vascular damage.

Sickle cell disease (SCD) is one of the most prevalent hereditary blood disorders characterized by aberrant hemoglobin synthesis that causes red blood cells (RBCs) to sickle and result in vaso-occlusion. The complex pathophysiological mechanisms that underlie SCD are explored in this study, including hemoglobin polymerization, the formation of fetal hemoglobin (HbF), and hemoglobin switching regulation. Notably, pharmaceutical approaches like hydroxyurea, l-glutamine, voxelotor, and crizanlizumab, in addition to therapeutic techniques like gene therapies like Casgevy and Lyfgenia, signify noteworthy advancements in the management of issues connected to SCD. Furthermore, the deciphering of the molecular mechanisms that dictate hemoglobin switching has revealed several potentially therapeutic targets, including key transcriptional repressors such as β-cell lymphoma/leukemia 11A (BCL11A), Zinc finger and BTB domain-containing 7A (ZBTB7A), Nuclear Factor IX (NFIX), and Nuclear Factor IA (NFIA), which play crucial roles in γ-globin silencing. Additionally, transcriptional activators such as Nuclear Factor Y (NF-Y), and Hypoxia-inducible factor 1α (HIF1α) have emerged as promising regulators that can disrupt repression and enhance HbF synthesis. Other epigenetic regulators, such as lysine-specific histone demethylase 1 (LSD1), euchromatic histone methyltransferases 1/2 (EHMT1/2), histone deacetylases (HDACs), DNA methyltransferases (DNMTs), and protein arginine methyltransferases (PRMTs). It has been demonstrated that inhibiting these targets can prevent the silencing of the gene encoding for the formation of γ-chains and, in turn, increase the synthesis of HbF, providing a possible treatment option for SCD symptoms. These approaches could pave the way for innovative, mechanism-driven therapies that address the unmet medical needs of SCD patients.

The pathophysiology of sickle cell disease (SCD) is linked to haemolysis and systemic inflammation. To determine the range of systemic alterations, we assessed the frequency of 12 immune populations and the levels of related cytokines in the peripheral blood of paediatric (n = 13) and adult (n = 12) patients with SCD prescribed hydroxyurea, as compared to paediatric (n = 5) and adult (n = 10) race-matched controls. Transcriptome analysis of the peripheral blood of paediatric SCD patients and controls was also performed. Flow cytometry showed a 3.5-fold increase (p = 0.005) in CD19+ B cells in paediatric SCD and a 52% decrease in central memory B cells (p = 0.002) in adult SCD. Paediatric transcriptomic data revealed significant upregulation of pro-B cell transcripts (27 genes, top 3: E2F2, RAD51, ASPM) and plasma cell transcripts (37 genes, top 3: IGF1, TNFRSF17, DERL3). Cytokine analyses showed significant increases in IL-2 (p = 0.013), IL-4 (p = 0.026), TNFβ (p = 0.039), IL-13 (p = 0.034) and BAFF (p = 0.041) in paediatric SCD, and an increase in CD40L and BAFF in adult SCD. In summary, our data showed alterations in the humoral immophenotype in paediatric SCD, specifically an increase in CD19+ B cells, suggesting there may be significant alterations of homeostasis in the humoral immune system in children with SCD treated with hydroxyurea.

It was an honor to be asked to deliver the Walter B. Cannon Lecture during the 2024 American Physiological Summit meeting. Dr. Cannon served as president of the American Physiological Society from 1914-1916. He coined the term "fight or flight" to describe an animal's response to threats and the concept of Homeostasis. He was the consummate physician-scientist, an outstanding mentor and teacher, a prolific writer, and a humanitarian. The title of my lecture is based on a poem entitled "Ithaca," written by the Greek poet C. P. Cavafy, who recounts the 10 yr travels of Ulysses, from Troy to his home, Ithaca. Odysseus had to overcome many obstacles to survive this long journey. Like Odysseus, I encountered myriad of professional and health problems. But, I also have experienced the thrill of contributing to scientific knowledge, the satisfaction of watching my mentees develop into independent scientists, the excitement of teaching respiration physiology to medical and professional students, and the pleasure of being of service to my discipline by serving as Editor of the American Journal of Physiology-Lung Cellular and Molecular Physiology and of Physiological Reviews. During my career, I have been interested in identifying the basic mechanisms by which oxidant gases and pathogens damage the blood gas barrier resulting in acute and chronic lung injury. In this brief review, I summarize the results of current studies implicating free heme as a major mediator of acute lung injury and our efforts to develop recombinant forms of human hemopexin, as a countermeasure.

To investigate use of asthma controller medications and their effect on lung function in pediatric patients with sickle cell disease (SCD).

Retrospective study in pediatric patients who self-identified as African American with SCD treated at Phoenix Children's between 2014 and 2021. Associations of asthma controller medications with changes in lung symptoms (cough, wheeze, chest pain, shortness of breath with exercise, sleep disturbance), Acute Chest Syndrome (ACS), and percent predicted probabilities (FEV1, FVC, FEV1/FVC, FEF25%-75%)) were examined (Fisher exact, Wilcoxon rank sum) in SCD patients overall, and by physician-diagnosed asthma.

Of the total 98 SCD patients, 28 (29%) had an asthma diagnosis and 76 (78%) were treatment naïve. During study follow-up, asthma controller medications were used by 57 (58%) patients (35 new prescriptions, 13 continued prescriptions and 9 with prescription escalation), with 41 patients remaining treatment naïve. Medication use vs non-use during follow-up improved cough (33% vs 7%, p = 0.002), chest pain (12% vs 5%, p = 0.03) and shortness of breath with exercise (32% vs 10%, p = 0.01) among all SCD patients. Medications also improved the mean relative percent change FEV1 (12.3 vs -3.6; p < 0.0001), FVC (10.5 vs -1.3; p < 0.0001), and FEF25%-75%, (20.6 vs -8.8; p < 0.0001), overall and in both asthmatics and non-asthmatics.

These findings demonstrate improved lung function and respiratory symptoms with asthma controller medications in pediatric patients with SCD, irrespective of an asthma diagnosis, and hold great promise for this undertreated population.

Despite advances in the treatment of sickle cell disease (SCD), an inherited disorder leading to abnormal sickle hemoglobin (HbS) polymerization, patients continue to have a shorter life expectancy comparatively to the general population. Increase in the concentration of oxygenated HbS in red blood cells (RBCs) has been considered as a novel approach to inhibit HbS polymerization and reduce RBC sickling and their complications, raising interest for novel oxygen affinity modulators.

This review summarizes the characteristics and primary results obtained with osivelotor, a novel oxygen affinity modulator, for the treatment of SCD. Osivelotor is presented with improved pharmacokinetic properties comparatively to voxelotor. It may enable higher hemoglobin (Hb) occupancy at lower doses potentially leading to significant improvements of clinical outcomes.

The first clinical phase 2/3 trial with osivelotor reported increases of Hb levels and RBC counts, and decrease of RBC sickling. The treatment was apparently well tolerated. However, osivelotor shares the same mechanism of action as voxelotor, and therefore similar limitations regarding its efficacy for which the improvement in Hb level appears misleading. Several issues remain to be resolved before considering any drug approval.

Sickle cell disease (SCD) is frequently associated with cardiovascular complications, with cardiac involvement being evident in the electrocardiogram (ECG). This study aimed to systematically research the literature to investigate factors associated with ECG abnormalities in SCD patients.

Five online databases of PubMed, Scopus, Web of Science, Embase, and Google Scholar were reviewed using a broad search strategy to identify original studies reporting factors associated with abnormal ECG findings among patients with SCD. Using Comprehensive Meta-Analysis software, various effect sizes-odds ratios (ORs), mean differences, and correlation coefficients-were analyzed, pooled with a random effects model, and visualized through forest plots.

Data analysis from 23 studies covering 2943 SCD patients revealed increased odds of prolonged QTc interval (2.54, 95% CI = 1.34-4.83), ST segment elevation (9.23, 95% CI = 3.15-27.07), and ST segment depression (3.82, 95% CI = 1.99-7.33) during crises. Patients with moderate SCD severity had higher odds of having any ECG abnormalities compared to those with mild severity (2.80, 95% CI = 1.47-5.33). No significant associations were observed between moderate and mild severity for left ventricular hypertrophy, sinus arrhythmia, and sinus tachycardia, or between male and female for having any ECG abnormalities, left ventricular hypertrophy, and prolonged QTc interval. Additionally, mean hemoglobin level was 0.60 mg/dL lower (95% CI = -0.91 to 0.28) in those with prolonged QTc interval.

This study highlights the link between SCD status and severity, and hemoglobin, and ECG abnormalities. It emphasizes the need for cardiac monitoring, caution with QTc-prolonging medications, and routine ECG assessments to prevent cardiovascular complications in these high-risk patients.

Intrahepatic accumulation of cell-free hemoglobin (Hb) is a significant pathology linked with hemolytic disorders such as sickle cell disease (SCD). In addition to hepatic Kupffer cells, liver sinusoidal endothelial cells (LSECs) were recently reported to contribute to Hb clearance in SCD mice and patients via currently unknown endocytic mechanism. Using small-molecule inhibitors of endocytic pathway components in primary human and mouse LSECs, we show that sickle-Hb (HbS) uptake by LSECs occurs predominantly through micropinocytosis or fluid-phase endocytosis. However, inhibiting clathrin-mediated endocytosis, receptor recycling, or drop in pH also significantly attenuated HbS uptake by LSECs. LSEC-driven HbS uptake was independent of haptoglobin. Finally, we found that the presence of lipid droplets promotes endothelial HbS internalization, whereas hypolipidemic condition inhibits it. In conclusion, this study identifies previously unknown alternative mechanism of LSEC-mediated HbS internalization. Our findings also inform the need to evaluate the therapeutic potential of blocking these mechanisms to ameliorate hemolysis-associated liver damage in SCD and other hemolytic disorders.

Sickle cell disease (SCD) is an autosomal recessive disorder of blood characterized by a mutation in the β chain of hemoglobin (Hb), leading to the production of sickle Hb (HbS). In SCD, under low oxygen conditions, red blood cells (RBCs) containing HbS form a characteristic "sickle" shape, resulting in chronic hemolytic anemia and acute vaso-occlusive crises. Current therapies for SCD have limitations in efficacy or availability, highlighting the need for new anti-sickling drugs. To facilitate the discovery of new anti-sickling compounds, we previously developed a high throughput sickling assay, which permits rapid screening of thousands of compounds for the ability to inhibit RBC sickling. In this study, we improved the sickling assay by optimizing the assay condition and expanded our screening efforts by evaluating the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) compound library, which contains approximately 2.5 times more compounds than previously screened. We were able to increase the number of blood samples that were adequate for identifying anti-sickling compounds in the improved sickling assay and identified voxelotor and SNS-314 as compounds that successfully prevented sickling. The improved sickling assay will increase access to valuable blood samples from SCD volunteers, providing more opportunities to develop anti-sickling compounds for treating SCD.

Sickle cell disease (SCD) is a monogenic blood disorder characterized by abnormal hemoglobin S production, which polymerizes under hypoxia conditions to produce chronic red blood cell hemolysis, widespread organ damage, and vasculopathy. As a result of vaso-occlusion and ischemia-reperfusion injury, individuals with SCD have recurrent pain episodes, infection, pulmonary disease, and fall victim to early death. Oxidative stress due to chronic hemolysis and the release of hemoglobin and free heme is a key driver of the clinical manifestations of SCD. The net result is the generation of reactive oxygen species that consume nitric oxide and overwhelm the antioxidant system due to a reduction in enzymes such as superoxide dismutase and glutathione peroxidase. The primary mechanism for handling cellular oxidative stress is the activation of antioxidant proteins by the transcription factor NRF2, a promising target for treatment development, given the significant role of oxidative stress in the clinical severity of SCD. In this review, we discuss the role of oxidative stress in health and the clinical complications of SCD, and the potential of NRF2 as a treatment target, offering hope for developing effective therapies for SCD. This task requires our collective dedication and focus.

Newly produced platelets acquire a low activation state, but whether the megakaryocyte plays a role in this outcome has not been fully uncovered. Mesenchymal stem cells (MSCs) were previously shown to promote platelet production and lower platelet activation. We found that healthy megakaryocytes transfer mitochondria to MSCs, which is mediated by connexin 43 (Cx43) gap junctions on MSCs and leads to platelets at a low energetic state with increased LYN activation, characteristic of resting platelets with increased LYN activation, characteristic of resting platelets. On the contrary, MSCs have a limited ability to transfer mitochondria to megakaryocytes. Sickle cell disease (SCD) is characterized by hemolytic anemia and results in heightened platelet activation, contributing to numerous disease complications. Platelets in SCD mice and human samples had a heightened energetic state with increased glycolysis. MSC exposure to heme in SCD led to decreased Cx43 expression and a reduced ability to uptake mitochondria from megakaryocytes. This prevented LYN activation in platelets and contributed to increased platelet activation at steady state. Altogether, our findings demonstrate an effect of hemolysis in the microenvironment leading to increased platelet activation in SCD. These findings have the potential to inspire new therapeutic targets to relieve thrombosis-related complications of SCD and other hemolytic conditions.

Sickle cell disease (SCD) is an inherited hemoglobin disorder that is widespread across the globe. It is characterized by a very complex pathogenesis, but at the basis of the disease is the mutation of the HBB gene, which determines the production of a mutated hemoglobin: sickle cell hemoglobin (HbS). The polymerization of HbS, which occurs when the protein is in a deoxygenated state, and the greater fragility of sickle cell red blood cells (sRBCs) determine the release of iron, free heme, and HbS in the blood, favoring oxidative stress and the production of reactive oxygen species (ROS). These features are common to the features of a new model of cell death known as ferroptosis, which is characterized by the increase of iron and ROS concentrations and by the inhibition of glutathione peroxidase 4 (GPx4) and the System Xc-. In this context, this review aims to discuss the potential molecular and biochemical pathways of ferroptosis involved in SCD, aiming to highlight possible tags involved in treating the disease and inhibiting ferroptosis.

Most of the current treatment modalities for sickle hemoglobinopathy are disease-modifying rather than curative. Therefore, there is a need for effective treatment of complications of sickle cell disease (SCD) that impair quality of life. This need drives the evaluation of preclinical therapeutics in search of new treatment modalities.

Interventions are likely to progress from research to clinical practice, their potential impact, and future directions in SCD care: HbF inducers, pyruvate kinase activators, anti-selectin P monoclonal antibodies, allosteric Hb modifiers, proactive treatment of cerebral artery conditional blood velocity, multimodal, and gene therapy. Established treatment modalities (e.g with hydroxyurea) are not included because these have advanced well beyond the preclinical stage of therapeutics. Information dated 2025 backward was obtained from Medline, PubMed, and other public sources.

Places for the conduct of preclinical studies ought to include areas of high SCD prevalence. Limited resources currently hinder universal accessibility of curative SCD therapies in these places. The recent approval of non-viral gene therapy for SCD and the number of preclinical therapeutics in development bring realistic expectation that curative and disease-modifying interventions, such as multimodal therapy and proactive treatment of cerebral artery conditional blood velocity to prevent stroke, will become standard care.

Plasma free hemoglobin (PFH) is a direct biomarker for hemolysis that has been associated with clinical complications such as pulmonary hypertension and death in patients with sickle cell disease (SCD). We sought to characterize the relationship between PFH and more clinically available hemolytic markers including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), bilirubin, reticulocyte percentage and to derive a composite hemolysis score derived from principal component analysis (PCA) of these biomarkers. In 68 adult patients (median age 31 years old, IQR 25-39) with HbSS or HbSβ0-thalassemia enrolled in the IMPROVE II study, median PFH was elevated at 21.9 mg/dL (IQR 9.9-44.9 mg/dL) at steady state. Using Pearson correlation analysis, PFH had a stronger relationship to LDH (R = 0.699), AST (R = 0.587), and total bilirubin (R = 0.475), compared to reticulocyte count (R = 0.316). The hemolysis score was significantly associated with PFH (R = 0.677). When compared with other laboratory measures, PFH correlated with hemoglobin (R= -0.275) and HbS (R = 0.277), but did not correlate with white blood cell count (WBC) or HbF. The hemolysis score was significantly associated with WBC (R = 0.307), hemoglobin (R = -0.393), HbF (R=- 0.424), and HbS (R = 0.423). This study confirms that the conventional hemolytic biomarkers LDH, AST, bilirubin, and reticulocyte percentage correlate with PFH. Additionally, the hemolysis score is a valid tool to measure hemolysis and that it may be a marker of global hemolysis as opposed to PFH, which quantifies intravascular hemolysis. Further studies will be needed to elucidate the role of PFH and intravascular hemolysis in the development of clinical complications of sickle cell disease. Statements and Funding Declarations: The research leading to these results received funding from the National Heart, Lung, and Blood Institute (NHLBI) R01 HL142671 Grant under J.G. J.G. has also served as a consultant for CSL Behring, Novartis, and Novo Nordisk synteract DSMB and is supported by NHLBI RO1HL159116, R01 HL142671, R01 ES030717, UG1 HL138645, UH3 HL143192, U01HL167036, and the Doris Duke Charitable Foundation Advancing Cures grant. S.C. has served as a consultant for Pfizer and is supported by the NHLBI 5K23HL151884 grant. A.L. is supported by the NHLBI 5T32HL129974-05. C.J.M is supported by the NHLBI 5T32HL129974-05. P.S., and S.M. declare no conflicts and/or funding.

Sickle cell anemia (SCA) presents a complex interplay of factors, with the production of high levels of reactive oxygen species (ROS) and the chronic inflammatory process leading to chronic oxidative stress. In this context, efficient action of antioxidant systems becomes crucial, with particular emphasis on peroxiredoxins (PRDXs) due to their abundance and vital roles. Our primary objective was to establish associations between gene and protein expression of PRDXs 1, 2, and 6, as well as their reducers TRX1, TRXR1, and SRX1, with the characteristic hyperoxidative status observed in SCA patients. Concomitantly, we assessed the production of other essential antioxidant enzymes (SOD1, CAT, and GPX1) in reticulocytes and erythrocytes and explored mRNA levels of the NRF2/KEAP1/PKCδ complex. Our comprehensive analysis revealed a ∼ 3-fold elevation in ROS levels in erythrocytes of patients compared to healthy individuals. However, the NRF2/KEAP1/PKCδ complex exhibited a significant reduction in gene expression, hinting that another transcription factor may regulate the antioxidant response among SCA patients. In addition, the pattern of increased transcript levels of antioxidants in SCA patients was not associated with their protein levels, indicating a possible degradation by proteasome. The protein content of PRDX2 showed a significant reduction, indicating an increased vulnerability of these cells to oxidative damage. Intriguingly, both PRDXs 1 and 2 exhibited significant increases in the plasma of SCA patients, indicating that, besides their well-known intracellular antioxidant role, these enzymes may also play a vital extracellular role in modulating inflammation in these individuals. Our findings unveil novel insights into the redox metabolism adaption of erythroid cells in response to the presence of HbS in homozygosity, thus, into the complex SCA pathophysiology. Moreover, our study reveals the simultaneous presence of both PRDXs 1 and 2 in the plasma of these patients, thereby offering valuable implications for potential prognostic and therapeutic avenues.

This review addresses oxidative stress and redox signaling in the pancreas under healthy physiological conditions as well as in acute pancreatitis, chronic pancreatitis, pancreatic cancer, and diabetes. Physiological redox homeodynamics is maintained mainly by NRF2/KEAP1, NF-κB, protein tyrosine phosphatases, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), and normal autophagy. Depletion of reduced glutathione (GSH) in the pancreas is a hallmark of acute pancreatitis and is initially accompanied by disulfide stress, which is characterized by protein cysteinylation without increased glutathione oxidation. A cross talk between oxidative stress, MAPKs, and NF-κB amplifies the inflammatory cascade, with PP2A and PGC1α as key redox regulatory nodes. In acute pancreatitis, nitration of cystathionine-β synthase causes blockade of the transsulfuration pathway leading to increased homocysteine levels, whereas p53 triggers necroptosis in the pancreas through downregulation of sulfiredoxin, PGC1α, and peroxiredoxin 3. Chronic pancreatitis exhibits oxidative distress mediated by NADPH oxidase 1 and/or CYP2E1, which promotes cell death, fibrosis, and inflammation. Oxidative stress cooperates with mutant KRAS to initiate and promote pancreatic adenocarcinoma. Mutant KRAS increases mitochondrial reactive oxygen species (ROS), which trigger acinar-to-ductal metaplasia and progression to pancreatic intraepithelial neoplasia (PanIN). ROS are maintained at a sufficient level to promote cell proliferation, while avoiding cell death or senescence through formation of NADPH and GSH and activation of NRF2, HIF-1/2α, and CREB. Redox signaling also plays a fundamental role in differentiation, proliferation, and insulin secretion of β-cells. However, ROS overproduction promotes β-cell dysfunction and apoptosis in type 1 and type 2 diabetes.

Sickle cell disease (SCD) is a hereditary hemolytic anemia associated with the alteration of the membrane composition of the sickle erythrocytes, the loss of glycolysis, dysregulation of the pyruvate phosphatase pathway, and changes in nucleotide metabolism of the sickle red blood cell (RBC). This review provides a comprehensive overview of the impact of the presence of Hb S, which leads to the disruption of the normal RBC metabolism. The intricate interplay between the redox and energetic balance in erythrocytic cells, where the glycolysis, pentose phosphate pathway, and methemoglobin reductase pathways are all altered in sickle RBC, is a key focus. Moreover, this review summarizes the current knowledge about the disease-modifying agents and their action mechanisms based on the sickle RBC alterations previously mentioned (i.e., their association with beneficial effects on the sickle cells' membrane, to their RBCs' energy metabolism, and to their oxidative status). Therefore, providing a comprehensive understanding of how sickle cells cope with the disruption of metabolic homeostasis and the most promising therapeutic agents able to ameliorate the various consequences of abnormal sickle RBC alterations.

Vaso-occlusive pain crisis (VOC) is recognized as a prominent complication of sickle cell disease, accompanied by debilitating pain and serious consequences for patients, making it the primary cause of visits to hospital emergency departments. In the etiology of VOC, the intricate interaction of endothelial cells, hypoxia, inflammation, and the coagulation system is pivotal. Hemoglobin S polymerization under hypoxic conditions leads to the formation of rigid and adhesive red blood cells that interact with vascular endothelial cells and other blood cells, causing occlusion and subsequent inflammation. Hemolysis of red blood cells results in anemia and heightened inflammation, whereas oxidative stress and involvement of the coagulation system further complicate matters. In this review, we strive to examine the pathophysiology of VOC from these mentioned aspects by consolidating findings from various studies, as a comprehensive understanding of the causes of VOC is essential for the development of targeted therapeutic interventions and the prevention and management of pain, ultimately improving the quality of life for patients.

Asthma is strongly associated with poor health amongst individuals with SCD. Adverse Childhood Experiences (ACEs) are traumatic experiences occurring before 18 years of age. ACEs occur at the individual and familial (original ACEs) levels and expand to the community level (additional ACEs). Chronic exposure to ACEs leads to toxic stress, inflammation, and risk for chronic illnesses, including asthma. This study examined the relationship between ACEs and asthma among children and adolescents with SCD.

Employing a cross-sectional study design, 75 children/adolescents with SCD were screened for ACEs. Prevalence ratios and logistic regressions were used to test the association and independent relationship between ACEs and asthma.

Fifty-nine (78%) participants reported exposure to at least 1 ACE. Adolescents exposed to ≥2 original- or ≥4 expanded ACEs (original + additional) were 1.15 times as likely to have asthma compared to those with no/low ACEs (95% CI: 1.03, 1.28, P = .01; 1.07, 1.24, P ≤ .001). Through logistic regression analyses, covarying age, sex, SCD genotype, income, and disease-modifying therapy, original (OR 1.52, 95% CI: 1.031, 2.242), additional (2.43, 95% CI: 1.335, 4.421), and expanded ACEs (1.529, 95% CI: 1.149, 2.036) were all shown to be independently associated with asthma.

The findings from this research support our hypothesis that children/adolescents with SCD exposed to a higher number of ACEs have a higher prevalence of asthma compared to low-exposed subjects. This study lays the foundation for future longitudinal and interventional studies aimed to improve SCD-asthma outcomes through ACE-protective mechanisms.

Hemoglobin (Hb) releases during hemorrhaging and causes oxidative damage, further exacerbates the development of multiple diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates cellular defenses against toxic and oxidative challenges. However, the regulation mechanism of Nrf2 in Hb-induced oxidative stress remains unclear in teleost. To accomplish this goal, a hemolysis model was established by injecting grass carp with phenylalanine (PHZ), and the immunofluorescence analysis (IFA) and hematoxylin and eosin (H&E) staining revealed that PHZ-induced hemolysis caused Hb accumulation and hepatic vacuolization, resulted in tissue damage. Prussian blue, Sirius red, and Masson staining results revealed significant iron deposition and extensive collagen fiber accumulation in the liver. IFA and immunohistochemical analyses demonstrated that PHZ-induced hemolysis markedly increased the production of reactive oxygen species (ROS), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). The quantitative real-time PCR (qRT-PCR) analysis data revealed that the PHZ-induced hemolysis also significantly upregulated the expression of antioxidant-related genes through activation of the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/Nrf2 signaling pathway. To further explore the molecule regulation mechanism of PHZ-induced hemolysis, the RNA-seq analysis was performed, and the data revealed that the AMPK/Nrf2 and multiple programmed cell death pathways, including ferroptosis, autophagy, apoptosis, and necroptosis in PHZ injection groups were significant upregulated. In vitro, the hemin supplementation activated the expression of target genes in the AMPK/Nrf2 pathway detected by qRT-PCR. To further verify the regulation function of Nrf2, an Nrf2 activator (4OI) was supplemented, and the flow cytometer analysis results suggested that the Hb-induced cell damage was significantly attenuated. However, the supplementary of ML385 down-regulated the AMPK/Nrf2 pathway and aggravated the hemin induced cell death. In conclusion, these findings highlight the critical regulatory role of the AMPK/Nrf2 signaling pathway in protecting against Hb-induced oxidative damage in the liver of grass carp.

Oxidative stress is considered as the root-cause of different pathological conditions. Transition metals, because of their redox-active states, are capable of free radical generation contributing oxidative stress. Hemoglobin and myoglobin are two major heme proteins, involved in oxygen transport and oxygen storage, respectively. Heme prosthetic group of heme proteins is a good reservoir of iron, the most abundant transition metal in human body. Although iron is tightly bound in the heme pocket of these proteins, it is liberated under specific circumstances yielding free ferrous iron. This active iron can react with H2O2, a secondary metabolite, forming hydroxyl radical via Fenton reaction. Hydroxyl radical is the most harmful free radical among all the reactive oxygen species. It causes oxidative stress by damaging lipid membranes, proteins and nucleic acids, activating inflammatory pathways and altering membrane channels, resulting disease conditions. In this review, we have discussed how heme-irons of hemoglobin and myoglobin can promote oxidative stress under different pathophysiological conditions including metabolic syndrome, diabetes, cardiovascular, neurodegenerative and renal diseases. Understanding the association of heme proteins to oxidative stress may be important for knowing the complications as well as therapeutic management of different pathological conditions.

To assess foetal circulating free foetal haemoglobin (fHbF) levels and heme defences, correlated to foetal circulatory biometry and foetal sex in severe early-onset foetal growth restriction (FGR).

A prospective study severe early-onset foetal growth restriction pregnancies with close clinical management (estimated foetal weight (EFW) < 3rd centile and < 600 g at 20-26 + 6 weeks; N = 20).

Temporal foetal vascular obstetric biometry was recorded. Cord blood fHbF and key heme-scavenger defences were measured and compared with normal term births (N = 26) and births with late-onset FGR (N = 12).

fHbF was elevated in early-onset FGR compared with normal pregnancy: 0.437(0.337/0.753) mg/mL; and 0.098 (0.045/0.264) mg/mL, respectively (p < 0.0001); whilst hemopexin was downregulated in early- (p < 0.001) and late-onset FGR (p < 0.0001), compared to normal pregnancy: 36(14/81) μg/mL, 25(19/40) μg/mL, and 155(132/219) μg/mL, respectively; median (interquartile ranges). Early-onset FGR male foetuses had higher HbF compared with the normal males: 0.710(0.433/0.857) mg/mL; (p < 0.001); 0.099(0.043/0.246) mg/mL, respectively; median (interquartile ranges). In early-onset FGR, ratios of mid-cerebral artery and umbilical artery pulsatility indices correlated positively with heme-scavenger levels (hemopexin and a heme-handling composite measure: p < 0.05, r = 0.672; and p < 0.01, r = 0.620; respectively), indicating lower levels are associated with cerebral vascular redistribution. These heme handling measures also positively correlated with gestational age at delivery (r = 0.713 and r = 0.642, respectively, p < 0.01, both) and birthweight (r = 0.742, p < 0.001; and r = 0.523, p < 0.05; respectively).

Overproduction of fHbF and an inadequate heme defence may contribute to foetal distress and poor umbilical arterial Dopplers in early onset FGR due to elevated placental vascular resistance and vascular inflammation.

The current study sought to determine the prevalence of elevated estimated pulmonary artery systolic pressure (ePASP) in clinically stable children and adults with sickle cell disease)SCD(worldwide.

The studies included were identified through a search of databases such as PubMed, Scopus, Science Direct, Web of Science, and Embase, as well as Google Scholar engine, adhering to specific inclusion and exclusion criteria. Heterogeneity among the primary study results was assessed using the I-squared index, while publication bias was evaluated through funnel plots, Egger's test, and trim and fill analysis. All statistical analyses were conducted using R software, version 4.3.0.

79 primary studies were included, comprising 6,256 children (<18 years old) and 6,582 adults (≥18 years old) with SCD from 22 countries. The prevalence of elevated ePASP was found to be 21.8% (95% confidence interval [CI]: 18.46 to 25.07) in children and 30.6% (95% CI: 27.1 to 34.1) in adults. The prevalence of elevated ePASP among studies with severe SCD genotypes including HbSS and HbS/β0 was found to be 19.45% (95% CI: 14.95 to 23.95) in children and 29.55% (95% CI: 24.21 to 34.89) in adults. Furthermore, sex-specific prevalence among SCD patients with elevated ePASP indicated the highest prevalence in male children at 60.35% (95% CI: 54.82 to 65.88) and adult female patients at 54.41% (95% CI: 47.3 to 61.5). A comparative analysis of the mean values of clinical and laboratory results revealed significant differences in several characteristics, including age, oxygen saturation, hemoglobin levels, fetal hemoglobin, white blood cell counts, platelet counts, and reticulocyte counts between patients with elevated ePASP and those without, in both children and adult SCD populations.

Our findings regarding clinically stable SCD patients highlight a high prevalence of elevated ePASP.

Sickle cell disease (SCD) is an inherited hematologic disease caused by sickle hemoglobin as the predominant RBC hemoglobin or by sickle hemoglobin in combination with other abnormal β-hemoglobin variants like HbC, HbD and others. Sickling of erythrocytes under deoxygenated conditions is the basis of inflammatory and thrombotic cascades which result in multiple serious complications, leading to early morbidity and mortality. While HLA-matched allogeneic bone marrow transplantation is potentially curative, it has considerable limitations due to potential severe toxicities. Despite slow progress towards novel therapeutic strategies for SCD and hydroxyurea being the sole medication that is shown to reduce vaso-occlusive events and mortality for almost 20 years, several pharmacological agents targeting different mechanisms have been examined in clinical trials and recently US- US-FDA-approved, including L-glutamine and crizanlizumab. Voxelotor was previously US-FDA-approved but has been voluntarily withdrawn from the market as the overall benefit did not outweigh the risks. Gene therapies based on CRISPR-Cas9 and lentiviral vectors have been very recently approved. However, excessive costs are a barrier to widespread use. Nonetheless, there is still a large area of unmet needs for patients with SCD, and further research towards better care is warranted.

Sickle cell disease (SCD) is a mono-genic disorder causing chronic hemolysis, anemia, and vaso-occlusion, leading to musculoskeletal complications such as osteonecrosis, osteoporosis, and bone fractures affecting 50-70% SCD patients. These complications result from a complex interplay of genetic and physiological factors, including abnormal hemoglobin production, chronic inflammation, and oxidative stress. This review discusses the pathophysiology, pre-clinical symptoms, and clinical manifestations of musculoskeletal complications in SCD, as well as current treatment options, including pharmacological interventions, surgical procedures, and bone marrow transplantation. Early detection of pre-clinical symptoms is crucial to prevent progression. Pharmacological interventions (analgesics, anti-inflammatory agents, bone-modifying agents and hydroxyurea), surgical interventions (core decompression, bone grafting, joint replacement and osteotomy) and supportive measures enhance mobility, strength and well-being. A multidisciplinary approach is essential for optimal care, and early diagnosis and management are crucial to prevent long-term damage and improve outcomes. Future research directions include targeted therapies, biomarker investigation and infrastructure development to improve outcomes for SCD individuals with musculoskeletal complications.

The transsulfuration (TSS) pathway is an alternative source of cysteine for glutathione synthesis. Little of the TSS pathway in antioxidant capacity in sickle cell disease (SCD) is known. Here, we evaluate the effects of TSS pathway activation through cystathionine beta-synthase (CBS) to attenuate reactive oxygen species (ROS) and ferroptosis stresses in SCD. A vital contribution of the TSS pathway in sustaining cysteine levels is detected only under hemin exposure or physiological but not supraphysiological cystine supplement. Mechanistic studies show that hemin suppresses CBS expression to inhibit the TSS pathway and de novo cysteine biosynthesis. By contrast, the expression of CBS is inducible by dimethyl fumarate (DMF) through nuclear factor erythroid 2-related factor 2 (NRF2) activation and CpG islands DNA hydroxymethylation. DMF induces the expression of L-2-hydroxyglutarate dehydrogenase (L2HGDH) to downregulate L-2-hydroxyglutarate (L2HG) and increase global and locus-specific DNA hydroxymethylation levels. This DMF-upregulated DNA hydroxymethylation affects CBS locus chromatin structure modifications and upregulates gene expression. Our results suggest that CBS of the TSS pathway plays an important role in maintaining cysteine levels under restricted cystine availability or excess hemin exposure, and CBS upregulation by DMF increases the cellular glutathione levels to protect against ROS and ferroptosis stress in SCD.

Pulmonary vascular disease is not a single condition; rather it can accompany a variety of pathologies that impact the pulmonary vasculature. Applying precision medicine strategies to better phenotype, diagnose, monitor, and treat pulmonary vascular disease is increasingly possible with the growing accessibility of powerful clinical and research tools. Nevertheless, challenges exist in implementing these tools to optimal effect. The 2023 Grover Conference Series reviewed the research landscape to summarize the current state of the art and provide a better understanding of the application of precision medicine to managing pulmonary vascular disease. In particular, the following aspects were discussed: (1) Clinical phenotypes, (2) genetics, (3) epigenetics, (4) biomarker discovery, (5) application of precision biology to clinical trials, (6) the right ventricle (RV), and (7) integrating precision medicine to clinical care. The present review summarizes the content of these discussions and the prospects for the future.

Individuals living with Sickle Cell Disease (SCD) are subject to numerous chronic complications, including disabling chronic pain, often dependent on opioids and with important repercussions on the quality of life. The use of Medicinal Cannabis in this scenario may be a promising strategy for mitigating this impact.

This work compiles current knowledge regarding the endocannabinoid system in humans and the role of this system in various organic functions. Articles were retrieved through a comprehensive search of the PubMed NCBI database, covering relevant studies up to 2024. These data bring important speculations on the potential role of the use of medicinal cannabis in modulating SCD chronic complications, and the preliminary results of clinical trials carried out in this condition are discussed.

The search for understanding the role of cannabis-derived products in the management of chronic complications of sickle cell disease could add resources to the serious challenge of dealing with the multiple aspects of the disease faced by patients. They range from the management of chronic pain itself to the risks of opioid dependence, in addition to other difficult scenarios, such as leg ulcers and chronic inflammation and its consequences.

The objective of this study was to explore the therapeutic effect of blueberries on hematological parameters, oxidative stress, and interleukin-10 levels in acute hemolytic anemia induced by the administration of an intraperitoneal injection of 40 mg/kg phenylhydrazine. Male Wistar rats were divided into three groups: Control, anemia (PHZ), and anemia plus blueberries (PHZ+BB). Blueberries were administered via oral gavage (250 mg/day). The erythrocyte osmotic fragility, splenomegaly, iron metabolism, hematological analysis, reactive oxygens species, sulfhydryl group, and interleukin-10 levels were evaluated. The erythrocyte osmotic fragility (in 0.85% and 0.55% sodium chloride solution) and spleen weight-to-body weight ratio (∼400%) were elevated in the PHZ and PHZ+BB Groups compared to the controls (p-value < 0.05). Increased transferrin and reactive oxygens species levels were found in the PHZ (15%) compared to the Control Group (p-value < 0.05). There was an immune inflammatory response in the PHZ Group due to increases in the total leukocyte (300%), lymphocyte (100%), and neutrophil (400%) counts compared to the Control Group (p-value < 0.05); the PHZ Group showed increased interleukin-10 levels (100%) compared to the Control Group (p-value < 0.05). Blueberries showed a partial protective effect on these parameters, since there were lower neutrophil and lymphocyte counts and diminished interleukin-10 levels in the PHZ+BB Group compared to the PHZ Group (p-value < 0.05). In addition, blueberries increased sulfhydryl group levels (p-value < 0.05). These data suggest a protective role of blueberries against inflammatory response and oxidative stress in an acute hemolytic anemia model.

Sickle cell disease, a debilitating, inherited haemolytic anaemia with premature morbidity and mortality, affects millions globally. Mitapivat, a first-in-class, oral, allosteric activator of pyruvate kinase, improves red blood cell survival by increasing ATP and diminishes sickling by decreasing 2,3-diphosphoglycerate. We aimed to evaluate the efficacy and safety of mitapivat in patients with sickle cell disease.

We report results from the phase 2, 12-week, double-blind period of RISE UP, a global, phase 2/3, double-blind, randomised, placebo-controlled trial. The phase 2 part of the study was conducted at 32 clinical study sites across 13 countries. Patients aged 16 years or older with a confirmed diagnosis of sickle cell disease (any genotype), baseline haemoglobin of 5·5-10·5 g/dL (inclusive), and two to ten sickle cell pain crises within 12 months before providing informed consent, were randomly assigned 1:1:1 to receive oral mitapivat 50 mg, 100 mg, or placebo twice daily, in this portion of the study which is now complete. Randomisation was performed using a permuted-block method and concealed with an interactive response system; patients, investigators, and individuals assessing outcomes were masked to treatment assignment. Primary efficacy and safety endpoints were haemoglobin response (≥1·0 g/dL increase from baseline in average haemoglobin concentration from week 10 through week 12), and type, severity, and relationship to study drug of adverse and serious adverse events. Efficacy and safety endpoints were evaluated in the full analysis set (all randomly assigned patients) and safety analysis set (all patients who received at least one dose of study drug), respectively. This study is registered with ClinicalTrials.gov as part of an ongoing phase 2/3 study (NCT05031780).

Between Jan 19, 2022, and April 25, 2023, 79 patients were randomly assigned (51 [65%] female, 28 [35%] male; 46 [58%] Black or African American, 26 [33%] White, five [6%] multiracial, two [3%] Asian); 26 received mitapivat 50 mg, 26 received mitapivat 100 mg, and 27 received placebo, twice daily. Both treatment groups showed a statistically significant haemoglobin response rate versus placebo (12 [46%] of 26 patients in the mitapivat 50 mg group and 13 [50%] of 26 patients in the mitapivat 100 mg group, versus one [4%] of 27 patients in the placebo group; two-sided p=0·0003 and p=0·0001, respectively). Mitapivat was generally well tolerated. Serious adverse events were reported in two (8%) of 26 patients in the mitapivat 50 mg group, four (15%) of 26 patients in the mitapivat 100 mg group, and three (11%) of 27 patients in the placebo group; grade 3 or worse adverse events occurred in three (12%), five (19%), and two (7%) patients, respectively. No serious or grade 3 or worse adverse events were considered treatment related and there were no treatment-related deaths. The most common grade 3 or worse adverse events were infections and infestations, and included one patient in the placebo group with an infected skin ulcer, one patient in the mitapivat 50 mg group with meningitis and one with pelvic inflammatory disease, and one patient each with malaria, pneumonia, and tonsillitis in the mitapivat 100 mg group.

Mitapivat, through its dual effect of increasing ATP and decreasing 2,3-diphosphoglycerate, could provide clinical benefit to patients with sickle cell disease. These results support continued evaluation of mitapivat in the phase 3 portion of the study.

Agios Pharmaceuticals.

Cell-free hemoglobin (CFH) is used to detect hemolysis and was recently suggested to trigger acute lung injury. However, its role has not been elucidated in severe acute respiratory distress syndrome (ARDS) patients undergoing extracorporeal membrane oxygenation (ECMO). We investigated the association of carboxyhemoglobin (COHb) and haptoglobin-two indirect markers of hemolysis-with mortality in critically ill patients undergoing veno-venous ECMO (VV-ECMO) with adjusted and longitudinal models (primary aim). Secondary aims included assessment of association between COHb and haptoglobin with the development of ventilator-associated pneumonia (VAP) and with hemodynamics. We retrospectively collected physiological, laboratory biomarkers, and outcome data in 147 patients undergoing VV-ECMO for severe ARDS. Forty-seven patients (32%) died in the intensive care unit (ICU). Average levels of COHb and haptoglobin were higher and lower, respectively, in patients who died. Higher haptoglobin was associated with lower pulmonary (PVR) and systemic vascular resistance, whereas higher COHb was associated with higher PVR. Carboxyhemoglobin was an independent predictor of VAP. Both haptoglobin and COHb independently predicted ICU mortality. In summary, indirect signs of hemolysis including COHb and haptoglobin are associated with modulation of vascular tone, VAP, and ICU mortality in respiratory ECMO. These findings suggest that CFH may be a mechanism of injury in this patient population.