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The peroxiredoxins (PRXs) constitute a ubiquitous antioxidant. Growing evidence in neurodegenerative disorders such as Parkinson’s disease (PD) or Alzheimer’s disease (AD) has highlighted a crucial role for PRXs against neuro-oxidation. Chorea-acanthocytosis/Vps13A disease (ChAc) is a devastating, life-shortening disorder characterized by acanthocytosis, neurodegeneration and abnormal proteostasis. We recently developed a Vps13a−/− ChAc-mouse model, showing acanthocytosis, neurodegeneration and neuroinflammation which could be restored by LYN inactivation. Here, we show in our Vps13a−/− mice protein oxidation, NRF2 activation and upregulation of downstream cytoprotective systems NQO1, SRXN1 and TRXR in basal ganglia. This was associated with upregulation of PRX2/5 expression compared to wild-type mice. PRX2 expression was age-dependent in both mouse strains, whereas only Vps13a−/− PRX5 expression was increased independent of age. LYN deficiency or nilotinib-mediated LYN inhibition improved autophagy in Vps13a−/− mice. In Vps13a−/−; Lyn−/− basal ganglia, absence of LYN resulted in reduced NRF2 activation and down-regulated expression of PRX2/5, SRXN1 and TRXR. Nilotinib treatment of Vps13a−/− mice reduced basal ganglia oxidation, and plasma PRX5 levels, suggesting plasma PRX5 as a possible ChAc biomarker. Our data support initiation of therapeutic Lyn inhibition as promptly as possible after ChAc diagnosis to minimize development of irreversible neuronal damage during otherwise inevitable ChAc progression.

Background It has been established that children with Autism Spectrum Disorders (ASD) are affected by oxidative stress, the origin of which is still under investigation. In the present work, we evaluated inflammatory and pro-oxidant soluble signature in non-syndromic ASD and age-matched typically developing (TD) control children. Methods We analyzed leukocyte gene expression of inflammatory cytokines and inflammation/oxidative-stress related molecules in 21 ASD and 20 TD children. Moreover, in another—comparable—group of non-syndromic ASD (N = 22) and TD (N = 21) children, we analyzed for the first time the protein expression of the four members of the antioxidant enzyme family of peroxiredoxins (Prx) in both erythrocyte membranes and in plasma. Results The gene expression of IL6 and of HSP70i, a stress protein, was increased in ASD children. Moreover, gene expression of many inflammatory cytokines and inflammation/oxidative stress-related proteins correlated with clinical features, and appeared to be linked by a complex network of inter-correlations involving the Aryl Hydrocarbon Receptor signaling pathway. In addition, when the study of inter-correlations within the expression pattern of these molecules was extended to include the healthy subjects, the intrinsic physiological relationships of the inflammatory/oxidative stress network emerged. Plasma levels of Prx2 and Prx5 were remarkably increased in ASD compared to healthy controls, while no significant differences were found in red cell Prx levels. Conclusions Previous findings reported elevated inflammatory cytokines in the plasma of ASD children, without clearly pointing to the presence of neuro-inflammation. On the other hand, the finding of microglia activation in autoptic specimens was clearly suggesting the presence of neuro-inflammation in ASD. Given the role of peroxiredoxins in the protection of brain cells against oxidative stress, the whole of our results, using peripheral data collected in living patients, support the involvement of neuro-inflammation in ASD, and generate a rational for neuro-inflammation as a possible therapeutic target and for plasma Prx5 as a novel indicator of ASD severity.

BackgroundChorea-acanthocytosis (ChAc) is a neurodegenerative disease caused by mutations in the VPS13A gene. It is characterized by several neurological symptoms and the appearance of acanthocytes. Elevated tyrosine kinase Lyn activity has been recently identified as one of the key pathophysiological mechanisms and therefore represents a promising drug target.MethodsWe evaluated an individual off-label treatment with the FDA-approved tyrosine kinase inhibitor dasatinib (100 mg/d, 25.8-50.4 weeks) of three ChAc patients. Alongside with a thorough safety monitoring, we assessed motor and non-motor scales (e.g. MDS-UPDRS, UHDRS, quality of life) as well as routine and experimental laboratory parameters (e.g. serum neurofilament, Lyn kinase activity, actin cytoskeleton in red blood cells).ResultsDasatinib appeared to be reasonably safe. The clinical parameters remained stable without significant improvement or deterioration. Regain of deep tendon reflexes was observed in one patient. Creatine kinase, serum neurofilament levels and acanthocyte count did not reveal consistent effects. However, reduction of initially elevated Lyn kinase activity and accumulated autophagy markers as well as partial restoration of actin cytoskeleton was found in red blood cells.DiscussionWe report on the first treatment approach with disease-modifying intention in ChAc. The experimental parameters indicate target engagement in red blood cells, while clinical effects on the central nervous system could not be proven within a rather short treatment time. Limited knowledge on the natural history of ChAc and the lack of appropriate biomarkers remain major barriers for “clinical trial readiness”. Here, we suggest a panel of outcome parameters for future clinical trials in ChAc.

Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a−/− mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a−/− basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a−/− Lyn−/− showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a−/− hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.

International audience; Anemia in β-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on β-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbb th3/+ mouse model for β-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbb th3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in β-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing β-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with β-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for β-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.

Mitapivat, an oral activator of pyruvate kinase (PK), was recently shown to improve b-thalassemic anemia with a reduction of ineffective erythropoiesis and an amelioration of b-thalassemic red cell features in a mouse model for b-thalassemia (Hbb 3th/+ mice).). These changes were also associated with a beneficial effect on iron homeostasis by modulation of duodenal DMT1 expression (Matte A et al JCI 2021). Two clinical studies have shown improvement of anemia and ineffective erythropoiesis with mitapivat treatment in patients with non-transfusion-dependent (NTD) thalassemia (Kuo et al. EHA 2021). Based on these results, Phase 3 studies in both NTD and TD thalassemia are currently on going. The objective of this preclinical study was to determine if treatment with mitapivat affects the length between transfusion of red blood cells (RBCs) and the liver iron concentration (LIC). Using a previously established murine model of RBCs transfusions (Park Y et al Blood 2020), in Hbb 3th/+ mice, we used Hb 10.5 g/dL as threshold for RBCs transfusion, with washed RBCs, at 40% Hct (400 uL total volume infused). The animals were divided into two groups: vehicle and mitapivat (50mg/Kg by gavage BID for up to 61 days).The length of the interval between transfusions increased in mitapivat treated compared to vehicle treated animals (transfusion interval: 13.8±1.0 days vs vehicle 10.5±1.0 days respectively n=4 and n=3). In both groups, the transfusion regimen induced a significant reduction in spleen weight/mouse weight ratio and in extramedullary erythropoiesis. We also found a significant reduction in liver iron content (LIC) in mitapivat treated compared to vehicle treated animals. We then evaluated the effects of mitapivat in combination with iron chelation using deferiprone (DFP,1.25 mg/mL, drinking water). Casu et al. have previously shown in the same mouse model for β-thal that DFP did not affect erythropoiesis. In the β-thal mice, we did not find negative effects on hematologic parameters when mitapivat (50 mg/Kg/d by gavage BID) was co-administrated with DFP for 28 days. LIC was reduced in mitapivat treated mice and in mitapivat +DFP treated β-thal mice was further decreased compared to vehicle treated animals. This allowed us to reduce DFP dosage from 1.25 to 0.8 mg/mL in mitapivat treated β-thal mice. These data show that in mouse model of transfused β-thalassemia, mitapivat increases the time interval between transfusions, reduces transfusion burden and allows a reduction of the dosing iron chelation with DFP. Thus, mitapivat might represent an interesting option in transfusion dependent β-thalassemic patients, being transfusion burden still an unmet need in this patient population. Disclosures Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. De Franceschi: F. Hoffmann-La Roche Ltd: Consultancy.

Cardiomyopathy due to iron-overload is a severe complication of patients undergoing chronic transfusion regimen such as β-thalassemia and myelodysplastic syndromes. Previous studies have shown the key role of Nrf2, a redox-related transcriptional factor, in both β-thalassemia erythropoiesis and iron homeostasis (Matte A et al. ARS 2018, 2019; Lim PJ et al Nat Metab, 2019). Here, we compared Nrf2 knockout male mice (Nrf2 -/-) and C57BL-6J as wild-type (WT) controls, with a focus on cardiac function. Nrf2 -/- mice were characterized by a mild chronic hemolytic anemia associated with ineffective erythropoiesis, similar to what observed in murineβ-thalassemia (Toya SCM et al., Blood, 2019). Aging Nrf2 -/- mice developed systolic and diastolic dysfunction, associated with increased cardiac oxidative stress, degradation of the calcium-dependent SERCA2A transporter and activation of metalloproteinase MMP9, involved in both SERCA2A degradation and heart remodeling. In Nrf2 -/- mice, we observed increased plasma NTBI, heart iron deposition and elevated expression of cardiac ferroportin when compared to WT animals. Moreover, cardiac Hamp mRNA levels were down-regulated in aging Nrf2 -/- mice when compared to WT mice. This pattern was consistent with progressive cardiac iron overload in absence of Nrf2. Interestingly, activation of TGF-b receptor and PDGF-B-related pathway as well as increased collagen deposition were observed in hearts from 12 months old Nrf2 -/- mice. Taken together our data suggest an aging-associated development of iron-overload cardiomyopathy in mice genetically lacking Nrf2. To evaluate the role of Nrf2 in iron overload cardiomyopathy, Nrf2 -/- and WT mice were exposed to dietary iron supplementation (2.5% w/w carbonyl iron for 28 days). Nrf2 -/- mice developed cardiac hypertrophy which was accompanied by a worsening in collagen deposition and persistent activation of PDGF-B pathway. This was associated with inflammatory vasculopathy. The biologic importance of Nrf2 is supported by the cardiac activation of Nrf2, degradation of SERC2A and activation of TGF-b receptor and PDGF-B pathway in a mouse model of beta thalassemia intermedia, the Hbb3th/+ mice. Collectively our data support the crucial role of Nrf2 in the protection of cardiomyocytes against iron cytotoxicity which significantly develops in aging as well as in β-thalassemia. Disclosures Vinchi: Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Vifor Pharma: Research Funding; PharmaNutra: Research Funding; Novartis: Research Funding. Ghigo: Kither Biotech: Other: Board member and Co-Founder. Iolascon: Bluebird Bio: Other: Advisory Board; Celgene: Other: Advisory Board.

Fyn is a tyrosine kinase belonging to the Src family (Src-Family-Kinase, SFK), ubiquitously expressed. Previously, we report that Fyn is important in stress erythropoiesis. Here, we show that in red cells Fyn specifically stimulates G6PD activity, resulting in a 3-fold increase enzyme catalytic activity (kcat) by phosphorylating tyrosine (Tyr)-401. We found Tyr-401 on G6PD as functional target of Fyn in normal human red blood cells (RBC), being undetectable in G6PD deficient RBCs (G6PD-Mediterranean and G6PD-Genova). Indeed, Tyr-401 is located to a region of the G6PD molecule critical for the formation of the enzymatically active dimer. Amino acid replacements in this region are mostly associated with a chronic hemolysis phenotype. Using mutagenesis approach, we demonstrated that the phosphorylation status of Tyr401 modulates the interaction of G6PD with G6P and stabilizes G6PD in a catalytically more efficient conformation. RBCs from Fyn-/−mice are defective in G6PD activity, resulting in increased susceptibility to primaquine-induced intravascular hemolysis. This negatively affected the recycling of reduced Prx2 in response to oxidative stress, indicating that defective G6PD phosphorylation impairs defense against oxidation. In human RBCs, we confirm the involvement of the thioredoxin/Prx2 system in the increase vulnerability of G6PD deficient RBCs to oxidation. In conclusion, our data suggest that Fyn is an oxidative radical sensor, and that Fyn-mediated Tyr-401 phosphorylation, by increasing G6PD activity, plays an important role in the physiology of RBCs. Failure of G6PD activation by this mechanism may be a major limiting factor in the ability of G6PD deficient RBCs to withstand oxidative stress., Graphical abstract Image 1, Highlights • In red cells, Fyn acts as an oxidative sensor. • Fyn specifically stimulates G6PD activity by phosphorylating tyrosine (Tyr)-401. • In Fyn-/-mice, failure of G6PD activation increases red cell vulnerability against oxidation. • Fyn targeting G6PD protects Prx2 recycling, which is required to remove peroxides.

Hereditary spherocytosis (HS) is the most common cause of inherited red cell membranopathy, due to mutations in genes encoding for membrane or cytoskeletal proteins, including band 3, ankyrin, spectrin, band 4.1 or band 4.2. Membrane instability results in membrane surface area loss and generation of spherocytic red cells with elevated MCHC, decreased cellular deformability and reduced red cell survival, due to splenic sequestration. Clinical management of the hemolytic anemia due to HS depends on the age of the patient and the severity of anemia. Splenectomy is indicated in children with symptomatic anemia. A classic diagnostic test for HS is the incubated osmotic fragility: this test highlights the crucial role that ATP content plays in maintenance of normal RBC function including membrane stability: it is generally believed that the increased fragility of HS is the result of abnormal ATP depletion over the 24hr incubation. We explored the hypothesis that pyruvate kinase activator, mitapivat, by modulating ATP content could have potential beneficial effects for HS RBCs in band 4.2-/- mice, a well-established model of HS (Peters LL et al JCI 103: 1527, 1999). 4.2-/- mice exhibit moderate anemia which recapitulates most of the features of typical human HS without showing the profound anemization seen in other mouse models. Oral AG-348 administration to band 4.2-/- mice at dosages of 200 mg/kg/day over 6 months resulted in (i) improvement of anemia with reduced reticulocyte count (Hb 11.6±0.035 g/dL, n=17 vs 13.104±0.09 g/dL, n=9; P Disclosures Kung: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kosinski:Agios Pharmaceuticals Inc: Current Employment, Current equity holder in publicly-traded company. Dang:Agios Pharmaceuticals Inc.: Current Employment, Current equity holder in publicly-traded company. Brugnara:Sysmex America Inc.: Consultancy; American Journal of Hematology: Other.

The signaling cascade induced by the interaction of erythropoietin (EPO) with its receptor (EPO-R) is a key event of erythropoiesis. We present here data indicating that Fyn, a Src-family-kinase, participates in the EPO signaling-pathway, since Fyn(−/−) mice exhibit reduced Tyr-phosphorylation of EPO-R and decreased STAT5-activity. The importance of Fyn in erythropoiesis is also supported by the blunted responsiveness of Fyn(−/−) mice to stress erythropoiesis. Fyn(−/−) mouse erythroblasts adapt to reactive oxygen species (ROS) by activating the redoxrelated-transcription-factor Nrf2. However, since Fyn is a physiologic repressor of Nrf2, absence of Fyn resulted in persistent-activation of Nrf2 and accumulation of nonfunctional proteins. ROS-induced over-activation of Jak2-Akt-mTOR-pathway and repression of autophagy with perturbation of lysosomal-clearance were also noted. Treatment with Rapamycin, a mTOR-inhibitor and autophagy activator, ameliorates Fyn(−/−) mouse baseline erythropoiesis and erythropoietic response to oxidative-stress. These findings identify a novel multimodal action of Fyn in the regulation of normal and stress erythropoiesis.

Sickle cell disease (SCD; ORPHA232; OMIM # 603903) is a chronic and invalidating disorder distributed worldwide, with high morbidity and mortality. Given the disease complexity and the multiplicity of pathophysiological targets, development of new therapeutic options is critical, despite the positive effects of hydroxyurea (HU), for many years the only approved drug for SCD. New therapeutic strategies might be divided into (1) pathophysiology-related novel therapies and (2) innovations in curative therapeutic options such as hematopoietic stem cell transplantation and gene therapy. The pathophysiology related novel therapies are: a) Agents which reduce sickling or prevent sickle red cell dehydration; b) Agents targeting SCD vasculopathy and sickle cell-endothelial adhesive events; c) Anti-oxidant agents. This review highlights new therapeutic strategies in SCD and discusses future developments, research implications, and possible innovative clinical trials.

Anemia of β-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of β-thalassemia (Hbb(th3/+) mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free α-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2α phosphorylation. The improvement of β-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of β-thalassemia.

Erythropoiesis is a dynamic and multistep process, resulting in generation of red cells from hematopoietic stem cells. Aging is characterized by increased oxidation and reduced efficiency of cytoprotective mechanisms. Nrf2 is a key transcription factor that participates in acute response to oxidative stress and controls the expression of anti-oxidant and cytoprotective systems. We previously documented activation of Nrf2 during β-thalassemic erythropoiesis. In addition, a previous study reported a mild chronic hemolytic anemia as a result of increased erythrophagocytosis in mice genetically lacking Nrf2 (PNAS, 2004). Here, we show an age-dependent worsening of anemia in Nrf2-/- mice characterized by accelerated senescence of circulating erythrocytes in association with reticulocytopenia, suggesting a perturbation of erythroblast maturation. Indeed, we found ineffective erythropoiesis in 12 months-old Nrf2-/- mice with extramedullary erythropoiesis, increased ROS levels, reduction in the expression of Nrf2 related anti-oxidant proteins and apoptosis of erythroid precursor cells. In agreement, we observed an age dependent sensitivity of Nrf2-/- mice to stress erythropoiesis induced by either phenyhydrazine (PHZ) or doxorubicine. In 12-month-old mice we observed (i) activation of the unfoldeld protein response system (UPR) due to endosomal reticulum stress and (ii) impaired autophagy. The cytoprotective processes were unable to fully counteract oxidation re-directing cells towards apoptosis as supported by the increased activity of caspase-3. To understand the impact of oxidation in this model of accelerated senescence, we treated Nrf2-/- mice with astaxanthin, a powerful anti-oxidant but with low oral bioavailability, by administrating it in the form of PLGA loaded nanoparticles (ATS-NP). Treatment of Nrf2-/- mice with ATS-NP ameliorated the age-dependent anemia and decreased ineffective erythropoiesis through (i) inactivation of UPR system; (ii) improvement of autophagy and (iii) reduction in caspase-3 activity. In future studies, we plan to evaluate the impact of ATS-NP administration in other models of pathologic erythropoiesis. In summary, we propose that Nrf2, a key transcriptional factor plays a key protective role in regulating aged related oxidation and ensures normal erythroid maturation and growth. Disclosures No relevant conflicts of interest to declare.

Resolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R-RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.

Erythropoiesis is a complex multistep process responsible of the production of circulating mature erythrocytes and involved the production of reactive oxygen species (ROS) during erythroid differentiation. Here, we document that Fyn, a Src-family-kinase, participates in erythropoietin (EPO) signaling pathway, by the reducing extent of Tyr-phosphorylation of EPO-R and by decreasing STAT5 activity. The importance of Fyn in EPO cascade is also supported by the increased sensitivity of Fyn−/−mice to stress erythropoiesis. Fyn−/−mouse erythroblasts adapt to the induced stress by the activation of the redox-related-transcription-factor Nrf2. However, the absence of the Nrf2 physiologic repressor Fyn resulted in the persistent activation of Nrf2 and accumulation of non-functional proteins. This is paralleled by ROS induced over-activation of Jak2-Akt-mTOR pathway and repression of autophagy and perturbation of lysosomal-clearance during Fyn−/−reticulocyte maturation. Treatment with Rapamycin, a mTOR inhibitor and autophagy activator, ameliorates Fyn−/−mouse baseline erythropoiesis and restored the erythropoietic response to phenylhydrazine. Taken together these findings have enabled to identify the novel multimodal action of Fyn in the developmental program of erythropoiesis.

Aims: Iron overload (IO) is a life-threatening complication of chronic hemolytic disorders such as ?-thalassemia. IO results in severe cellular oxidative damage, leading to organ failure. Peroxiredoxin-2 (Prx2), a typical 2-cysteine-(Cys)-peroxiredoxin, is an important component of the cytoprotective system, but its response to IO is still to be fully defined. Results: We studied the effects of IO on Prx2-knockout mice (Prx2-/-). The absence of Prx2 enhanced toxicity due to IO on erythropoiesis. We found that IO failed to induce the typical hepcidin (Hamp) upregulation in Prx2-/- mice due to its failure to activate the signal transducer and activator of transcription-3 (STAT3) with intact Jak2 signaling. In Prx2-/- mice, the loss of Hamp response was also observed after administration of a single dose of oral iron. When lipopolysaccharide (LPS) was used to explore IL6-STAT3 activation in Prx2-/- mice, STAT3 activation and Hamp upregulation were once again defective. Treatment with PEP-fusion-recombinant-Prx2 (PEP Prx2) significantly increased STAT3 activation with upregulation of Hamp expression in both IO- and LPS-exposed Prx2-/- mice. We also confirmed the beneficial effects of PEP Prx2 on Hamp expression through STAT3 activation in ?-thalassemic mice. Innovation: We propose that Prx2 plays a key role in responding to cytotoxicity of IO, directly targeting STAT3-transcriptional factor in a Jak2-independent fashion and regulating Hamp in response to canonical stimuli. Conclusion: Collectively, our data highlight a novel role of Prx2 in iron homeostasis. Prx2 is a key cytoprotector against IO that is induced either by iron supplementation or due to chronic hemolysis as in ?-thalassemia. Prx2 is required to support STAT3 transcriptional activity and regulation of Hamp expression. Antioxid. Redox Signal. 28, 1-14.

Pulmonary-artery-hypertension (PAH) is a life-threatening and highly invalidating chronic disorder. Chronic oxidation contributes to lung damage and disease progression. Peroxiredoxin-2 (Prx2) is a typical 2-cysteine (Cys) peroxiredoxin but its role on lung homeostasis is yet to be fully defined. Here, we showed that Prx2-/- mice displayed chronic lung inflammatory disease associated with (i) abnormal pulmonary vascular dysfunction; and (ii) increased markers of extracellular-matrix remodeling. Hypoxia was used to induce PAH. We focused on the early phase PAH to dissect the role of Prx2 in generation of PAH. Hypoxic Prx2-/-mice showed (i) amplified inflammatory response combined with cytokine storm; (ii) vascular activation and dysfunction; (iii) increased PDGF-B lung levels, as marker of extracellular-matrix deposition and remodeling; and (iv) ER stress with activation of UPR system and autophagy. Rescue experiments with in vivo the administration of fused-recombinant-PEP-Prx2 show a reduction in pulmonary inflammatory vasculopathy and in ER stress with down-regulation of autophagy. Thus, we propose Prx2 plays a pivotal role in the early stage of PAH as multimodal cytoprotector, targeting oxidation, inflammatory vasculopathy and ER stress with inhibition of autophagy. Collectively, our data indicate that Prx2 is able to interrupt the hypoxia induced vicious cycle involving oxidation-inflammation-autophagy in the pathogenesis of PAH.

The data presented in this article are related to the research paper entitled "peroxiredoxin-2 plays a pivotal role as multimodal cytoprotector in the early phase of pulmonary hypertension" (Federti et al., 2017) [1]. Data show that the absence of peroxiredoxin-2 (Prx2) is associated with increased lung oxidation and pulmonary vascular endothelial dysfunction. Prx2-/- mice displayed activation of the redox-sensitive transcriptional factors, NF-kB and Nrf2, and increased expression of cytoprotective system such as heme-oxygenase-1 (HO-1). We also noted increased expression of both markers of vascular activation and extracellular matrix remodeling. The administration of the recombinant fusion protein PEP Prx2 reduced the activation of NF-kB and Nrf2 and was paralleled by a decrease in HO-1 and in vascular endothelial abnormal activation. Prolonged hypoxia was used to trigger pulmonary artery hypertension (PAH). Prx2-/- precociously developed PAH compared to wildtype animals.

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by chronic hemolytic anemia in association with acute and chronic life-threatening complications mainly related to acute vaso-occlusive events (VOCs) due to amplified inflammatory response and defective pro-resolving events. Imatinib is an oral Tyrosine (Tyr)-kinase inhibitor, developed for the treatment of chronic myeloid leukemia (CML). Few case reports on SCD patients with CML undergoing imatinib treatment highlight the beneficial impact of imatinib on severity and recurrence of acute VOCs in SCD. In red blood cells (RBCs), Imatinib has been shown to interfere with Tyr-phosphorylation state of the integral membrane protein band 3, affecting RBC microparticle formation. Here, we study the actions of Imatinib on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)TowHbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6-7 animals in each group) with Imatinib 50 mg/Kg/d for 2 weeks before hypoxia/Reoxygenation (H/R) stress used to mimic acute VOCs. Under normoxia, we found that in SCD mice Imatinib significantly reduced (i) Tyr-phosphorylation state of sickle red cell membrane proteins; (ii) the amount of phosphatidyl-serine (PS)+ sickle RBCs; and (iii) the release of erythroid microparticles, which was associated with accumulation of hemichromes and a more efficient erythrophagocytosis compared to vehicle treated animals. In SCD mice exposed to H/R stress, imatinib significantly decreased the H/R-induced (i) increase in peripheral neutrophil count; (ii) lung inflammatory cell infiltrate; (iii) kidney inflammatory cell infiltrate. In the lung of H/R SCD mice, Imatinib inhibited the H/R induced NF-kB activation and prevented the up-regulation of (i) pro-inflammatory cytokines such as ET-1; (ii) vascular endothelial activation markers (VCAM-1, ICAM-1); (iii) pro-fibrotic markers such as PDGF-B and (iv) the activation of UPR system. In the kidney of H/R SCD mice, Imatinib significantly reduced H/R induced expression of ET-1, VCAM-1 and E-selectin, which were again associated with inhibition of NF-kB. In addition, Imatinib significantly upregulated the expression of the microRNAs miR200a/b, which has been described to reduce renal fibrosis. Finally, in isolated aorta from Imatinib treated SCD exposed to H/R stress, a significant reduction in vascular activation markers was observed compared to vehicle treated animals. Collectively, our data indicate that Imatinib acts on multiple targets, modulating signal transduction and reducing inflammatory vasculopathy and extracellular matrix remodeling process related to VOC in SCD. Thus, Imatinib might represent a new therapeutic tool in clinical management of SCD patients. Disclosures El Nemer: Imara: Research Funding.

Sickle bone disease (SBD) is a severe and invalidating complication related to recurrent bone vaso-occlusive events affecting patients with sickle cell disease (SCD). In a humanized mouse model for SCD, we previously showed that SBD is sustained by impaired osteoblast function and increased osteoclast activity associated with local up-regulation of pro-inflammatory cytokines and anti-oxidant systems (Dalle Carbonare L., Blood 2015;126:2320-2328). Growing evidence supports a role of ω-3 fatty acid supplementation in improving bone homeostasis (Fallon EM, et al J Surg Res 2014;191:148-155.). A diet enriched with ω-3 fatty acids beneficially impacts SCD inflammatory vasculopathy, and blunts the acute and chronic SCD-related organ damage in humanized SCD mice (Kalish BT et al Haematologica 2015;100:870-880). Here, we sought to compare the dietary effects of ω-6 (soybean oil-based, SD)- vs ω-3 (fish oil-based, FD)-enriched diets on SBD in SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). Treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6 animals in each group) were exposed to recurrent hypoxia/reoxygenation (rH/R) stress, which closely mimics SBD natural history (Dalle Carbonare L., Blood 2015). In SCD mice, FD prevented rH/R-induced bone loss compared to animals exposed to SD by decreasing osteoclast and increasing osteoblast activities. Up-regulation of molecular osteogenic markers such as Runx2 and Sparc and down-regulation of Rank and RankL, molecular markers of osteoclast recruitment and activity were observed in FD SCD mice exposed to rH/R compared to SD SCD animals. Similar changes, but to a lesser extent, were also observed in AA control mice exposed to rH/R stress. Expression of IL-6 (Il6) and matrix-metalloproteinase-9 (Mmp9) regulators which interact with RankL on osteoclastic activity and bone resorption were studied. In SCD mice, FD markedly reduced the up-regulation of both genes compared to SD SCD animals in conjunction with down-regulation of peroxiredoxin-2 (Prx2) gene expression, an important cytoprotective and antioxidant system. We also evaluated bone adipogenesis, which is believed to be an important contributor to bone impairment in SBD. Bone immunohistochemistry for Peripilin-1 which coats storage lipid droplets revealed increased adipogenesis in SD SCD mice compared to either FD SCD animals or AA controls, in association with downregulation of miR205, which decreases adipogenesis and enhances osteogenic activity. Our data thus indicate that in SCD exposed to rH/R, FD (i) improves osteoblastogenesis; (ii) decreases osteoclast activity; (iii) modulates the bone inflammatory response; iv) reduces adipogenesis. These findings provide new insights on the mechanism of action of ω-3 fatty acid supplementation on the pathogenesis of SBD and strengthen the rationale for ω-3 fatty acid dietary supplementation in SCD as a complementary therapeutic intervention targeting an amplified inflammatory response and sickle cell-related bone impairment. Disclosures No relevant conflicts of interest to declare.

The unbalanced hemoglobin chain synthesis in beta-thalassemias leads to hemichrome-induced damage that contributes to ineffective erythropoiesis, hemolysis and reduced red cell survival. Iron overload related to ineffective erythropoiesis and abnormally low Hepcidin (Hamp), combined with the cytotoxic effects of free heme with free-alpha-chains play a key role in the increased generation of reactive oxygen species (ROS) in beta thalassemias.[2][HA{3] Here we used a specific and selective inhibitor of the plasma membrane expressed glycine transporter GlyT1 (Ro4917838). Use of Ro4917838 has been associated with a dose-dependent decrease in MCH, Hb, soluble transferrin receptor, and increase in absolute reticulocytes and RBC counts in several animal species, attributed to reduce glycine bioavailability in erythroblasts and decreased heme synthesis. In rats, Ro4917838 reduces heme synthesis, and down-regulates erythroid transferrin receptor, but does not interfere with hepcidin regulation and systemic iron homeostasis (Winter et al. Exp Hematol, DOI: 10.1016/j.exphem.2016.07.003). We aimed to determine if reduced cellular availability of glycine in erythroblasts may reduce heme synthesis, and impact pathologic erythropoiesis in a mouse model for b-thalassemia. Wild-type control (WT) C57B6/2J, and beta-thalassemia Hbbth3/+ mice were treated with either vehicle or Ro4917838 at dosages of 3, 10, 30 mg/kg/d administered over 4 weeks once daily by gavage. [6]RO4917838 administration was associated with significant improvements of central hallmarks of the b-thalassemia pathology. Reduced erythrocyte destruction was seen bydemonstrated by [8][WM{9]as significant improvements in erythrocyte morphology and amelioration of hemoglobin reduction in reticulocytes. We also observed an impressively quick reduction of the circulating erythroblast count within 1 week of initiating treatment. This was also associated with decreased hemolysis biomarkers. Ro4917838 induced a significant reduction in extramedullary erythropoiesis and reduction in orthochromatic erythroblasts as well as insoluble alpha chain aggregates in circulating red cells. Red cell survival of b-thal mice treated with 30 mg/kg/day Ro4917838 significantly increased by more than 50%. CD71+ erythroid precursors significantly decreased in WT mice treated with Ro4917838 at 30 mg/kg and in b-thal mice at the dosage of 30 mg/kg/ d. [10]These data suggest that Ro4917838 ameliorates anemia in a b-thalassemia mouse model and positively affects ineffective erythropoiesis and red cell survival in peripheral circulation. Ro4917838 may represent a novel therapeutic approach for the treatment of anemia in b-thalassemia patients. [1] I do see the point for beta-thalassemia, but this does not apply for all thalassemias, I mean the free alpha chains. The ROS yes, they have in common. In this sentence it seems to be that the alpha chains are culprit for ROS in all thalassemias. [HA{3]I do see the point for beta-thalassemia, but this does not apply for all thalassemias, I mean the free alpha chains. The ROS yes, they have in common. In this sentence it seems to be that the alpha chains are culprit for ROS in all thalassemias. ROS was not measured, therefore I would refrain mentioning it. No data is shown. [HA{5]ROS was not measured, therefore I would refrain mentioning it. No data is shown. I propose deletion of the PK info. Although the information is correct I don't think this is the level of detail needed for the abstract. It may even be confusing as it is not the focus of the abstract to do a cross species comparison. [AK7]I propose deletion of the PK info. Although the information is correct I don't think this is the level of detail needed for the abstract. It may even be confusing as it is not the focus of the abstract to do a cross species comparison. Dosing and dosing frequency is already described above, we do not need to repeat this here. [WM{9]Dosing and dosing frequency is already described above, we do not need to repeat this here. Was the liver expression determined or the serum Hepcidin levels measured? I would rephrase it if latter is the case. [HA{11]Was the liver expression determined or the serum Hepcidin levels measured? I would rephrase it if latter is the case. Disclosures Brugnara: F. Hoffmann-La Roche Ltd, Basel, Switzerland: Consultancy. Harmeier:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. De Franceschi:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Matte:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Winter:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Koerner:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Khwaja:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Singer:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Employment. Beneduce:Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Federti:Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding. Siciliano:Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding.

Peroxiredoxin-2 (Prx2), a typical 2-cysteine (Cys) peroxiredoxin, is a key anti-oxidant system in both normal and pathological erythropoiesis characterized by oxidative stress such as b-thalassemia. We recently showed that the absence of Prx2 worsens ß-thalassemic erythropoiesis and related iron-overload (Matte A et al. Antioxid Redox Signal, 2015). Here, we studied the effects of iron overload in a mouse model genetically lacking Prx2 (Prx2-/-). Two months old female wild-type (WT) and Prx2-/- pure bred mice were fed with a diet containing 2.5% carbonyl-iron compared to standard diet treated mice. We evaluated hematologic parameters, red cell indices and reticulocyte count in both mouse strains at baseline and at 30, 49, 60 and 90 days of treatment with carbonyl-iron. We observed a rapid drop in Hct and reticulocyte count in Prx2-/- mice compared to wild-type mice between 30-49 days of iron supplementation with the appearance of severe hyporegenerative anemia at 60 days of treatment in Prx2-/- characterized by a significant reduction in CD44+TER119+Fsc high cells. This was associated with marked increases in apoptotic Prx2-/- orthochromatic erythroblast compared to either baseline values or WT treated mice. In sorted erythroid precursors from iron overload WT mice, Prx2 expression was significantly increased compared to WT under standard diet. We observed a modulation of Erythropherrone (Erfe) expression during erythropoiesis with upregulation of Erfe in WT orthochromatic erythroblast compared to Prx2-/- erythroblasts. In liver from Prx2-/- mice exposed to iron-overload, we found liver iron content similar to WT mice but Pearls stain analysis showed differential iron distribution in cellular components of liver. While iron accumulated in hepatocytes and Kuppfer cells in Prx2-/- mice, iron-deposits were present only in hepatocytes of WT liver. Oxyblot analysis and liver MDA levels were significantly higher in Prx2-/- mice indicating that the absence of Prx2 promotes a severe liver oxidative stress. In WT liver, Prx2 expression was marked increased in a time depend way during iron supplementation, indicating that Prx2 is part of an adaptive cellular response to iron overload. This is in agreement with increased levels of ferritin-H in Prx2-/- mice compared to WT mice. Hepcidin (HAMP) expression was markedly increased in iron-overload WT mice compared to untreated control group, while no major changes were observed in Prx2-/- mice. Tfr2 expression was significantly increased only in livers of iron-overload WT mice, whereas phospho smad 1-5 was significantly increased in both mouse strains in response to iron-overload. The activation of the signaling pathway through Erk-1/2 only in iron-overload WT mice but not in Prx2-/- mice is most likely related to severe oxidative stress in Prx2-/- resulting in switching off of the Erks pathway. Importantly, administration of PEP1-Prx2 fusion protein rescue almost completely the hematologic phenotype with modulation of Erk signaling pathway towards Tfr2 and the smad system, validating our hypothesis of a role for Erk signaling for the observed phenoytpes. Our data highlight Prx2 as novel factor involved in iron homestasis through the control of oxidative stress modulating signaling pathway towards hepcidin expression. Disclosures No relevant conflicts of interest to declare.

The anemia of sickle cell disease is associated with a severe inflammatory vasculopathy and endothelial dysfunction, which leads to painful and life-threatening clinical complications. Growing evidence supports the anti-inflammatory properties of ω-3 fatty acids in clinical models of endothelial dysfunction. Promising but limited studies show potential therapeutic effects of ω-3 fatty acid supplementation in sickle cell disease. Here, we treated humanized healthy and sickle cell mice for 6 weeks with ω-3 fatty acid diet (fish-oil diet). We found that a ω-3 fatty acid diet: (i) normalizes red cell membrane ω-6/ω-3 ratio; (ii) reduces neutrophil count; (iii) decreases endothelial activation by targeting endothelin-1 and (iv) improves left ventricular outflow tract dimensions. In a hypoxia-reoxygenation model of acute vaso-occlusive crisis, a ω-3 fatty acid diet reduced systemic and local inflammation and protected against sickle cell-related end-organ injury. Using isolated aortas from sickle cell mice exposed to hypoxia-reoxygenation, we demonstrated a direct impact of a ω-3 fatty acid diet on vascular activation, inflammation, and anti-oxidant systems. Our data provide the rationale for ω-3 dietary supplementation as a therapeutic intervention to reduce vascular dysfunction in sickle cell disease.

Sickle cell disease (SCD) is characterized by hemolytic anemia in association with acute and chronic life-threatening clinical complications. Acute vaso-occlusive crisis (VOCs) are the main cause of hospitalization for SCD patients. In VOCs, amplified inflammatory response plays a key role in acute organ damage. Pro-Resolving lipid mediators such as resolvins (Rv) accelerate resolution of acute inflammation in different models, indicating that stimulation of endogenous resolution of inflammatory processes may be an additional strategy in limiting tissue damage. Recent data suggest beneficial effects of Rvs in hypoxia/reoxygenation (H/R) related tissue injury. Here, we study the effects of Rvs on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n =6-7 animals in each group) with RvD1, 7S, 8R, 17S- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid, 17R- RvD1, 7S, 8R, 17R- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid; RvD2, 7S, 16R, 17S-trihydroxy-docosa-4Z, 8E, 10Z, 12E, 14E, 19Z-hexaenoic acid. Mice were treated with RvD1 and RvD2 at the dose of 2.5 ug/Kg by gavage 1 hour (hr) before H/R stress (10 hrs 8% oxygen followed by 3 hrs reoxygenation), which we have used in the past to mimic acute VOCs. We found that RvD1 and RvD2 significantly reduced the H/R-induced (i) increase in neutrophil count; (ii) lung inflammatory cell infiltrate, mucus and thrombi formation; (iii) glomerular inflammatory cell infiltration, glomerular sclerosis and thrombi formation. In the lung of H/R SCD mice, RvD1 prevented the H/R induced up-regulation of (i) cytokines such as MCP2, IL-6 and ET-1; (ii) vascular endothelial activation markers (VCAM-1 and ICAM-1; (iii) cytoprotective systems such as Prx6 and HO-1. In the kidney of H/R SCD mice, RvD1 significantly reduced H/R induced expression of IL-6 and ET-1 as well as HO-1. Our data indicate that RvD1 and RvD2 modulating inflammatory responses related to H/R in SCD, protect sickle cell target organs, and foster resolution. Thus, RvD1 and RvD2 might represent a novel therapeutic approach for acute VOCs in SCD. Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD) is a genetic red cell disorder characterized by hemolytic anemia in association with acute and chronic life-threatening clinical complications mainly related to inflammatory vasculopathy. Promising, but limited, human studies have shown potential therapeutic effects of ω-3 fatty acid supplementation in SCD. Here, we sought to compare the effects of 6 weeks ω-6 (soybean oil diet, SD) vs ω-3 (fish oil diet, FD) diet on vasculopathy in SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6-7 animals in each group). In SCD mice, FD was associated with a decreased the ratio of ω-6 to ω-3 fatty acids in red cell membranes, as well as a decrease in neutrophil count compared to SD. We then evaluated the effects of FD on cardiovascular system of SCD mice. We found that FD resulted in significantly (i) lower diastolic blood pressure; (ii) reduced cardiac output; (iii) down-regulation of VCAM-1, ET-1 and HO-1 expression in isolated aorta compared to SD, reaching levels similar to those observed in AA mice. In the lung of SCD mice, FD significantly reduced ET-1 expression compared to SD. FD was also associated with reduced bronchoalveolar lavage leukocyte and protein contents compared to SD, suggesting protection against pulmonary leak. We then studied the effect of FD during vaso-occusive crises (VOCs), mimicked by exposing SCD mice to hypoxia/reoxygenation (H/R) stress. In SCD mice, FD significantly (i) ameliorated the H/R-induced reduction in hematocrit and hemoglobin levels; (ii) reduced the fraction of dense red cells and the H/R-induced increase in neutrophil count. Histologic analysis revealed that H/R stress recapitulates the SCD acute organ damage. In the lung of H/R SCD mice, FD reduced inflammatory cell infiltrates, bronchial mucus, thrombosis, and ET-1 and ET1-1R expression. In isolated aorta from SCD mice exposed to H/R, FD prevented the H/R-induced ET-1 and VCAM-1 expression and the H/R-induced up-regulation of anti-oxidant systems compared to SD. These findings provide new insight into the mechanism of action of ω-3 supplementation on the pathogenesis of SCD and strengthen the rationale for ω-3 dietary supplementation as a simple therapeutic intervention to reduce vascular dysfunction in SCD. Disclosures No relevant conflicts of interest to declare.