Peroxiredoxin 2 Plays a Pivotal Role Against Oxidation in the Early Phase of Pulmonary Artery Hypertension

Background. Peroxiredoxin 2 (Prx2) is a typical 2-cysteine (Cys) peroxiredoxin ubiquitously expressed. Prx2 is able to efficiently scavenge H2O2 and other hydro-peroxides to maintain intracellular redox balance. Prx2 has been largely studied in red cells, where it acts as antioxidant system and chaperone-like molecule. Although growing evidences indicate the important role of Prx2 in different cell models, much still remains to be investigated in complex disease scenario such as chronic lung disease or stress erythropoiesis. Aim. The major aim of my work is to understand the functional role of Peroxiredoxin 2 in a mouse model of pulmonary artery hypertension. Section 1. I focus on the functional role of peroxiredoxin-2 (Prx2) in lung homeostasis. Here, I studied the events related to the generation of pulmonary artery hypertension (PAH), a life threatening and highly invalidating chronic disorder. Hypoxia was used as trigger factor. I found that Prx2-/- mice displayed chronic lung inflammatory disease associated with (i) abnormal pulmonary vascular dysfunction; and (ii) increased markers of extracellular-matrix remodelling. Rescue experiments with in vivo the administration of fused-recombinant-PEP-Prx2 to hypoxic mice show a reduction in pulmonary inflammatory vasculopathy and down-regulation of autophagy. Thus, we propose Prx2 plays a pivotal role in the early stage of PAH as multimodal anti-oxidant system targeting oxidation, inflammatory vasculopathy and indirectly autophagy. Section 2. I have studied the effect of iron-overload (IO) diet on Prx2-/- mouse erythropoiesis and its link with iron homeostasis. In Prx2-/- mice, IO displayed a potent cytotoxic effect on erythropoiesis and it was associated with lacking in liver STAT3 activation. This results in low hepcidin expression. Treatment with PEP Prx2 ameliorates IO-induced anemia and restored liver STAT3 activation, with adequate hepcidin expression in relation to IO. The importance of Prx2 on the axis between erythropoiesis and iron metabolism is also supported by the beneficial effects of PEP Prx2 on hepcidin levels a mouse model of \u3b2-thalassemia. Section 3. I have studied a humanized mouse model for sickle cell disease (SCD, Tim Townes mouse model). Based on the revision of the literature, SCD and healthy mice were supplemented with \u3c9-3 fatty acids. Hypoxia/reoxygenation stress was used to mimic SCD acute vaso-occlusive crisis. We found that \u3c9-3 fatty supplementation reduces: (i) sickle cell related oxidative stress, (ii) systemic and local (lung and liver) inflammatory response, (iii) vascular endothelial activation and (iv) vascular dysfunction. Our data generate a rationale for \u3c9-3 fatty supplementation in clinical management of SCD patients

Emerging drugs in randomized controlled trials for sickle cell disease: are we on the brink of a new era in research and treatment?

ABSTRACT Introduction: Sickle cell disease (SCD) is caused by a mutation in the HBB gene which is key for making a component of hemoglobin. The mutation leads to the formation of an abnormal hemoglobin molecule called sickle hemoglobin (HbS). SCD is a chronic, complex disease with a multiplicity of pathophysiological targets; it has high morbidity and mortality. Hydroxyurea has for many years been the only approved drug for SCD; hence, the development of new therapeutics is critical. Areas covered: This article offers an overview of the key studies of new therapeutic options for SCD. We searched the PubMed database and Cochrane Database of Systemic Reviews for agents in early phase clinic trials and preclinical development. Expert opinion: Although knowledge of SCD has progressed, patient survival and quality of life must be improved. Phase II and phase III clinical trials investigating pathophysiology-based novel agents show promising results in the clinical management of SCD acute events. The design of longterm clinical studies is necessary to fully understand the clinical impact of these new therapeutics on the natural history of the disease. Furthermore, the building of global collaborations will enhance the clinical management of SCD and the design of primary outcomes of future clinical trials

NEW THERAPEUTIC OPTIONS FOR THE TREATMENT OF SICKLE CELL DISEASE

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

Impaired pro‐resolving mechanisms promote abnormal NETosis , fueling autoimmunity in sickle cell disease

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorders with still high mortality and morbidity and limited therapeutic options. SCD is characterized by anemia, chronic hemolysis, and acute vaso-occlusive painful crises. The biocomplexity of SCD goes beyond red cells, involving neutrophils and soluble factors such as cytokines or alternative complement pathway intensively cross-talking with vascular endothelial cells. In addition, in SCD, the overactivation of neutrophils contributes to the production of neutrophil extracellular traps (NETs) (1, 2). This might trigger endothelial vascular injury, promoting acute sickle cell related events and increasing the risk of infections in patients with SC

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of \u3b2-thalassemia

Anemia of \u3b2-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 \u3b2-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free \u3b1-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2\u3b1 phosphorylation. The improvement of \u3b2-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 \u3b2-thalassemia

Tyrosine phosphorylation modulates peroxiredoxin-2 activity in normal and diseased red cells

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

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in M-thalassemic mice

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Epeleuton, a novel synthetic ω-3 fatty acid, reduces hypoxia/ reperfusion stress in a mouse model of sickle cell disease

Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to the progression of SCD. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel, orally administered, second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory responses and improves red cell features, resulting in reduced hemolysis and sickling compared with that in vehicle-treated SCD mice. In addition, epeleuton prevents hypoxia/reoxygenation-induced activation of nuclear factor-κB with downregulation of the NLRP3 inflammasome in lung, kidney, and liver. This was associated with downregulation of markers of vascular activation in epeleuton-treated SCD mice when compared to vehicle-treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis

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, \u3b3-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

Oxidation Impacts the Intracellular Signaling Machinery in Hematological Disorders

The dynamic coordination between kinases and phosphatases is crucial for cell homeostasis, in response to different stresses. The functional connection between oxidation and the intracellular signaling machinery still remains to be investigated. In the last decade, several studies have highlighted the role of reactive oxygen species (ROS) as modulators directly targeting kinases, phosphatases, and downstream modulators, or indirectly acting on cysteine residues on kinases/phosphatases resulting in protein conformational changes with modulation of intracellular signaling pathway(s). Translational studies have revealed the important link between oxidation and signal transduction pathways in hematological disorders. The intricate nature of intracellular signal transduction mechanisms, based on the generation of complex networks of different types of signaling proteins, revealed the novel and important role of phosphatases together with kinases in disease mechanisms. Thus, therapeutic approaches to abnormal signal transduction pathways should consider either inhibition of overactivated/accumulated kinases or homeostatic signaling resetting through the activation of phosphatases. This review discusses the progress in the knowledge of the interplay between oxidation and cell signaling, involving phosphatase/kinase systems in models of globally distributed hematological disorders