SnoRNAs: Exploring Their Implication in Human Diseases.

Small nucleolar RNAs (snoRNAs) are earning increasing attention from research communities due to their critical role in the post-transcriptional modification of various RNAs. These snoRNAs, along with their associated proteins, are crucial in regulating the expression of a vast array of genes in different human diseases. Primarily, snoRNAs facilitate modifications such as 2'-O-methylation, N-4-acetylation, and pseudouridylation, which impact not only ribosomal RNA (rRNA) and their synthesis but also different RNAs. Functionally, snoRNAs bind with core proteins to form small nucleolar ribonucleoproteins (snoRNPs). These snoRNAs then direct the protein complex to specific sites on target RNA molecules where modifications are necessary for either standard cellular operations or the regulation of pathological mechanisms. At these targeted sites, the proteins coupled with snoRNPs perform the modification processes that are vital for controlling cellular functions. The unique characteristics of snoRNAs and their involvement in various non-metabolic and metabolic diseases highlight their potential as therapeutic targets. Moreover, the precise targeting capability of snoRNAs might be harnessed as a molecular tool to therapeutically address various disease conditions. This review delves into the role of snoRNAs in health and disease and explores the broad potential of these snoRNAs as therapeutic agents in human pathologies.

Keap1-Nrf2 Heterodimer: A Therapeutic Target to Ameliorate Sickle Cell Disease.

Sickle cell disease (SCD) is a monogenic inheritable disease characterized by severe anemia, increased hemolysis, and recurrent, painful vaso-occlusive crises due to the polymerization of hemoglobin S (HbS)-generated oxidative stress. Up until now, only four drugs are approved for SCD in the US. However, each of these drugs affects only a limited array of SCD pathologies. Importantly, curative therapies, such as gene therapy, or hematopoietic stem cell transplantation are not available for every patient because of their high costs, availability of donor matching, and their serious adverse effects. Therefore, there is an unmet medical need for novel therapeutic strategies that target broader SCD sequelae. SCD phenotypic severity can be alleviated by increasing fetal hemoglobin (HbF) expression. This results in the inhibition of HbS polymerization and thus sickling, and a reduction in oxidative stress. The efficacy of HbF is due to its ability to dilute HbS levels below the threshold required for polymerization and to influence HbS polymer stability in RBCs. Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein-1 (Keap1)-complex signaling is one of the most important cytoprotective signaling controlling oxidative stress. Nrf2 is present in most organs and, after dissociation from Keap1, it accumulates in the cytoplasm, then translocates to the nucleus where it binds to the antioxidant response element (ARE) sequences and increases the expression of various cytoprotective antioxidant genes. Keeping this in mind, various researchers have proposed a role of multiple agents, more importantly tert-Butylhydroquinone (tBHQ), curcumin, etc., (having electrophilic properties) in inhibiting keap1 activity, so that Nrf2 can translocate to the nucleus to activate the gamma globin gene, thus maintaining alpha-hemoglobin-stabilizing protein (AHSP) and HbF levels. This leads to reduced oxidative stress, consequently minimizing SCD-associated complications. In this review, we will discuss the role of the Keap-1-Nrf2 complex in hemoglobinopathies, especially in SCD, and how this complex might represent a better target for more effective treatment options.

Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability.

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1-2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability.

The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the ß-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Cilia proteins are biomarkers of altered flow in the vasculature.

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation-the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.

The Role of RBC Oxidative Stress in Sickle Cell Disease: From the Molecular Basis to Pathologic Implications.

Sickle cell disease (SCD) is an inherited monogenic disorder and the most common severe hemoglobinopathy in the world. SCD is characterized by a point mutation in the ß-globin gene, which results in hemoglobin (Hb) S production, leading to a variety of mechanistic and phenotypic changes within the sickle red blood cell (RBC). In SCD, the sickle RBCs are the root cause of the disease and they are a primary source of oxidative stress since sickle RBC redox state is compromised due to an imbalance between prooxidants and antioxidants. This imbalance in redox state is a result of a continuous production of reactive oxygen species (ROS) within the sickle RBC caused by the constant endogenous Hb autoxidation and NADPH oxidase activation, as well as by a deficiency in the antioxidant defense system. Accumulation of non-neutralized ROS within the sickle RBCs affects RBC membrane structure and function, leading to membrane integrity deficiency, low deformability, phosphatidylserine exposure, and release of micro-vesicles. These oxidative stress-associated RBC phenotypic modifications consequently evoke a myriad of physiological changes involved in multi-system manifestations. Thus, RBC oxidative stress in SCD can ultimately instigate major processes involved in organ damage. The critical role of the sickle RBC ROS production and its regulation in SCD pathophysiology are discussed here.

Mn porphyrins as a novel treatment targeting sickle cell NOXs to reverse and prevent acute vaso-occlusion in vivo.

In sickle cell disease (SCD), adhesion of sickle red blood cells (SSRBCs) and activated leukocytes in inflamed venules affects blood rheology, causing vaso-occlusive manifestations and vital reduction in microvascular blood flow. Recently, we found that NADPH oxidases (NOXs) create a vicious feedback loop within SSRBCs. This positive feedback loop mediates SSRBC adhesion to the endothelium. We show for the first time the therapeutic effectiveness of the redox-active manganese (Mn) porphyrins MnTnBuOE-2-PyP5+ (MnBuOE; BMX-001) and MnTE-2-PyP5+ (MnE; BMX-010, AEOL10113) to treat established vaso-occlusion in a humanized sickle mouse model of an acute vaso-occlusive crisis using intravital microscopy. These Mn porphyrins can suppress SSRBC NOX activity. Subcutaneous administration of only 1 dose of MnBuOE or MnE at 0.1 to 2 mg/kg after the inflammatory trigger of vaso-occlusion, or simultaneously, reversed and reduced leukocyte and SSRBC adhesion, diminished leukocyte rolling, restored blood flow, and increased survival rate. Furthermore, MnBuOE and MnE administered to sickle mice subcutaneously at 0.1 to 1 mg/kg for 28 days (except on weekends) did not exacerbate anemia, which seemed to be due to downregulation of both SSRBC reactive oxygen species production and exposure of the eryptotic marker phosphatidylserine. In addition, Mn porphyrins ameliorated leukocytosis, venous blood gases, endothelial activation, and organ oxidative damage. Our data suggest that Mn porphyrins, likely by repressing NOX-mediated adhesive function of SSRBCs and activated leukocytes, could represent a novel, safe therapeutic intervention to treat or prevent the establishment of acute pain crises. These NOX-targeted antioxidants merit further assessment in SCD clinical trials.

Oxidative Stress and Thrombosis during Aging: The Roles of Oxidative Stress in RBCs in Venous Thrombosis.

Mid-life stage adults are at higher risk of developing venous thrombosis (VT)/thromboembolism (VT/E). Aging is characterized by an overproduction of reactive oxygen species (ROS), which could evoke a series of physiological changes involved in thrombosis. Here, we focus on the critical role of ROS within the red blood cell (RBC) in initiating venous thrombosis during aging. Growing evidence has shifted our interest in the role of unjustifiably unvalued RBCs in blood coagulation. RBCs can be a major source of oxidative stress during aging, since RBC redox homeostasis is generally compromised due to the discrepancy between prooxidants and antioxidants. As a result, ROS accumulate within the RBC due to the constant endogenous hemoglobin (Hb) autoxidation and NADPH oxidase activation, and the uptake of extracellular ROS released by other cells in the circulation. The elevated RBC ROS level affects the RBC membrane structure and function, causing loss of membrane integrity, and decreased deformability. These changes impair RBC function in hemostasis and thrombosis, favoring a hypercoagulable state through enhanced RBC aggregation, RBC binding to endothelial cells affecting nitric oxide availability, RBC-induced platelet activation consequently modulating their activity, RBC interaction with and activation of coagulation factors, increased RBC phosphatidylserine exposure and release of microvesicles, accelerated aging and hemolysis. Thus, RBC oxidative stress during aging typifies an ultimate mechanism in system failure, which can affect major processes involved in the development of venous thrombosis in a variety of ways. The reevaluated concept of the critical role of RBC ROS in the activation of thrombotic events during aging will help identify potential targets for novel strategies to prevent/reduce the risk for VT/E or VT/E recurrences in mid-life stage adults.

MEK1/2 as a Therapeutic Target in Sickle Cell Disease.

Identification of novel therapeutic targets has improved diagnostics and treatment of many diseases. Many innovative treatment strategies have been developed based on the newly identified biomarkers and key molecules. Most of the research focused on ways to manipulate signaling pathways by activating or suppressing them, validate new therapeutic targets for treatment, and epigenetic treatment of diseases. With the identification of aberrations in multiple growth pathways, the focus then shifted to the small molecules involved in these pathways for targeted therapy. In this communication/short review, we highlight the importance of identification of abnormal activation of the mitogen-activated protein kinase (MAPK), ERK1/2, and its upstream mediator MEK1/2, in erythrocytes in patients with sickle cell disease (SCD) critical for the adhesive interactions of these cells with the endothelium, and leukocytes promoting circulatory obstruction leading to tissue ischemia and infraction. We also discuss how targeting this signaling cascade with MEK1/2 inhibitors can reverse acute vasoocclusive crises in SCD.

Nitric oxide loading reduces sickle red cell adhesion and vaso-occlusion in vivo.

Sickle red blood cells (SSRBCs) are adherent to the endothelium, activate leukocyte adhesion, and are deficient in bioactive nitric oxide (NO) adducts such as S-nitrosothiols (SNOs), with reduced ability to induce vasodilation in response to hypoxia. All these pathophysiologic characteristics promote vascular occlusion, the hallmark of sickle cell disease (SCD). Loading hypoxic SSRBCs in vitro with NO followed by reoxygenation significantly decreased epinephrine-activated SSRBC adhesion to the endothelium, the ability of activated SSRBCs to mediate leukocyte adhesion in vitro, and vessel obstruction in vivo. Because transfusion is frequently used in SCD, we also determined the effects of banked (SNO-depleted) red blood cells (RBCs) on vaso-occlusion in vivo. Fresh or 14-day-old normal RBCs (AARBCs) reduced epinephrine-activated SSRBC adhesion to the vascular endothelium and prevented vaso-occlusion. In contrast, AARBCs stored for 30 days failed to decrease activated SSRBC adhesivity or vaso-occlusion, unless these RBCs were loaded with NO. Furthermore, NO loading of SSRBCs increased S-nitrosohemoglobin and modulated epinephrine's effect by upregulating phosphorylation of membrane proteins, including pyruvate kinase, E3 ubiquitin ligase, and the cytoskeletal protein 4.1. Thus, abnormal SSRBC NO/SNO content both contributes to the vaso-occlusive pathophysiology of SCD, potentially by affecting at least protein phosphorylation, and is potentially amenable to correction by (S)NO repletion or by RBC transfusion.

Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion.

In sickle cell disease (SCD), recurrent painful vasoocclusive crisis are likely caused by repeated episodes of hypoxia and reoxygenation. The sickle erythrocyte (SSRBC) adhesion plays an active role in vasoocclusion. However, the effect of prolonged reoxygenation after hypoxic stress on the molecular mechanisms in SSRBCs involved in onset of episodic vasoocclusion remain unclear. Exposure of human SSRBCs to hypoxia followed by 2 h reoxygenation, increased reactive oxygen species (ROS) production. Using specific pharmacological inhibitors, we show that excess ROS production in both reticulocytes and mature SSRBCs is regulated by NADPH oxidases (NOXs), the mitogen-activated protein kinase (ERK1/2), and G-protein coupled-receptor kinase 2 (GRK2). Consequently, SSRBC ROS create an intracellular positive feedback loop with ERK1/2 and GRK2 to mediate SSRBC adhesion to endothelium in vitro, and vasoocclusion in a mouse model of vasoocclusion in vivo. Importantly, reducing ROS levels in SSRBCs with redox-active manganese (Mn) porphyrins, commonly known as mimics of superoxide dismutase (SOD), disrupted the cycle created by ROS by affecting NOX and GRK2 activities and ERK1/2 phosphorylation, thus abrogating RBC-endothelial interactions. Inhibition adhesion assays show that LW (ICAM-4, CD242) blood group glycoprotein and CD44 are the RBC adhesion molecules mediating endothelial binding. Conversely, hypoxia/reoxygenation of normal RBCs failed to activate this feedback loop, and adhesion. These findings provide novel insights into the pathophysiological significance of the deleterious cycle created by NOX-dependent ROS, GRK2 and ERK1/2 within SSRBCs activated by hypoxia/reoxygenation, and involved in SSRBC adhesion and vasoocclusion. Thus, this loop in SSRBCs, which can be disrupted by Mn porphyrins, likely drives the profound SCD vasculopathy, and may point to new therapeutic targets to prevent chronic vasoocclusive events.

Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell-induced vaso-occlusion.

Sevuparin is a novel drug candidate in phase II development as a treatment for vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Here, we demonstrate that sevuparin inhibits the adhesion of human sickle red blood cells (SS-RBCs) to stimulated cultured endothelial cells in vitro. Importantly, sevuparin prevents vaso-occlusion and normalizes blood flow in an in vivo mouse model of SCD vaso-occlusion. Analyses by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) demonstrate that sevuparin binds to P- and L-selectins, thrombospondin, fibronectin and von Willebrand factor, all of which are thought to contribute to vaso-occlusion in SCD. Despite low anticoagulation activity, sevuparin has anti-adhesive efficacy similar to the low molecular weight heparin tinzaparin both in vitro and in vivo. These results suggest that the anti-adhesive properties rather than the anticoagulant effects of heparinoids are critical for the treatment of vaso-occlusion in SCD. Therefore, sevuparin is now being evaluated in SCD patients hospitalized for treatment of VOC.

MEK1/2 inhibitors reverse acute vascular occlusion in mouse models of sickle cell disease.

In sickle cell disease (SCD), treatment of recurrent vasoocclusive episodes, leading to pain crises and organ damage, is still a therapeutic challenge. Vasoocclusion is caused primarily by adherence of homozygous for hemoglobin S (SS) red blood cells (SSRBCs) and leukocytes to the endothelium. We tested the therapeutic benefits of MEK1/2 inhibitors in reversing vasoocclusion in nude and humanized SCD mouse models of acute vasoocclusive episodes using intravital microscopy. Administration of 0.2, 0.3, 1, or 2 mg/kg MEK1/2 inhibitor to TNF-α-pretreated nude mice before human SSRBC infusion inhibited SSRBC adhesion in inflamed vessels, prevented the progression of vasoocclusion, and reduced SSRBC organ sequestration. By use of a more clinically relevant protocol, 0.3 or 1 mg/kg MEK1/2 inhibitor given to TNF-α-pretreated nude mice after human SSRBC infusion and onset of vasoocclusion reversed SSRBC adhesion and vasoocclusion and restored blood flow. In SCD mice, 0.025, 0.05, or 0.1 mg/kg MEK1/2 inhibitor also reversed leukocyte and erythrocyte adhesion after the inflammatory trigger of vasoocclusion and improved microcirculatory blood flow. Cell adhesion was reversed by shedding of endothelial E-selectin, P-selectin, and αvß3 integrin, and leukocyte CD44 and ß2 integrin. Thus, MEK1/2 inhibitors, by targeting the adhesive function of SSRBCs and leukocytes, could represent a valuable therapeutic intervention for acute sickle cell vasoocclusive crises.

Gαs proteins activate p72(Syk) and p60-c-Src tyrosine kinases to mediate sickle red blood cell adhesion to endothelium via LW-αvß3 and CD44-CD44 interactions.

G protein-coupled receptors (GPCRs) have been suggested as new drug targets to treat a variety of diseases. In sickle cell disease (SCD), the LW erythrocyte adhesion receptor can be activated by stimulation of ß2 adrenergic receptors (ß2ARs), to mediate sickle erythrocyte (SSRBC) adhesion to endothelium. However, the involvement of tyrosine protein kinases in ß2AR signaling to activate SSRBC adhesion to endothelium has not been thoroughly elucidated. Either direct activation with Cholera toxin of Gαs protein, which acts downstream of ß2ARs, or inhibition with Pertussis toxin of Gαi, mediating suppression of adenylyl cyclase, increased SSRBC adhesion to endothelium over baseline adhesion. This effect involved the non-receptor tyrosine kinases, p72(Syk) and p60-c-Src, which were more abundant in SSRBCs than in normal erythrocytes. In contrast, Pertussis toxin and Cholera toxin failed to increase adhesion of normal erythrocytes. SSRBC Gαi inhibition also increased phosphorylation of p72(Syk) and p60-c-Src. Further, we investigated the relevance of activation of p72(Syk) and p60-c-Src, and identified LW (ICAM-4, CD242) and CD44 as the erythroid adhesion molecules both physically interacting with activated p60-c-Src. As a result, SSRBC LW underwent increased tyrosine phosphorylation, leading to SSRBC LW and CD44 binding to endothelial αvß3 integrin and CD44, respectively. These data provide in vitro mechanistic evidence that p60-c-Src, which could act downstream of Gαs/p72(Syk), associates with LW and CD44 on SSRBCs leading to their interactions with endothelial αvß3 and CD44, respectively. Thus, increased activation of these signaling mechanisms in SSRBCs could initiate or exacerbate vascular occlusion, the hallmark of SCD.

MEK inhibitors, novel anti-adhesive molecules, reduce sickle red blood cell adhesion in vitro and in vivo, and vasoocclusion in vivo.

In sickle cell disease, sickle erythrocyte (SSRBC) interacts with endothelial cells, leukocytes, and platelets, and activates coagulation and inflammation, promoting vessel obstruction, which leads to serious life-threatening complications, including acute painful crises and irreversible damage to multiple organs. The mitogen-activated protein kinase, ERK1/2, is abnormally activated in SSRBCs. However, the therapeutic potential of SSRBC ERK1/2 inactivation has never been investigated. I tested four different inhibitors of MEK1/2 (MEK), the kinase that activates ERK1/2, in a model of human SSRBC adhesion to TNFα-activated endothelial cells (ECs). SSRBC MEK inhibition abrogated adhesion to non-activated and TNFα-activated ECs to levels below baseline SSRBC adhesion to non-activated ECs in vitro. SSRBC MEK inhibition also prevented SSRBCs from activating naïve neutrophils to adhere to endothelium. To determine the effect of MEK inhibitors on SSRBC adherence in vivo, sham-treated or MEK inhibitor-treated SSRBCs were infused to nude mice previously treated with TNFα. Sham-treated SSRBCs displayed marked adhesion and occlusion of enflamed vessels, both small and large. However, SSRBC treatment with MEK inhibitors ex vivo showed poor SSRBC adhesion to enflamed vessels with no visible vasoocclusion in vivo. In addition, MEK inhibitor treatment of SSRBCs reduced SSRBC organ trapping and increased the number of SSRBCs circulating in bloodstream. Thus, these data suggest that SSRBC ERK1/2 plays potentially a critical role in sickle pathogenesis, and that MEK inhibitors may represent a valuable intervention for acute sickle cell crises.

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation.

BACKGROUND: In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown. RESULTS: To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2. CONCLUSIONS: These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.

Sickle erythrocytes target cytotoxics to hypoxic tumor microvessels and potentiate a tumoricidal response.

Resistance of hypoxic solid tumor niches to chemotherapy and radiotherapy remains a major scientific challenge that calls for conceptually new approaches. Here we exploit a hitherto unrecognized ability of sickled erythrocytes (SSRBCs) but not normal RBCs (NLRBCs) to selectively target hypoxic tumor vascular microenviroment and induce diffuse vaso-occlusion. Within minutes after injection SSRBCs, but not NLRBCs, home and adhere to hypoxic 4T1 tumor vasculature with hemoglobin saturation levels at or below 10% that are distributed over 70% of the tumor space. The bound SSRBCs thereupon form microaggregates that obstruct/occlude up to 88% of tumor microvessels. Importantly, SSRBCs, but not normal RBCs, combined with exogenous prooxidant zinc protoporphyrin (ZnPP) induce a potent tumoricidal response via a mutual potentiating mechanism. In a clonogenic tumor cell survival assay, SSRBC surrogate hemin, along with H(2)O(2) and ZnPP demonstrate a similar mutual potentiation and tumoricidal effect. In contrast to existing treatments directed only to the hypoxic tumor cell, the present approach targets the hypoxic tumor vascular environment and induces injury to both tumor microvessels and tumor cells using intrinsic SSRBC-derived oxidants and locally generated ROS. Thus, the SSRBC appears to be a potent new tool for treatment of hypoxic solid tumors, which are notable for their resistance to existing cancer treatments.

Effect of propranolol as antiadhesive therapy in sickle cell disease.

Sickle red blood cells (SSRBCs) adhere to both endothelial cells (ECs) and the extracellular matrix. Epinephrine elevates cyclic adenosine monophosphate in SSRBCs and increases adhesion of SSRBCs to ECs in a ß-adrenergic receptor and protein kinase A-dependent manner. Studies in vitro as well as in vivo have suggested that adrenergic stimuli like epinephrine may contribute to vaso-occlusion associated with physiologic stress. We conducted both animal studies and a Phase I dose-escalation study in sickle cell disease (SCD) patients to investigate whether systemically administered propranolol inhibits SSRBC adhesion and to document the safety of propranolol in SCD. Systemically administered propranolol prevented SSRBC adhesion and associated vaso-occlusion in a mouse model. In patients receiving a single oral dose of 10, 20, or 40 mg propranolol, SSRBC adhesion to ECs was studied before and after propranolol, with and without stimulation with epinephrine. Propranolol administration significantly reduced epinephrine-stimulated SSRBC adhesion in a dose dependent manner (p = 0.03), with maximum inhibition achieved at 40 mg. Adverse events were not severe, did not show dose dependence, and were likely unrelated to drug. No significant heart rate changes occurred. These results imply that ß-blockers may have a role as antiadhesive therapy for SCD.

Erythrocyte plasma membrane-bound ERK1/2 activation promotes ICAM-4-mediated sickle red cell adhesion to endothelium.

The core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. ß(2)-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72(syk)-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4-mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and ß-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.

Impaired adenosine-5'-triphosphate release from red blood cells promotes their adhesion to endothelial cells: a mechanism of hypoxemia after transfusion.

OBJECTIVE: Transfusion of red blood cells has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that red blood cell storage impairs the ability of red blood cells to release adenosine-5'-triphosphate and that impaired adenosine-5'-triphosphate release was injurious in vivo, in part through increased red blood cell adhesion. DESIGN: Prospective, controlled, mechanistic study. SETTING: University research laboratory. SUBJECTS: Human and mouse blood donors; nude mouse transfusion recipients. INTERVENTIONS: Manipulation of adenosine-5'-triphosphate release, supplemental adenosine-5'-triphosphate, and antibodies to red blood cell and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released adenosine-5'-triphosphate and adhesion in responses to (transfused) red blood cells. MEASUREMENTS AND MAIN RESULTS: The ability of stored red blood cells to release adenosine-5'-triphosphate declined markedly within 14 days after collection despite relatively stable levels of adenosine-5'-triphosphate within the red blood cells. Inhibiting adenosine-5'-triphosphate release promoted the adhesion of stored red blood cells to endothelial cells in vitro and red blood cell sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human red blood cells, stored red blood cell transfusion in mice decreased blood oxygenation and increased extravasation of red blood cells into the lung's alveolar air spaces. Similar findings were seen with transfusion of fresh red blood cells treated with the adenosine-5'-triphosphate release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either coinfusion of an adenosine-5'-triphosphate analog or pretransfusion incubation of the red blood cells with an antibody against the erythrocyte adhesion receptor Landsteiner-Wiener (intercellular adhesion molecule-4). CONCLUSIONS: The normal flow of red blood cells in pulmonary microvessels depends in part on the release of antiadhesive adenosine-5'-triphosphate from red blood cells, and storage-induced deficiency in adenosine-5'-triphosphate release from transfused red blood cells may promote or exacerbate microvascular pathophysiology in the lung, in part through increased red blood cell adhesion.