Fatty acid nitroalkenes – Multi-target agents for the treatment of sickle cell disease

Sickle cell disease (SCD) is a hereditary hematological disease with high morbidity and mortality rates worldwide. Despite being monogenic, SCD patients display a plethora of disease-associated complications including anemia, oxidative stress, sterile inflammation, vaso-occlusive crisis-related pain, and vasculopathy, all of which contribute to multiorgan dysfunction and failure. Over the past decade, numerous small molecule drugs, biologics, and gene-based interventions have been evaluated; however, only four disease-modifying drug therapies are presently FDA approved. Barriers regarding effectiveness, accessibility, affordability, tolerance, and compliance of the current polypharmacy-based disease-management approaches are challenging. As such, there is an unmet pharmacological need for safer, more efficacious, and logistically accessible treatment options for SCD patients. Herein, we evaluate the potential of small molecule nitroalkenes such as nitro-fatty acid (NO2-FA) as a therapy for SCD. These agents are electrophilic and exert anti-inflammatory and tissue repair effects through an ability to transiently post-translationally bind to and modify transcription factors, pro-inflammatory enzymes and cell signaling mediators. Preclinical and clinical studies affirm safety of the drug class and a murine model of SCD reveals protection against inflammation, fibrosis, and vascular dysfunction. Despite protective cardiac, renal, pulmonary, and central nervous system effects of nitroalkenes, they have not previously been considered as therapy for SCD. We highlight the pathways targeted by this drug class, which can potentially prevent the end-organ damage associated with SCD and contrast their prospective therapeutic benefits for SCD as opposed to current polypharmacy approaches.


Introduction to sickle cell disease
Sickle Cell Disease (SCD) is a congenital blood disorder with a worldwide incidence of 300,000 to 400,000 births annually, 75% of which prevails in Sub-Saharan Africa [1].SCD originates from the substitution of glutamic acid by valine at the sixth amino acid position in the β-chain of adult hemoglobin (HbA) [2,3].This results in hemoglobin S (HbS), which polymerizes in the setting of hypoxia, low pH, and decreased temperature [2][3][4].The reduced oxygen affinity of Hb, due to elevated levels of 2,3-diphosphoglycerate, and cellular dehydration, a result of altered cation/anion homeostasis, also stimulate Hb polymerization.In aggregate, polymerized Hb fibers will sickle erythrocytes and change cell shape, flexibility, and rheology, both reversibly and irreversibly [1,5,6].Linked with this, accelerated generation of reactive oxygen species (ROS) and iron-heme complexes generated by Hb auto-oxidation promote erythrocyte membrane lipid oxidation and cytoskeletal damage [1,5,7] (Fig. 1A).These diverse pathogenic assaults on sickled erythrocytes confer susceptibility to hemolysis, shortening their lifespan by more than 75% [1].
A toxic mélange of heme, Hb, ROS, arginase, serum lactate 1A.Hemoglobin S (HbS) undergoes polymerization under conditions of low oxygen, pH and temperature, causing erythrocytes to sickle.The sickled erythrocytes undergo hemolysis due to cellular alterations and release hemoglobin (Hb) into the circulation.Free Hb ((Ferrous Fe (II) Hb) either undergoes auto-oxidation or reacts with nitric oxide (NO) to form methemoglobin (Ferric Fe (III) Hb).The heme supports reactive oxygen species (ROS) generation causing erythrocytic membrane peroxidation, cytoskeletal damage and phosphatidylserine (PS), CD36, and CD47-induced erythrophagocytosis.Extra-erythrocytic events follow, stimulating further ROS generation.1B: High levels of heme and Hb saturate hemopexin and haptoglobin, respectively.Redox imbalance occurs due to a downregulation of antioxidants: glutathione (GSH), glutathione peroxidase (GPx), vitamin E, catalase, peroxiredoxin; and upregulation of pro-oxidants: nicotinamide dinucleotide phosphate (NADPH) oxidase, xanthine oxidase (XO), myeloperoxidase (MPO), uncoupled endothelial nitric oxide synthase (eNOS).ROS cause activation of leukocytes and platelets and the Toll-like receptor 4-nuclear factor-ĸB (TLR4-NFĸB) signaling pathway.The TLR4-NFĸB pathway stimulates the release of proinflammatory cytokines, including Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor (TNF) and Prostaglandin E2 (PG-E 2 ).Adhesion mediators including vascular cell adhesion molecule-1 (VCAM1), intracellular adhesion molecule-1 (ICAM1) and P-selectin are highly expressed on endothelium and secreted into the circulation.The cytokines and adhesion mediators promote adhesion of sickled erythrocytes and activated leukocytes and platelets to the vascular wall.These events, in addition to NO depletion, impair endothelial function and blood vessel relaxation.Blood flow encounters cellular and vascular obstacles that result in vaso-occlusive crisis (VOC).Repeated episodes of VOC become widespread across the body due to persistent oxidative stress, promoting tissue ischemia, inflammation and infarction.1C: Multiple end-organ injuries take place as a result, which makes SCD a multi-factorial systemic phenomenon.* and text in red indicate the SCD-associated disorders that can be targeted by NO 2 -FA based upon defined mechanisms of action and relevant published experimental results.
The vasculopathy associated with SCD generates a multitude of acute and chronic complications in vital organs (Fig. 1C).Patients with higher Hb levels have increased frequency of pain episodes, due to an inability to compensate for the higher blood viscosity or vaso-occlusion.Patients with lower Hb levels due to severe hemolytic anemia show higher levels of free heme, serum bilirubin, and LDH [1,28].In this case, reduced •NO levels and persistent inflammatory stress due to hemolysis and oxidative stress increase risk for further vascular complications, including pulmonary hypertension, cardiomegaly, cardio-ventricular dysfunction, ischemic stroke, leg ulcers, nephropathy, and gallstones [28].These and other associated comorbidities shorten the life expectancy of people having SCD by an average of 30 years [29,30].

Challenges with current therapeutic management strategies
For almost two decades hydroxyurea (HU) was the only FDAapproved drug for SCD [29].It induces fetal hemoglobin (HbF) induction 2% to over 30% [31][32][33] and reduces VOC, acute chest syndrome, infections, hospital admissions and risk for death [7,34].Despite the benefits HU provides, adverse side effects include myelosuppression, teratogenicity, and a growing concern that long-term use of HU can lead to DNA damage, impaired spermatogenesis [35], oxidative stress, and leukemogenesis [36,37].The considerable interpatient variability with regards to efficacy and maximum tolerated dose complicates clinical management, requiring frequent dose adjustments [38].All these factors reduce patient adherence to HU therapy.
Blood transfusions are also frequently used as a treatment for SCD [39].The newly transfused healthy erythrocytes dilute the percentage of erythrocytes containing HbS, improving cellular rheology in the circulation and suppressing further sickling of HbS erythrocytes [40].However, blood transfusions can cause volume overload, iron overload, and even adverse neurological events as a result of hyperviscosity [39,41].Although exchange transfusion can overcome these problems, the procedure is expensive and requires proper venous access (an issue in very young and old populations), skilled operators, and a high number of red cell units to reach hemoglobin goals [39,42].Additionally, transfusions carry the risks of alloimmunization, hemolytic transfusion reactions, and hyperhemolysis in patients with SCD [36,41].
Another FDA-approved therapeutic option for SCD is hematopoietic stem cell transplantation (HSCT).This is the only clinically available curative treatment strategy for SCD, with matched related donor transplants having the best outcomes.This procedure can initiate donorderived erythropoiesis and repair dysfunctional organs [43,44].In addition to the significant limitations posed by donor matching, there is an increased risk for acquiring graft versus host disease (GVHD).Also, the overall complexity of the procedure and daunting economic barriers, such as a lack of sophisticated health care availability in poorer countries replete with rural areas, greatly limit widespread use of HSCT [7,29].
Four new drugs for treating SCD have received FDA approval in the last 6 years: L-glutamine, voxelotor, crizanlizumab, and deferiprone.Lglutamine is a substrate for the synthesis of nicotinamide adenine dinucleotide (NAD+) and GSH, with these metabolites viewed to improve redox state and mitigate the erythrocyte membrane damage and adhesion events that are promoted by glutamine depletion [45,46].
Voxelotor binds covalently to the N-terminal valine of the α-globin chain of HbS, where it stabilizes the oxygenated Hb state to limit the polymerization of deoxy-HbS [45,47].Crizanlizumab is a monoclonal antibody that binds to P-selectin, thereby inhibiting the adherence of erythrocyte microparticles and activated leukocytes and platelets to the endothelium [45,48].Clinical trial results indicate that these new therapeutic agents only partially address the complex pathophysiology of SCD.For instance, a reduced incidence of vaso-occlusion and acute chest syndrome (ACS) has been reported for crizanlizumab and L-glutamine therapy respectively, regardless of concomitant HU therapy [6,7,43,47].There were no substantial changes in Hb, hematocrit, or reticulocyte levels [6,7,29,49].Voxelotor increased the Hb level by a mean of 1.1 g/dl and reduced hemolytic markers, but did not reduce the frequency of VOC [7,45,47].Shifting the Hb-oxygen affinity profile with voxelotor may have disadvantages due to impaired off-loading of bound oxygen in highly metabolic tissues where more oxygen is required [50].Deferiprone, an oral iron chelator, is a newly approved drug for the treatment of iron overload associated with frequent blood transfusions in SCD patients [51].Although the newly approved drugs have good safety margins, it is still unknown whether these drugs impact end-organ damage or are disease modifying [29,46].
Contemporary SCD management calls for a multi-agent therapeutic approach [1,6]; thus, combinations of prescription analgesics [52,53], antibiotics, steroids, and bronchodilators [54], along with HU and blood transfusions, are common.SCD patients require frequent doctor visits, hospital stays, blood transfusions, surgeries, and constant screening to help improve their quality of life [1,54,55].Although advances in therapeutic management have significantly improved quality of life and F.A. Chowdhury et al. the lifespan of SCD patients in high-income countries, life expectancy still lags far behind the general population.The disease burden and mortality rates for SCD worldwide are unchanged, as the poorer regions of Sub-Saharan Africa, where the majority of SCD sufferers reside [45], have restricted access to emergency care with only 19-50% of hospitals having the ability to provide 24-h medical attention and treatment [56].It was also found that 18-41% of the facilities have expired drugs in their inventories [56].Along with drug availability issues, the significantly lower income of many SCD patients limits access to HU and in particular newer therapeutic agents [45].The benefits of new therapies are not cost-effective and thus disadvantageous in low-income settings [45,57,58].Additionally, these drugs target specific features of SCD and do not have broad mechanisms of action that address the multiple pathological facets of SCD [27].Given the current scenario, choosing expensive multi-agent treatment options which do not significantly change the course of the disease or improve the quality of life or lifespan [29,45,59], thus does not provide a rational alternative from health and economic perspectives.

The unmet clinical need for better therapeutic management of SCD
As noted, there are challenges regarding effectiveness, accessibility, affordability, tolerance, and compliance of the current polypharmacybased approach to the therapeutic management of SCD.Herein, we detail the current state of therapeutic approaches for managing SCD in Section 2, as well as the ongoing clinical studies that aim to expand and improve future treatment options for the disease in Table 1.By doing so, our intent is to highlight the loopholes in the general concept of targeting and designing therapeutics for a multi-factorial disease like SCD.
Table 1 lists drugs at different stages of current clinical trials for SCD and does not include the trials in recruiting phase and the upcoming gene modification therapies, as these are beyond the scope of this review.The data have been collected from some recent review articles [1,6,7,29,60,61] and clinicaltrials.gov,and organized by the targets or proposed mechanisms of each drug.The table highlights the therapeutic effect of each drug at the molecular, cellular, and organ levels.The reason behind differentiating these three levels of biological complexity is to emphasize different SCD-related actions of the drugs in pipeline.Since SCD is a systemic phenomenon, targeting a specific pathway upstream or downstream of the critical inciting pathophysiological events can attenuate modification of the disease course.The table is meant to show how a majority of the drugs in the pipeline, rather than being multi-target, are restricted to single pathological events of SCD.Consequently, most therapies do not display broadly significant clinical benefits.This highlights an unmet clinical needthe application of multi-target drugs to limit disease symptoms, crises, and progression by concomitantly modulating multiple pathways.
Here, we introduce a drug class that has not been previously tested in SCD -electrophilic fatty acid nitroalkenes or nitro-fatty acid (NO 2 -FA).Given the multi-target actions of NO 2 -FA, we are the first to consider the suitability of the drug for targeting the multiple dysregulated pathways of SCD that lead to widespread end-organ damage: sickling and hemolysis, endothelial dysfunction, oxidative stress, inflammation, vasoocclusion, and pain crises (Table 1).We rationalize the potential for NO 2 -FA therapy to prevent or mitigate much of the systemic pathology of SCD.Figs. 1 and 2 also highlight NO 2 -FA as a prospective multi-target therapy for SCD.

Multi-target small molecule nitroalkenes as therapeutic agents for treating SCD
NO 2 -FA are endogenously generated lipid mediators normally found in plasma, urine, cell membranes and tissues in free, esterified form or adducted to nucleophilic residues in proteins [62][63][64].NO 2 -FA are readily formed during digestion of foods such as vegetables, dairy products, and both plant and marine oils.The acidic conditions of digestion catalyzes the formation of nitrogen dioxide from nitrite, that in turn mediates the nitration of dietary unsaturated fatty acids [65].NO 2 -FA are also formed as a consequence of inflammation, where multiple convergent reactions of superoxide, hydrogen peroxide, •NO, nitrite and heme-based catalysts ultimately give rise to the proximal unsaturated FA nitrating species, nitrogen dioxide [62][63][64].
Nitrated fatty acids have been studied both preclinically and clinically [63].Endogenously, unsaturated fatty acids with 2 or more conjugated double bonds are the primary substrates for nitration and the endogenous generation of NO 2 -FA (Fig. 2).Thus, nitro derivatives of conjugated linoleic and linolenic acid and their metabolites are readily detectable in healthy human urine and plasma [66,67].Naturally-occurring NO 2 -FA homologs and non-natural small molecule nitroalkenes can be synthesized by nitrosenylation or nitro-aldol condensation followed by acetylation and elimination steps, the latter approach allowing the synthesis of specific positional isomers [63].All nitroalkenes are characterized by double bonds containing an electron-withdrawing nitro substituent that confers an electrophilic reactivity to the other carbon of the double bond.This reactivity in turn promotes Michael addition with nucleophilic (electron-rich) residues such as cysteine [68] and histidine [68][69][70].Michael addition can also occur with lipid electrophiles having an, substituents (e.g., 4-hydroxy-2-nonenal, 4-oxo-2-nonenal, 15-deoxy-prostaglandin-J 2 ) [67,71].As a consequence of these reactions, post-translational protein modifications (PTM) occur that can trigger changes in the activity, localization, structure and function of proteins [62,72].These PTMs, when induced by nM concentrations of reversibly-reactive small molecule electrophiles, typically promote adaptation to inflammatory, oxidative and metabolic stress [63,65](Fig.2).NO 2 -FA impact the function of 100-150 target proteins, all having hyper-reactive cysteines, that regulate diverse signaling pathways important in regulating inflammation and vascular function.This includes nuclear factor erythroid 2-related factor (Nrf2)/KEAP1, NF-ĸB, heat shock response (HSR), peroxisome proliferator-activated receptor gamma (PPARγ), Janus kinase/signal transducers and activators of transcription (JAK/STAT), stimulator of interferon genes (STING), epoxide hydrolase, calcineurin A and angiotensin II receptor function, among others [63,64,73,74].The protein reactions and functional impact of synthetic and endogenously occurring NO 2 -FA have been studied in multiple preclinical disease models (reviewed in Table 2), demonstrating both anti-fibrotic and anti-inflammatory activities.
NO 2 -FAs synthesized and characterized to date include nitro-oleic acid (NO 2 -OA), nitro-linoleic acid (NO 2 -LA), nitro-arachidonic acid (NO 2 -AA), and nitro-conjugated linoleic acid (NO 2 -cLA) [62,66,67,[70][71][72][73][74][75][76][77][78][79].NO 2 -OA is structurally the simplest and most studied in terms of biochemistry, signaling responses, preclinical effects, and clinical safety.The metabolism, signaling, and mechanisms of action of both endogenous and pharmacological nitroalkenes have been discussed in recent reviews [62,63].The most studied NO 2 -OA regioisomer is 10-NO 2 -OA, with preclinical toxicology, Phase I clinical trials (n = 5) and ongoing Phase II clinical trials [67] not revealing safety concerns at therapeutic doses.In addition to the value of NO 2 -FA drug-based treatments, dietary approaches that increase the endogenous levels of NO 2 -FA have been proposed.This gains relevance as dietary supplementation of cLA (3 g) + 15 NO 2 -(20 mg) increased plasma NO 2 -cLA in healthy volunteers to levels that parallel concentrations attained in Phase 1 clinical studies of NO 2 -OA [80,81].cLA and 15 NO 2 -supplementation reached similar plasma concentrations of 15 NO 2 -cLA (C max 8 nM), comparable to NO 2 -OA at the target dose defined, while conducting Phase 2 clinical trials (pulmonary arterial hypertension, chronic kidney diseases, and asthma, 150 mg dose, C max 7.6 nM).These levels agree with those associated with protective pharmacological actions in murine models of disease (ranging from 5 to 30 nM) [80] [-] [82].Overall, this highlights the potential for dietary approaches to reach pharmacological levels expected to promote beneficial actions and induce protection in SCD.Given the multi-target reactivity of NO 2 -FA and other small molecule nitroalkenes, this class of mediators may represent an effective therapy for diseases having a multifactorial pathophysiology, such as SCD.A single drug targeting several disease-causing pathways obviates the need for multi-agent treatment regimens and potentially improving both the disease course and the quality of life for patients with SCD.Based on the mechanisms of action and pharmacological evidence collected so far for NO 2 -FA and the complex pathology of SCD, we propose that a low dose of NO 2 -FA could provide protection early on, and delay or preclude the onset of many of the pathologic manifestations of SCD.Moreover, the acute crises characteristic of SCD could be treated with higher doses of NO 2 -FA.A promising alternative might also be to use dietary supplementation of NO 2 -FA precursors (nitrite, nitrate, CLA) to achieve pharmacological levels in SCD.Of significance, and as opposed to many aldehydic or α,β-unsaturated ketone-containing fatty acid electrophiles, nitroalkene reactivity towards soft nucleophiles (predominantly cysteine) is both rapid and reversible.Thus, drug accumulation should not be dose-limiting concern [68,72].The therapeutic potential for NO 2 -FA as a modulator of the signaling pathways associated with organ damage in SCD is described in Figs. 1 and 2.

Erythropoiesis
SCD patients suffer from acute and chronic anemia that is precipitated by a dysfunctional bone marrow, splenic sequestration, and hemolysis of erythrocytes [29,83].The continuous production of erythrocytes in response to the anemic stress exhausts the bone marrow, impairing erythropoiesis [83,84].Extramedullary erythropoiesis and VOC in the spleen cause splenic sequestration and splenomegaly.During splenic sequestration, there is a drop in overall levels of Hb, circulating blood volume, erythrocytes, and platelets [83,85].Functional hyposplenia and asplenia, due to subclinical splenic infarction, predispose to systemic infections as the spleen fails to clear encapsulated bacteria [83,85].The Nrf2-signaling pathway enhances HSC function in the bone marrow, promotes HSC homing and quiescence, and is required for stress erythropoiesis [86,87].Nrf2 also induces HbF synthesis that protects against erythrocyte sickling and hemolysis [88][89][90].It has been shown that knocking out Nrf2 aggravates the pathophysiology of SCD in mice [91].NO 2 -FA are robust activators of the transcription factor Nrf2 [63,69,71], having the potential to improve erythrocyte production and longevity, lessening the deleterious effects of hemolysis and anemia.

Cardiovascular disorders
Approximately 32% of SCD-associated deaths are attributed to cardiovascular events [30].In SCD, •NO depletion causes recurrent episodes of inflammation, generation of ROS, and vaso-occlusion [5,10,24].Persistent anemia results in cardiomegaly and left ventricular hypertrophy and dysfunction, which can lead to acute myocardial infarction, arrythmia, congestive heart failure, and death [92].NO 2 -FA provide cardiovascular protection in ischemic heart disease, in part, by inhibiting the proinflammatory NF-ĸB signaling and impeding the transcription of pro-inflammatory cytokines [65,70].Further vascular protection comes from the ability of NO 2 -FA to suppress ROS generation by inflammatory cells, mitochondria, and cardiomyocytes, thereby simultaneously limiting •NO consumption and the generation of secondary oxidants catalyzed by ROS and oxidase, oxygenase and peroxidase reactions [93].In concert with the upregulation of eNOS expression, this leads to a more stable and functional vascular milieu [94][95][96].
NO 2 -FA upregulate heme-oxygenase 1 (HO-1) and heat shock protein (HSP) expression by inducing Nrf2 [97] and heat shock factor (HSF) [71] dependent responses.HO-1 catabolizes heme and hinders smooth muscle cell migration after vascular injury, inhibiting restenosis [98].The HSPs are responsible for the proper folding of proteins in cells that become denatured under stressed conditions [71].NO 2 -FA also increase the expression of other Nrf2-regulated genes such as GPx, glutathione reductase, and superoxide dismutase [62,99], as well as non-competitively inhibit XO [70], thereby attenuating vascular ROS generation and its sequelae.NO 2 -OA and NO 2 -LA mediate inhibition of JAK/STAT pathways and maintain vascular homeostasis by regulating the immune responses, macrophage polarization and phagocytosis, as shown in lipopolysaccharide (LPS) injected murine models [100,101].NO 2 -OA also lowers the expression of adhesion mediators, limiting vascular infiltration of inflammatory cells [102] and inhibiting venular leukocyte sequestration [103].Finally, NO 2 -AA inhibits platelet aggregation via irreversible inactivation of prostaglandin endoperoxide H synthase I (PGHS-I) [104].These protective effects of NO 2 -FA are of relevance to cardiac function as well, including: reduced ischemic heart injury with low neutrophil accumulation and MPO in the infarct zone [105]; blunted cardiac remodeling with limited matrix-metalloproteinase activity [66]; decreased risk for acute ventricular tachycardia (VT) with homeostatic regulation of calcium [75] and attenuation of myocardial infarct-induced cardiac hypertrophy with NO 2 -cLA and nitrite therapy [106].These multi-target actions of NO 2 -FA have the potential to significantly impact cardiovascular function and inflammatory stress in SCD patients.

Pulmonary complications
Pulmonary disorders are responsible for 28% of mortality in SCD [30], with ACS and pulmonary hypertension (PH) being the most common [83,107].ACS can arise from pulmonary infections and edema, pulmonary embolism and infarction, pulmonary vaso-occlusion, and fat emboli from the bone marrow [92,108], all manifested by pulmonary infiltrates accompanied by chest pain, fever, tachypnea, wheeze, and cough.Pulmonary hypertension can develop from an altered immune response, endothelial dysfunction, vascular bed damage, parenchymal fibrosis, and smooth muscle hypertrophy [83,92].The ability of NO 2 -FA to protect against PH has been shown in several studies.NO 2 -OA and NO 2 -LA reduced PH in LPS-treated mice by depleting circulatory and pulmonary levels of 5-lipoxygenase (5-LO) and its downstream products leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-HETE [109].In an insulin-resistance model, treatment with NO 2 -FA improved PH by reducing ROS, XO, and cytokine levels [70].NO 2 -FA administration also increases the survival of lung endothelial cells by promoting PPARγ and β-catenin complexation and upregulation of pro-survival molecules such as apelin in the endothelium [110].10-NO 2 -OA also inhibits transforming growth factor-β (TGF-β) signaling, attenuating vascular fibrosis and right ventricular pressure [78].Thus, there are several avenues by which NO 2 -FA may protect against pulmonary complications traditionally associated with SCD.

Renal and hepatobiliary complications
Renal failure due to vaso-occlusion-induced ischemia, infarction,      [76,111].In a multi-organ endotoxemia model, inflammatory markers including TNFα, ICAM-1, VCAM-1 and PGE 2 were reduced after treatment with NO 2 -OA [112].Moreover, NO 2 -OA reduces ischemic injury to mouse kidneys by reducing renal MPO levels [111].NO 2 -FA also antagonize inflammation-induced carcinogenesis by modulating oxidative stress via inhibition of NFĸB and 5-LO pathways [62], a mechanism relevant to renal medullary carcinoma.This disorder is rare, but when associated with SCD and sickle cell trait its course is extremely aggressive in the young [83,113,114].
In the liver, acute VOC results in hepatic sequestration, sinusoidal obstruction, intracanalicular cholestasis, ischemia, and necrosis [83,115], all of which may be limited by the anti-inflammatory and antioxidant activities that are instigated by downstream NO 2 -FA signaling.Common SCD-associated hepato-pathologies include intrasinusoidal sickling and dilation, erythrophagocytosis with Kupffer cell hyperplasia, cirrhosis, and hemosiderosis.Multiple nitroalkenes are effective activators of Nrf2-mediated HbF synthesis, which can blunt erythrocyte sickling and hemolysis [88][89][90].NO 2 -FA can also limit CD36-mediated erythrophagocytosis [6,62].In SCD, long-term hepato-fibrosis resulting from oxidative injury leads to liver cirrhosis [116], a process that NO 2 -FA can counteract and even reverse [117].NO 2 -OA treatment of a murine non-alcoholic fatty liver disease (NAFLD) model improved energy metabolism in concert with blocking steatosis and fibrosis [118].Lastly, in the gallbladder, the chronic hemolysis associated with SCD leads to gallstone formation and bile-stained infarcts [115,119].NO 2 -FA also favorably regulate bile acid biosynthetic enzyme expression via upregulation of hepatic farnesoid X receptor levels [120].

Other tissues and organs
SCD also affects the nervous, musculoskeletal, and ophthalmic systems [29,30,83].Neuro-vascular occlusion, caused by sickling, hemolysis, and inflammation, promotes ischemic stroke and silent cerebral infarction, particularly in children.For adults with SCD, hemorrhagic strokes may be more common [121,122].NO 2 -FA have neuroprotective activity, as demonstrated by PPARγ activation [62] and regulation of nociceptive neuronal cells that control inflammation and pain in the central nervous system [123,124].Osteo-vascular and retino-vascular occlusion also occur, with the former provoking infarct, infection, necrosis, and degeneration [83] and the latter causing retinopathy and blindness via peripheral retinal ischemia [125].To date, the potential therapeutic effects of NO 2 -FA in osteopathy and visual systems have yet to be explored.

Conclusions
In the last few decades, remarkable advances have been made in discerning the pathophysiological mechanisms of SCD.A plethora of therapeutic agents have been proposed and tested to combat this hereditary hematological disorder [6,7].However, only four new disease-modifying agents have FDA approval, and these agents do not replace conventional therapeutic regimens in terms of cost-benefit ratio and effectiveness [45].It is possible that current drugs in the development pipeline may also fall short of significant therapeutic responses, due to an inability to limit the multiple ongoing pathophysiological insults in SCD.NO 2 -FA, being Michael acceptors, appear to modulate the most significant pro-inflammatory reactions and signaling pathways associated with the complex pathophysiology of SCD [62,63,70,73].This unique multi-target property suggests that NO 2 -FA could be efficacious in treating not only the symptoms and end organ damage of SCD but also other hemoglobinopathies.It is noteworthy that preclinical studies coming from multiple labs show NO 2 -FA are proficient in treating cardiovascular, pulmonary, renal, and hepatobiliary complicationsthe leading causes of death in SCD [30].Hence, disease modifying opportunities can stem from developing specific nitroalkenes screened to optimize the targeting of both the causes and consequences of SCD.This would involve tracking screening and efficacy criteria that includes the induction of HbF, the activation of key protective signaling pathways and the limitation of inflammation, thus promoting a significant improvement in the morbidity and mortality of this multi-systemic disorder that affects millions of individuals worldwide.

Sources of funding
We thank the University of Pittsburgh and the ARCS foundation for their continued support.

Fig. 1 .
Fig. 1.Pathophysiological effects and consequences of SCD and the pharmacological targets of NO 2 -FA.1A.Hemoglobin S (HbS) undergoes polymerization under conditions of low oxygen, pH and temperature, causing erythrocytes to sickle.The sickled erythrocytes undergo hemolysis due to cellular alterations and release hemoglobin (Hb) into the circulation.Free Hb ((Ferrous Fe (II) Hb) either undergoes auto-oxidation or reacts with nitric oxide (NO) to form methemoglobin (Ferric Fe (III) Hb).The heme supports reactive oxygen species (ROS) generation causing erythrocytic membrane peroxidation, cytoskeletal damage and phosphatidylserine (PS), CD36, and CD47-induced erythrophagocytosis.Extra-erythrocytic events follow, stimulating further ROS generation.1B: High levels of heme and Hb saturate hemopexin and haptoglobin, respectively.Redox imbalance occurs due to a downregulation of antioxidants: glutathione (GSH), glutathione peroxidase (GPx), vitamin E, catalase, peroxiredoxin; and upregulation of pro-oxidants: nicotinamide dinucleotide phosphate (NADPH) oxidase, xanthine oxidase (XO), myeloperoxidase (MPO), uncoupled endothelial nitric oxide synthase (eNOS).ROS cause activation of leukocytes and platelets and the Toll-like receptor 4-nuclear factor-ĸB (TLR4-NFĸB) signaling pathway.The TLR4-NFĸB pathway stimulates the release of proinflammatory cytokines, including Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor (TNF) and Prostaglandin E2 (PG-E 2 ).Adhesion mediators including vascular cell adhesion molecule-1 (VCAM1), intracellular adhesion molecule-1 (ICAM1) and P-selectin are highly expressed on endothelium and secreted into the circulation.The cytokines and adhesion mediators promote adhesion of sickled erythrocytes and activated leukocytes and platelets to the vascular wall.These events, in addition to NO depletion, impair endothelial function and blood vessel relaxation.Blood flow encounters cellular and vascular obstacles that result in vaso-occlusive crisis (VOC).Repeated episodes of VOC become widespread across the body due to persistent oxidative stress, promoting tissue ischemia, inflammation and infarction.1C: Multiple end-organ injuries take place as a result, which makes SCD a multi-factorial systemic phenomenon.* and text in red indicate the SCD-associated disorders that can be targeted by NO 2 -FA based upon defined mechanisms of action and relevant published experimental results.

Fig. 2 .
Fig.2.Formation, mechanism of action and potential effects of NO 2 -FA on major pathophysiological complications of SCD The endogenous formation of NO 2 -FA proceeds by the rapid addition of nitrogen dioxide to double bonds of conjugated fatty acids.Initial nitrated intermediates continue to react with oxygen and nitrogen radicals present in the reaction system but are eventually reduced to the resonance stabilized NO 2 -FA.In the free acid form, NO 2 -FA reversibly adduct to redox-sensitive cysteine residues in regulatory proteins and transcription factors.This chemical modification (i.e., nitroalkylation) introduces structural changes to the protein's native form, causing altered regulation and differential signaling responses.Important signaling pathways associated with SCD can be potentially modulated by NO 2 -FA, attenuating the major complications that cause organ-wide damage, including sickling and hemolysis, endothelial dysfunction, oxidative stress, inflammation, vaso-occlusion, and pain crises.Prostaglandin reductase 1 (PtGR-1) inactivates NO 2 -FA by disrupting its electrophilic character, thereby diminishing protein target adduction and modulation of signaling pathways.The green arrow shows induction, and the red arrow shows inhibition or down-regulation of the corresponding pathways/molecules (in bold) by NO 2 -FA.Nrf2, Nuclear factor erythroid 2-related factor; eNOS, endothelial nitric oxide synthase; ROS, reactive oxygen species; PPAR γ, peroxisome proliferator-activated receptor gamma; GPx, glutathione peroxidase; XO, xanthine oxidase; MPO, myeloperoxidase; HO-1, heme-oxygenase I; NF ĸB, nuclear factor-ĸB; JAK/STAT, Janus kinase/ signal transducers and activators of transcription; 5-LO, 5-lipoxygenase; VCAM-I, vascular cell adhesion molecule-1; ICAM-I, intracellular cell adhesion molecule-1; PGE 2 , prostaglandin E2; PGHS-I, prostaglandin endoperoxide H synthase I Financial support for this work was provided by the National Institutes of Health grants: R35 HL161177 (A C. Straub), R01 HL 149825 (A C. Straub), R01 HL 153532 (A C. Straub), American Heart Association grants: Established Investigator Award 19EIA34770095 (A C. Straub), R01 GM125944 (F.J. Schopfer), R33 HL157609, (B.A. Freeman) and R01 HL162787 (B A. Freeman).This work was also supported in part by the National Institutes of Health Grant 5KL2TR001856-07.

Table 1
Potential therapeutic agents in pipeline for SCD.

Table 1 (
continued ) (continued on next page) F.A.Chowdhury et al.

Table 2
NO 2 -FA evaluation in preclinical animal models.