DISRUPTED ARGININE–NITRIC OXIDE SIGNALING IN SICKLE CELL DISEASE: MOLECULAR MECHANISMS, PATHOPHYSIOLOGICAL CONSEQUENCES AND EMERGING THERAPEUTIC TARGETS

Emmanuel Ifeanyi Obeagu1*image, Christian C. Ezealaimage

 Department of Biomedical and Laboratory Science, Africa University, Mutare, Zimbabwe.

 

 

Abstract

Sickle cell disease (SCD) is marked by ongoing hemolysis and blood vessel dysfunction, with the arginine–nitric oxide (NO) pathway being crucial to its pathophysiology. This review examines the molecular mechanisms that contribute to the disruption of the arginine–NO axis in SCD, emphasizing how hemolysis-mediated arginase release reduces L-arginine, the precursor for NO production, while cell-free hemoglobin captures bioactive NO. Moreover, increased concentrations of natural NOS inhibitors like asymmetric dimethylarginine (ADMA) and oxidative stress play a role in the uncoupling of nitric oxide synthase (NOS), which further diminishes NO bioavailability. Impaired citrulline–arginine recycling worsens substrate shortage, together resulting in endothelial dysfunction, vasoconstriction, and inflammation. Therapeutic strategies aimed at this pathway, such as arginine and citrulline supplementation, arginase inhibition, antioxidants, and NO donors, are examined, highlighting their ability to restore vascular balance.

Keywords: Nitric oxide, nitrogen metabolism, sickle cell disease, therapeutic strategies, vascular dysfunction.

 

 

INTRODUCTION

 

Sickle cell disease (SCD) is a genetic hemoglobin disorder marked by the existence of abnormal hemoglobin S, which clumps together in low oxygen situations, resulting in red blood cell (RBC) distortion, ongoing hemolysis, and blood vessel blockage. These abnormal characteristics lead to a wide range of clinical issues, such as painful episodes, acute chest syndrome, stroke, and ongoing organ impairment. Even with progress in comprehending the genetic foundations of SCD, the complex molecular processes that lead to vascular problems and inflammation continue to be a focus of ongoing research. At the core of these mechanisms lies the dysregulation of the arginine–nitric oxide (NO) pathway, which is essential for regulating vascular tone and homeostasis1-3. Nitric oxide is a gaseous signaling molecule produced from L-arginine by nitric oxide synthase (NOS) enzymes, notably endothelial NOS (eNOS) in the vascular endothelium. NO produces strong vasodilatory, anti-inflammatory, and anti-thrombotic effects, preserving endothelial integrity and inhibiting abnormal vessel constriction. In individuals without health issues, this pathway rigorously regulates blood circulation and restricts vascular damage. In SCD, the availability of NO is significantly diminished, leading to endothelial dysfunction and the prothrombotic condition characteristic of the disease4-6.

The disturbance of NO balance in SCD arises from several interrelated molecular irregularities. Hemol-ysis, a characteristic of SCD, causes the release of intracellular substances like arginase-1 and free hemoglobin into the plasma. Arginase-1 facilitates the breakdown of L-arginine into urea and ornithine, reducing the substrate needed for NO synthesis. Simultaneously, free hemoglobin eagerly captures NO, producing inert nitrate and methemoglobin, thus diminishing NO’s ability to induce vasodilation. These two processes result in a significant functional deficiency of arginine and a swift reduction in NO availability. Additionally, increased levels of endogenous inhibitors like asymmetric dimethyl-arginine (ADMA) further suppress NOS activity in patients with SCD. Oxidative stress, caused by repeated ischemia reperfusion injury and ongoing inflammation, facilitates the uncoupling of NOS. In this uncoupled condition, NOS generates reactive oxygen species instead of NO, worsening oxidative harm and impairing endothelial function7-10.

The downstream effects of arginine–NO pathway dysfunction is significant. Decreased NO signaling promotes vasoconstriction, platelet clumping, leukocyte sticking, and smooth muscle growth, resulting in a vascular environment susceptible to blockage, inflammation, and remodeling. These pathological alterations are associated with numerous serious complications observed in SCD, such as pulmonary hypertension, stroke, and chronic organ ischemia11,12. Recent years have seen significant interest in therapeutic strategies that focus on the arginine–NO axis. L-arginine or L-citrulline supplementation seeks to enhance substrate availability, while arginase inhibitors aim to maintain natural arginine levels by blocking its breakdown. Antioxidants and NOS cofactors, including tetrahydrobiopterin (BH₄), are investigated to lessen oxidative stress and reestablish NOS coupling13,14. Moreover, inhaled nitric oxide and novel gene-targeted therapies indicate pioneering methods to directly enhance NO bioactivity or address fundamental genetic issues

The primary aim of this review is to comprehensively examine the role of NO and nitrogen metabolism in the pathophysiology of SCD and to explore current and emerging therapeutic strategies targeting these pathways.

An overview of the arginine–nitric oxide pathway

The arginine–nitric oxide (NO) pathway is central to many physiological processes, controlling vascular tone, immune responses, neurotransmission, and cellular signaling. At the heart of it is the amino acid L-arginine, a semi-essential compound that acts as the main substrate for NOS enzymes, which facilitate the transformation of arginine into nitric oxide and L-citrulline. This enzymatic process is fundamental to endothelial wellness and overall vascular balance. In the vascular endothelium, endothelial eNOS is crucial. In response to shear stress or different agonists (such as acetylcholine, bradykinin), eNOS is activated through a calcium-calmodulin–dependent mechanism. With vital cofactors like tetrahydrobiopterin (BH₄), oxygen, and NADPH, eNOS facilitates the oxidation of L-arginine, generating nitric oxide, a gaseous signaling molecule, along with L-citrulline15,16. 

In addition to vasodilation, NO has anti-inflammatory, antithrombotic, and anti-proliferative effects. It prevents platelet adhesion, decreases leukocyte-endothelial interactions, and regulates oxidative stress by opposing reactive oxygen species (ROS). These defensive functions are particularly important in conditions marked by vascular damage and inflammation17. Nevertheless, the supply of L-arginine is closely controlled and affected by competing processes. A significant rival is arginase, which breaks down arginine into ornithine and urea, redirecting it from NO synthesis. This competitive interaction is especially significant in pathological states like sickle cell disease, where hemolysis-driven release of arginase-1 reduces arginine levels, hindering NO synthesis and facilitating vascular issues18. Additionally, there is another regulatory layer from endogenous inhibitors like asymmetric dimethylarginine (ADMA), which competes with L-arginine for NOS binding, further limiting NO production. Additionally, in situations of oxidative stress or lack of cofactors, NOS may become uncoupled, resulting in the production of superoxide rather than NO-thereby increasing vascular harm. The arginine-NO pathway is consequently a delicate, well-regulated system. Any disruption in its components such as arginine availability, NOS function, cofactor integrity, or competing metabolic pathways can shift the balance toward endothelial dysfunction, a key feature of numerous cardiovascular and hemolytic diseases19.

Mechanisms of arginine–nitric oxide pathway impairment in sickle cell disease

The arginine–nitric oxide pathway, essential for maintaining vascular integrity and regulating the immune system, is significantly altered in SCD, leading to the endothelial dysfunction, vasculopathy, and inflammation characteristic of this hemoglobin-pathy. Nitric oxide synthesis is crucial for physiological functions, yet its dysfunction in SCD arises from a mix of biochemical, oxidative, and inflammatory factors that limit substrate availability, impede enzymatic activity, and promote NO degradation20. This disruption primarily involves L-arginine, the precursor substrate for NOS. In people with SCD, hemolysis significantly contributes to the reduction of systemic arginine levels. When red blood cells break apart, they discharge arginase-1 into the plasma. This enzyme quickly converts L-arginine into ornithine and urea, thereby decreasing the arginine accessible for NO production. Increased plasma arginase activity has consistently been linked to reduced vasodilation, heightened pulmonary pressures, and worse clinical outcomes in SCD. Besides substrate depletion, SCD also creates conditions for NO scavenging. Hemoglobin released into the bloodstream during intravascular hemolysis interacts with and deactivates NO in a stoichiometric reaction, resulting in the formation of methemoglobin and nitrate21,22.

A significant factor in disrupting the arginine–NO pathway is the buildup of natural NOS inhibitors, especially ADMA. In SCD patients, elevated ADMA competes with arginine for binding to NOS, thereby inhibiting NO production. Concurrently, oxidative stress common in SCD due to ongoing inflammation, ischemia reperfusion damage, and ROS production may cause NOS uncoupling. In its uncoupled condition, NOS stops producing NO and instead produces superoxide anions, which interact with any leftover NO to create peroxynitrite, a strong and harmful oxidant. Moreover, citrulline recycling the mechanism by which L-citrulline, a byproduct of NO synthesis, is reverted to L-arginine is similarly impaired in SCD. This recycling relies on the coordinated function of argininosuccinate synthase and argininosuccinate lyase, enzymes that are frequently downregulated in inflammatory or hypoxic conditions typical in SCD. The inability to convert citrulline back to arginine further restricts NO synthesis and continues vascular dysfunction23,24.

These interconnected processes arginase mediated substrate depletion, hemoglobin's NO scavenging, ADMA-induced inhibition, oxidative stress related NOS uncoupling, and disrupted citrulline recycling collaborate to establish a condition of nitric oxide resistance in SCD. The resulting impacts are clinically relevant: heightened vascular tone, platelet clumping, leukocyte sticking, and smooth muscle growth—all factors that lead to the vaso-occlusive episodes, pulmonary hypertension, and organ impairment observed in this condition25. Treatment approaches like arginine supplementation, arginase blockers, citrulline treatment, and NO donors are being studied to reestablish NO equilibrium. Nonetheless, addressing this intricate metabolic dysfunction necessitates a comprehensive strategy focused on the fundamental hemolysis, oxidative stress, and enzymatic dysregulation that hinder the arginine–NO axis in sickle cell disease26.

A molecular perspective

The pathophysiology of SCD is deeply rooted in molecular disturbances that extend beyond the well known hemoglobin S polymerization. Central among these is the dysregulation of the arginine–nitric oxide pathway a biochemical axis crucial for maintaining vascular tone, endothelial health, and immune balance. A detailed molecular perspective reveals a cascade of interrelated mechanisms that converge to impair NO bioavailability and drive the complex clinical phenotype of SCD27. At the core of this dysfunction is arginine metabolism. Under physiological conditions, L-arginine serves as the principal substrate for NOS, which catalyzes the conversion of arginine into NO and L-citrulline. In SCD, however, chronic intravascular hemolysis leads to the release of erythrocyte contents, notably arginase-1, into the plasma. This enzyme competes with NOS by converting arginine into ornithine and urea, thereby depleting circulating and intracellular arginine pools. The consequence is a “functional arginine deficiency” that restricts NO synthesis at the endothelial interface28.

In parallel, the release of cell free hemoglobin into the bloodstream represents a potent NO scavenger. Free hemoglobin binds NO with high affinity, leading to the formation of inactive nitrate and methemoglobin, and diminishing NO’s vasodilatory effects. This mechanism exacerbates vasoconstriction and contri-butes to the development of pulmonary hyper-tension and vaso-occlusive crises, key complications in SCD29. The molecular environment is further destabilized by elevated levels of ADMA, an endogenous NOS inhibitor. ADMA interferes with NOS activity, reducing NO output and promoting endothelial dysfunction. Compounding this, oxidative stress, a hallmark of SCD due to recurrent ischemia-reperfusion injury and chronic inflammation, leads to NOS uncoupling. In this uncoupled state, NOS generates superoxide anions instead of NO, which not only fails to fulfill NO's vasoprotective functions but also reacts with residual NO to form peroxynitrite a highly reactive and damaging oxidant30. Moreover, the citrulline–arginine recycling pathway, mediated by argininosuccinate synthase (ASS) and arginino-succinate lyase (ASL), becomes compromised in SCD due to oxidative and inflammatory signaling. This disruption impairs the regeneration of arginine from citrulline, weakening intracellular arginine replenishment and further suppressing NO production in endothelial and immune cells31.

The downstream effects of these molecular events are far reaching. Impaired NO signaling contributes to leukocyte adhesion, platelet activation, smooth muscle proliferation, and vascular remodeling, all of which foster a pro-inflammatory and pro-thrombotic milieu. This biochemical cascade links molecular dysfunction to clinical manifestations such as stroke, acute chest syndrome, priapism, and chronic organ damage.32 Importantly, this molecular understanding has guided the development of targeted therapies. Agents such as L-arginine, L-citrulline, and arginase inhibitors aim to replenish substrate availability and reduce enzymatic competition. Concurrently, antioxidants and NOS cofactors such as tetrahydrobiopterin (BH₄) are being explored to restore NOS coupling and enhance NO production. Inhaled NO and gene based therapies further reflect the shift toward precision medicine rooted in molecular insight33.

Pathophysiological role of nitric oxide in sickle cell disease

NO plays a central role in maintaining vascular integrity, modulating inflammation, and preventing thrombotic complications. In SCD, the dysregulation of NO bioavailability is a major contributor to disease pathology, exacerbating vascular dysfunction, oxidative stress, and inflammatory processes19.

1. NO bioavailability and hemolysis

In SCD, chronic hemolysis releases large amounts of free hemoglobin into the plasma. This cell-free hemoglobin avidly scavenges NO, converting it to inactive nitrate and reducing its availability. Additionally, hemolysis releases arginase, an enzyme that depletes L-arginine, the substrate required for NO synthesis. The combined effects of NO scavenging and L-arginine depletion create a state of NO deficiency, impairing vascular function and promoting disease complications20.

2. Endothelial dysfunction

NO is essential for maintaining endothelial homeostasis by promoting vasodilation and inhibiting endothelial activation. In SCD, reduced NO levels lead to endothelial dysfunction, characterized by the upregulation of adhesion molecules such as ICAM-1, VCAM-1, and E-selectin. These molecules enhance the adhesion of sickled erythrocytes and leukocytes to the vascular endothelium, initiating vaso-occlusion and microvascular ischemia. The dysfunctional endothelium also becomes a source of pro-inflamm-atory cytokines, amplifying vascular inflammation and further impairing blood flow21.

3. Vaso-occlusion and ischemia

The interaction between sickled erythrocytes, leukocytes, and the endothelium is central to VOCs. NO deficiency exacerbates these interactions by promoting vasoconstriction, increasing vascular resistance, and reducing blood flow. The resultant ischemia contributes to severe pain episodes, tissue damage, and organ dysfunction. The role of NO in preventing such events underscores its importance in the pathophysiology of SCD22.

4. Oxidative stress and reactive oxygen species

In SCD, the chronic inflammatory state and hemolysis generate ROS, which further deplete NO and impair its signaling. NO reacts with superoxide, forming peroxynitrite, a reactive nitrogen species that damages cellular components and exacerbates oxidative stress. This creates a vicious cycle where oxidative stress and NO deficiency perpetuate endothelial damage and vascular dysfunction23.

5. Pulmonary hypertension and cardiovascular complications

Pulmonary hypertension (PH) is a severe complication of SCD associated with high mortality rates. NO deficiency contributes to PH by promoting vasoconstriction, vascular remodeling, and increased pulmonary arterial pressure. Chronic NO depletion also predisposes patients to cardiovascular complications, such as stroke and heart failure, by impairing vascular elasticity and increasing systemic vascular resistance24.

6. Immune dysregulation

NO plays an immunomodulatory role by regulating leukocyte activity and suppressing excessive inflammation. In SCD, reduced NO levels impair immune regulation, leading to heightened leukocyte activation and increased production of pro-inflammatory cytokines. This inflammatory milieu exacerbates vascular damage and promotes VOCs, further complicating the disease course.25

7. Renal and neurological implications

The kidneys and brain are particularly vulnerable to the effects of NO deficiency in SCD. In the renal vasculature, reduced NO bioavailability leads to vasoconstriction, glomerular injury, and progression to chronic kidney disease (CKD). Similarly, in the cerebral vasculature, NO dysregulation contributes to ischemic strokes, a common complication in pediatric SCD patients26.

Clinical implications of NO deficiency in sickle cell disease

NO deficiency in SCD has profound clinical consequences that underpin many of the disease’s most debilitating complications. NO’s critical role in maintaining vascular homeostasis means that its depletion contributes directly to the widespread endothelial dysfunction, vasoconstriction, and inflammation observed in SCD patients. These pathophysiological changes translate into a clinical phenotype marked by recurrent vaso-occlusive episodes, progressive organ damage, and increased morbidity34,35. One of the most prominent clinical manifestations linked to NO deficiency is vaso-occlusive crisis (VOC), characterized by episodes of severe pain due to microvascular occlusion and ischemia. NO deficiency exacerbates vasoconstriction by impairing endothelial relaxation, reducing blood flow through already compromised microcirculation. This creates a vicious cycle where ischemia and reperfusion injury further elevate oxidative stress, fueling more NO depletion and endothelial injury. As a result, patients experience frequent painful crises, which significantly impact quality of life and increase healthcare utilization36,37. Beyond VOC, NO deficiency is implicated in the development of pulmonary hypertension (PH), a severe and life-threatening complication of SCD. Reduced NO bioavailability impairs pulmonary vasodilation, leading to increased vascular resistance and right ventricular strain. PH in SCD patients is associated with increased mortality, emphasizing the critical need for therapeutic strategies that restore NO signaling to improve pulmonary vascular function38,39.

Another critical clinical consequence of impaired NO metabolism is end-organ damage. The kidneys, brain, and heart are particularly vulnerable to ischemia driven by microvascular dysfunction. NO deficiency contributes to a prothrombotic state, promoting platelet aggregation and leukocyte adhesion, which can precipitate strokes and chronic kidney disease. In the brain, endothelial dysfunction compromises cerebral blood flow, increasing the risk of silent cerebral infarcts and overt stroke, particularly in pediatric populations40. Chronic NO depletion also affects immune regulation and inflammation, further complicating disease progression. NO possesses anti-inflammatory properties; its deficiency facilitates leukocyte activation and endothelial adhesion molecule expression, amplifying systemic inflammation. This heightened inflammatory state exacerbates vascular injury and contributes to the chronic pain and fatigue experienced by many SCD patients41. Importantly, the clinical implications of NO deficiency extend to therapeutic considerations. Conventional treatments, such as hydroxyurea, indirectly modulate NO pathways by reducing hemolysis and inflammation. Meanwhile, emerging therapies directly targeting NO bioavailability-through supplementation with arginine or citrulline, arginase inhibitors, and inhaled NO aim to correct the underlying biochemical defects. Understanding the clinical ramifications of NO deficiency is vital for optimizing these interventions and tailoring treatment to individual patient needs42.

Therapeutic strategies targeting the arginine–NO axis in sickle cell disease

The disruption of the arginine–nitric oxide axis in SCD has emerged as a pivotal mechanism driving vascular dysfunction, inflammation, and end-organ damage. As research uncovers the biochemical underpinnings of this impaired pathway—marked by reduced arginine availability, NO scavenging, and endothelial injury several therapeutic strategies have been developed or proposed to restore NO bioavailability and reestablish vascular homeostasis. These interventions aim to correct substrate deficiencies, modulate enzymatic activity, and prevent NO inactivation, offering new hope in the management of SCD related complications43,44. One of the most extensively studied approaches is L-arginine supplementation. By replenishing systemic arginine levels, this strategy seeks to overcome substrate depletion caused by hemolysis-induced arginase release. Clinical studies have shown that oral or intravenous L-arginine can improve endothelial function, reduce pulmonary pressures, and potentially alleviate the severity of vaso-occlusive crises. However, the effectiveness of arginine therapy is influenced by concurrent arginase activity and the presence of endogenous inhibitors such as ADMA, which may limit its clinical impact45,46.

Recognizing the competitive consumption of arginine by arginase, arginase inhibitors have been developed as a complementary or alternative therapeutic strategy. These agents block the enzymatic conversion of arginine to ornithine and urea, preserving arginine for NO synthesis. Preclinical models of SCD have demonstrated that arginase inhibition enhances NO production, improves vascular reactivity, and reduces inflammation. Early phase clinical trials are underway to evaluate their safety and efficacy in human subjects47,48. Another promising intervention is L-citrulline supplementation, which leverages the citrulline–arginine recycling pathway. Unlike arginine, citrulline is not a substrate for arginase and is more efficiently taken up by cells. Once inside, citrulline is converted to arginine by argininosuccinate synthase and lyase, providing a sustained intracellular source of arginine for NOS-mediated NO production. This strategy not only circumvents extracellular arginase activity but also enhances the local availability of NO where it is most needed within the vascular endothelium49. Inhaled nitric oxide (iNO) has also been explored for acute vaso-occlusive episodes. As a direct NO donor, inhaled NO offers the potential to rapidly dilate constricted blood vessels and mitigate pain. While some studies have shown short term hemodynamic benefits, larger clinical trials have yielded mixed results, highlighting the challenges of delivering NO effectively in a systemically deficient and inflamed vascular environment50.

Beyond these direct approaches, antioxidant therapies such as N-acetylcysteine and tetrahydrobiopterin (BH₄) aim to prevent NOS uncoupling and reduce the oxidative inactivation of NO. These agents stabilize NOS function and scavenge reactive oxygen species, indirectly enhancing NO bioavailability and endothelial protection. Furthermore, statins, traditionally used for lipid management, have shown pleiotropic effects, including upregulation of endothelial NOS and improvement of endothelial function in SCD models51. By addressing the root cause of NO pathway dysregulation, these approaches represent a paradigm shift toward curative strategies in SCD52.

Emerging therapeutic targets

The increasing recognition of nitric oxide (NO) deficiency as a central contributor to sickle cell disease (SCD) pathology has catalyzed the development of targeted therapies aimed at restoring NO bioavailability and improving vascular function. Therapeutic interventions now extend beyond symptom management to focus on correcting the molecular imbalances driving disease progression. These emerging strategies aim to address the specific disruptions in the arginine–NO pathway, offering hope for more effective and personalized treatments.53 One of the most direct approaches involves arginine supplementation, intended to replenish the substrate necessary for NO synthesis. Clinical studies have demonstrated that intravenous or oral L-arginine can improve endothelial function, reduce pulmonary pressures, and shorten the duration of vaso-occlusive crises (VOC). However, systemic arginase activity remains a significant barrier, rapidly metabolizing administered arginine and limiting its therapeutic impact. To overcome this, researchers are increasingly exploring arginase inhibitors as a complementary or alternative strategy. By blocking the activity of arginase particularly arginase-1 released during hemolysis these agents preserve endogenous arginine levels, promoting sustained NO production54.

In addition to arginine based therapies, L-citrulline supplementation offers another promising route. Citrulline, a precursor to arginine in the urea cycle, bypasses arginase degradation and is efficiently converted into arginine intracellularly. This not only supports NO generation but may also have a longer lasting effect on plasma arginine levels. Combined citrulline and arginine therapy is currently being investigated to determine whether it provides synergistic benefits in restoring vascular NO signaling55. Another important area of exploration involves targeting oxidative stress, a key factor in nitric oxide synthase (NOS) uncoupling and vascular injury. Agents such as N-acetylcysteine (NAC) and vitamin C have been evaluated for their ability to scavenge reactive oxygen species and stabilize NOS function. Additionally, supplementation with tetrahydrobiopterin (BH₄), an essential cofactor for NOS activity, has shown potential in reducing NOS uncoupling and enhancing NO production. These antioxidant based approaches aim to improve NO bioefficacy by correcting the redox imbalances that impair enzyme function56.

Inhaled nitric oxide (iNO) represents a more direct intervention, delivering exogenous NO to the pulmonary vasculature to alleviate hypoxia and pulmonary hypertension. While iNO has demonstrated benefits in acute settings, particularly during VOC or acute chest syndrome, its utility as a long term therapy remains under investigation. Advances in NO delivery systems, including sustained release formulations and NO donors with longer half lives, may expand its role in chronic SCD management57. 

 

CONCLUSIONS

 

The disruption of the arginine–nitric oxide pathway in sickle cell disease serves as a key mechanism connecting hemolysis, vascular issues, and organ damage. Persistent arginine scarcity, increased arginase activity, nitric oxide depletion by free hemoglobin, and oxidative stress come together to disturb endothelial balance and sustain the pathophysiological features of SCD, such as vaso-occlusion, inflammation, and pulmonary hyper-tension. Grasping these molecular disruptions not only clarifies disease mechanisms but also paves the way for targeted therapeutic approaches focused on reinstating NO bioavailability and vascular function.

 

ACKNOWLEDGEMENTS 

 

The authors would like to thank Africa University, Zimbabwe to provide necessary facilities for this work.

 

AUTHOR'S CONTRIBUTION

 

Obeagu EI: conceived the idea, writing the manuscript, literature survey. Ezeala CC: formal analysis, critical review. Final manuscript was checked and approved by the both authors.

  

DATA AVAILABILITY

 

Data will be made available on request.                        

      

CONFLICT OF INTEREST

  

None to declare.

 

REFERENCES

 

  1. Kim-Shapiro DB, Gladwin MT. Nitric oxide pathology and therapeutics in sickle cell disease. Clin Hemo Micro 2018;68(2-3):223-237.https://doi.org/10.3233/CH-189009  
  1. Reiter CD, Gladwin MT. An emerging role for nitric oxide in sickle cell disease vascular homeostasis and therapy. Curr Opin Hemat 2003;10(2):99-107.https://doi.org/10.1097/00062752-200303000-00001 
  1. Gladwin MT, Schechter AN. Nitric oxide therapy in sickle cell disease. Semi Hemat 2001;38(4):333-342.https://doi.org/10.1016/s0037-1963(01)90027-7
  1. Gladwin MT, Schechter AN, Shelhamer JH, et al. The acute chest syndrome in sickle cell disease: Possible role of nitric oxide in its pathophysiology and treatment. Ameri J Respi Criti Care Med 1999;159(5):1368-1376.https://doi.org/10.1164/ajrccm.159.5.9810094
  1. Gupta P, Kumar R. Nitric oxide: A potential etiological agent for vaso-occlusive crises in sickle cell disease. Nitric Oxide 2024. https://doi.org/10.1016/j.niox.2024.01.008
  2. Wood KC, Hsu LL, Gladwin MT. Sickle cell disease vasculopathy: A state of nitric oxide resistance. Free Radi Bio Medicine 2008;44(8):1506-1528. https://doi.org/10.1016/j.freeradbiomed.2008.01.008
  1. Wajih N, Basu S, Jailwala A, et al. Potential therapeutic action of nitrite in sickle cell disease. Redox Bio 2017;12:1026-1039.https://doi.org/10.1016/j.redox.2017.05.006  
  1. Akinsheye I, Klings ES. Sickle cell anemia and vascular dysfunction: The nitric oxide connection. J Cell Physi 2010;224(3):620-625. https://doi.org/10.1002/jcp.22195
  2. Pereira DA, Calmasini FB, Costa FF, et al. Nitric oxide resistance in priapism associated with sickle cell disease: Mechanisms, therapeutic challenges, and future directions. J Pharm Exper Ther 2024;390(2):203-212.https://doi.org/10.1124/jpet.123.001962
  1. Hallmark L, Almeida LE, Kamimura S, et al. Nitric oxide and sickle cell disease—Is there a painful connection? Exper Bio Med 2021;246(3):332-341.https://doi.org/10.1177/1535370220976397 
  1. Maley JH, Lasker GF, Kadowitz PJ. Nitric oxide and disorders of the erythrocyte: Emerging roles and therapeutic targets. Cardio Haem Dis-Drug Targ 2010;10(4):284-291.https://doi.org/10.2174/187152910793743878  
  1. Obeagu EI, Obeagu GU. Living with sickle cell in Uganda: A comprehensive perspective on challenges, coping strategies, and health interventions. Medi (Baltimore) 2024;103(51):e41062. PMID: 39705436https://doi.org/10.1097/MD.0000000000041062  
  1. Obeagu EI, Adias TC, Obeagu GU. Advancing life: Innovative approaches to enhance survival in sickle cell anemia patients. Ann Med Surg (Lond) 2024; 86(10):6021-6036. PMID: 39359845https://doi.org/10.1097/MS9.0000000000002534  
  1. Obeagu EI, Obeagu GU. Malnutrition in sickle cell anemia: Prevalence, impact, and interventions: A Review. Med (Baltimore) 2024;103(20):e38164. PMID: 38758879https://doi.org/10.1097/MD.0000000000038164
  1. Obeagu EI, Obeagu GU. Managing gastrointestinal challenges: Diarrhea in sickle cell anemia. Med (Baltimore) 2024;103(18):e38075. PMID: 38701274.https://doi.org/10.1097/MD.0000000000038075
  1. Obeagu EI, Obeagu GU. Management of diabetes mellitus patients with sickle cell anemia: Challenges and therapeutic approaches. Med (Baltimore) 2024; 103(17):e37941. PMID: 38669382. https://doi.org/10.1097/MD.0000000000037941  
  1. Obeagu EI, Obeagu GU. Immunization strategies for individuals with sickle cell anemia: A narrative review. Med (Baltimore) 2024;103(38):e39756. PMID: 39312357.https://doi.org/1097/MD.0000000000039756
  2. Dilli PP, Obeagu E, Tamale A, et al. Update on the practice of premarital screening for sickle cell traits in Africa: A systematic review and meta-analysis. BMC Pub Health 2024;24(1):1467. PMID: 38822327.https://doi.org/10.1186/s12889-024-19001-y    
  1. Reiter CD, Gladwin MT. An emerging role for nitric oxide in sickle cell disease vascular homeostasis and therapy. Curr Opin hema 2003;10(2):99-107.https://doi.org/10.1097/00062752-200303000-00001 
  1. Akinsheye I, Klings ES. Sickle cell anemia and vascular dysfunction: The nitric oxide connection. J Cell Phys 2010;224(3):620-625. https://doi.org/10.1002/jcp.22195
  2. Kim-Shapiro DB, Gladwin MT. Nitric oxide pathology and therapeutics in sickle cell disease. Clin Hemo Micro 2018;68(2-3):223-237.https://doi.org/10.3233/CH-189009    
  1. Conran N, Franco-Penteado CF, Costa FF. Newer aspects of the pathophysiology of sickle cell disease vaso-occlusion. Hemo 2009;33(1):1-6.https://doi.org/10.1080/03630260802625709
  1. Nader E, Grau M, Fort R, et al. Hydroxyurea therapy modulates sickle cell anemia red blood cell physiology: Impact on RBC deformability, oxidative stress, nitrite levels and nitric oxide synthase signalling pathway. Nitric Oxide 2018;81:28-35.https://doi.org/10.1016/j.niox.2018.10.003  
  1. Bunn HF, Nathan DG, Dover GJ, et al. Pulmonary hypertension and nitric oxide depletion in sickle cell disease. Blood J Amer Soci Hema 2010;116(5):687-692.https://doi.org/10.1182/blood-2010-02-268193
  1. Aboderin FI, Oduola T, Davison GM, et al. A review of the relationship between the immune response, inflammation, oxidative stress, and the pathogenesis of sickle cell anaemia. Bio Med 2023;11(9):2413.https://doi.org/10.3390/biomedicines11092413
  1. Hallmark L, Almeida LE, Kamimura S, et al. Nitric oxide and sickle cell disease—Is there a painful connection? Experi Bio Med 2021;246(3):332-341.https://doi.org/10.1177/1535370220976397 
  1. Pereira DA, Calmasini FB, Costa FF, et al. Nitric oxide resistance in priapism associated with sickle cell disease: Mechanisms, therapeutic challenges, and future directions. J Pharm Exper Ther 2024;390(2):203-212.https://doi.org/10.1124/jpet.123.001962
  1. Mondoro TH, Ryan BB, Hrinczenko BW, et al. Biological action of nitric oxide donor compounds on platelets from patients with sickle cell disease. Brit J Haem 2001;112(4):1048-1054.https://doi.org/10.1046/j.1365-2141.2001.02623
  1. Jaja SI, Ogungbemi SO, Kehinde MO, et al. Supplementation with l-arginine stabilizes plasma arginine and nitric oxide metabolites, suppresses elevated liver enzymes and peroxidation in sickle cell anaemia. Patho 2016;23(2):81-85.https://doi.org/10.1016/j.pathophys.2016.04.004 
  1. Taylor CM, Kasztan M, Sedaka R, et al. Hydroxyurea improves nitric oxide bioavailability in humanized sickle cell mice. Ameri J Physi-Regu, Integ Comp Physi 2021;320(5):R630-640.https://doi.org/10.1152/ajpregu.00205.2020
  1. Buehler PW, Schaer DJ. Haptoglobin therapeutics and compartmentalization of cell-free hemoglobin toxicity. Trend Mole Med 2020;26(7):683-697.https://doi.org/10.1016/j.molmed.2020.02.004
  1. Vona R, Sposi NM, Mattia L, et al. Sickle cell disease: Role of oxidative stress and antioxidant therapy. Antioxidants 2021;10(2):296.https://doi.org/10.3390/antiox10020296
  1. Anurogo D, YuliPrasetyo Budi N, Thi Ngo MH, et al. Cell and gene therapy for anemia: Hematopoietic stem cells and gene editing. Inter J Mole Sci 2021;22(12):6275. https://doi.org/10.3390/ijms22126275
  2. Al Hajeri A, Serjeant GR, Fedorowicz Z, et al. Inhaled nitric oxide for acute chest syndrome in people with sickle cell disease. Coch Data Syst Rev 1996;2013(2).https://doi.org/10.1002/14651858.CD006957
  1. Kato GJ. Novel small molecule therapeutics for sickle cell disease: nitric oxide, carbon monoxide, nitrite, and apolipoprotein AI. ASH Edu Prog Book 2008;2008(1):186-192. https://doi.org/10.1182/asheducation-2008.1.186
  2. McCarty MF. Potential utility of full-spectrum antioxidant therapy, citrulline, and dietary nitrate in the management of sickle cell disease. Med Hypo 2010;74(6):1055-1058. https://doi.org/10.1016/j.mehy.2009.12.020
  3. Aliyu ZY, Tumblin AR, Kato GJ. Current therapy of sickle cell disease. Haem 2006 91(1):7. PMID: 16434364, PMCID: PMC2204144.
  4. Osunkwo I, Manwani D, Kanter J. Current and novel therapies for the prevention of vaso-occlusive crisis in sickle cell disease. Thera Adv Hema 2020; 11:2040620720955000.https://doi.org/10.1177/2040620720955000
  1. Mack AK, Kato GJ. Sickle cell disease and nitric oxide: A paradigm shift? Intern J Bio Cell Bio 2006;38(8):1237-1243. https://doi.org/10.1016/j.biocel.2006.01.010
  2. Aboursheid T, Albaroudi O, Alahdab F. Inhaled nitric oxide for treating pain crises in people with sickle cell disease. Coch Data Syst Rev 2022(7).https://doi.org/10.1002/14651858.CD011808.pub3  
  1. Sagi V, Mittal A, Tran H, et al. Pain in sickle cell disease: Current and potential translational therapies. Trans Res 2021;234:141-158. https://doi.org/10.1016/j.trsl.2021.03.007
  2. Umeh NI, Ajegba B, Buscetta AJ, et al. The psychosocial impact of leg ulcers in patients with sickle cell disease: I don’t want them to know my little secret. PLoS One 2017;12(10): e0186270.https://doi.org/10.1371/journal.pone.0186270
  1. Weiner DL, Hibberd PL, Betit P, et al. Preliminary assessment of inhaled nitric oxide for acute vaso-occlusive crisis in pediatric patients with sickle cell disease. Jama 2003;289(9):1136-1142. https://doi.org/10.1001/jama.289.9.1136
  2. Patel S, Patel R, Mukkala SR, et al. Emerging therapies and management approaches in sickle cell disease (SCD): A critical review. J Phyto Pharma Sci 2023;3(3):1-1. https://doi.org/10.54085/jpps.2023.3.3.3
  3. Raghupathy R, Billett HH. Promising therapies in sickle cell disease. Cardio Haem Dis-Drug Targ (Form Curr Drug Tar-Cardio Hema Dis) 2009;9(1):1-8.https://doi.org/10.2174/187152909787581354
  1. Carden MA, Little J. Emerging disease-modifying therapies for sickle cell disease. Haem 2019; 104(9):1710.https://doi.org/10.3324/haematol.2018.207357
  1. Sagi V, Mittal A, Tran H, et al. Pain in sickle cell disease: Current and potential translational therapies. Trans Res 2021;234:141-158.https://doi.org/ 10.1016/j.trsl.2021.03.007
  1. Demirci S, Uchida N, Tisdale JF. Gene therapy for sickle cell disease: An update. Cyto 2018;20(7):899-910.https://doi.org/10.1016/j.jcyt.2018.04.003
  1. Newman TV, Yan g J, Suh K, et al. Use of disease-modifying treatments in patients with sickle cell disease. JAMA Net Open 2023; 6(11): e2344546.https://doi.org/10.1001/jamanetworkopen.2023.44546
  1. El Hoss S, El Nemer W, Rees DC. Precision medicine and sickle cell disease. Hema 2022;6(9):e762.https://doi.org/10.1001/jamanetworkopen.2023.44546
  1. Thompson LM, Ceja ME, Yang SP. Stem cell transplantation for treatment of sickle cell disease: bone marrow versus cord blood transplants. Amer J Health-Sys Phar 2012; 69(15):1295-1302.https://doi.org/10.2146/ajhp110308  
  1. Colombatti R, Martella M, Cattaneo L, et al. Results of a multicenter universal newborn screening program for sickle cell disease in Italy: A call to action. Pediatric Blood Cancer 2019; 66(5): e27657.https://doi.org/ 10.1002/pbc.27657
  1. Evangelidis P, Evangelidis N, Kalmoukos P, et al. Genetic susceptibility in endothelial injury syndromes after hematopoietic cell transplantation and other cellular therapies: Climbing a steep hill. Curr Issu Mole Biol 2024;46(5):4787-4802.https://doi.org/10.3390/cimb46050288
  1. Chang JC, Matsubara D, Morgan RW. Skewed cytokine responses rather than the magnitude of the cytokine storm may drive cardiac dysfunction in multisystem inflammatory syndrome in children. J Amer Heart Asso 2021;10(16): e021428. https://doi.org/10.1161/JAHA.121.021428
  2. Minoia F, Tibaldi J, Muratore V, et al. Thrombotic microangiopathy associated with macrophage activation syndrome: A multinational study of 23 patients. J Pedi 2021; 235:196-202.https://doi.org/10.1016/j.jpeds.2021.04.004
  1. Mazzierli T, Allegretta F, Maffini E, et al. Drug-induced thrombotic microangiopathy: An updated review of causative drugs, pathophysiology, and management. Front Pharm 2023;13:1088031.https://doi.org/10.3389/fphar.2022.1088031
  1. Thompson GL, Kavanagh D. Diagnosis and treatment of thrombotic microangiopathy. Inter J Labo Hema 2022; 44:101-113. https://doi.org/10.1111/ijlh.13954