Lack of Clustering of SCD Complications Into Two Distinct Subphenotypes In An Adult Patient Population.

Abstract Abstract 1643 The complex pathophysiologic mechanisms that contribute to disease pathology in sickle cell disease (SCD) include microvascular occlusion secondary to deoxy-Hb S polymerization, interaction of sickle RBCs with vascular endothelium and other blood cells, hemolysis with resultant nitric oxide (NO) scavenging, endothelial activation with inflammation, and activation of coagulation. It has been recently hypothesized that there may be two distinct sub-phenotypes in SCD: one where hemolysis and NO depletion predominates (hemolysis/endothelial dysfunction) and the other where vaso-occlusion and increased whole blood viscosity plays a more prominent role. The clinical complications of SCD thus cluster into one of the two subphenotypes: pulmonary hypertension, priapism, leg ulcers and stroke appear to be more commonly associated with the hemolysis/NO depletion/endothelial dysfunction subphenotype, whereas frequent pain episodes, acute chest syndrome, osteonecrosis and retinopathy tend to be more common in the viscosity/vaso-occlusion group. We had previously analyzed the records of 124 patients with Hb SS or S-b° thalassemia followed at the Medical College of Georgia Adult Sickle Cell Clinic to validate the clustering of disease complications into the aforementioned two sub-phenotypes and found no significant associations between phenotypes within each sub-group, as well as between phenotypes across the groups (all p values >0.1). The following criteria were used to define complications: We have now extended our analyses to 203 patients with Hb SS or S-b° thalassemia (ages 12–60; 101 males, 102 females). We performed principal component analysis on the data from 203 patients. A total of 10 sub-phenotype variables were used in the analysis. The variables are VOE, ACS, retinopathy, gallstones, AVN, stroke, nephropathy, pulmonary hypertension, leg ulcers, and priapism. The individual factor map based on the first 2 principal components is shown below. Each dot represents a patient in the figure. If the hypothesis of two groups of sub-phenotypes were correct, we would see two clusters of patients. However, from this figure, there is no clear clustering of patients. We also plotted the sub-phenotype variables factor map based on the results from the principal components analysis. As shown in the following figure, the sub-phenotypes are not clustered into two groups as predicted by the hypothesis. In particular, we have retinopathy in one extreme and gallstones in the other. They are not clustered in one group. Our results indicate that the complications of sickle cell disease do not cluster into two distinct subphenotypes as previously hypothesized. While this hypothesis may provide a useful conceptual framework in deconstructing and understanding various pathophysiologic mechanisms operative in sickle cell disease, such clear cut distinction is not applicable in clinical practice. Disclosures: No relevant conflicts of interest to declare.

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The Arginine-to-Ornithine Ratio: Biomarker of Arginase Activity and Predictor of Mortality in Sickle Cell Disease.

Blood ◽

10.1182/blood.v104.11.237.237 ◽

2004 ◽

Vol 104 (11) ◽

pp. 237-237 ◽

Cited By ~ 2

Author(s):

Claudia R. Morris ◽

Gregory Kato ◽

Mirjana Poljakovic ◽

William C. Blackwelder ◽

Stan Hazen ◽

...

Keyword(s):

Nitric Oxide ◽

Pulmonary Hypertension ◽

Endothelial Dysfunction ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Risk Ratio ◽

Arginase Activity ◽

Nitric Oxide Production ◽

Cell Disease ◽

Arginine Metabolism

Abstract Sickle cell disease (SCD) is characterized by a state of nitric oxide resistance and limited bioavailability of L-arginine, the substrate for nitric oxide synthesis. While nitric oxide resistance occurs secondary to inactivation of nitric oxide by plasma hemoglobin released during intravascular hemolysis and by reactive oxygen species, mechanisms that limit L-arginine are not known. We hypothesized that increased arginase activity in patients with SCD would shift arginine metabolism away from nitric oxide production and towards ornithine metabolism, contributing to endothelial dysfunction and the development of pulmonary hypertension. Furthermore, since arginine and ornithine compete for the same transport system for cellular uptake, a decrease in the Arginine-to-Ornithine ratio resulting from increased arginase activity could also impair arginine bioavailability for nitric oxide production. Our goal was to evaluate associations between plasma arginase, arginine metabolism and pulmonary hypertension and prospective mortality in SCD. Plasma and erythrocyte arginase activity and amino acid levels were determined for patients with SCD and compared to ethnically matched control subjects. A diagnosis of pulmonary hypertension by Doppler-echocardiogram and prospective mortality were determined over 30 months of sequential patient enrollment. Plasma arginase activity was significantly elevated in patients with SCD compared to controls (2.2±2, n=140 vs. 0.4±2 μmol/ml/hr, n=45, p=0.007), trending higher in subjects with pulmonary hypertension. Plasma arginase activity correlated with the Arginine-to-Ornithine ratio (r=−0.33, p=0.0004), and lower ratios were associated with greater severity of pulmonary hypertension (1.1±0.4 vs. 0.8±0.4 vs. 0.6±0.3, controls vs. SCD without pulmonary hypertension vs. SCD with pulmonary hypertension, respectively, p=0.01) and independently associated with mortality (0.7±0.4 vs. 0.5±0.2, alive vs. dead, p=0.003; Risk Ratio = 4.9 [CI: 1.4, 17.1], p=0.002, for a low Arginine-to-Ornithine ratio; 13 deaths total). The mortality risk ratio increased to 7.0 ([CI: 1.6, 31.6], p=0.01), when the Arginine-to-Ornithine ratio was adjusted for creatinine, likely reflecting the impact of renal disease. Plasma arginase activity correlated with markers of increased hemolytic rate, including LDH (r=0.44, p<0.001), AST (r=0.39, p<0.002), reticulocyte count (r=0.25, p<0.001), and Hct (r= −0.25, p<0.001), and was higher in erythrocytes of SCD patients compared to controls (37.7±2.9, n=16 vs 23.5±1.7 nmol/mg/min, n=45, p<0.0001), consistent with hemolytic release of erythrocyte arginase. These data support a novel mechanism of disease whereby hemolysis not only liberates vasoactive hemoglobin but also releases erythrocyte arginase, which contributes to impaired nitric oxide bioavailability, endothelial dysfunction, pulmonary hypertension and death. The Arginine-to-Ornithine ratio, a reflection of arginase activity, may represent a useful biomarker of disease severity and risk of death in patients with SCD.

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Hemolysis in Sickle Cell Mice Causes Pulmonary Hypertension Due to Global Impairment in Nitric Oxide Bioavailability.

Blood ◽

10.1182/blood.v108.11.785.785 ◽

2006 ◽

Vol 108 (11) ◽

pp. 785-785

Author(s):

Lewis L. Hsu ◽

Hunter C. Champion ◽

Sally A. Campbell-Lee ◽

Trinity J. Bivalacqua ◽

Elizabeth A. Manci ◽

...

Keyword(s):

Nitric Oxide ◽

Animal Model ◽

Pulmonary Hypertension ◽

Endothelial Dysfunction ◽

Sickle Cell Disease ◽

Sickle Cell ◽

No Production ◽

Cell Disease ◽

Sickle Hemoglobin ◽

Pulmonary Vasodilators

Abstract Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiological mechanisms by which sickle hemoglobin leads to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model of severe sickle cell disease. Using micro-cardiac catheterization we found that all mice expressing exclusively human sickle hemoglobin develop pulmonary hypertension. Recognizing that the NO pathways can have complex abnormalities in other conditions of pulmonary hypertension, the NO axis in sickle mice was assessed by multiple methods. From a mechanistic standpoint the mice exhibit profound pulmonary and systemic endothelial dysfunction and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors and endothelium-dependent pulmonary vasodilators, and enhanced responses to vaso-constrictors. However, endothelium-independent vasodilation in the sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO-axis: impaired constitutive nitric oxide synthase activity with loss of eNOS coupling (dimerization), increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Consistent with a functional rather than structural defect, light microscopy and computed tomography of the lungs revealed no plexogenic arterial remodeling, thrombi/emboli, or inflammation. Transplanting sickle marrow into wild-type mice conferred the same phenotype. Similar pathobiology was observed in a non-sickle mouse model of acute alloimmune hemolysis, supporting a major role of hemolysis as a mechanism for this dysregulation of NO and vasculopathy. In this study, alloimmune hemolytic mice were chosen for comparison in order to generalize beyond hemoglobinopathies. Future analogous studies with thalassemic mice may be useful to model pulmonary hypertension in human thalassemia intermedia. In conclusion, this animal model extends the evidence for global impairment in NO responsiveness and NO production in sickle cell disease, and suggests that hemolytic anemia is associated with endothelial dysfunction and pulmonary hypertension.

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Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability

Blood ◽

10.1182/blood-2006-08-039438 ◽

2006 ◽

Vol 109 (7) ◽

pp. 3088-3098 ◽

Cited By ~ 140

Author(s):

Lewis L. Hsu ◽

Hunter C. Champion ◽

Sally A. Campbell-Lee ◽

Trinity J. Bivalacqua ◽

Elizabeth A. Manci ◽

...

Keyword(s):

Nitric Oxide ◽

Pulmonary Hypertension ◽

Endothelial Dysfunction ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Time Course ◽

Cell Disease ◽

Plasma Hemoglobin ◽

Constitutive Nitric Oxide Synthase ◽

Oxide Synthase

Abstract Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiologic mechanisms leading to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model. By microcardiac catheterization, all mice expressing exclusively human sickle hemoglobin had pulmonary hypertension, profound pulmonary and systemic endothelial dysfunction, and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors, and endothelium-dependent vasodilators and enhanced responses to vasoconstrictors. However, endothelium-independent vasodilation in sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO axis: impaired constitutive nitric oxide synthase activity (NOS) with loss of endothelial NOS (eNOS) dimerization, increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Light microscopy and computed tomography revealed no plexogenic arterial remodeling or thrombi/emboli. Transplanting sickle marrow into wild-type mice conferred the same phenotype, and similar pathobiology was observed in a nonsickle mouse model of acute alloimmune hemolysis. Although the time course is shorter than typical pulmonary hypertension in human sickle cell disease, these results demonstrate that hemolytic anemia is sufficient to produce endothelial dysfunction and global dysregulation of NO.

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Detection of the mRNA Transcription Level of Several Genes of the HIF and NO Metabolic Pathways in PBMCs of Sickle Cell Disease Patients Using Quantitative RT-PCR Assay.

Blood ◽

10.1182/blood.v110.11.844.844 ◽

2007 ◽

Vol 110 (11) ◽

pp. 844-844

Author(s):

Tatyana Ammosova ◽

Min Xu ◽

Sergei Nekhai ◽

Gregory J. Kato ◽

Mark T. Gladwin ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Primary Pulmonary Hypertension ◽

Soluble Guanylate Cyclase ◽

Large Subunit ◽

Housekeeping Gene ◽

The Other ◽

Cell Disease ◽

Rt Pcr

Abstract Background. Pulmonary hypertension is a common complication of sickle cell disease but the pathophysiology is not completely understood. Methods. Using quantitative RT-PCR we studied mRNA from PBMCs of seven patients with sickle cell disease having pulmonary hypertension, five patients without pulmonary hypertension and 10 controls with neither sickle cell disease nor pulmonary hypertension. We investigated transcripts of several HIF regulated genes including endothelin-1 (ET1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS), several genes of the NO metabolic pathway including cGMP-specific phosphodiesterase 5A (PDE5), soluble guanylate cyclase large subunit (sGC), and arginase 2 (Arg2), and a gene implicated in primary pulmonary hypertension, bone morphogenetic protein receptor type II (BMPR2). β-actin and diacylglycerol kinase alpha (DGKA) were used as housekeeping gene controls. Results. HIF-regulated genes: Levels of ET1 transcripts were higher in patients with sickle cell disease and pulmonary hypertension than the other two groups while VEGF transcripts were higher in both sickle cell disease groups than controls and levels of iNOS transcripts were low and did not differ among the groups. Upregulation of ET1 is associated with the development of hypoxic pulmonary hypertension in both experimental animals and humans. NO pathway genes: PDE5, sGC, Arg2 transcripts were higher in patients with sickle cell disease and PH than the other two groups. Upregulation of PDE5 and Arg2 might be expected to contribute to reduced NO signaling. Primary pulmonary hypertension gene: BMPR2 transcripts were higher in patients with sickle cell disease and PH than the other two groups. Discussion. These results are consistent with the possibility that complex molecular pathways, including dysregulation of HIF and NO pathways, may contribute to the development of pulmonary hypertension in sickle cell disease.

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Treatment dilemmas: strategies for priapism, chronic leg ulcer disease, and pulmonary hypertension in sickle cell disease

Hematology ◽

10.1182/hematology.2021000275 ◽

2021 ◽

Vol 2021 (1) ◽

pp. 411-417

Author(s):

Roberta C.G. Azbell ◽

Payal Chandarana Desai

Keyword(s):

Pulmonary Hypertension ◽

Sickle Cell Disease ◽

Hemolytic Anemia ◽

Sickle Cell ◽

Acute Chest Syndrome ◽

Leg Ulcers ◽

Cell Disease ◽

Ulcer Disease ◽

Multiorgan Disease ◽

Chronic Hemolytic Anemia

Abstract Sickle cell disease is a disorder characterized by chronic hemolytic anemia and multiorgan disease complications. Although vaso-occlusive episodes, acute chest syndrome, and neurovascular disease frequently result in complication and have well-documented guidelines for management, the management of chronic hemolytic and vascular-related complications, such as priapism, leg ulcers, and pulmonary hypertension, is not as well recognized despite their increasing reported prevalence and association with morbidity and mortality. This chapter therefore reviews the current updates on diagnosis and management of priapism, leg ulcers, and pulmonary hypertension.

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PPARγ increases HUWE1 to attenuate NF-κB/p65 and sickle cell disease with pulmonary hypertension

Blood Advances ◽

10.1182/bloodadvances.2020002754 ◽

2021 ◽

Vol 5 (2) ◽

pp. 399-413

Author(s):

Andrew J. Jang ◽

Sarah S. Chang ◽

Changwon Park ◽

Choon-Myung Lee ◽

Raymond L. Benza ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Endothelial Dysfunction ◽

Sickle Cell Disease ◽

Signaling Pathways ◽

Sickle Cell ◽

Peroxisome Proliferator ◽

Cell Disease ◽

Littermate Control

Abstract Sickle cell disease (SCD)-associated pulmonary hypertension (PH) causes significant morbidity and mortality. Here, we defined the role of endothelial specific peroxisome proliferator-activated receptor γ (PPARγ) function and novel PPARγ/HUWE1/miR-98 signaling pathways in the pathogenesis of SCD-PH. PH and right ventricular hypertrophy (RVH) were increased in chimeric Townes humanized sickle cell (SS) mice with endothelial-targeted PPARγ knockout (SSePPARγKO) compared with chimeric littermate control (SSLitCon). Lung levels of PPARγ, HUWE1, and miR-98 were reduced in SSePPARγKO mice compared with SSLitCon mice, whereas SSePPARγKO lungs were characterized by increased levels of p65, ET-1, and VCAM1. Collectively, these findings indicate that loss of endothelial PPARγ is sufficient to increase ET-1 and VCAM1 that contribute to endothelial dysfunction and SCD-PH pathogenesis. Levels of HUWE1 and miR-98 were decreased, and p65 levels were increased in the lungs of SS mice in vivo and in hemin-treated human pulmonary artery endothelial cells (HPAECs) in vitro. Although silencing of p65 does not regulate HUWE1 levels, the loss of HUWE1 increased p65 levels in HPAECs. Overexpression of PPARγ attenuated hemin-induced reductions of HUWE1 and miR-98 and increases in p65 and endothelial dysfunction. Similarly, PPARγ activation attenuated baseline PH and RVH and increased HUWE1 and miR-98 in SS lungs. In vitro, hemin treatment reduced PPARγ, HUWE1, and miR-98 levels and increased p65 expression, HPAEC monocyte adhesion, and proliferation. These derangements were attenuated by pharmacological PPARγ activation. Targeting these signaling pathways can favorably modulate a spectrum of pathobiological responses in SCD-PH pathogenesis, highlighting novel therapeutic targets in SCD pulmonary vascular dysfunction and PH.

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