Hemolysis-associated Pulmonary Hypertension Research Articles

Experimental intravascular hemolysis induces hemodynamic and pathological pulmonary hypertension: association with accelerated purine metabolism.

Pulmonary hypertension (PH) is emerging as a serious complication associated with hemolytic disorders, and plexiform lesions (PXL) have been reported in patients with sickle cell disease (SCD). We hypothesized that repetitive hemolysis per se induces PH and angioproliferative vasculopathy and evaluated a new mechanism for hemolysis-associated PH (HA-PH) that involves the release of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) from erythrocytes. In healthy rats, repetitive administration of hemolyzed autologous blood (HAB) for 10 days produced reversible pulmonary parenchymal injury and vascular remodeling and PH. Moreover, the combination of a single dose of Sugen-5416 (SU, 200 mg/kg) and 10-day HAB treatment resulted in severe and progressive obliterative PH and formation of PXL (Day 26, right ventricular peak systolic pressure (mmHg): 26.1 ± 1.1, 41.5 ± 0.5 and 85.1 ± 5.9 in untreated, HAB treated and SU+HAB treated rats, respectively). In rats, repetitive administration of HAB increased plasma ADA activity and reduced urinary adenosine levels. Similarly, SCD patients had higher plasma ADA and PNP activity and accelerated adenosine, inosine, and guanosine metabolism than healthy controls. Our study provides evidence that hemolysis per se leads to the development of angioproliferative PH. We also report the development of a rat model of HA-PH that closely mimics pulmonary vasculopathy seen in patients with HA-PH. Finally, this study suggests that in hemolytic diseases released ADA and PNP may increase the risk of PH, likely by abolishing the vasoprotective effects of adenosine, inosine and guanosine. Further characterization of this new rat model of hemolysis-induced angioproliferative PH and additional studies of the role of purines metabolism in HA-PH are warranted.

Glutathione and arginine levels: Predictors for acetaminophen-associated asthma exacerbation?

Glutathione and arginine levels: Predictors for acetaminophen-associated asthma exacerbation?

NT-Pro Brain Natriuretic Peptide Levels and the Risk of Stroke and Death in the Cooperative Study of Sickle Cell Disease.

Abstract 1541Poster Board I-564 BackgroundElevations in NT-proBNP levels are associated with hemolysis-associated pulmonary hypertension and mortality in adults with sickle cell disease. The association of this vasculopathy with the risk of stroke has not been explored. The Cooperative Study of Sickle Cell Disease (CSSCD) is the largest cohort of adult and pediatric patients with sickle cell disease with the longest median follow-up. Using stored plasma samples from patients enrolled in the CSSCD, we tested the hypothesis that adult patients with high levels of NT-proBNP would be at a high risk of death and stroke and that pediatric patients with a high BNP might be at a high risk of stroke. MethodsA threshold NT-pro-BNP value previously identified to predict mortality in adults with sickle cell disease was used to determine the association between the risk of stroke and mortality in a cohort of 758 participants (pediatric cohort, n=428 and adult cohort, n=330) in the CSSCD. ResultsThe prevalence of an abnormal NT-proBNP level ≥ 160 pg/ml was 27.6 % in patients in the adult CSSCD cohort. The prevalence of a NT-proBNP level ≥ 160 pg/ml was 27.4 % in the pediatric cohort, which is at least in part explained by known higher normal NT-proBNP levels in children, especially during the first year of life. The incidence of the development of a high NT-proBNP level in subjects with a normal baseline level was 6 % over a mean follow-up time of 5.9 years (incidence rate per 100-person years of 1.03) in the pediatric cohort and 16.5 % over a mean follow-up time of 1.9 years in the adult cohort (incidence rate per 100-person years of 8.59). In subjects with normal baseline levels, multivariate logistic regression analysis of clinical and laboratory factors associated with the development of a high NT-proBNP level included increasing age (OR 3.43, 95% CI 1.6-7.2, P = 0.001), low hemoglobin (OR 0.47, 95% CI 0.3-0.7, P < 0.001), high white blood cell count (OR 1.69, 95% CI 1.05-2.7, P = 0.03), high creatinine (OR 2.22, 95% CI 1.1-4.3, P = 0.02), blood urea nitrogen (OR 1.64, 95% CI 1.2-2.3, P = 0.004), alanine aminotransferase (OR 1.26, 95% CI 1.01-1.6, P = 0.04) and uric acid levels (OR 2.32, 95% CI 1.4-3.8, P = 0.001), and history of leg ulcers (OR 7.46, 95% CI 2.0-28.5, P = 0.003). Elevated NT pro-BNP levels were associated with indices of hemolytic anemia (hemoglobin: OR 0.33, 95% CI 0.2-0.4, P < 0.001) and a history of stroke (OR 1.86, 95% CI 0.9-3.7, P = 0.02). An NT-pro-BNP level ≥160 pg/ml was a predictor of mortality (RR 6.24, 95% CI 2.9-13.3, P < 0.001) and stroke (RR 3.84, 95 % CI 1.0-14.3, P = 0.05) in this cohort. ConclusionsA high NT-proBNP level is a major risk factor for death in patients with sickle cell disease. These findings provide further support for a mechanistic link between hemolytic anemia and the development of cardiovascular complications in this patient population. Finally, we have identified a novel widely available biomarker of the risk of death and stroke in sickle cell disease. DisclosuresNo relevant conflicts of interest to declare.

Free Arginase-I in Human Pulmonary Microvascular Endothelial Cell Culture Leads to Endothelial Dysfunction and Increased Reactive Oxygen Species.

Abstract 3041Poster Board II-1017 IntroductionEndothelial dysfunction in chronic hemolytic diseases (e.g., sickle cell disease) is believed to lead to hemolysis-associated pulmonary hypertension. Dysregulated arginine metabolism, via free arginase-I released during hemolysis, is one mechanism proposed in prior studies. However, the effects of free arginase-I on endothelial cell function have not previously been reported. We hypothesized that free arginase-I would decrease hPMVEC nitric oxide (NO) production and therefore limit NO bioavailability. MethodsCultured human pulmonary microvascular endothelial cells (hPMVEC), at passage 6-8 and approximately 80% confluence, with increasing arginase-I concentrations added to the media (CN: 0 ng prot/mL, A30: 30 ng prot/mL, A90: 90 ng prot/mL) were studied. To maximize arginase-I activity, 0.1 mM MnCl2 (an important cofactor for arginase-I) and 3mM of L-arginine were added to each condition. The hPMVEC were incubated for 24h in normoxia (n=8) or hypoxia (1% O2; n=4), then media was harvested for urea/nitrite analyses and cell protein was harvested for Western blot analyses. ANOVA analyses were conducted to determine statistical significance. ResultsUrea production significantly increased with increasing media arginase-I in both normoxia (CN: 1.47±0.22, A30: 1.46±0.15, A90: 2.09±0.20 umol/mgPr; p<0.05) and hypoxia (CN: 0.913±0.22, A30: 1.01±0.04, A90: 1.99±0.14 umol/mgPr; p<0.005); interestingly, hypoxia means were significantly lower than those in normoxia (p<0.01). However, the percent change from controls in hypoxia (CN: 100±24, A30: 110±4, A90: 218±15) was significantly higher (p<0.05) than in normoxia conditions (CN: 100±15, A30: 99±10, A90: 142±13). Nitrite production showed no significant change with increasing media arginase-I in normoxia (CN: 17.6±2.4, A30: 20.8±4.6, A90: 18.8±2.1 nmol/mgPr; p=0.79); however, in hypoxia nitrites trended down with increasing media arginase-I (CN: 5.26±0.42, A30: 4.35±0.27, A90: 3.69±0.51 nmol/mgPr; p=0.068). In addition, means for nitrite production in hypoxia were significantly less than in normoxia conditions (p<0.0001). In normoxia, Western blot analyses showed significantly decreased 3-nitrotyrosine (3-NT) levels with increasing media arginase-I (CN: 1.00±0.03, A30: 0.88±0.07, A90: 0.77±0.07 [fold change]; p<0.05). 3-NT expression in hypoxia showed no differences with increasing media arginase-I (CN: 1.90±0.16, A30: 1.88±0.19, A90: 1.75±0.27 [fold change, normalized to normoxia control]; p=0.87); however, hypoxic means were significantly higher than in normoxia conditions (p<0.0001). In normoxia, NAD(P)H oxidase isoform 4 (Nox4) show a decreasing trend in protein levels with increasing media arginase-I (CN: 1.00±0.03, A30: 0.93±0.10, A90: 0.86±0.06 [fold change]), with an increase in Nox4 protein levels in hypoxia with increasing media arginase-I (CN: 1.18±0.02, A30: 1.33±0.08, A90: 1.47±0.01 [fold change, normalized to normoxia control]). The hypoxic levels of Nox4 were also higher than in normoxia conditions. ConclusionsAs expected, increasing free media arginase-I concentrations in hPMVEC culture resulted in significant increases in urea production in both normoxia and hypoxia. Hypoxia resulted in lower nitrite production by endothelial nitric oxide synthase (eNOS). However, increasing amounts of free arginase-I only affected nitrite production in hypoxia, suggesting that increased arginase-I serum concentrations may also become an important regulator of NO production in clinical cases of hypoxia (e.g., acute chest syndrome and vaso-occlusive crises in sickle cell patients). The concurrent increased expression of 3-NT in hypoxia, despite lower nitrites, implies an increase in reactive oxygen species (ROS). Our preliminary data for Nox4 expression, the predominant source of ROS in endothelial cells, indicates that ROS production via this pathway may increase in hypoxia with increasing free arginase-I. Further studies on how free arginase-I in the media affects endothelial function are warranted, particularly in regards to the mechanism by which free arginase-I leads to greater Nox4 expression and ROS production. We speculate that arginase-I inhibitors may represent a potential therapeutic target in patients with chronic hemolysis and acute hypoxic events. DisclosuresNo relevant conflicts of interest to declare.

Other Causes of PAH (Schistosomiasis, Porto-Pulmonary Hypertension and Hemolysis-Associated Pulmonary Hypertension)

Recent years have witnessed a significant increase in the knowledge about the pathophysiology of pulmonary arterial hypertension (PAH) and the availability of multiple drugs specifically aimed at pulmonary circulation. Although most of this is related to the idiopathic form of PAH, this development has also turned attention to other forms of pulmonary hypertension such as schistosomiasis-associated PAH (Sch-PAH), portopulmonary hypertension (POPH), and hemolysis-associated pulmonary hypertension. The importance of these different forms of pulmonary hypertension spans their epidemiology and the multiple pathophysiological mechanisms associated with their development and complications. Taken together, Sch-PAH and hemolysis-associated pulmonary hypertension may represent the most prevalent forms of PAH worldwide. Portopulmonary hypertension is particularly important if the morbidity and mortality that it adds to patients with liver disease is considered. Although clear progress has been reached in these various forms of PAH, there are many different aspects yet to be addressed that may contribute to the basis of specific treatment applied to these subgroups of patients.

Adenosine deaminase–adenosine pathway in hemolysis-associated pulmonary hypertension

Hemolysis-associated pulmonary hypertension (HA-PH) is a serious clinical complication of various hemolytic disorders, and pulmonary hypertension (PH) is considered the greatest risk factor for death in patients with a hemolytic disorder. It is now well established that hemolysis causes the release of soluble hemoglobin and arginase from injured erythrocytes into plasma. This leads to nitric oxide (NO) deficiency, oxidative stress and a state of endothelial dysfunction that is associated with clinical development of PH. We challenge this concept and propose that in addition to the NO-arginase pathway, the adenosine deaminase-adenosine pathway plays a significant role in HA-PH and that modulation of this pathway may offer protective/therapeutic effects in HA-PH. Our preliminary data suggest that in HA-PH adenosine deaminase (ADA) is released from injured erythrocytes into plasma and that metabolic conversion of adenosine (ADO) to inosine by ADA reduces extracellular ADO levels. Adenosine, mainly via activation of adenosine A(2A) receptors, mediates a number of biological responses that may reduce hemolysis-induced vasculopathy and the risk of PH. Hypoxia is the strongest stimulus for ADO synthesis, and this increased ADO production counteracts some of the tissue/vascular injury caused by hypoxia itself. Unfortunately, under hypoxic conditions (anemia, vasoconstriction, and vaso-occlusion) in HA-PH, this "ADO negative-feed back" is abolished and the vascular protective effects of ADO are severely diminished by ADA released from injured erythrocytes. We hypothesize that in hemolytic anemia the repetitive release of ADA increases the risk for vaso-occlusive events and PH. We also propose that increase in extracellular ADO levels or activation of adenosine A(2A) receptors attenuates HA-PH, and we suggest further preclinical and clinical investigation of ADA inhibitors and ADO agonists in HA-PH.

Pulmonary Hypertension as a Risk Factor for Death in Patients with Sickle Cell Anemia

Pulmonary hypertension develops in most forms of hereditary and chronic hemolytic anemia, including sickle cell disease, thalassemia, hereditary spherocytosis, and paroxysmal nocturnal hemoglobinuria, suggesting that there is a clinical syndrome of hemolysis-associated pulmonary hypertension. Retrospective studies from tertiary care referral centers suggest a prevalence of pulmonary hypertension in adults with sickle cell disease ranging from 20 to 40%. Despite the fact the elevations in pulmonary artery pressures are slight, morbidity and mortality are high. In adult sickle cell anemia patients, pulmonary hypertension is emerging as a major risk factor for death. We performed Doppler echocardiographic assessments of pulmonary-artery systolic pressure in 80 consecutive patients (20 men and 60 women; mean [±SD] age, 30 ± 10.8 years) between 1/20/2006 and 1/20/2008. The genotype on the basis of hematologic and hemoglobin characteristics was hemoglobin SS in all patients. Pulmonary hypertension was prospectively defined as a tricuspid regurgitant Jet velocity (TFJV) of at least 2.5 m per second. Patients were followed for a mean of 18 months (6–24 months), and data were censored at the time of death or loss to follow-up. Doppler-defined pulmonary hypertension occurred in 37.5 percent of patients (30/80). Multiple logistic-regression analysis, with the use of the dichotomous variable of a tricuspid regurgitant jet velocity of less than 2.5 m per second or 2.5 m per second or more, identified age, female sex, deferasirox therapy, left ventricular mass index, pulmonary artery systolic pressure, reticulocytes, white-cell count, platelet count, lactate dehydrogenase (a marker of hemolysis), blood urea nitrogen, creatinine, uric acid and self-reported history of cardiovascular complication, billiary stones, retinopathy and acute chest syndrome, as significant independent correlates of pulmonary hypertension. The hemoglobin level, fetal hemoglobin level, hydroxyurea therapy and serum ferritin level were unrelated to pulmonary hypertension. Hazard rate for death according to the TFJV of at least 2.5 m per second, as compared with a velocity of less than 2.5 m per second, was associated with an increased risk of death (0.00 versus 2.54; P=0.998). Mortality rate in 24 months was 6.7% (2/30) for patients with TRJ velocity ≥ 2.5 m/sec versus 0.0% (0/50) for patients without pulmonary hypertension. Pulmonary hypertension, diagnosed by Doppler echocardiography, is common in adults with sickle cell disease. It appears to be a complication of chronic hemolysis, is resistant to hydroxyurea therapy, and confers a high risk of death. Large trials evaluating the effects of treatment for pulmonary hypertension in the sickle cell anemia population are indicated.

Abstract 3574: A Unique Model and Mechanisms of Hemolysis-Associated Pulmonary Hypertension

Pulmonary hypertension is a common complication of intravascular hemolysis and is suspected to be a strong mortality determinant of hemolytic disorders. However, the pathophysiologic mechanisms leading to hemolytic anemia-associated pulmonary hypertension remains unclear. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses only human cells due to its unique receptor specificity for human CD59 (hCD59). We recently generated a novel intravascular hemolysis mouse model in which ILY exclusively lyses erythrocytes transgenically expressing hCD59 ( ThCD59RBC mice) ( Nature Medicine, 2008, 14: 98 –103 ). Using this model, we investigated the pathogenesis of hemolytic anemia-associated pulmonary hypertension. In ThCD59RBC mice, but not wild type mice, intravenous injection of ILY with a 1.5-fold lethal dose induced rapid intravascular hemolysis with acute death (less than 1 minute) that was preceded by a sharp increase in the pulmonary pressure (10.40 ± 0. 89 mmHg increase, n = 5) with 0.65 ± 0.36 mmHg/second rate of rise of systolic pulmonary artery pressure as measured by right-heart catheterization. We also observed decreased NO levels indirectly measured by plasma nitrate + nitrite concentration [10.01 ± 2.47μM in ThCD59RBC (n=3) vs 22.16 ± 2.20 μM in WT (n=3); P &lt;0.001] and increased plasma P-selectin levels [445.65 ± 54.85 ng/ml in ThCD59RBC (n=4) vs 186.95 ± 18.64 ng/ml in WT (n=4); P &lt;0.001]. These results suggest that the lethality of acute intravascular hemolysis may result from a sudden increase in pulmonary pressure likely a consequence of the reduced bioavailability of NO and resulting pulmonary vasoconstriction and platelet activation. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Platelet activation in patients with sickle disease, hemolysis-associated pulmonary hypertension, and nitric oxide scavenging by cell-free hemoglobin

AbstractIncreased platelet activation is recognized in patients with sickle cell disease (SCD), but its pathogenesis and clinical relevance remain uncertain. Pulmonary arterial hypertension (PAH), an important complication of SCD, is characterized by a proliferative pulmonary vasculopathy, in situ thrombosis, and vascular dysfunction related to scavenging of nitric oxide (NO) by hemoglobin released into blood plasma during intravascular hemolysis. We investigated links between platelet activation, PAH and NO scavenging in patients with SCD. Platelet activation marked by activated fibrinogen receptor correlated to the severity of PAH (r = 0.58, P < .001) and to laboratory markers of intravascular hemolysis, such as reticulocyte count (r = 0.44, P = .02). In vitro exposure of platelets to pathologically relevant concentrations of cell-free hemoglobin promoted basal- and agonist-stimulated activation and blocked the inhibitory effects on platelet activation by an NO donor. In patients with SCD, administration of sildenafil, a phosphodiesterase-5 inhibitor that potentiates NO-dependent signaling, reduced platelet activation (P = .01). These findings suggest a possible interaction between hemolysis, decreased NO bioavailability, and pathologic platelet activation that might contribute to thrombosis and pulmonary hypertension in SCD, and potentially other disorders of intravascular hemolysis. This supports a role for NO-based therapeutics for SCD vasculopathy. This trial was registered at www.clinicaltrials.gov as no. NCT00352430.

Hemolysis-Associated Pulmonary Hypertension in Sickle Cell Disease: Global Disruption of the Arginine-Nitric Oxide Pathway

Hemolysis-Associated Pulmonary Hypertension in Sickle Cell Disease: Global Disruption of the Arginine-Nitric Oxide Pathway

Pulmonary Hypertension in Thalassemia: Association with Hemolysis, Arginine Metabolism Dysregulation, and a Hypercoagulable State

Pulmonary Hypertension in Thalassemia: Association with Hemolysis, Arginine Metabolism Dysregulation, and a Hypercoagulable State

Dysregulated Arginine Metabolism, Hemolysis-Associated Pulmonary Hypertension, and Mortality in Sickle Cell Disease

Dysregulated Arginine Metabolism, Hemolysis-Associated Pulmonary Hypertension, and Mortality in Sickle Cell Disease

Hemolysis‐Associated Pulmonary Hypertension in Thalassemia

Accumulating evidence supports the existence of a condition involving hemolysis-associated pulmonary hypertension (PHT). Hemolysis-induced release of cell-free hemoglobin and red blood cell arginase, resulting in impaired nitric oxide bioavailability, endothelial dysfunction, and PHT, has been reported in sickle cell disease. Since thalassemia is also a condition of chronic hemolysis, these patients are at risk. The data demonstrate that hemolysis-induced dysregulation of arginine metabolism and PHT also occurs in thalassemia. Erythrocyte release of arginase during hemolysis contributes to the development of PHT. Therapies that maximize arginine and nitric oxide bioavailability may benefit patients with thalassemia.

Dysregulated Arginine Metabolism, Hemolysis-Associated Pulmonary Hypertension, and Mortality in Sickle Cell Disease

Sickle cell disease is characterized by a state of nitric oxide resistance and limited bioavailability of l-arginine, the substrate for nitric oxide synthesis. We hypothesized that increased arginase activity and dysregulated arginine metabolism contribute to endothelial dysfunction, pulmonary hypertension, and patient outcomes. To explore the role of arginase in sickle cell disease pathogenesis, pulmonary hypertension, and mortality. Plasma amino acid levels, plasma and erythrocyte arginase activities, and pulmonary hypertension status as measured by Doppler echocardiogram were prospectively obtained in outpatients with sickle cell disease. Patients were followed up for survival up to 49 months. Urban tertiary care center and community clinics in the United States between February 2001 and March 2005. Two hundred twenty-eight patients with sickle cell disease, aged 18 to 74 years, and 36 control participants. Plasma amino acid levels, plasma and erythrocyte arginase activities, diagnosis of pulmonary hypertension, and mortality. Plasma arginase activity was significantly elevated in patients with sickle cell disease, with highest activity found in patients with secondary pulmonary hypertension. Arginase activity correlated with the arginine-ornithine ratio, and lower ratios were associated with greater severity of pulmonary hypertension and with mortality in this population (risk ratio, 2.5; 95% confidence interval [CI], 1.2-5.2; P = .006). Global arginine bioavailability, characterized by the ratio of arginine to ornithine plus citrulline, was also strongly associated with mortality (risk ratio, 3.6; 95% CI, 1.5-8.3; P<.001). Increased plasma arginase activity was correlated with increased intravascular hemolytic rate and, to a lesser extent, with markers of inflammation and soluble adhesion molecule levels. These data support a novel mechanism of disease in which hemolysis contributes to reduced nitric oxide bioavailability and endothelial dysfunction via release of erythrocyte arginase, which limits arginine bioavailability, and release of erythrocyte hemoglobin, which scavenges nitric oxide. The ratios of arginine to ornithine and arginine to ornithine plus citrulline are independently associated with pulmonary hypertension and increased mortality in patients with sickle cell disease.