Association of asymmetric dimethylarginine with sickle cell disease-related pulmonary hypertension

Pulmonary hypertension (PHT) occurs in approximately 30% of adult sickle cell patients and is associated with a high risk of early death. Hemolysis driven reductions in nitric oxide (NO) bioavailability resulting from NO scavenging by cell free hemoglobin and increased arginase activity are of importance in the pathophysiology of SCD related PHT.1

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) contribute to limiting NO bioavailability in SCD.2 ADMA and symmetric dimethylarginine (SDMA) derive from the irreversible post-translational methylation of arginine residues by protein arginine methyltransferases (PRMT) and are released as free amino acids upon proteolysis. ADMA (but not SDMA) competitively inhibits NO synthase (NOS) enzymes, thereby limiting NO production. ADMA is degraded by dimethylarginine dimethylaminohydrolases (DDAH) whereas SDMA is mainly cleared renally.3 Elevated plasma ADMA concentrations occur in several forms of PHT and are associated to PHT outcome.4,5 We investigated whether ADMA concentrations are associated with PHT in SCD.

Serum and EDTA plasma samples were available from adult sickle cell patients consecutively screened for PHT with echocardiography as previously reported.6 Mild and moderate-severe PHT are defined as tricuspid regurgitant jet flow velocity (TRV) of 2.5–2.9 m/s and TRV≥3 m/s respectively, with pulmonary-artery pressures considered normal in patients with trace or no tricuspid regurgitation (with TRV assigned 1.3 m/s).1 Plasma concentrations of ADMA, SDMA, amino acids and serum soluble vascular cell adhesion molecule-1 (sVCAM-1) levels were determined as previously described.2,7 For analysis, HbSS and HbSβ-thalassemia patients were grouped together, as were HbSβ-thalassemia and HbSC patients. p-values <0.05 were considered statistically significant (SPSS 12.0.2, SPSS , Chicago, IL, USA). The study was carried out in accordance with the principles of the Declaration of Helsinki.

Table 1.Demographics and laboratory parameters in sickle cell patients with and without pulmonary hypertension.

Two out of 19 PHT patients had moderate-severe PHT. Hydroxyurea use did not differ between patients with and without PHT and no patients used anticoagulation, calcium antagonists, endothelin receptor blockers or sildenafil. Between group comparisons were only performed in HbSS/HbSβ°-thalassemia patients as only 3 HbSC/HbSβ-thalassemia patients had PHT of whom one had blood samples drawn.

ADMA concentrations in patients without PHT were high compared to previously reported values in healthy race-matched controls. Irrespective of PHT, HbSS/HbSβ°-thalassemia patients were characterized by lower hemoglobin, higher LDH, ADMA and sVCAM-1 concentrations than HbSC/HbSβ-thalassemia patients (all p<0.001). ADMA and sVCAM-1 were higher in HbSS/HbSβ°-thalassemia patients with PHT than those without PHT, with a significant correlation between ADMA and TRV as well (see correlation studies in Online Supplementary Table 2). sVCAM-1 and hemoglobin were significantly correlated to TRV in HbSS/HbSβ-thalassemia patients (r_s=0.49, p=0.002, r_s=−0.30, p=0.04, respectively). SDMA, but not ADMA, was significantly correlated to GFR (r_s=−0.66, p<0.001, r_s=−0.08, p=0.60, respectively) in HbSS/HbSβ°-thalassemia patients. Given the relation between hemolysis and methylarginine concentrations, it is likely that the hemolytic rate is an important determinant of their production in SCD (likely due to the increased protein turn-over in the stress erythropoiesis), also explaining the higher concentrations in HbSS/HbSβ-thalassemia patients. A relative decrease in renal function (generally more evident in HbSS/HbSβ-thalassemia patients) could contribute especially to SDMA elevations. Contributing factors related to the pulmonary vasculature could be shear stress induced PRMT activity8 and hypoxia induced DDAH downregulation.9 Although difference in ADMA between patients with and without PHT seems modest, even small increases in extra-cellular ADMA lead to significant intra-cellular NOS inhibition through preferred cellular ADMA uptake over arginine.3 Indeed, plasma ADMA concentrations ≥0.64μmol/L are associated with strongly reduced pulmonary artery endothelial NOS expression and early death in PHT patients.5 Based upon the strong correlation of sVCAM-1 to ADMA, it would be interesting to hypothesize that chronic hemolysis induced ADMA elevations significantly contribute to endothelial activation and dysfunction in SCD via NOS inhibition, and that patients with higher ADMA concentrations are more prone to develop a vasculopathy leading to complications such as PHT over time.

Arginase activity (reflected by arginine to ornithine ratios) is elevated in sickle cell patients with moderate-severe PHT but, in agreement with previous studies,1,10 did not differ between patients with mostly mild PHT and those without PHT. Conceding the fact that we did not determine plasma arginase activity directly, these data suggest that ADMA could play a role of pathophysiological importance at a relatively earlier stage than arginase activity.

The relatively small number of patients needs to be taken into account when interpreting these data and no conclusions can be drawn about HbSC/HbSβ-thalassemia patients. Also, right heart catheterization remains the gold standard diagnostic test for PHT and is recommended in sickle cell patients with moderate-severe PHT detected with echocardiography. However, given the excellent correlation between pulmonary artery pressure and TRV in SCD,1 and the fact that an elevated TRV is the result of solely left-sided heart disease in only a minority of cases,11 the lack of right heart catheterization is unlikely to have significantly affected our results. Lastly, our data are largely limited to patients with mild PHT. Nonetheless, mortality is high in these patients1 and plasma ADMA concentrations were well in the range associated with death in other forms of PHT.4,5

Taken together, our data identify an association of plasma ADMA concentrations to PHT in SCD, possibly identifying a novel factor of importance in its pathophysiology. Also, ADMA induced limitation of NO production may well provide an important new mechanistic link between hemolysis and the characteristic endothelial activation of SCD.

Ackowledgments

we gratefully acknowledge the expert technical assistance of Sigrid de Jong.

Footnotes

• ↵* The CURAMA study group is a collaborative effort studying sickle cell disease in the Netherlands Antilles and The Netherlands. Participating centers: The Red Cross Blood Bank Foundation, Curaçao, Netherlands Antilles; The Antillean Institute for Health Research, Curaçao, Netherlands Antilles, The Department of Internal Medicine, Slotervaart Hospital, Amsterdam, The Netherlands; the Department of Vascular Medicine and the Department of Haematology, Academic Medical Center, Amsterdam, The Netherlands; the Department of Haematology, Erasmus Medical Center, Rotterdam, The Netherlands; the Department of Pathology, Groningen University Hospital, The Netherlands; the Department of Internal Medicine, Laboratory of Clinical Thrombosis and Haemostasis, and the Cardiovascular Research Institute, Academic Hospital Maastricht, The Netherlands.