Published ahead of print on September 13, 2007, doi:10.1164/rccm.200702-278OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200702-278OC
Asymmetric Dimethylarginine Is Increased in Chronic Thromboembolic Pulmonary Hypertension1 Division of Cardiology, Department of Internal Medicine II, 2 Department of Clinical Pharmacology, and 3 Department of Cardiothoracic Surgery, Vienna General Hospital, Medical University of Vienna, Vienna, Austria; and 4 Wilhelminenspital der Stadt Wien, Vienna, Austria Correspondence and requests for reprints should be addressed to Irene M. Lang, M.D., Professor of Vascular Biology, Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. E-mail: irene.lang{at}meduniwien.ac.at
Rationale: Asymmetric dimethylarginine (ADMA), a potent endogenous nitric oxide synthase (NOS) inhibitor, is increased in idiopathic pulmonary arterial hypertension and associated with unfavorable outcome. Objectives: Chronic thromboembolic pulmonary hypertension (CTEPH), although principally amenable to surgical removal of major pulmonary arterial obstructions by pulmonary endarterectomy, may show a small-vessel pulmonary arteriopathy similar to idiopathic pulmonary arterial hypertension. We hypothesized that ADMA plasma levels are increased in patients with CTEPH. Methods: We measured ADMA by high-performance liquid chromatography at the time of diagnosis in 135 patients with CTEPH. Inoperability in 66 patients was based on an imbalance between severity of pulmonary hypertension and morphologic lesions.
Measurements and Main Results: ADMA plasma levels were significantly elevated in patients, compared with 40 matched control subjects (0.62 [0.51–0.73] vs. 0.51 [0.45–0.6] µmol/L, P = 0.0002). At baseline, ADMA plasma concentrations correlated with mixed venous saturation (r = –0.25, P = 0.005), right atrial pressure (r = 0.35, P < 0.0001), and cardiac index (r = –0.21, P = 0.01). Patients who underwent surgery demonstrated lower ADMA levels at baseline than inoperable patients (0.60 [0.5–0.68] vs. 0.63 [0.53–0.85] µmol/L, P = 0.02), with a further decrease 12 ± 1 months after pulmonary endarterectomy (P = 0.02). Endothelial NOS expression in endothelial cells was low in patients with elevated ADMA plasma levels. Survival of patients with ADMA plasma levels Conclusions: ADMA plasma levels correlate with the severity of pulmonary vascular disease and predict outcome in patients with CTEPH. Measurement of ADMA plasma levels may be useful for estimating the degree of small-vessel arteriopathy in CTEPH.
Key Words: chronic thromboembolic pulmonary hypertension • nitric oxide • nitric oxide synthase
Chronic thromboembolic pulmonary hypertension (CTEPH) is believed to result from single or recurrent pulmonary thromboemboli arising from sites of venous thrombosis (1). For reasons still unclear, the lysis of blood clots does not occur in up to 3.8% of acute pulmonary thromboemboli (2), evolving to organized obstructions inside the pulmonary artery. Increased vascular resistance results in right heart strain and remodeling. Although acute pulmonary embolism is generally accepted as the main initiating event of CTEPH, small-vessel arteriopathy is believed to appear later in the course of the disease, contributing to the progression of hemodynamic and symptomatic decline (3). Histopathologic studies of microvascular changes in CTEPH have identified vascular lesions indistinguishable from those observed in idiopathic pulmonary arterial hypertension (IPAH) (4). Endothelial dysfunction attributable to reduced bioavailability of endogenous vasodilator substances such as nitric oxide (NO) is believed to play an important role in the pathogenesis of pulmonary hypertension (PH) (5, 6). NO is synthesized in the endothelium from L-arginine by NO synthase (NOS), with endothelial NOS (eNOS) and inducible NOS (iNOS) representing important vascular isoforms. The most abundant endogenous NOS inhibitor is asymmetric dimethylarginine (ADMA) (7). Plasma levels of ADMA are increased in patients with congenital heart disease and PH (8). In IPAH, increased ADMA plasma levels are associated with unfavorable pulmonary hemodynamics and outcome (9). However, ADMA plasma levels and their relationship to outcome have not been assessed in patients with CTEPH. We hypothesized that ADMA plasma levels are increased in patients suffering from CTEPH. Some of the results of these studies have been previously reported in the form of an abstract (10, 11).
Patients and Clinical Assessments Patients were included if they had CTEPH in the absence of unstable atherosclerotic vascular disease, renal dysfunction, untreated hyperlipidemia, obesity, or smoking within the past year, and were diagnosed between February 1992 and February 2004, at the time of first diagnostic right heart catheterization. Diagnosis and indication for surgery were based on published standards (12). Right heart catheterization was performed at baseline as previously described (13). Patients did not undergo surgery if there was a mismatch between the degree of hemodynamic compromise and the distality of most proximal thrombi. Each case was reviewed by a cardiologist, pulmonologist, radiologist, and a pulmonary endarterectomy (PEA) surgeon blinded to the study results. CTEPH type (14, 15) was classified according to the surgical specimen. Patients not undergoing PEA because of comorbidities were excluded. None of the patients was under treatment with vasodilators at the time of blood sampling. We performed ambulatory follow-up routines every 3 to 6 months. Forty healthy nonobese nonsmoking control subjects (25 females; age, 56.5 [50–65] yr) without a history of hypertension were selected from a preexisting pool with a normal echocardiogram. The ethics committee of the Medical University of Vienna approved the study, and patients signed informed consent.
Sample Preparations and Measurements Blood samples drawn into ethylenediaminetetraacetic acid tripotassium (K3) were centrifuged at 2,500 x g (4°C, 15 min), and plasma was stored in 1-ml aliquots at –80°C. L-arginine and ADMA were measured by high-performance liquid chromatography (16). Plasma nitrate/nitrite (NOx) was determined with an ultrasensitive colorimetric assay (Oxford Biomedical Research, Oxford, MI).
Immunohistochemical Analyses
Statistical Analysis Patients were stratified as those who did or did not undergo surgery. L-arginine, ADMA, hemodynamic parameters, sex, and age were entered into a stepwise Cox regression model to assess prognostic factors of survival. Survival of patients with CTEPH was analyzed using the log-rank test, and was described by Kaplan-Meier curves, after dichotomization in patients with ADMA above and below 0.64 µmol/L. Study endpoint was death from right heart failure or lung transplantation. Patients who died within 28 days postoperatively were censored at the date of death. ADMA and survival were depicted by a receiver operating characteristic curve analysis. Cutoffs were chosen from the optimal sensitivity to specificity relation. The significance level was set at P < 0.05 (two-sided). All prognostic data were correlated with baseline ADMA.
Overall Group Of 168 patients with CTEPH diagnosed between February 1992 and February 2004, we were able to include 135 patients. Thirty-three patients (18 not operated on and 15 patients who underwent PEA later) were excluded from the study. The reasons for the exclusion were as follows: untreated hyperlipidemia in 13 patients, the presence of unstable atherosclerotic vascular disease in 2 patients, renal dysfunction in 7 patients, obesity in 1 patient, elevated blood glucose in 3 patients, and smoking within the past year before study start in 7 patients. Among the excluded patients, specific reasons for inoperability were advanced age in 9 patients, and severe chronic obstructive pulmonary disease in another 4 patients. No patients were lost to follow-up. Hemodynamic parameters and patient baseline characteristics are summarized in Table 1. Patients were in functional classes II–IV according to diagnostic criteria defined by the World Health Organization (18). Patients received conventional therapy consisting of digoxin, oxygen, and oral anticoagulants at an international normalized ratio (INR) of 2 to 3. There was no difference between patients with CTEPH (59 [49–70] yr, 64 females) and control subjects with regard to serum cholesterol and triglycerides, blood glucose levels, and renal function (Table 2). Patients with CTEPH tended to have a higher median homocysteine level (12.5 [10.5–15.6] vs. 10.0 [8.3–12.8] µmol/L, P < 0.05), with no significant difference in homocysteine levels between patients undergoing surgery and patients who did not undergo surgery.
In the control group, there were 25 females (62.5%) compared with 64 females in the CTEPH patient group (47.4%). There was no statistical difference between control subjects and patients concerning sex (P = 0.09). Mean plasma ADMA concentrations were significantly higher in patients with CTEPH (0.62 [0.51–0.73] [n = 135] vs. 0.51 [0.45–0.6] µmol/L in controls, P = 0.0002). L-arginine plasma concentration was significantly decreased in patients with CTEPH (62.6 [49–86.9] vs. 80.4 [68.6–96.7] µmol/L in controls, P = 0.008). Baseline plasma ADMA levels correlated with mean pulmonary arterial pressure (r = 0.24, P = 0.005), mean right atrial pressure (r = 0.36, P < 0.0001), cardiac output (r = –0.24, P = 0.008), cardiac index (r = –0.21, P = 0.01), pulmonary vascular resistance (r = 0.24, P = 0.006), and mixed venous oxygen saturation (r = –0.25, P = 0.006 ) (Figure 1).
Plasma ADMA levels were not correlated with serum creatinine (r = 0.06, P = 0.6). Median follow-up was 37 months (range, 0.3–149 mo). During this period, 31 patients who did not undergo surgery and 22 patients who did undergo surgery died; 2 patients not operated on and 2 patients operated on underwent lung transplantation.
Patients Who Did and Did Not Undergo Surgery ADMA plasma levels at the time of diagnosis were higher in patients who remained unoperated upon during follow-up compared with patients subsequently undergoing PEA (0.63 [0.53–0.85] vs. 0.60 [0.5–0.68] µmol/L, P = 0.02; Figure 2A). ADMA plasma levels decreased after PEA within 12 ± 1 months from 0.60 (0.5–0.68) to 0.51 (0.48–0.56) µmol/L (P = 0.02), whereas plasma L-arginine did not change. ADMA plasma levels in patients who underwent surgery with distal thrombi were higher (CTEPH types 3 and 4; n = 13; 0.68 [0.5–0.88] µmol/L) than in patients with proximal thrombi (CTEPH type 1; n = 20; 0.62 [0.51–0.69] µmol/L (P = 0.01) (Figure 2B).
The analysis of NOx revealed significantly higher levels in patients undergoing surgery (281 [250.0–304.4] µM) compared with patients who did not undergo surgery (261.1 [248–281.9] µM) (P = 0.04).
NOS Expression Analysis in Endothelial Cells of Patients with CTEPH
Survival ADMA was the only predictor of survival in the overall group (P < 0.0001), but both mean right atrial pressure and ADMA were found predictive of survival in patients who did not undergo surgery (P < 0.0001, respectively) (Table 4). Receiver operating curve analysis revealed that baseline ADMA was a good predictor of outcome (P < 0.0001). ADMA can predict death with a sensitivity of 81.1% and a specificity of 79.3% at a cutoff value of 0.64 µmol/L (Figure 3).
Patients with plasma ADMA levels of 0.64 µmol/L or greater had a worse survival (P = 0.02 for patients who underwent surgery, and P < 0.0001 for patients who did not have surgery) than patients with plasma ADMA levels below 0.64 µmol/L (Figures 4A and 4B).
Plasma ADMA levels are increased in patients with CTEPH. ADMA plasma levels correlate with hemodynamic parameters and thus with the severity of pulmonary vascular disease. ADMA is a predictor of mortality in patients with CTEPH. Patients with ADMA plasma levels below 0.64 µmol/L have a better outcome. The lungs are a major source of NOS and ADMA, which are in a delicate balance and determine net NO production (20). ADMA is metabolized by dimethyl-arginine dimethylaminohydrolase (DDAH), with DDAH II representing the predominant endothelial isoform. Its expression and activity have been shown to be reduced in PH induced by hypobaric hypoxia (21). Suppression of endothelial DDAH II expression and function was identified as an important underlying mechanism in monocrotaline-induced PH, in hypoxia (22) and in iPAH (23). Most recent work has demonstrated that loss of DDAH I activity is another important contributor to accumulation of ADMA and reduction in NO signaling (24, 25). Acute administration of ADMA increases pulmonary vascular resistance and decreases stroke volume in healthy subjects (26). ADMA is increased in IPAH (9) and in patients with congenital heart defects and PH (8). The finding of elevated ADMA levels in patients with CTEPH strengthens the concept that CTEPH and PAH share pathophysiologic characteristics (23). By contrast, acute venous thromboembolism does not appear to affect ADMA plasma levels (27). CTEPH is an important cause of PH. The disease is underdiagnosed, and its true prevalence is still unclear. It is characterized by intraluminal thrombus organization and fibrotic stenoses or complete obliteration of pulmonary arteries. Pulmonary embolism, either as single or recurrent episodes, is believed to be the initiating event, followed by progressive vascular remodeling (28). PEA has become the classical therapeutic solution for CTEPH, due to clinically relevant and long-lasting functional improvement conferred by this procedure (29). Currently, there exists no general definition for inoperability, although a disproportionately high pulmonary vascular resistance in relation to the most proximal location of thrombi, comorbidities and associated medical conditions, patient wishes, and the functional status of the affected pulmonary parenchyma contribute to the decision. In the present study, patients who did not undergo surgery were those with a mismatch between hemodynamic compromise and distality of thromboembolic material. At our institution, this surgical practice, combined with further exclusion criteria as listed, determines that approximately 50% of patients are classified as not being candidates for surgery. Furthermore, approximately 10% of patients who undergo PEA obtain no relief and maintain a pulmonary hypertensive state or experience recurrent PH, likely due to concomitant small-vessel arteriopathy in addition to distal thromboembolic disease (30). Persistent PH is a critical and consistent determinant of perioperative risk. Although traditional diagnostic modalities are adequate in identifying the presence of proximal disease in CTEPH, they provide very limited information on microcirculatory dysfunction (30). Therefore, preoperative assessment of the degree and contribution of small-vessel disease is important. In our study, we found increased ADMA levels in patients with CTEPH compared with matched control subjects (0.62 [0.51–0.73] vs. 0.51 [0.45–0.6] µmol/L, P = 0.0002). Despite the variability induced by dietary nitrites, plasma NOx levels were in accord with the observations of elevated ADMA. Lower NOx plasma levels could be explained through diminished NO production as a consequence of elevated ADMA. Despite elevated homocysteine plasma levels in patients with CTEPH compared with control subjects, levels were within the normal range (i.e., between 5 and 15 µmol/L), and the significance of this observation with regard to ADMA plasma levels is uncertain (31, 32). Methylarginine-mediated inhibition of NOS activity has been implicated in endothelial dysfunction (24, 33), and may be an important mechanism determining outcomes of CTEPH. Although it is unknown whether ADMA influences NOS expression, decreased eNOS protein as found in vascular tissues from patients with CTEPH may enhance the negative effects of elevated ADMA. We acknowledge that the data obtained by counting positive cells yield only semiquantitative information. However, in patients with ADMA levels above 0.64 µmol/L, endothelial NOS immunoreactivity was virtually absent (Figure 2C). ADMA concentrations of 0.64 µmol/L or greater in patients with CTEPH could serve as a surrogate marker for small-vessel arteriopathy. After PEA, ADMA plasma levels decrease, indicating that endothelial dysfunction is grossly reversible with normalization of pressures. Further studies will clarify whether pharmacologic treatments have a similar impact on ADMA plasma levels. In conclusion, the results of this study promise that ADMA measurement may be useful for estimating the degree of small-vessel disease in CTEPH. The levels of circulating ADMA add prognostic information in CTEPH.
Supported by the Austrian fellowship grants Fonds zur Förderung der wissenschaftlichen Forschung S9406-B11 (to I.M.L.), Österreichischer Herzfonds (to D.B.), Österreichischer Selbsthilfeverein Lungenhochdruck, and the Ludwig Boltzmann Institute for Cardiovascular Research. Originally Published in Press as DOI: 10.1164/rccm.200702-278OC on September 13, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form February 18, 2007; accepted in final form September 12, 2007
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0.64 µmol/L was worse than in patients with ADMA plasma levels < 0.64 µmol/L. 
