Sumiaki Tanaka, Yu Matsueda, Tatsuhiko Wada, Junichi Tanaka, Tatsuo Nagai, Shunsei Hirohata

Original Contribution Kitasato Med J 2017; 47: 52-61 Long-term survival of patients with pulmonary arterial hypertension associated with connective tissue disease: beneficial effects of beraprost sodium, an oral prostacyclin analog Sumiaki Tanaka, Yu Matsueda, Tatsuhiko Wada, Junichi Tanaka, Tatsuo Nagai, Shunsei Hirohata Department of Rheumatology and Infectious Diseases, Kitasato University School of Medicine Objectives: To reveal the long-term survival of patients with pulmonary arterial hypertension (PAH) associated with connective tissue disease (CTD) and to evaluate the effect of beraprost sodium (BPS), a prostacyclin analog. Methods: We performed a retrospective cohort study of all the patients with PAH and CTD treated in our hospital from April 1975 through June 2005. Results: Seventy-four PAH patients with CTD were analyzed, including 27 systemic sclerosis (SSc), 16 systemic lupus erythematosus (SLE), 5 SSc + SLE, 1 SLE + dermatomyositis, 12 mixed connective tissue disease, and 13 other CTDs. The survival rates at 1, 2, 5, 10, and 30 years from the diagnosis of PAH were 90.5%, 78.5%, 37.3%, and 22.3%, respectively. Long-term survival was significantly correlated with World Health Organization functional class and pulmonary vascular resistance from the diagnosis of PAH. Among the 40 patients treated with BPS, 20 patients who improved within 6 months had much better prognosis than did those who were not treated with BPS (HR: 0.12, 95% CI: 0.03 0.41). Conclusion: The results demonstrate that CTD-PAH is chronically progressive. Moreover, the data also demonstrate that BPS is effective and that the response to BPS in the first 6 months is the strongest prognostic factor for long-term survival. Key words: pulmonary arterial hypertension, mixed connective tissue diseases, beraprost sodium, systemic sclerosis, systemic lupus erythematosus Introduction C onnective tissue disease (CTD) is frequently complicated with various pulmonary manifestations, including pulmonary arterial hypertension (PAH). The reported ranges for the prevalence of PAH is 5% 50% in patients with systemic sclerosis (SSc), 1 25% in patients with mixed connective tissue disease (MCTD) and 20% in patients with systemic lupus erythematosus (SLE). 2 A well-designed (multicenter, prospective, retrospective) UNCOVA study showed that the prevalence of PAH in patients with either MCTD or SSc was 13.3% based on estimated right ventricular systolic pressure (ERVSP) more than 40 mmhg by Doppler echocardiogram. 3 Such high prevalence of PAH in patients with CTD has led to its recognition as an established clinical subgroup, PAH associated with CTD (CTD-PAH), in the Dana Point Classification. 4 It is well known that survival of CTD-PAH patients is poor. For example, 5-year survival rate in PAH associated with SSc has been reported to be approximately 10%, 5 which is worse than that in patients with idiopathic PAH (IPAH). 6 In many studies on PAH, however, survival was measured within 5 years and sometimes compared with predicted survival based on the d'alonzo equation. 7,8 Likewise, previous studies that assessed 3 7 years survival of CTD-PAH have not reported over 7 years survival. 6,9-11 On the other hand, potent effective PAH drugs have been recently developed, updating the treatment algorithm and possibly resulting in a better survival in patients with IPAH and CTD-PAH. It is now more important to confirm the long-term survival of PAH based on observation but not on prediction, for correct evaluation of the efficacy of various forms of treatment Received 8 December 2016, accepted 26 December 2016 Correspondence to: Sumiaki Tanaka, Department of Rheumatology and Infectious Diseases, Kitasato University School of Medicine 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan E-mail: sumi@med.kitasato-u.ac.jp 52

Long-term survival in CTD-PAH for PAH. Currently, many potent effective drugs for PAH are available, including prostacyclin analogs, including epoprostenol 12,13 and beraprost sodium (BPS), 14,15 an endothelin receptor antagonist, including bosentan, 16,17 ambrisentan 17 and macitentan, 18 phosphodiesterase-5 inhibitors, including sildenafil 19 and tadalafil. 20 It should be pointed out that oral BPS therapy had been the only choice of treatment for PAH in patients with CTD in Japan until 2005. Although efficacy of oral BPS appears modest on PAH under current circumstance where potent effective drugs for PAH therapy have been available, some clinical trials showed the efficacy of BPS in patients with PAH. 15,21,22 In this study, we performed a retrospective cohort study to analyze the long-term survival of 74 patients with CTD-PAH based on observation for as long as 30 years. In addition, we evaluated the effect of BPS monotherapy between 1975 and 2005. Materials and Methods Patients Data on 74 Japanese CTD-PAH patients were collected from the database of patients with CTD who were treated in Department of Rheumatology and Infectious Diseases, Kitasato University Hospital from April 1975 through June 2005. Patients were selected if they fulfilled all of the following criteria: 1. satisfaction of diagnostic criteria of CTDs including SSc, 23 MCTD, 24 SLE, 25 and dermatomyositis (DM) 26 was confirmed on their medical records; 2. chronic thromboembolic pulmonary hypertension was ruled out using ventilation-perfusion scintigram and pulmonary angiography; 3. PAH was diagnosed after the onset of CTD; and 4. PAH was diagnosed based on right cardiac catheterization (RHC) and/or echocardiography. Briefly, the hemodynamic definition of PAH on RHC is mean pulmonary artery pressure (PAP) greater than 25 mmhg, pulmonary capillary wedge pressure less than or equal to 15 mm Hg, and pulmonary vascular resistance (PVR) greater than 3 Wood units. 4 PAH was diagnosed with echocardiography alone when the following findings were obtained: 40 mmhg or higher systolic PAP estimated from maximum velocity of tricuspid regurgitation flow measured by Doppler echocardiography (estimated mean right atrial pressure, 10 mmhg), and right ventricular wall thickening or right ventricle enlargement by 2D echocardiography. 4 We excluded pulmonary hypertension associated with left heart disease when the following findings were observed: pulmonary capillary wedge pressure greater 53 than 15 mmhg by RHC or left atrial and/or ventricular dilatation or mitral and/or aortic valve disorder revealed by echocardiography. We also excluded pulmonary venoocclusive disease and pulmonary hypertension associated with severe interstitial lung disease (ILD) revealed using high-resolution computed tomography. Study design Survival of PAH associated with CTD The present study design was a retrospective cohort study based on medical records in a single center. The primary endpoint was total death. The starting date of the followup period was a date at the diagnosis of PAH. Because BPS therapy had been the only alternative of CTD-PAH treatment through June 2005 in Japan, we also evaluated the effects of BPS monotherapy on the long-term outcome in patients with CTD-PAH from April 1975 through June 2005. This study was approved by ethical committee in Kitasato University. The risk factors for total death were examined based on the age at PAH diagnosis, PAH severity according to World Health Organization functional class (WHO-FC), and the results of RHC. Other parameters included gender, types of CTD and antinuclear antibodies, the presence of Raynaud's phenomenon and ILD, high-dose corticosteroid (CS) therapy (prednisolone greater than 1 mg/kg weight, or methylprednisolone pulse therapy), warfarin therapy, oxygen therapy, and BPS monotherapy. Long-term survival of CTD-PAH patients treated with BPS monotherapy We next explored predictive factors for the long-term outcome in CTD-PAH patients treated with BPS monotherapy in a retrospective cohort study design. The primary endpoint was total death or discontinuation of BPS monotherapy. Follow-up periods were measured from the dates when BPS monotherapy was started. BPS responders were: patients in whom WHO-FC II or higher improved in their WHO-FC in the first 6 months and patients in whom WHO-FC I did not worsen in the first 6 months. We compared the event-free period between the BPS responder group and the BPS non-responder group. Statistical analysis Continuous variables were summarized as the means ± SD or median (interquartile range) and compared using Student's t-test or Wilcoxon's rank sum test where appropriate. Categorical variables were summarized by frequencies with 95% confidence intervals (CIs) and

Tanaka S. et al. Figure 1. General demographic features of the 74 patients with PAH associated with CTD MCTD, mixed connective tissue disease; SjS, Sjöegren's syndrome; RA, rheumatoid arthritis; MPA, microscopic polyangiitis; APS, anti-phospholipid syndrome; AOSD, adult onset Still's disease; SSc, systemic sclerosis; SLE, systemic lupus erythematosus; PM/DM, polymyositis/ dermatomyositis. Closed circles indicate each subject. In SSc: 5 diffuse cutaneous SSc patients; 5 limited cutaneous SSc patients Table 1. Characteristics of the 74 patients with PAH associated with CTD n % Age at the diagnosis of PAH, years (mean ± SD) 48.0 ± 14.5 Gender (female) 66/74 89.2 Scl-70 positive 9/74 12.2 ACA positive 8/74 10.8 SS-A positive 44/74 54.0 U1-RNP positive 36/74 48.6 Raynaud's phenomenon positive 63/74 85.1 Area ratio of ILD on Chest radiograph <1/4 (absent-mild) 52/74 70.3 1/4 (moderate-severe) 22/74 29.7 WHO-FC at diagnosis I and II 32/74 43.2 III and IV 42/74 56.7 Mean PAP, mmhg (mean ± SD) 36.7 ± 12.4 PVR, Wood units/m 2 (mean ± SD) 9.50 ± 5.86 Corticosteroid( 1 mg/kg weight of PSL ) 27/74 36.5 Warfarine 36/74 48.6 Oxygen 33/74 44.6 Calcium-channel blocker 12/74 16.2 BPS 40/74 54.1 ACA, anti-centromere antibody; WHO-FC, World Health Organization functional class; ILD, interstitial lung disease; PAP, pulmonary arterial pressure; PVR, pulmonary vascular resistance; PSL, prednisolone; BPS, beraprost sodium 54

Long-term survival in CTD-PAH compared using Fisher's exact test. P values less than 0.05 (two-tailed) were considered significant. Time-toevent analyzes were performed using the Kaplan-Meier product-limit estimation model and Cox proportional hazards model. Factors associated with survivals were ascertained by univariable Cox proportional hazards model. Variables, which were found to be significant in univariable analysis (P 0.10) or otherwise to be clinically important (e.g., age at diagnosis of PAH or age at start of BPS monotherapy) were incorporated into a multivariable model. The proportional hazards assumption was examined for all covariates using the log minus log test of proportionality test and Schoenfeld residuals proportional hazards test. For statistical analysis, we used SPSS v15 (SPSS Inc., IL, USA) and JMP v7 (SAS Institute Inc., NC, USA). Results Patient characteristics Figure 1 and Table 1 show the demographic features of the 74 patients at the diagnosis of PAH. Of the 74 patients, 27 had SSc, 16 had SLE, 5 had SSc + SLE, 1 had SLE + DM, 12 had MCTD and 13 had other CTDs. There were some patients with overlapping CTDs. Raynaud's phenomenon was observed in 68% of the patients. The age at the diagnosis of PAH was 48.0 ± 14.5 years. The patients with SSc were older than those with other CTDs (53.0 ± 11.7 years vs. 41.2 ± 14.4 years, respectively, P < 0.0008). Fifty-five patients and 19 patients were diagnosed as PAH based on RHC and on echocardiography, respectively. In the latter group, the estimated spap was 59.4 ± 19.3 mmhg. The numbers of patients in WHO-FC I, II, III, and IV at diagnosis of PAH were 13, 19, 26, and 16, respectively. Treatment As shown in Table 1, 40 patients (54.1%) received 60 mg b.i.d. or t.i.d. of BPS. Twelve patients were also given suboptimal doses 27 of calcium-channel blockers. Thirty-six patients received warfarin anticoagulant therapy. Thirty-three patients received oxygen therapy, of which 27 patients were in the status of WHO-FC III/ IV at the diagnosis of PAH. Twenty-seven of the 74 patients received high-doses of corticosteroids. In 2 of the 40 patients, BPS monotherapy was discontinued. And bosentan was added in 1 patient after 7 months, in which BPS was switched to intravenous epoprostenol therapy after 8 months. Survival Thirty-seven patients died during observation after the diagnosis of PAH. Of those 37 patients, 34 patients died of PAH, and 3 patients died of malignancy, one each of: lung cancer, stomach cancer, and colon cancer. These 3 patients were handled as "events" in this survival analysis. Figure 2. Survival of CTD-PAH patients Kaplan-Meier survival curves of CTD-PAH patients 55

Tanaka S. et al. Figure 2 shows the survival curve calculated using the Kaplan-Meier method. The survival rates after 1, 2, 5, 10, and 30 years were 90.5%, 78.5%, 37.3%, and 22.3%, respectively. This survival curve indicates that PAH is a chronically progressive and fatal complication in CTD. Another notable finding was that 4 patients survived over 15 years after the diagnosis of PAH, and 2 patients survived more than 30 years. Table 2A lists the selected variables in the univariable analysis of hazard ratio for total death. The results disclosed that WHO-FC III/IV, PVR, oxygen administration, and lack of BPS therapy are risk factors for total death, whereas types of CTD, types of autoantibodies, and presence of ILD did not significantly contribute to total death. Since WHO-FC was significantly correlated with PVR (data not shown), as was consistent with previous studies, 7 we selected WHO- FC as a parameter of PAH severity for powerful statistical analysis. Oxygen therapy was administered in patients whose WHO-FC status was III or IV to assuage dyspnea, accounting for the negative-correlation between status of WHO-FC and oxygen therapy. We, therefore, exclude oxygen therapy from our multivariate model. Thus, WHO-FC III/IV remained a strong risk factor for mortality in this multivariable model (Table 2B). Of note, BPS monotherapy still significantly reduced hazard ratio for total death in the multivariable model. Effects of BPS monotherapy on survival Although BPS monotherapy was found to reduce the hazard ratio for total death significantly, the improvement of long-term survival by BPS appears to be modest (Figure 3A). We next analyzed 40 patients treated with BPS to find out such factors that affect the efficacy of BPS. Events were observed in 18 patients of those 40 patients. Of the 18 patients with events, 16 patients (BPS responder Table 2A. Univariate analysis of hazard ratio for death in patients with CTD- PAH Variable Hazard ratio 95% CI P value n Age, y 1.02 0.99 1.04 0.148 74 SSc 1.02 0.53 1.99 0.945 74 SLE 1.67 0.86 3.26 0.130 74 MCTD 0.53 0.20 1.39 0.200 74 Scl-70 Ab 2.47 0.98 6.22 0.055 74 ACA 1.28 0.39 4.23 0.689 74 U1RNP Ab 0.85 0.44 1.63 0.849 74 Raynaud's phenomenon 1.17 0.45 3.02 0.747 74 ILD (>1/4) 0.92 0.45 1.86 0.808 74 WHO-FC (III/IV) 3.09 1.44 6.64 0.004 74 Mean PAP, mhg 1.04 1.01 1.07 0.020 55 PVR, Wood units/m 2 1.10 1.03 1.18 0.008 55 Corticosteroid 0.65 0.33 1.29 0.218 74 Warfarin 0.84 0.44 1.62 0.604 74 Oxygen 2.56 1.29 5.05 0.007 74 BPS 0.50 0.26 0.98 0.043 74 Table 2B. Multivariate analysis of hazard ratio for death in patients with CTD-PAH Variable Hazard ratio 95% CI P value n Age, y 1.02 0.99 1.05 0.189 74 Scl-70 Ab 2.89 0.98 8.56 0.054 74 WHO-FC (III/IV) 3.19 1.44 7.05 0.004 74 BPS 0.40 0.15 0.75 0.007 74 ACA, anti-centromere antibody; WHO-FC, World Health Organization functional class; ILD, interstitial lung disease; PAP, pulmonary arterial pressure; PVR, pulmonary vascular resistance; PSL, prednisolone; BPS, beraprost sodium 56

Long-term survival in CTD-PAH Figure 3. Effects of BPS monotherapy on survival A. Kaplan-Meier survival curves of CTD-PAH patients with or without BPS monotherapy (P = 0.0392, Log-rank test). B. Kaplan-Meier survival curves of CTD-PAH patients in the BPS responder and non-responder groups (P < 0.001, Log-rank test). BPS responders were: patients with WHO-FC II or higher who improved in the first 6 months; patients with WHO-FC I who did not worsen in the first 6 months. 57

Tanaka S. et al. group: 3 patients in the BPS responder group, 13 patients in the non-responder group) died, and in 2 patients in the BPS non-responder group BPS monotherapy was discontinued due to clinical exacerbation of PAH. Survival rates in the BPS responder group and in the BPS non-responder group were analyzed using the Kaplan-Meier method (Figure 3B). The data showed that the survival curve of the BPS responder group was significantly better than that of the BPS non-responder group. After univariate analysis, which revealed several risk factors for total death, including anti-scl-70 antibody, PVR, and BPS non-responder state (Table 3A), we created a multivariate model. As shown in Table 3B, the response to BPS in the first 6 months was the strongest independent prognostic factor for long-term survival in CTD-PAH patients treated with BPS (hazard ratio of 0.12, 95% CI: 0.03 to 0.41, P = 0.001). This model also suggested that WHO-FC III/IV at the start of BPS therapy might predict poor long-term survival, although it was not statistically significant. Discussion The results in the present study revealed, to our knowledge, for the first time, the long-term survival of longer than 5 years in patients with CTD-PAH. And to our knowledge, there have been no studies in the literature that report survival in patients with PAH based on observation for as long as 30 years. The long-term survival rates can be predicted using the D'Alonzo Table 3A. Univariate analysis of hazard ratio for death in CTD-PAH patients treated with BPS Variable Hazard ratio 95% CI P value n Age, y 1.03 0.99 1.07 0.109 40 SSc 2.46 0.88 6.85 0.085 40 SLE 1.62 0.52 5.03 0.408 40 MCTD 0.35 0.08 1.55 0.168 40 Scl-70 Ab 3.59 1.33 9.70 0.012 40 ACA 1.82 0.24 14.32 0.559 40 U1RNP Ab 0.63 0.24 1.63 0.341 40 Raynaud's phenomenon 4.51 0.58 35.23 0.151 40 ILD (>1/4) 1.76 0.66 4.52 0.266 40 WHO-FC (III/IV) 2.80 0.98 9.07 0.055 40 Mean PAP, mhg 1.02 0.98 1.07 0.322 29 PVR, Wood units/m 2 1.13 1.00 1.26 0.045 29 Corticosteroid 0.36 0.11 1.16 0.088 40 Warfarin 0.66 0.26 1.68 0.368 40 Oxygen 4.23 1.38 12.93 0.011 40 BPS responder* 0.11 0.31 0.39 0.001 40 Table 3B. Multivariate analysis of hazard ratio for death in CTD-PAH patients with BPS Variable Hazard ratio 95% CI P value n Age, y 1.04 0.99 1.08 0.132 40 Scl-70 Ab 1.67 0.53 5.28 0.380 40 WHO-FC (III/IV) 3.96 0.88 17.90 0.073 40 BPS responder* 0.12 0.03 0.41 0.001 40 ACA, anti-centromere antibody; WHO-FC, World Health Organization functional class; ILD, interstitial lung disease; PAP, pulmonary arterial pressure; PVR, pulmonary vascular resistance; PSL, prednisolone; BPS, beraprost sodium *BPS responders were defined as: patients in whom WHO-FC II or higher improved in their WHO-FC in the first 6 months; patients in whom WHO-FC I did not worsen in their WHO-FC in the first 6 months. 58

Long-term survival in CTD-PAH equation used for IPAH. 7 Moreover, the predicted survival rates of the CTD-PAH patients in the present study calculated by this equation at 1, 2, and 5 years were 88.3%, 76.6% and 60.4%, respectively. The actual survival rates at 1 and 2 years that we observed were almost the same as these predicted survival rates. However, the actual survival rate at 5 years (37.3%) was apparently lower than the predicted survival rate (60.4%). These results suggest that the survival of CTD-PAH might be lower than that of IPAH at 5 years after the diagnosis of PAH or alternatively that the D'Alonzo equation might not be applied to CTD-PAH patients. It should be also noted that 4 patients survived longer than 15 years after diagnosis of PAH in the present long-term observation. It is, therefore, suggested that the clinical courses of CTD- PAH patients may be different from those of IPAH patients. In Europe and the United States, limited cutaneous SSc (lc-ssc) has a higher risk for PAH than does diffuse cutaneous SSc (dc-ssc). 28 Steen et al. reported that anti Scl-70 antibody, which is associated with dc-ssc, appeared to be protective for developing PAH. 9 The data in the present study demonstrated that SSc did not significantly affect the long-term survival in CTD-PAH patients. Moreover, anti-scl-70 antibody did not affect the long-term survival, either. Of the 33 patients with SSc in the present study, only 9 patients were lc-ssc positive. Anti-Scl-70 antibody was positive in only 9 patients with SSc. Therefore, a study with larger numbers of SSc patients would be necessary to confirm the relationship of subtypes of SSc with PAH in Japan. Mathai et al. showed that survival was significantly poor in SSc patients complicated with ILD-associated PH than in SSc patients complicated with PAH. 11 In the present study, moderate or severe ILD did not significantly affect the survival rate. This result might have been partly because this study also included patients with SLE and MCTD, although further studies are required to clarify the reason for this discrepancy. Currently, potent effective drugs for PAH are available, and the treatment algorithm has been updated. 29 Because it remains difficult to evaluate the efficacy of the long-term survival of monotherapy under the current circumstances, our data on the long-term survival in CTD- PAH patients receiving BPS monotherapy provided important information on the efficiency of BPS in the treatment of CTD-PAH patients. Our multivariable model showed that BPS monotherapy modestly but significantly improved survival. Moreover, stratified analysis has also demonstrated that CTD-PAH patients who responded to BPS within the first 6 months had favorable survival 59 rates. Both BPS therapy and the response to BPS within the first 6 months were independent prognostic factors for the survival of CTD-PAH patients. It should be noted that BPS has an acute vasodilatation effect. However, it should be also pointed out that PAH is a chronic disease based on the pulmonary vascular remodeling responder 30 and that acute pulmonary vasodilatation induced by nitric oxide is not correlated with chronic effects on hemodynamics. 31 It is noteworthy that the survival rate at 4 years after starting BPS monotherapy was 100% in the BPS responder group but only 25.2% in the BPS nonresponder group. It is evident that BPS might have prevented progression of pulmonary vascular remodeling in these patients. Modified release formulation of BPS (BPS-MR) has been available in Japan and enabled CTD- PAH patients to receive high doses of BPS (120 180μg b.i.d.). 22 Phase II global study using high-doses of BPS- MR (120 600μg b.i.d.) demonstrated the efficacy of the treatment for PAH. 32 Therefore, our results may support that BPS is a potent effective choice in the treatment algorithm for PAH. 33 More importantly, the data also indicate that additional drugs for PAH should be considered for those patients who do not show adequate response to BPS even after 6 months. Several studies have suggested the efficacy of immunosuppressive therapy for CTD-PAH patients. 34,35 Kawaguchi et al. 34 reported the efficacy of steroid pulse therapy in CTD-PAH patients, while Sanchez et al. 35 showed the efficacy of intravenous cyclophosphamide pulse therapy. The hazard ratio for total death of patients receiving CS therapy in the present study indicated that the long-term survival in CTD-PAH patients was independent of high doses of CS therapy. It should be pointed out that survival was usually evaluated between 1 and 5 years in these previous reports, whereas the survival in the present study was evaluated for a much longer period, of up to and longer than 30 years. Alternatively, there might be other factors than PAH that affect the efficacy of CS or immunosuppressants in the previous studies. 34,35 Further studies with a larger number of patients are required to determine the efficacy of immunosuppressive therapy for CTD-PAH patients. Finally, the limitations of this study included that the study design was a retrospective cohort study. We could not analyze parameters of PAH severity such as a 6- minute walking distance and serum BNP levels. Moreover, we could not obtain enough data of respiratory function tests including diffusing capacity of the lung for carbon monoxide. Further studies designed in a prospective manner with these parameters of PAH severity would be important for the correct evaluation of

Tanaka S. et al. the treatment for CTD-PAH patients. In summary, we have reported long-term survival, for as long as 30 years, in patients with CTD-PAH, to our knowledge, for the first time. Under the current circumstances, where the development of potent effective drugs for the treatment of PAH patients is improving their long-term prognosis, the survival curve for longer periods based on observation is becoming more important for the establishment of the most appropriate treatment strategies for PAH. The data also showed that the response to BPS in the first 6 months is the strongest prognostic factor for long-term survival of patients with CTD-PAH. Acknowledgment We thank Yoshiyuki Makita, Keiko Fukuoka, Chiaki Kusanagi, Rumiko Endo, Yumi Hiramoto, and Mitsugu Yoshida, laboratory technicians in Kitasato University Hospital, for conducting the echocardiographic studies; we also thank Hirobumi Kondo, M.D., Ph.D., and Jun Okada, M.D., Ph.D., for their support to accomplish this long-term study. References 1. Denton CP, Black CM. Pulmonary hypertension in systemic sclerosis. Rheum Dis Clin North Am 2003; 29: 335-49, vii. 2. Simonson JS, Schiller NB, Petri M, et al. Pulmonary hypertension in systemic lupus erythematosus. J Rheumatol 1989; 16: 918-25. 3. Wigley FM, Lima JA, Mayes M, et al. The prevalence of undiagnosed pulmonary arterial hypertension in subjects with connective tissue disease at the secondary health care level of community-based rheumatologists (the UNCOVER study). Arthritis Rheum 2005; 52: 2125-32. 4. McLaughlin VV, Archer SL, Badesch DB, et al. ACCF/AHA 2009 expert consensus document on pulmonary hypertension a report of the American College of Cardiology Foundation Task Force on Expert Consensus Documents and the American Heart Association developed in collaboration with the American College of Chest Physicians; American Thoracic Society, Inc.; and the Pulmonary Hypertension Association. J Am Coll Cardiol 2009; 53: 1573-619. 5. McLaughlin VV, Presberg KW, Doyle RL, et al. Prognosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 2004; 126: 78S-92S. 6. Kuhn KP, Byrne DW, Arbogast PG, et al. Outcome in 91 consecutive patients with pulmonary arterial hypertension receiving epoprostenol. Am J Respir Crit Care Med 2003; 167: 580-6. 7. D'Alonzo GE, Barst RJ, Ayres SM, et al. Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Intern Med 1991; 115: 343-9. 8. Humbert M, Sitbon O, Yaici A, et al. Survival in incident and prevalent cohorts of patients with pulmonary arterial hypertension. Eur Respir J 2010; 36: 549-55. 9. Steen V, Medsger TA, Jr. Predictors of isolated pulmonary hypertension in patients with systemic sclerosis and limited cutaneous involvement. Arthritis Rheum 2003; 48: 516-22. 10. McLaughlin VV, McGoon MD. Pulmonary arterial hypertension. Circulation 2006; 114: 1417-31. 11. Mathai SC, Hummers LK, Champion HC, et al. Survival in pulmonary hypertension associated with the scleroderma spectrum of diseases: impact of interstitial lung disease. Arthritis Rheum 2009; 60: 569-77. 12. Badesch DB, Tapson VF, McGoon MD, et al. Continuous intravenous epoprostenol for pulmonary hypertension due to the scleroderma spectrum of disease. A randomized, controlled trial. Ann Intern Med 2000; 132: 425-34. 13. Sitbon O, Humbert M, Nunes H, et al. Long-term intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and survival. J Am Coll Cardiol 2002; 40: 780-8. 14. Nagaya N, Uematsu M, Okano Y, et al. Effect of orally active prostacyclin analogue on survival of outpatients with primary pulmonary hypertension. J Am Coll Cardiol 1999; 34: 1188-92. 15. Galie N, Humbert M, Vachiery JL, et al. Effects of beraprost sodium, an oral prostacyclin analogue, in patients with pulmonary arterial hypertension: a randomized, double-blind, placebo-controlled trial. J Am Coll Cardiol 2002; 39: 1496-502. 16. Rubin LJ, Badesch DB, Barst RJ, et al. Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 2002; 346: 896-903. 17. McLaughlin VV, Sitbon O, Badesch DB, et al. Survival with first-line bosentan in patients with primary pulmonary hypertension. Eur Respir J 2005; 25: 244-9. 18. Pulido T, Adzerikho I, Channick RN, et al. Macitentan and morbidity and mortality in pulmonary arterial hypertension. N Engl J Med 2013; 369: 809-18. 19. Galie N, Ghofrani HA, Torbicki A, et al. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 2005; 353: 2148-57. 60

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