PLOS ONE RESEARCH ARTICLE Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers Ivan Castellvı́ ID1☯*, Carmen Pilar Simeón2☯, Monica Sarmiento1‡, Jordi Casademont3‡, Hèctor Corominas ID1‡, Vicenç Fonollosa2‡ 1 Department of Rheumatology, Hospital Universitari de la Santa Creu i Sant Pau, Barcelona, Spain, 2 Department of Internal Medicine, Hospital Universitari de Vall Hebron, Barcelona, Spain, 3 Department of Internal Medicine, Hospital Universitari de la Santa Creu i Sant Pau, Barcelona, Spain a1111111111 ☯ These authors contributed equally to this work. a1111111111 ‡ These authors also contributed equally to this work. a1111111111 * icastellvi@santpau.cat a1111111111 a1111111111 Abstract Systemic sclerosis is a disease where microcirculation damage is critical in their beginning and vascular complications have similar pathogenic findings. Digital ulcers are a frequent OPEN ACCESS complication in systemic sclerosis patients and pulmonary hypertension is one of the lead- Citation: Castellvı́ I, Simeón CP, Sarmiento M, ing causes of death. The use of bosentan has been shown to be useful for the treatment of Casademont J, Corominas H, Fonollosa V (2020) Effect of bosentan in pulmonary hypertension pulmonary arterial hypertension and to prevent new digital ulcers. However, is unknown if development in systemic sclerosis patients with bosentan can prevent pulmonary hypertension. Our objective was to determine if bosentan digital ulcers. PLoS ONE 15(12): e0243651. https:// is useful to prevent pulmonary hypertension in SSc patients. A retrospective study in 237 doi.org/10.1371/journal.pone.0243651 systemic sclerosis patients with digital ulcers history treated or not with bosentan to prevent Editor: Luca Navarini, Universita Campus Bio- it was made. We analyzed the occurrence of pulmonary hypertension defined by an echo- Medico di Roma, ITALY cardiogram pulmonary arterial pressure > 40 mmHg in the entire cohort. Demographic, clini- Received: June 25, 2020 cal, and treatment variables were recorded for all patients. Statistical significance was Accepted: November 20, 2020 denoted by p values < 0.05. Fifty-nine patients were treated with bosentan a median of 34 Published: December 10, 2020 months. 13.8% of treated patients had pulmonary hypertension vs 23.7% of untreated patients (p 0.13) during the follow up. In multivariate analysis patients not treated with Peer Review History: PLOS recognizes the benefits of transparency in the peer review bosentan had 3.9fold-increased risk of pulmonary hypertension compared with patients process; therefore, we enable the publication of under bosentan treatment (p < 0.02). Moreover the percentage carbon monoxide diffusing all of the content of peer review and author capacity (DLCO) in bosentan treated patients did not decrease from baseline to the end of responses alongside final, published articles. The editorial history of this article is available here: follow-up (61.8±14% vs 57±20.1%, p = 0.89). We concluded that Systemic sclerosis https://doi.org/10.1371/journal.pone.0243651 patients with digital ulcers treated with bosentan seems to have less risk to develop pulmo- Copyright: © 2020 Castellvı́ et al. This is an open nary hypertension and to stabilize DLCO access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Data Availability Statement: All relevant data are Systemic Sclerosis (SSc), also known as scleroderma, is an autoimmune disease characterized within the manuscript and supporting information by microangiopathy, fibrotic disfunction and immune disregulation. Currently we have strong file. evidence of microvascular involvement at the beginning of the disease [1]. The most important PLOS ONE | https://doi.org/10.1371/journal.pone.0243651 December 10, 2020 1/9 PLOS ONE Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers Funding: The authors received no specific funding vascular complications are digital ulcers (DUs) and pulmonary hypertension (PH). DUs are a for this work. frequent severe vascular complication with a prevalence of 40–50% [2–5] being an important Competing interests: The authors have declared cause of disability [6–8]. The endotelin receptor antagonist bosentan (BOS) demonstrated to that no competing interests exist. prevent DUs episodes in two randomized controlled trials (RCT) [6,9]. Pulmonary Arterial Hypertension (PAH) is the most frequent cause of PH in SSc and a leading cause of death [10,11]. A recent meta-analysis showed a 9% prevalence of PAH in SSc [12]. The prognosis of PAH improved since the emergence of new treatments with one, two and three years survival rates of 78%, 58% and 47% respectively [13]. Bosentan has been dem- onstrated to improve the New York Heart Association Functional Class (NYHA-FC), exercise capacity, haemodynamic parameters, survival [14] and to delay the clinical worsening of patients with PAH [15–17]. Bosentan was also demonstrated to be useful in the initial stages of PAH [17] and it has been indicated to treat PAH in NYHA-FCII/III. It is noteworthy that compared with idiopathic PAH the survival in SSc is worse [18] and that screening programs of PAH for SSc patients were associated with a better outcomes [13]. In scleroderma is hypothesized that therapies used to treat different vascular involvements could be useful to prevent other complications [1]. In SSc exist a shared damage mechanisms [19]. Some studies investigate the clinical vascular complications in patients with and without DUs. The Canadian Scleroderma Research Group investigated in 998 patients the findings associated with DUs [20]. They found no association between DUs and PAH. No association was found between DUs and PAH in a Spanish study with 1326 SSc patients (552 with previ- ous history of DUs) [21]. On the other hand, the German Network in SSc found PAH as a risk factor to present DUs [22]. The ItinérAIR-Sclérodermie French cohort (599 SSc patients) found not increased frequency of PAH in patients with history of DUs, but digital ulceration was associated in patients with diffusing capacity for carbon monoxide (DLCO) of <60% [23]. This was an important finding because a reduced DLCO without impairment in pulmonary function may be a surrogate marker of vasculopathy. Our purpose was to determine if bosentan could prevent PH in a large cohort of patients with DUs. Material and methods A retrospective case-control study that was carried out in 237 SSc patients with previous his- tory of DUs from cohorts of two scleroderma reference centers. All patients full-filed ACR/ EULAR 2013 classification criteria for scleroderma [24] and were followed since January 1980 to December 2012 or up the lack of the follow-up or patient’s decease. Case definition was for all patients who received bosentan treatment to prevent DUs for at least one month and con- trols were those naïve for bosentan. All patients had documented at least one DU. We excluded patients without history of DU or with PH/PAH diagnosed previously. Definition of DU We evaluated patients rather with the presence of active DU or with a history of DU related to digital ischemia, according to current definition of DU [25]. Ulcers overlying bony promi- nence secondary to trauma at the site of joint contractions, areas of calcium extrusion and skin fissures were excluded. Definition of pulmonary hypertension Our study was performed in patients from two historical cohorts, we did have not right heart catheterization (RHC) of most patients available, thus we assumed an echocardiographic PLOS ONE | https://doi.org/10.1371/journal.pone.0243651 December 10, 2020 2/9 PLOS ONE Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers definition of PH. We considered that patients with estimated systolic pulmonary arterial pres- sure (sPAP) > 40mmHg are at higher risk to present PH. Dataset We obtained epidemiological and clinical parameters of the patients in the study. In those exposed to bosentan, we analysed the use, dose and treatment duration. Echocardiography and pulmonary function test of all patients were both recorded from the beginning till the end of the study. Data of the patients were anonymized previously to start the study. Statistical analysis Descriptive statistics of all available data were made. We detailed the number of cases and per- centage in qualitative analysis and average, median, standard deviation, 25–75 percentile and 95% confidence interval (CI) for quantitative data. Fisher’s exact test to analyse qualitative data and Student test to analyse quantitative inde- pendent variables were used. To identify the sensitivity change of echocardiographic and pulmonary function test from the beginning to the end of follow-up for all patients we applied T test for paired data and McNemar test. To compare the change between case and control groups the U Mann-Whitney and exact Fisher test were applied. A multivariate logistic regression model (stepwise regression) were used between the presence of pulmonary hypertension (dependent variable) respect to the administration of Bosentan and those variables with bivariate association (p <0.05) in patients treated or not with bosentan (inde- pendent variables), for detect possible confounding factors between the association of pulmonary hypertension and administration of bosentan. Taking into consideration our design a post-hoc analysis was not performed. Once the variables with a statistically significant association (p <0.05) were obtained with respect to the presence of PH Odds Ratio (OR) was calculated for each variable with respect to the risk of presenting pulmonary hypertension according to the defi- nition of the study. A p value under 0.05 was considered significant. All statistical analyses were carried out with the statistics software SAS program V9.2SAS Institute Inc., Cary, NC, USA. Ethics statement Approval of Ethics Committee was not necessary based on the Spanish Biomedical Investiga- tion Law 14/2007 (Spanish Ministry of Health) by the characteristics of the our study because data was retrieved using previous existing information present in the historical database from the two cohorts fully anonymized previously to our analysis. Authors had access to the data from March 2013 to July 2013 included. Results After to exclude fifteen patients with previous suspicion (estimated sPAP > 40 mmHg) or PH/ PAH diagnosis, a total of 222 patients were included in the analysis. 201 (91%) were women with a mean age of 63.9 ± 19.6 years and with a median age at first SSc manifestation of 40 (95%CI 30–52) years. 138 patients were classified as limited cutaneous subset (lcSSc). Baseline characteristics are shown in Table 1. The bosentan group included 59 patients (26.6%) and 163 were considered as a control group. An estimated sPAP > 40 mmHg was observed in 21% of patients and RHC was performed in 14 patients. We observed a higher percentage of patients with left ventricle diastolic dysfunction (13.9% vs 32.2%; p<0.001) and higher tricuspid regur- gitation velocity (TRV) (1.93±1.05 vs 2.42±0.87 m/s; p<0.05) at the end of the follow-up. PLOS ONE | https://doi.org/10.1371/journal.pone.0243651 December 10, 2020 3/9 PLOS ONE Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers Values of forced vital capacity (FVC), carbon monoxide diffusion capacity (DLCO) and DLCO adjusted by alveolar volume (KCO) was also impaired (83.5±19.2% vs 77.8±21.2%; p < 0.0001, DLCO 64.4±18.5% vs 59.2±19.9% KCO 75±20% vs 67.5±18.7; p <0.0001). We did not find differences in FVC/DLCO ratio at end of study. Treatment characteristics 59 patients were at least one month under bosentan treatment, with a median of 34 (95% CI 5–59) months. The most conventional dose was 250mg bid (60%). 35.6% of patients had at least one adverse effect (Fig 1) with a withdrawal in 27.1% of patients. The other concomitant vasodilators were calcium channel blockers in 85% of patients, phosphodiesterase type 5 (PDE-5) inhibitors in 14.3% of patients and 12.7% of patients were treated with prostanoids. Clinical characteristics No differences were found between the proportion of patients treated with bosentan in both DUs patients cohorts (35% vs 24%; p = 0.14). The percentage of patients with diffuse cutaneous involvement (dcSSc) was higher in bosentan group (47.5% vs 22.7%; p<0.01). Not significant differences were observed within other organs involvements between treated or no treated. Pulmonary hypertension in patients with or without bosentan treatment. During the follow-up, 13.8% of treated patients with bosentan and 23.7% of no treated developed pulmo- nary hypertension (PH) based on echocardiography definition (OR = 0.52; CI95% = 0.22– 1.19) (Fig 2). Although this data did not reach the statistical significance (p >0.05). Right Heart Catheterism (RHC). Only had RHC data of 14 patients in both retrospective cohorts. PAH was confirmed in 7 patients. We did not observe haemodynamic differences between patients with or without treatment. Risk to develop PH. Interestingly, patients without history of bosentan treatment had 3.9fold-increased risk to present PH compared with patients treated with an Odds Ratio (OR) of 3.91 (IC95%:1.3–11.6; p<0.02). The risk of PAH observed in patients who did not take PDE5-inhibitors were similar (OR 3.74; IC95%: 1.2–11.5; p<0.03) and less evident in patients who had never taken prostanoids (OR 2.65; IC95%:1.0–7.0; p<0,05). No differences were found among other treatments (Fig 3). Pulmonary function test and echocardiographic findings. We found that %DLCO pre- dicted levels was stabilized in the bosentan group. In contrast the control group presented lower levels of %DLCO at the end of the follow-up (65.5±20.2% vs 60.5±19.9%; p<0.04) (Fig 4). In echocardiographic parameters we found a trend with more LV diastolic disfunction in the control group. We did not find any difference in other echocardiogram values (Table 2). Discussion Microvascular involvement is the main feature in SSc, and overexpression of type 1 endotelin (ET-1) is involved as a trigger for complications [26]. It is reasonable to consider that blocking ET-1 could be useful to treat SSc complications [15]. We found a possible protective effect of bosentan to develop PH in SSc. Patients with bosentan presented a prevalence of suspected PH of 13.8% vs 23.7% of patients without after adjusting to confounding factors. No statistical sig- nificance was acchieved, but taking into consideration that we used Fisher exact test, a 10% dif- ference between groups could be clinically outstanding. We estimated a number of 240 patients in each group to obtain a 80% of statistical power to find differences between groups. The multivariate results support a protective effect of bosentan. We found that patients treated with bosentan had 3.9 time-fold decrease to develop suspected PH by echo during the follow- up. Moreover we found that treated patients did not present an impairment of DLCO levels. PLOS ONE | https://doi.org/10.1371/journal.pone.0243651 December 10, 2020 4/9 PLOS ONE Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers This result is remarkable because worse values of % DLCO are associated with PAH higher risk [27,28]. To stabilize % DLCO values could be protective against PAH development. Echo parameters did not show significant differences between both groups. Considering all our results together a randomized controlled trial to confirm our data is strictly essential. The preventive effect of bosentan in SSc vascular complications has been only analyzed in two randomized controlled trials (RCT) involving DU prevention [6,9] that supports the use to prevent DUs in patients with SSc and previous history of DU. None other vascular compli- cations were analyzed in both trials. None RCTs that analyze the use of drugs for primary prevention of vascular complications in SSc exist. Our present retrospective case-control study is the first to demonstrate the lower risk to develop PAH (estimated by echocardiogram) in patients treated with bosentan. Never- theless, other authors analyzed if bosentan could be a protective effect of PAH in SSc. Only one study analyses the effect of bosentan to prevent PAH in the literature. Murdaca et al. followed 69 patients with SSc 25 of them with DUs treated with bosentan and compared them with the remaining 44. They did not found cases of PAH in bosentan group and 7 PAH-SSc cases in the control group. Indeed the investigators found an increase in echocardi- ography parameters in the control group and lower values in bosentan group and better parameters of terminal N of brain natriuretic peptide (NT-proBNP) at the end of the follow- up [29]. Some differences exist between those results and our study: firstly, no details of the number of patients with DUs in control group were given and they did not give a full compari- son with the other treatments. Moreover, they analyzed the effect of bosentan in NT-proBNP values but they did not study the effect of the treatment in %DLCO, a powerful predictive fac- tor for PAH. Romaniello et al. studied PAH and sPAP on echocardiogram in 54 SSc with DUs treated with bosentan without previous echocardiografic evidence of PH [30]. They did RHC in patients with sPAP � 45mmHg and compared sPAP of patients at the beginning and at the end of the study. They did not find differences in sPAP values and only one patient was diag- nosed of PAH by RHC. Similarly to our results, they did not find differences in echocardio- gram sPAP, but they did not compare treated group with control group and they did not analyze the effect of concomitant medication. In our study we lowered the bias produced by other treatments by control group and multivariate analysis. An observational retrospective study with 89 patients with SSc in treatment with bosentan to prevent DUs was published recently [31]. The authors found a lower prevalence of PH (3,4%) compared with other cohorts. This could imply a preventive effect of bosentan in PH. Unlike our study, the authors did not analyze the PH presence or echo data before treatment initiation and they did not have control group. Other authors compared 30 PAH-SSc patients treated with bosentan at least 6 months versus 30 SSc patients without PAH and without bosentan use [32] and demonstrated a significant lower frequency of DU in bosentan group (20% vs 53.3%; p<0.01). More interesting has appeared to explore the effect of bosentan treatment to stabilize % DLCO. A recent work in 10 PAH-SSc patients treated with bosentan authors found no decrease of %DLCO during treatment period [33]. Those data are similar with our findings. Seibold et al. found no differences in %DLCO between bosentan vs placebo group [34], but the design was made to study patients with interstitial lung disease that had low initial levels of %DLCO (%DLCO bosentan: 45.3±12.5 vs %DLCO placebo: 45.1±12.4), so this finding com- plicate the interpretation. The protective effect in SSc-PAH development was studied with other drugs. Caramaschi et al. analyzed retrospectively the incidence of severe vascular complications in 115 patients (10 of whom with bosentan) treated with iloprost for Raynaud’s phenomenon or DUs at least PLOS ONE | https://doi.org/10.1371/journal.pone.0243651 December 10, 2020 5/9 PLOS ONE Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers of 3 years [35]. They found an incidence rate of PAH during the follow-up lower than inci- dence published previously [36]. Thus, a possible protector effect of iloprost in development of PAH in SSc could exist. In our study, we found an OR 2.7 times fold-risk of PH in patients without prostanoids treatment. It is remarkable that in Caramaschi et al. study did not have a control group, and they had a group of patients treated with bosentan also who were not ana- lyzed. In addition, they did not study DLCO. Our data support a possible protector effect of prostanoids by the multivariate analysis. Nevertheless, not all data published are favorable for a protective effect of prostanoids. Airò et al. did not find differences in PH existence in 56 patients treated with iloprost when compared with 56 matched controls [37]. Another secondary endpoint in our study was if PDE-5 inhibitor treatment could be pro- tective against PAH development. In our study the OR to develop suspected PH was 3.75 times lower in comparison with no history of taking sildenafil. We had only 8 patients in the sildenafil group who never took bosentan. Unlike to bosentan there are no studies in the litera- ture that demonstrated a protective effect of sildenafil in a PAH. Our study presents several limitations. First the retrospective design limits the collection data, therefore we can not determine the effect of intermediate variables during the follow-up. Another limitation is the scarce epidemiological data and the no standardized length of the fol- low-up of the patients. Other confounding factor is the PH definition by echo. We know that the gold standard is RHC, but unfortunately we do not have RHC of all patients by our retro- spective design. Some parallel studies between RHC and echo demonstrated that sPAP and TRV correlates with haemodynamic parameters [38], but we cannot exclude false positive cases. We only include patients with DUs for two interesting reasons: the indication of use bosentan in Spain (prevention of DU or treatment of PAH) and the intention to have homoge- neous groups. Another lack for our study is that we did not analyze serological biomarkers than could be useful in PAH [39–41]. Sadly, we did not have values of most patients in both historical cohorts. It has been demonstrated a inverse correlation between DLCO and NT- proBNP [42–44] and taking into consideration the current evidence about DLCO role in PAH prognosis [27,41,43] our data suggest that DLCO stabilization in the case group could be also any kind of effect in NT-proBNP values. As a major concern, we fully consider the importance of DLCO as a predictor factor for DU and PAH in SSc. Steen et al. matched 106 patients who had PAH-SSc with 106 SSc patients [27]. They found that DLCO levels can predict the devel- opment of PAH-SSc. It suggests that a stabilization of DLCO could influence to develop PAH. Finally, another limitation is that our group did not study the survival fact that it would be interesting. In summary, our study demonstrates that patients who initiated bosentan to prevent DU have a lower risk to present PH estimated by echocardiography and stabilizes DLCO. We observed a modest effect with prostanoids and sildenafil as well. A randomized control trial is warranted to demonstrate a protective effect of these specific drugs. Supporting information S1 File. (XLSX) Author Contributions Conceptualization: Ivan Castellvı́, Carmen Pilar Simeón, Jordi Casademont, Hèctor Corominas. Data curation: Ivan Castellvı́, Carmen Pilar Simeón, Monica Sarmiento. PLOS ONE | https://doi.org/10.1371/journal.pone.0243651 December 10, 2020 6/9 PLOS ONE Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers Formal analysis: Ivan Castellvı́, Carmen Pilar Simeón, Monica Sarmiento, Hèctor Corominas. Investigation: Ivan Castellvı́, Carmen Pilar Simeón, Monica Sarmiento, Jordi Casademont. Methodology: Ivan Castellvı́, Carmen Pilar Simeón, Jordi Casademont, Hèctor Corominas. Project administration: Ivan Castellvı́, Carmen Pilar Simeón. Resources: Ivan Castellvı́, Carmen Pilar Simeón. Software: Ivan Castellvı́. Supervision: Ivan Castellvı́, Carmen Pilar Simeón. 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