Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers.

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 complication in systemic sclerosis patients and pulmonary hypertension is one of the leading causes of death. The use of bosentan has been shown to be useful for the treatment of pulmonary arterial hypertension and to prevent new digital ulcers. However, is unknown if bosentan can prevent pulmonary hypertension. Our objective was to determine if bosentan is useful to prevent pulmonary hypertension in SSc patients. A retrospective study in 237 systemic sclerosis patients with digital ulcers history treated or not with bosentan to prevent it was made. We analyzed the occurrence of pulmonary hypertension defined by an echocardiogram pulmonary arterial pressure > 40 mmHg in the entire cohort. Demographic, clinical, and treatment variables were recorded for all patients. Statistical significance was denoted by p values < 0.05. Fifty-nine patients were treated with bosentan a median of 34 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 bosentan had 3.9fold-increased risk of pulmonary hypertension compared with patients under bosentan treatment (p < 0.02). Moreover the percentage carbon monoxide diffusing capacity (DLCO) in bosentan treated patients did not decrease from baseline to the end of follow-up (61.8±14% vs 57±20.1%, p = 0.89). We concluded that Systemic sclerosis patients with digital ulcers treated with bosentan seems to have less risk to develop pulmonary hypertension and to stabilize DLCO.

Introduction

Systemic Sclerosis (SSc), also known as scleroderma, is an autoimmune disease characterized by microangiopathy, fibrotic disfunction and immune disregulation. Currently we have strong evidence of microvascular involvement at the beginning of the disease [1]. The most important vascular complications are digital ulcers (DUs) and pulmonary hypertension (PH). DUs are a frequent severe vascular complication with a prevalence of 40–50% [2-5] being an important cause of disability [6-8]. The endotelin receptor antagonist bosentan (BOS) demonstrated to 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 demonstrated 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 previous 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 history 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 controls 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 prominence 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 definition of PH. We considered that patients with estimated systolic pulmonary arterial pressure (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 percentage 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 independent 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 (independent 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 definition 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 Investigation 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 regurgitation velocity (TRV) (1.93±1.05 vs 2.42±0.87 m/s; p<0.05) at the end of the follow-up. 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.

Table 1

Baseline characteristics.

Characteristic	Total	Bosentan Group	Control Group	P-Value
	222	59	163
Sex
Female	201 (91)	56 (94.9)	145 (89.5)	0.29
Age years	63.9 (19.6)	49.2 (18.7)	69.2 (17.1)	< 0.0001
SSc duration	17.3 (9.3)	13.2 (8.3)	19.2 (9.1)	< 0.0001
dcSSc	65 (29.27)	28 (47.5)	37 (22.7)	< 0.01
Anticentromere Abs.	84 (35.4)	23 (38.9)	61 (37.4)	< 0.01
Antitopoisomerase Abs.	52 (21.9)	31 (52.4)	21 (12.9)	< 0.01
Raynaud’s Syndrome	222 (100)	59 (100)	163 (100)	1.00
Digital Ulcers (active or history)	222 (100)	59 (100)	163 (100)	1.00
Telangiectasias	150 (71.8)	37 (62.7)	113 (75.3)	0.09
Calcinosis	66 (31.7)	23 (39)	43 (28.9)	0.18
ILD involvement	109 (49.1)	29 (49.2)	80 (49.1)	1.00
Renal involvement	9 (4.1)	3 (5.1)	6 (3.7)	0.70
Gastrointestinal involvement	135 (64.9)	36 (61.0)	99 (66.4)	0.52
Heart involvement	19 (9.2)	4 (6.8)	15 (10.1)	0.59
Concomitant treatments
Calcium channel blockers	175 (85)	57 (96.6)	131 (80.4)	<0.01
PDE-5i	31 (14.3)	23 (39)	8 (5.1)	<0.0001
Prostanoids	28 (12.7)	13 (22)	15 (9.3)	<0.03
Statins	34 (16.5)	15 (25.4)	21 (12.9)	<0.04
Corticosteroids	85 (41.1)	28 (47.5)	57 (38.5)	0.27
Methotrexate	5 (2.4)	2 (3.4)	3 (2)	0.63
Cyclophosphamide	23 (11.1)	12 (20.3)	11 (7.4)	<0.02
Mycophenolate	22 (10.6)	15 (25.4)	7 (4.7)	<0.0001
Rituximab	4 (1.9)	3 (5.1)	1 (0.7)	0.08
First sPAP, mmHg	36.5 (14.6)	30.5 (5.3)	37.9 (15.8)	<0.04
Last sPAP, mmHg	39.1 (18.8)	34.4 (18.8)	41 (18.6)	<0.02
First FVC, %	83.5 (19.2)	82.5 (18.2)	83.8 (19.5)	0.83
Last FVC, %	77.8 (21.2)	79.8 (19)	77.2 (21.9)	0.52
First DLCO, %	64.4 (18.5)	61.8 (14)	65.5 (20.2)	0.22
Last DLCO, %	59.2 (19.9)	57.3 (20.1)	60.5 (19.9)	0.54

Data presented as n (%) of patients or mean ± S.D. SSc: Systemic sclerosis, dSSc: Diffuse cutaneous systemic sclerosis, ILD: Interstitial lung disease, sPAP: Systolic pulmonary arterial pressure by echocardiography, PDE-5i: Phosphodiesterase 5 inhibitors.

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.

Fig 1

Adverse effects related to bosentan.

AE: Adverse effect.

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 pulmonary 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).

Fig 2

Pulmonary hypertension development during the follow-up in bosentan and in control group.

PH: Estimated pulmonary hypertension by sPAP> 40mmHg in echocardiogram. OR (IC95%): 0,52 (0,22–1,19); p>0,1.

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).

Fig 3

Odds ratio (OR) to no develop pulmonary hypertension on echocardiography during the follow-up.

PDE-5 inh: Phosphodiesterase type 5 inhibitor. * p < 0.02; ¶ p < 0.03; ¥ p < 0.05.

Pulmonary function test and echocardiographic findings

We found that %DLCO predicted 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).

Fig 4

Change of % DLCO during the follow-up.

* p < 0.04.

Table 2

Echocardiographic parameters between groups.

Characteristic	Bosentan Group		Control Group		p value bosentan vs control
	Baseline	Follow-up	Baseline	Follow-up
% LVEF (SD)	65.5 (7.3)	62.7 (7.8)	63.8 (8.2)	63.7 (8.6)	0.29
sPAP, mmHg (SD)	30.5 (5.3)	34.4 (18.8)	37.9 (15.8)	41 (18.6)	0.016
TRV, m/s (SD)	1.59 (1.10)	2.29 (0.88)	2.05 (1.02)	2.48 (0.86)	0.068
TAPSE, mm (SD)	23.9 (3)	20.7 (5)	21.8 (2.8)	21.4 (3.9)	0.72
DD (%)	4 (12.1)	10 (25.6)	19 (14.3)	38 (34.5)	0.32
PE (%)	0	5 (12.8)	10 (7.5)	12 (11)	0.77

%LVEF: Percentage left ventricular ejection fraction; SD: Standard deviation; sPAP: Systolic pulmonary arterial pressure; TRV: Tricuspid regurgitation velocity; DD: Diastolic dysfunction; PE: Pericardic effusion.

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 significance was acchieved, but taking into consideration that we used Fisher exact test, a 10% difference 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. 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 complications 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. Nevertheless, 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 echocardiography 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 comparison 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 factor 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 diagnosed of PAH by RHC. Similarly to our results, they did not find differences in echocardiogram 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 complicate 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 of 3 years [35]. They found an incidence rate of PAH during the follow-up lower than incidence 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 analyzed. 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 protective 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 literature 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 follow-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 retrospective 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 homogeneous 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 development 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.

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2 Sep 2020

PONE-D-20-18616

Protective effect of bosentan against pulmonary hypertension development in systemic sclerosis patients with digital ulcers

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Reviewer #1: The authors report an important research regarding “Protective effect of bosentan against pulmonary hypertension development in systemic sclerosis patients with digital ulcers.”

The major weaknessess are lack of novelty and right heart catheterization to better define pulmonary artery hypertension.

Minor points:

- The authors report in the introduction “…Pulmonary Arterial Hypertension (PAH) is the most frequent cause of PH in SSc and a leading cause of death” buti t is not clear in the article if they are evaluating PAH or PH. Moreover any patients showed left ventricular dysfunction, are those patients affected by PH group II? What about group III?

-There were patients who were treated only with f PDE-5 inhibitor for treatment of digital ulcers?

Reviewer #2: A retrospective study investigating the role of bosentan in the prevention of Systemic Sclerosis- Pulmonary Arterial Hypertension (SSc-PAH) is of high interest even though there are lots of bias explaining that the title should be less affirmative. There are several issues/comments:

Major comments:

1.Title:

This study did not demostrates the effect of bosentan in the prevention of SSc-PAH. It described the occurrence of PAH in different groups of patients taken bosentan or not. The observational nature of the study does not allow to prospect any cause/effect relationship. Well-designed randomised controlled trials (RCTs) are needed to either support or refuse this hypothesis.

2.Material and methods (page 5):

-The variable, non-standardised length of follow-up represents a limitation.

-The incidental finding of high systolic pulmonary artery pressure on echocardiography is common and is not enough to diagnose pulmonary hypertension. Conditions associated with high systolic PA pressure on echocardiogram are not necessarily associated with PAH. Have you excluded patients with hemodynamically significant valvular disease, a history of uncontrolled systemic hypertension, hyperlipidaemia, cardiac failure, hepatic failure, diabetes, cerebrovascular diseases, peripheral vascular diseases, coagulopathy, smokers and pregnant or breastfeeding women?

3.Statistical analysis (page 6):

-Please, clarify more clearly which variables are included in multivariate regression analysis.

4.Results (page 7):

-Please specify in the results section why 15 of 237 enrolled patients were excluded from the study.

5.Table 1 (page 9):

- Please indicate the age, the prevalence of anticentromere and anti-topoisomerase I antibodies in both treatment and control group and the respective p-values.

- Although it is known, please explain the acronym dcSSc in the table.

- Please, add the p-value (i.e. 1.00) to the digital ulcers row and edit the term “Digital ulcers” to “Active digital ulcers or history of digital ulcers” as defined in Material and Methods section.

- I suggest using the term Renal involvement instead of SRC.

- Risk factors for SSc-PAH such as ACA, older age, longer disease duration, limited cutaneous SSc subset and presence of ILD might be considered (Y. Jianga et al. Autoimmunity Rev. 2020). I suggest therefore to add disease duration to baseline characteristics.

- About half of the cohort (49.1%) presented ILD. I suggest to specify other treatments (i.e. corticosteroids, immunosuppressive agents, cytotoxic drugs, or antifibrotic drugs) that could affects the study results.

Minor comments:

1.Abstract (page 2 line 3):

- In the Introduction I suggest to add the acronym PH (third line) if you want to use it later in the objective section.

2.Figure 3:

-Please edit the IC of Prostanoids to (1,0-7,0) as reported in the text (page 11).

3.Discussion (page 13):

-Please edit Discusion in Discussion.

**********

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Reviewer #1: No

Reviewer #2: No

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20 Oct 2020

Response to Editor and Reviewers

Dear Dr. Navarini, I hope you are well. First of all I would like to thank you and the reviewers your comments about our manuscript. We have read all the considerations with high interest. We are happy to notify that we modified the manuscript and answered your suggestions in the next lines. Please don't hesitate to contact with us if you need more requirements.

All the best

Ivan Castellví, MD; PhD.

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1. We reviewed all the manuscript to meet PLOS ONE’s style requirements.

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2. Done. Taking into consideration our design A post-hoc analysis was not performed. We included those explanation in material and methods

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3. We clarified it in Material and Methods part of the manuscript. Data of the patients were encrypted before to start our retrospective study.

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5. Thank you for your clarification. We modified it.

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6. Corrected. There was a mistake in one of author affiliation (sorry).

Thank you again.

Response to Reviewers:

Reviewer #1:

The authors report an important research regarding “Protective effect of bosentan against pulmonary hypertension development in systemic sclerosis patients with digital ulcers.”

The major weaknessess are lack of novelty and right heart catheterization to better define pulmonary artery hypertension.

Thank you for your comments. It's true that the main weakness of our study was the absence of the majority of RHC to design better than using an estimated sPAP by echocardiography. But our retrospective design and to use a historical cohort (with patients followed previous to current PAH detection recommendations by RHC) limits our work in this point.

Minor points:

- The authors report in the introduction “…Pulmonary Arterial Hypertension (PAH) is the most frequent cause of PH in SSc and a leading cause of death” but it is not clear in the article if they are evaluating PAH or PH. Moreover any patients showed left ventricular dysfunction, are those patients affected by PH group II? What about group III?

- Your comment is true definitely. Some patients can present group II or III (or combined I-II / I-III) of PH in SSc. But it is know that PAH (group I) is the most important cause of PH in SSc and is critical to find or to treat in the first phases of the (PH) disease. On the other hand bosentan only demonstrated be useful to treat group I PH (PAH), and our intention was in this retrospective study to investigate if (as in DUs) it drug can prevent PAH also. But (and in association with your comment previous your minor points) we can not are sure what kind of PH is in our patients without an haemodynamic study. Taking in consideration that we used echocardiography we use PH term. But our intention was evaluate the risk of PAH. Linked with this explanation we investigated and showed in the manuscript the effect of bosentan in other risk factors associated with PAH in SSc (DLCO).

-There were patients who were treated only with f PDE-5 inhibitor for treatment of digital ulcers?

- Thank you for your question. A total of 31 patients were in treatment for DUs with PDE-5 inhibitors. (23 concomitantly in bosentan group and eight in non bosentan group) as we shown in table 1.

Thank you again for your constructive comments.

Reviewer #2:

A retrospective study investigating the role of bosentan in the prevention of Systemic Sclerosis- Pulmonary Arterial Hypertension (SSc-PAH) is of high interest even though there are lots of bias explaining that the title should be less affirmative. There are several issues/comments:

Major comments:

1.Title:

This study did not demostrates the effect of bosentan in the prevention of SSc-PAH. It described the occurrence of PAH in different groups of patients taken bosentan or not. The observational nature of the study does not allow to prospect any cause/effect relationship. Well-designed randomised controlled trials (RCTs) are needed to either support or refuse this hypothesis.

1.Thank you for your observation. we only can agree with you. We changed the title for other more appropriate with our study.

2.Material and methods (page 5):

-The variable, non-standardised length of follow-up represents a limitation.

- It’s true. We included in the limitations of our study (page 15).

-The incidental finding of high systolic pulmonary artery pressure on echocardiography is common and is not enough to diagnose pulmonary hypertension. Conditions associated with high systolic PA pressure on echocardiogram are not necessarily associated with PAH. Have you excluded patients with hemodynamically significant valvular disease, a history of uncontrolled systemic hypertension, hyperlipidaemia, cardiac failure, hepatic failure, diabetes, cerebrovascular diseases, peripheral vascular diseases, coagulopathy, smokers and pregnant or breastfeeding women?

- Again you are absolutely on right. We did not exclude the major part of this risk factors by missing data in reviewed database (with an important number of historical patients). We also cited the lack of some epidemiological data in the limitations of our study (pages 15 & 16). We are sorry but we can’t recover this kind of missing data. On the other hand we think that, taking into consideration all those limitations (and the difficult to obtain data about this complication in patients with a rare disease), are relevant to take into account.

3.Statistical analysis (page 6):

-Please, clarify more clearly which variables are included in multivariate regression analysis.

- We re-edited the explanation with the intention to do this part more clear (page 6).

4.Results (page 7):

-Please specify in the results section why 15 of 237 enrolled patients were excluded from the study.

- We included the explanation in Results (page 7).

5.Table 1 (page 9):

- Please indicate the age, the prevalence of anticentromere and anti-topoisomerase I antibodies in both treatment and control group and the respective p-values.

- Done.

- Although it is known, please explain the acronym dcSSc in the table.

- Done.

- Please, add the p-value (i.e. 1.00) to the digital ulcers row and edit the term “Digital ulcers” to “Active digital ulcers or history of digital ulcers” as defined in Material and Methods section.

- Done.

- I suggest using the term Renal involvement instead of SRC.

- Thank you for the suggestion. Done.

- Risk factors for SSc-PAH such as ACA, older age, longer disease duration, limited cutaneous SSc subset and presence of ILD might be considered (Y. Jianga et al. Autoimmunity Rev. 2020). I suggest therefore to add disease duration to baseline characteristics.

- Thank you for your suggestion. We included disease duration in table 1.

- About half of the cohort (49.1%) presented ILD. I suggest to specify other treatments (i.e. corticosteroids, immunosuppressive agents, cytotoxic drugs, or antifibrotic drugs) that could affects the study results.

- We added the immunosuppressive agents in the table 1.

Minor comments:

1.Abstract (page 2 line 3):

- In the Introduction I suggest to add the acronym PH (third line) if you want to use it later in the objective section.

- Thank you for you comment. We changed “PH” for pulmonary hypertension ( in order to avoid acronyms in the abstract). We also modified the abstract according style requirements of PLOS ONE.

2.Figure 3:

-Please edit the IC of Prostanoids to (1,0-7,0) as reported in the text (page 11).

- Thank you for your observation. Corrected.

3.Discussion (page 13):

-Please edit Discusion in Discussion.

- We apologize by mistake. Corrected.

Thank you again for your constructive comments and suggestions.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

25 Nov 2020

Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers

PONE-D-20-18616R1

Dear Dr. Castellví,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Luca Navarini

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper is improved and all the suggestions have been raised. The paper is well written and the results are important in the management of systemic sclerosis complications

Reviewer #2: (No Response)

**********

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Reviewer #1: Yes: antonietta gigante

Reviewer #2: No

1 Dec 2020

PONE-D-20-18616R1

Effect of bosentan in pulmonary hypertension development in systemic sclerosis patients with digital ulcers

Dear Dr. Castellví:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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