Cover Page. The handle holds various files of this Leiden University dissertation

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1 Cover Page The handle holds various files of this Leiden University dissertation Author: Meijs, Jessica Title: Systemic sclerosis : from science to clinical practice Issue Date:

2 CHAPTER Blood flow in the hands of a predefined homogeneous systemic sclerosis population: the presence of digital ulcers and the improvement with bosentan Jessica Meijs, Alexandre E Voskuyl, Joanne PJ Bloemsaat-Minekus, Madelon C Vonk Rheumatology 2015;54:262-9.

3 ABSTRACT Objectives The aim of the study was to examine the effect of bosentan on blood flow in the hand in a subset of patients who had reduced blood flow relative to healthy subjects. Additionally, the relationship between blood flow in the hands of systemic sclerosis (SSc) patients and the presence of digital ulcers (DUs) was assessed. Methods SSc patients with a recent history of DUs and healthy subjects were included. Patients were classified into 4 subgroups: no current DUs or pitting scars; pitting scars only; new DUs; or persistent DUs. The hand was categorised into three regions of interest (ROI) and blood flow was measured by laser Doppler perfusion imaging at baseline, 4 and 12 weeks. Patients who had a reduction in blood flow of more than 50% relative to healthy control subjects in ROI 1 on baseline, were treated with bosentan for 12 weeks. Results Fifty-two SSc patients and 51 healthy subjects and were included in the analysis. There was no significant difference in blood flow in the hand across the patient subgroups at baseline. Sixteen SSc patients had a reduction of blood flow of 50% versus healthy subjects and received bosentan. Bosentan significantly (p<0.05) increased the blood flow in the whole hand after 12 weeks compared with baseline. Conclusions No relationship was found between blood flow in the hands and presence of DUs. After 12 weeks of bosentan treatment the blood flow had increased in the SSc patients but had not normalised to that of healthy subjects. 116 Chapter

4 INTRODUCTION Digital ulcers (DUs) are complications of SSc and arise as a result of ischaemia due to vasculopathy of the digital arteries(1). Nearly half of SSc patients are affected at some point with DUs (2). DUs cause pain and impair hand function, making it difficult to carry out normal daily activities such as eating and dressing, and have a severe impact on quality of life (3-5). In addition, DUs are susceptible to infection which, if not treated in the early stages, might lead to gangrene, osteomyelitis, septicaemia and amputation (2;3;6). Most investigations into pharmacological agents for DU prevention and healing have been small, pilot or open-label studies (-10). There have only been two large-scale randomized controlled clinical trials (RCTs) that have investigated the endothelin receptor antagonist (ERA) bosentan. In the Randomized Placebo-controlled Investigation of DUs in Scleroderma (RAPIDS) 1 and 2 studies, bosentan significantly reduced the number of new DUs in SSc patients (11;12). Bosentan has been shown to affect fibrosis (13;14) and vascular resistance in the lung capillaries (15). Nailfold capillaroscopy has also shown bosentan to have a positive effect on the vasculopathy of SSc patients (16;1). By blocking the receptors of endothelin, a potent vasoconstrictor, the observed effect on new DU occurrence with bosentan would be expected, at least in part, to be due to increased hand blood flow. However, neither RAPIDS 1 nor 2 included endpoints examining change in blood perfusion. Rosato et al. (18) showed that blood perfusion in the hands can be assessed in SSc patients using the non-invasive technique of laser Doppler perfusion imaging (LDPI). This technique demonstrated that patients with SSc had significantly reduced hand blood flow of 50% compared with healthy subjects. Additionally, they found that SSc patients who had pulmonary arterial hypertension (PAH) and were treated with bosentan had improved hand blood flow over time (18). However, as patients with active DUs were excluded from their study they were unable to relate blood flow to DU presence. Further research on the relationship between hand blood flow and DU presence is scare, as there has been limited research performed on bosentan s effects on hand blood flow. To further improve therapeutic management for DU patients, it is important to understand the relationship between blood flow restriction in the hands and DU status. Therefore the primary aim of this study was to determine the effect of bosentan on blood flow of the hands measured at 4 and 12 weeks after treatment initiation. Bosentan was allocated to patients who had 50% reduced blood flow relative to healthy subjects at baseline in at least one hand. Second, the aim was also to assess the relationship between blood flow of the hands, measured by LDPI, and DU status in SSc patients at baseline. METHODS Study design This was a prospective, non-randomized, open-label study involving seven centres in The Blood flow in the hands: digital ulcers and the improvement with bosentan 11

5 Netherlands. It was conducted in accordance with the International Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki. The protocol was approved by the ethics committees and institutional review boards of all centres and all patients provided written informed consent before study-related procedures. The trial is registered at (EudraCT number: ). Patients Patients were screened for eligibility from April to December Inclusion criteria were diagnosis of dcssc or LcSSc classified according to the ACR (19) or LeRoy (20) criteria of early SSc, a history of DU within the previous 2 years and age 18 years. Sex, age and smoking status for SSc patients and healthy subjects and SSc classification according to ACR or LeRoy criteria [19,20] and number of DUs for patients were recorded at baseline. DUs were defined as a loss of epithelialization and involving the epidermis, dermis, subcutaneous tissue and sometimes the bone located on the fingers or on the hands (21). This definition does not include fissures, paronychia, pitting scars or ulcers located over the MCP joints or elbows. Pitting scars were defined as small-sized hyperkeratosis (21). SSc patients were classified into four subgroups: no current DUs or pitting scars, pitting scars only, new DUs (i.e. present for <3 months) and persistent DUs (i.e. present >3 months). These subgroups were created post hoc as the DU subset and characteristics may significantly influence time to healing (21). Patients were excluded if they had previously used bosentan, had received parenteral prostanoid DU treatment within the 3 months prior to study initiation or were currently being treated with a phosphodiesterase type 5 inhibitor or an ERA. Other exclusion criteria included irreversible and significant limitation of hand function (e.g. amputation of more than one finger), a second significant peripheral vascular disease and serious medical co-morbidities. Patients were also excluded if they had a contraindication for bosentan. Medications, in particular vasoactive medications and those for DUs (other than those prohibited in the exclusion criteria), were allowed at a steady dose 1 month prior to inclusion and during the study. Antibiotic and analgesic doses were allowed to vary during the study. Healthy subjects Healthy adult control subjects were recruited via a flyer at participating hospitals. They were subject to the same inclusion and exclusion criteria as the patients, if relevant. LDPI assessment Blood flow [in arbitrary units (a.u.)] of both hands was measured using a LDPI scanner (PeriScan PiM 3, Perimed, Stockholm, Sweden). LDPI was performed in a quiet, air-conditioned room (the temperature was 20 C in all hospitals) after subjects had adjusted to the room temperature for at least 30 min. Patients and controls did not smoke and drank no coffee or alcoholic beverages for 4 h before the examination. Blood flow of the hands was 118 Chapter

6 measured by one observer blinded for patient characteristics (J. B). In order to measure the effect of bosentan on the blood flow of the hands, blood flow of the hands was measured by LDPI at 4 and 12 weeks after treatment initiation in a subset of patients who had a reduction of 50% in blood flow compared to healthy subjects. In order to evaluate the relationship between blood flow of both hands and DUs, blood flow was measured in all patients and healthy subjects at baseline. The LDPI scanner was placed perpendicularly 15 cm away from the hands. The laser sequentially scans 4096 (64 x 64 pixels) measurement points over the tissue under study, covering a maximal surface area of ~12 x 12cm. Two-dimensional images of superficial tissue blood perfusion were acquired at the highest time and spatial resolution. All perfusion signals were combined to form a colour-coded image using a scale ranging from dark blue (lowest value) to red (highest value) (18). The dorsal surface of the hand was categorized into three regions of interest (ROIs) for analysis, since blood flow perfusion differs between the proximal and distal regions of the hand (22). ROI 1 included the second, third and fourth finger of the hand from distal to the PIP joint. ROI 2 included the area between the PIP and MCP joint. ROI 3 included only the dorsal surface of the hand without the fingers. The perfusion gradient was defined as the difference between ROI 1 and ROI 3 (ROI 1 - ROI 3). A post hoc semi-quantitative assessment categorized the hand type by perfusion status according to LDPI into normal, low, high and mixed perfusion categories (see Fig. 1 for typical examples of each hand type). Fig. 1. Typical examples of hands as categorised by perfusion status ROI, region of interest; ROI 1, distal to the PIP joint of the middle 3 fingers; ROI 2, distal to the metacarpal joints and proximal to the PIP joint; ROI 3, the hand proximal to the metacarpal joint. Blood flow in the hands: digital ulcers and the improvement with bosentan 119

7 Treatment allocation Patients with a reduction in blood flow of >50% relative to healthy control subjects in ROI 1 in at least one hand were given bosentan for 12 weeks. Bosentan was administered at 62.5 mg twice daily for 4 weeks and 125 mg twice daily thereafter, tolerability permitting. Statistical analysis According to their distribution, continuous variables were presented as either mean (S.D.) or medians [interquartile range (IQR) 25th-5th percentiles]. Categorical variables were calculated as frequencies with percentages. All variables were compared between healthy subjects and the four SSc patient subgroups by using the Student s t-test. In order to analyse the differences in blood flow, healthy subjects and all SSc patients at baseline were compared post hoc using analysis of covariance (ANCOVA). Subject status, sex and smoking status were fixed effects and age was a covariate in the statistical model. In order to analyse the association of blood flow across SSc subgroups at baseline, regression analysis was used. A linear regression analysis was performed to determine the association between the four different DU stages as independent variables, with blood flow as the main outcome and adjusting for sex, age and smoking status. In order to measure an effect of bosentan on blood flow of the hands, a power was calculated based on the study of Rosato et al. (22), which showed that in patients with SSc-PAH bosentan significantly improved hand blood flow from 0.95 (S.D ) to (S.D ), resulting in a mean improvement of 14.6%, attaining values comparable to those observed in healthy subjects. This current study predicted a lower improvement in DU patients with a mean improvement of 10% (resulting in a mean difference of 0.08) as a result of differences in study population and a larger variation due to the multicentre design. The sample size needed for this study was 50 patients, calculated by using a paired t-test, assuming an alpha α of 0.05 and a power of 0.9 and an S.D. of the difference of 0.1 (based on the S.D. in Rosato et al. (18) and an assumed correlation of 0.3 between baseline and follow-up). Target recruitment was 56 SSc patients and a similar number of healthy subjects, since it was expected that 10% of the patients would not fulfil the inclusion criteria for treatment allocation or would be lost to follow-up. The change in hand blood flow from baseline to week 12 was analysed with a paired t-test and any missing values were replaced by the last value carried forward method (23). Healthy subjects blood flow at baseline was compared with blood flow at baseline and week 12 in SSc patients who received bosentan post hoc using ANCOVA to test significance. All data (including LDPI images) were analysed by one operator blinded for clinical data and diagnosis according to the intention-to-treat (ITT) principle. Two-sided P-values <0.05 were considered to be statistically significant. Data entry was performed using Microsoft Office Access 2003 (Microsoft, Redmond, WA, USA). All statistical analyses were executed using SPSS 1.0 software (SPSS, Chicago, IL, USA). 120 Chapter

8 RESULTS Demographics and study population Fifty-five SSc patients and 51 healthy subjects were screened. The demographics of SSc patients and healthy subjects are shown in Table 1. The healthy subjects were significantly (p=0.006) younger than the SSc patients. There was no difference in age within the SSc patient subgroups. No other demographic differences were observed. Of all 55 patients, three SSc patients had a DU in the past 2 years, but data on current DU status were missing. These three patients were included in the determination of the baseline blood flow only, but were excluded from the treatment allocation as they had a blood flow above the cut-off value. In total 52 patients were included in the analysis to investigate the relationship of hand blood flow and DU presence at screening. Of these 52 patients, 10 had no current DUs or pitting scars, 29 had pitting scars only, had new DUs and 6 had persistent DUs. Table 1. Demographics of healthy subjects and SSc patients at baseline Healthy All SSc SSc patients subgroups* subjects patients No DU Pitting scars New DU Persistent DU N=51 N=55 N=10 N=29 N= N=6 Mean±SD 45±16 53±12 55±14 50±12 59±11 51±8 age, years Sex Male Female Smoking Never Former Current Missing Diffuse SSc Not Limited applicable SSc Other *Data on number of DU was missing for three patients; DU, digital ulcers; SD, standard deviation The mean perfusion in ROI 1 among healthy subjects was 5.6 a.u. (S.D. 24.0). Eighteen patients had a reduction of blood flow 50% (i.e. <3.0 a.u.) in at least one hand and thus fulfilled the inclusion criteria for bosentan initiation. Of these 18 patients, 2 refused treatment. Sixteen patients started bosentan and were included in the ITT analyses; at week 12, Blood flow in the hands: digital ulcers and the improvement with bosentan 121

9 13 patients had completed 12 weeks of treatment, 2 had discontinued treatment but returned for the week 12 visit and 1 patient was lost to follow-up (week 4 data were imputed). The mean age of the 16 patients who received bosentan was 50 years (S.D. 11, range 36 66). Eleven patients were female and 11 were classified as having lcssc. Twelve patients had never smoked, one was an ex-smoker and three were current smokers. Current DU status was no DUs in two patients, pitting scars in eight patients and active DUs in six patients. Figure 2. Blood flow in healthy subjects and systemic sclerosis patient subgroups ROI: region of interest; DU: digital ulcer. Blood flow at baseline The mean blood flow in the whole hand was lowest in the SSc subgroup with persistent DUs [42. (S.D..)] and highest in the new DU group [61.4 (S.D..2)] (Fig. 2), although this difference was not statistically different. Similarly, blood flow tended to be higher for patients with new DUs compared with persistent DU patients in ROI 1, ROI 2 and ROI 3 (Figure 2). Linear regression analysis showed neither age, sex nor smoking had an effect on whole-hand blood flow in SSc patients (data not shown). Whole-hand blood flow and all ROIs varied widely within the SSc patients and healthy subjects (Fig.2 and Table 2). A post hoc analysis showed blood flow in ROI 1 was significantly lower for all SSc patients compared with healthy subjects (P=0.013). There were no statistically significant differences in the blood flow of ROI 2 or ROI 3 between the SSc patients and healthy control groups. The perfusion gradient was significantly (P<0.001) less pronounced in SSc patients [mean 13.6 (S.D. 21.6)] vs healthy subjects [mean 28.8 (S.D. 22.1)] and was absent in persistent DU patients [mean -3.4 (S.D. 13.5)]. A higher proportion of healthy subjects hands 122 Chapter

10 showed normal perfusion on LDPI than SSc patients (Table 3). SSc patients perfusion profiles showed a high proportion of abnormal perfusion patterns in the hands (Table 3). Table 2. Blood flow in healthy subjects and all SSc patients at baseline and SSc patients who received bosentan Healthy subjects N=51 SSc patients N=55 SSc patients who received bosentan N=16 Baseline Week 12 Whole hand Mean±SD 50.4± ± ± ±18.8 Median (range) ( ) ( ) ( ) ( ) P value compared to healthy < subjects ROI 1 Mean±SD 5.6± ± ± ±26.6 Median (range) ( ) ( ) ( ) ( ) P value compared to healthy < subjects ROI 2 Mean±SD 53.4± ± ± ±20.1 Median (range) ( ) ( ) ( ) ( ) P value compared to healthy < subjects ROI 3 Mean±SD 46.8± ± ± ±13.6 Median (range) ( ) ( ) ( ) ( ) P value compared to healthy subjects < ROI, region of interest; SD, standard deviation Change of blood flow in SSc patients treated with bosentan At baseline, the 16 SSc patients treated with bosentan had significantly lower blood flow in the whole hand and the three ROIs compared with healthy subjects (Table 2) (ANCOVA modelling P<0.001 for the whole hand and the three ROI comparisons). Compared with baseline, the blood flow increased significantly (P<0.05) in the Blood flow in the hands: digital ulcers and the improvement with bosentan 123

11 whole hand at week 12 (P<0.05) (Fig. 3). An increase in whole-hand blood flow was observed as early as week 4 (P<0.05). The largest change in blood flow was in ROI 1 at week 4 [5.3% (P<0.01)] and increased to 85.0% (P<0.001) at week 12. There was a significant change at week 12 in ROI 2 but not ROI 3 (Fig. 3). After 12 weeks of bosentan, the blood flow in the SSc patients was comparable to healthy control values in the whole hand and ROI 2 and ROI 3 (Table 2). In contrast, blood flow in ROI 1 after 12 weeks of bosentan increased but remained significantly reduced compared with healthy subjects (P=0.016). Table 3. Number and percentage of hand types categorised by perfusion status according to laser Doppler perfusion image Hand type category Number (%) of hands Healthy subjects SSc patients Total N=102 hands N=110 hands N=212 hands Normal perfusion (5.5) 40 (36.4) 11 (55.2) Low perfusion 13 (12.) 36 (32.) 49 (23.1) High perfusion 4 (3.9) 12 (10.9) 16 (.5) Mixed perfusion 8 (.8) 22 (20.0) 30 (14.2) Fig. 3. Change in blood flow from baseline to week 4 and 12 with bosentan treatment ROI: region of interest. 124 Chapter

12 DISCUSSION This study s main aim was to evaluate the effect of bosentan on the blood flow change in the whole hand from baseline to 12 weeks in patients who had a reduction in blood flow of 50% in the distal region of the fingers in at least one hand. Blood flow was shown to increase significantly after 12 weeks in the whole hand in SSc patients treated with bosentan compared with baseline. This improvement was found primarily in the fingers. The second aim was to investigate the relationship between hand blood flow of SSc patients and the presence of DUs. However, no such relationship was found. Using LDPI, Rosato et al. (18) showed that blood flow in the hands of SSc patients was significantly reduced compared with healthy controls. However, as patients with active DUs were excluded in that study, it was not possible to examine a relationship between blood flow and the presence of DUs. In our study, LDPI measured the hand blood flow of patients with and without current DUs, who had previously had DUs, and who had either new or persistent DUs or pitting scars. No significant relationship was found between blood flow and DU presence; a key finding was the wide individual variability in hand blood flow in each subgroup, which may explain the absence of a relationship. The highest restriction in blood flow was observed in persistent DU patients and the lowest in those with new DUs. There are several hypotheses for this phenomenon. First, it could be that the microvasculopathy is more impaired in patients with persistent DUs compared with those with new DUs. Second, it could be that blood may be directed to new DUs to aid healing. Increases in microcirculation, measured using LDPI, have been shown to occur during the healing process of venous ulcers (24). Additionally, blood flow measured by LDPI has been shown to be associated with burn wound healing, with high blood flow positively correlating with healing and, conversely, low blood flow correlating with a lack of healing (25). With persistent DUs, vasculopathy may be present to such an extent that it decreases blood flow to the fingertips and thus prevents DU healing. In line with previous studies, a wide individual variability in blood flow was also found in healthy control subjects (22;26;2). This result should be considered in future studies using LDPI to compare hand blood flow in healthy subjects and those with DUs. Despite individual variation in blood flow, SSc patients, compared with healthy subjects, did show a significant reduction in blood flow in the distal fingers, the region where blood flow is most pronounced in the hand. However, in the other ROIs and in the whole hand, blood flow was similar between SSc patients and healthy subjects. While blood flow was similar, the profile of perfusion images, when categorized as normal, low, high or mixed perfusion, were very different between SSc patients and healthy subjects. In a large proportion of SSc patients, normal perfusion was absent while abnormal perfusion patterns were more apparent, in particular low perfusion. The different perfusion patterns by patient could explain why we found no significant difference between SSc patients and healthy subjects in blood perfusion of the hand. Blood flow in the hands: digital ulcers and the improvement with bosentan 125

13 Bosentan has been shown to reduce the occurrence of new DUs (11;12). Studies with nailfold capillaroscopy have shown bosentan to have a positive effect on the vasculopathy in SSc patients (16;1). While it can be hypothesized that this may be partly due to increased blood flow through vasodilatory actions on the microvasculature, this has not been confirmed. In contrast to our results, Hettema et al. (28) did not find a significant change in vasodilator responses after 12 weeks of bosentan treatment. This can be explained by a different method of measuring blood flow and different inclusion criteria. In our study only patients with a blood flow <50% of normal were included. This study has several limitations that should be taken into account. First, based on the Rosato et al. data (18), the assumption was made that SSc patients enrolled in our study would have a marked reduction in blood flow in comparison with healthy subjects. However, this does not seem to be the case in an SSc population with either a history of DUs, or current DUs. Therefore a large number of patients were excluded from receiving bosentan due to the cut-off of a 50% reduction in blood flow in the distal fingers vs healthy subjects and so the calculated power of the study was not met. In particular, this cut-off excluded patients with new DU who had the highest levels of perfusion in the distal fingers. Since only patients with a high reduction in perfusion of hand blood flow were included to start bosentan, possibly regression to the mean was influencing the effect of bosentan on hand blood flow after 4 and 12 weeks. As such, this study has to be considered as a pilot study and additional research is advocated. Second, no reproducibility was measured. The studies of Correa et al, Cutolo et al. and Murray et al. (26;29;30) found a high reproducibility of the blood flow of the hands. The strength of this study is that this is the first study investigating blood flow in SSc patients with DUs (18;31). Our small study s data provide a rationale for further research, ideally in larger placebo-controlled RCTs, into the relationship between drugs that increase hand blood flow and DU prevention and healing. In particular, the absence of a perfusion gradient in patients with persistent DUs is of interest for future studies. In conclusion, blood flow in the fingertips of SSc patients was reduced compared with healthy subjects. However, no relationship with the presence of a DU or pitting scar could be found. We did observe different distribution patterns of blood flow by SSc patients compared with healthy subjects. No relationship was found between hand blood flow in SSc patients and DU presence. However, we did show that in patients with an initial low perfusion status, blood flow to the fingertips increased during bosentan treatment over 12 weeks. 126 Chapter

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16 (2) Rosato E, Molinaro I, Rossi C, Pisarri S, Salsano F. The combination of laser Doppler perfusion imaging and photoplethysmography is useful in the characterization of scleroderma and primary Raynaud s phenomenon. Scand J Rheumatol 2011;40(4): (28) Hettema ME, Zhang D, Stienstra Y, Smit AJ, Bootsma H, Kallenberg CG. No effects of bosentan on microvasculature in patients with limited cutaneous systemic sclerosis. Clin Rheumatol 2009 July;28(): (29) Correa MJ, Andrade LE, Kayser C. Comparison of laser Doppler imaging, fingertip lacticemy test, and nailfold capillaroscopy for assessment of digital microcirculation in systemic sclerosis. Arthritis Res Ther 2010;12(4):R15. (30) Cutolo M, Ferrone C, Pizzorni C, Soldano S, Seriolo B, Sulli A. Peripheral blood perfusion correlates with microvascular abnormalities in systemic sclerosis: a laser-doppler and nailfold videocapillaroscopy study. J Rheumatol 2010 June;3(6): (31) Rosato E, Rossi C, Molinaro I, Giovannetti A, Pisarri S, Salsano F. Laser Doppler perfusion imaging in systemic sclerosis impaired response to cold stimulation involves digits and hand dorsum. Rheumatology (Oxford) 2011 September;50(9): Blood flow in the hands: digital ulcers and the improvement with bosentan 129

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