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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Increased endothelin activity may play a role in the pathogenesis of vascular injury, a primary feature of systemic sclerosis (SSc; scleroderma). Our goal was to test the hypothesis that treatment with the oral endothelin receptor antagonist bosentan might improve vascular endothelial function in SSc patients.

Methods

A 4-week, prospective, parallel-group study compared 12 SSc patients who did not receive bosentan treatment with 12 patients who did receive treatment (125 mg/day) for pulmonary hypertension and/or digital ulcers. There were no differences in demographic and clinical characteristics or medications between the 2 groups. Baseline endothelial dysfunction was documented by decreased brachial artery ultrasound-derived flow-mediated dilation (FMD%; <5.5). Pulse wave analysis, venous occlusion plethysmography, and measurement of serum vascular markers were performed in parallel.

Results

FMD%, the main end point, increased significantly from a mean ± SD of 3.1 ± 1.3% to 8.4 ± 2.6% after 4 weeks of bosentan treatment (P < 0.001, compared with a change from 2.4 ± 1.6% to 2.4 ± 2.2% in control patients). Arterial blood pressure, endothelium-independent vascular function, augmentation index, peripheral flow reserve, as well as circulating intercellular adhesion molecule 1, E-selectin, vascular endothelial growth factor, and endothelin 1 were not significantly affected by bosentan treatment. In patients continuously treated for 4 months, during which the dosage of bosentan remained at 125 mg/day (n = 5) or increased to 250 mg/day (n = 5), the 4-week results remained unchanged.

Conclusion

Small doses of bosentan improve endothelial function without affecting hemodynamic parameters or endothelial activation–related processes, thus supporting a direct, reversible effect of endothelin in SSc-associated vascular injury. A long-term, controlled trial to examine the potentially global clinical benefit of endothelin receptor blockade in patients with early SSc may be warranted.

Systemic sclerosis (SSc; scleroderma) is a complex, immune-mediated disease associated with high mortality rates (1, 2). Although SSc has vascular, immunologic, and fibrotic components that are pathologically interconnected (1, 2), one hypothesis suggests that endothelial damage and vascular dysfunction may be some of the earliest pathogenetic alterations (3, 4). Functional abnormalities of the blood vessels, e.g., vasoconstriction, and structural changes, including intimal proliferation and obstruction, are expressed clinically as Raynaud's phenomenon, digital ulcers, renal and myocardial disease, and pulmonary hypertension (5).

Endothelial damage in SSc, whether caused by immunologic stimuli, ischemia-reperfusion injury, or other pathways, has many consequences, including increased production of endothelin (1, 2, 6). This endothelium-derived peptide, which is involved in the regulation of vascular function under normal physiologic conditions (7), plays a key role in vascular pathologies by exerting various deleterious effects, including hypertrophy of the vascular smooth muscle cells, cellular proliferation and fibrosis, increased vascular permeability, activation of leukocytes, and induction of cytokine and adhesion molecule expression (6, 8). Moreover, endothelin is the most potent naturally occurring vasoconstrictive mediator and when exogenously administered to healthy volunteers, a marked dose-dependent reduction of the forearm blood flow occurs (9). The effects of endothelin are transmitted upon binding to 2 cognate receptors, ETA and ETB, which are mainly expressed on endothelial cells (ETB), smooth muscle cells, and fibroblasts (6, 8).

Bosentan is a specific, orally active, dual endothelin receptor antagonist (10) that has recently been approved for the treatment of pulmonary hypertension. Two randomized, controlled studies have shown that bosentan reduces symptoms, improves exercise capacity and hemodynamics, and delays clinical worsening in patients with pulmonary hypertension (11, 12). A subgroup analysis of these trials and their open-label extensions in patients with connective tissue diseases showed that bosentan treatment may have a positive effect on outcome (13). Additional retrospective studies also suggest that bosentan treatment is clinically beneficial in SSc patients with pulmonary hypertension (14, 15), including patients with restrictive lung disease (15). Moreover, in a randomized, placebo-controlled trial of bosentan for SSc-related digital ulcers, although there was no difference between treatment groups in the healing of existing ulcers, bosentan prevented new digital ulcers and improved hand function (16).

Elevated circulating levels of endothelin have been repeatedly observed in SSc (for review, see ref.6), as well as in various other pathologies of the vascular endothelium (17), but whether increased endothelin activity is a causal factor of endothelial dysfunction and/or damage or an epiphenomenon remains unclear (17–19). Experimental studies have shown an improvement in the endothelial function of large arteries, assessed ex vivo, following short-term administration of endothelin receptor antagonists in animal models of hypertension (20, 21) and atherosclerosis (22), suggesting that some of the endothelin-mediated deleterious effects on the vasculature are reversible.

To the best of our knowledge, the direct or indirect effects of oral bosentan treatment on vascular endothelial dysfunction have not been previously addressed. To further study the effects of this treatment approach in SSc, we tested the hypothesis that bosentan, administered for pulmonary hypertension or digital ulcers in these patients, would improve endothelial function assessed by means of noninvasive, high-resolution brachial artery ultrasound.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Patient selection and study design.

Twenty-four patients with SSc who fulfilled the various American College of Rheumatology (formerly, the American Rheumatism Association) SSc classification criteria (23) participated in this prospective, parallel-group, interventional study. The main inclusion criterion was the presence of endothelial dysfunction at baseline, documented by decreased brachial artery ultrasound-derived flow-mediated dilation (FMD%) (24, 25). To avoid any possible mismatch between groups, patients with previous myocardial infarctions or stroke, valvular or congenital heart disease, congestive heart failure, diabetes mellitus, hypertrophic cardiomyopathy, dyslipidemia, history of smoking, or history of arterial hypertension before the diagnosis of SSc were excluded. Patients were randomly allocated into 2 groups, those who received 62.5 mg bosentan twice daily for 1 month (bosentan-treated group; n = 12) and those who received no treatment during the same period (control patients; n = 12). The randomization was carried out using the dynamic random allocation system. There were no significant differences between the 2 groups in demographic and clinical characteristics or in medications received (Table 1).

Table 1. Demographic and clinical characteristics of the SSc patients, by group*
 Bosentan-treated group (n = 12)Untreated control group (n = 12)
  • *

    Except where indicated otherwise, values are the number of patients. SSc = systemic sclerosis; FMD% = flow-mediated dilation; ACE = angiotensin-converting enzyme; NSAIDs = nonsteroidal antiinflammatory drugs.

Females/males11/110/2
Age, mean ± SD (range) years53 ± 15 (27–75)54 ± 12 (28–75)
Disease duration, mean ± SD (range) years10.4 ± 7.7 (2–30)10.1 ± 9.7 (1–30)
Diffuse SSc99
Limited SSc33
FMD%, mean ± SD (range)3.1 ± 1.3 (0.1–5.1)2.4 ± 1.7 (0.1–5.1)
Pulmonary hypertension75
Digital ulcers910
Concomitant treatment  
 Nifedipine98
 Amlodipine34
 ACE inhibitors45
 Furosemide21
 Corticosteroids67
 NSAIDs53
 Methotrexate21
 Levothyroxine11
 Domperidone1110
 Omeprazole109

Vascular studies were performed in a blinded manner in all patients at baseline and after 4 weeks. Serum levels of soluble intercellular adhesion molecule 1 (ICAM-1), E-selectin, vascular endothelial growth factor (VEGF), and endothelin 1 (ET-1) were measured in parallel. Clinical evaluation and ultrasound recordings of systolic pulmonary pressure were performed in all patients, as previously described (26). The primary end point was the possible bosentan-induced changes in brachial artery FMD% at the end of the first 4 weeks of treatment. Secondary end points included possible changes in brachial artery endothelium-independent, nitroglycerin-induced dilation (NTG%) and other hemodynamic parameters described below, as well as in serum levels of the studied vascular markers. The dosages of all concomitant medications remained unchanged during the study.

Vascular studies and measurements of circulating ICAM-1 and E-selectin were repeated in 10 of the 12 patients in the bosentan-treated group after 4 months of continuous treatment, while the remaining 2 patients did not return for followup at this point. The bosentan dosage during these months remained stable in the 5 patients for whom the drug was provided for skin ulcers (125 mg/day), or increased in 5 patients in the second month to 250 mg/day, according to the standard guidelines for the treatment of pulmonary hypertension. The study protocol was approved by the Ethics Committee, Alexandra Hospital, and all patients provided informed consent.

Vascular studies.

All blood sampling and vascular studies were performed at the same time of day (9:00 AM to 12:00 PM). All patients abstained from food, smoking, caffeine, and alcohol for 12 hours before the studies were performed. All medications were discontinued 12 hours before each study. Each patient rested in a supine position for 10 minutes in a quiet room with the temperature controlled at 20–25°C. All vascular studies were subsequently performed by the same experienced physician (CP), who was blinded to the case or control assignment of the patient examined. Premenopausal women (n = 3) were examined during any day of their menstrual period except during the menstrual phase. Studies were performed in the following fixed order: applanation tonometry, plethysmography, brachial artery FMD%, and NTG%, in order to avoid bias due to the hemodynamic effects of nitroglycerin.

Brachial artery reactivity.

Brachial artery reactivity was assessed by measuring both endothelium-dependent and endothelium-independent vasodilation (24, 25). Briefly, during each visit, scans of the brachial artery were recorded while the patient was at rest, during reactive hyperemia, and after administration of nitrate, using B-mode ultrasound imaging with a 7.0-MHz linear array transducer (Acuson 128XP; Siemens, Mountain View, CA). FMD% was induced in response to reactive hyperemia after deflation of a wrist cuff, and was expressed as the percentage change in the internal diameter of the brachial artery from the baseline diameter. The degree of reactive hyperemia was considered the stimulus for FMD% and was defined as the percentage increase in postischemic blood flow over resting blood flow. NTG% was measured 4 minutes after sublingual administration of 400 μg nitroglycerin, provided that no contraindication existed, and was expressed as the percentage change in brachial artery diameter from the baseline diameter. NTG% is considered to be independent of endothelial function (24, 25). The mean ± SD intraobserver variability for measurements of brachial artery diameter was 0.1 ± 0.2 mm, while the FMD% variability measured in the same patient on 2 different days was mean ± SD 1.1 ± 1.0%.

Venous occlusion plethysmography.

Strain gauge venous occlusion plethysmography, a well-known and highly accurate method for the study of small arterioles (27), was used to study forearm blood flow. These studies were performed using a strain gauge plethysmograph (model EC5R; Hokanson, Bellevue, WA). Mercury-filled Silastic strain gauges of the appropriate size for each patient were placed 5 cm below the antecubital crease, while a rapid cuff inflator (model E20; Hokanson) and an air source (model AG101; Hokanson) were used to inflate a vascular cuff to 50 mm Hg; this was used to occlude venous outflow from the extremity at the level of the upper arm. Reactive hyperemia was induced by inflating a wrist cuff to suprasystolic pressure for 5 minutes. It is known that arterial occlusion of such duration produces almost maximal vasodilation of the blood vessels and maximal peak reactive hyperemia (28). Forearm blood flow was calculated as the relative percentage change in limb volume (ml/minute per 100 ml), while peripheral flow reserve was calculated as the ratio of maximal forearm blood flow during hyperemia after forearm ischemia to the resting forearm blood flow.

Pulse wave analysis.

Radial artery applanation tonometry was used to obtain and analyze the pulse waveform of the aorta (SphygmoCor waveform analysis system; AtCor Medical, Sydney, New South Wales, Australia). Peripheral pressure waveforms were recorded at the radial artery using a hand-held high-fidelity tonometer (Millar Instruments, Houston, TX) and calibrated by using arterial pressures measured at the brachial artery. Aortic pressure waveforms were then calculated by applying generalized transfer functions, as previously described (29). Analysis of the derived aortic waveform allows for the calculation of indices, such as the augmentation index (AI), which partly correspond to the measure of arterial stiffness. The AI is defined as the ratio of the augmentation of systolic pressure induced by the reflected waves to the aortic pulse pressure. Pulse wave analysis is considered a highly reproducible method by which to assess AI (30).

Measurements of circulating vascular markers.

Circulating levels of soluble ICAM-1 and E-selectin were measured by quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) (Bender Medsystems, Vienna, Austria) of serum that had been kept at −70°C. Control serum samples were obtained from 20 female age-matched blood donors. The ICAM-1 ELISA uses 2 different mouse monoclonal antibodies against extracellular domain D4 (CL203.4) and domain D2 (R6.5) of the soluble molecule. This assay is highly specific for measuring human soluble ICAM-1 and has a minimum sensitivity of 0.625 ng/ml. The ELISA for soluble E-selectin uses 2 different mouse monoclonal antibodies against the soluble molecule. This method has a minimum sensitivity of 0.5 ng/ml and an average recovery of 95% and 101%, respectively, with the 2 mouse monoclonal antibodies.

Serum levels of VEGF and ET-1 in all samples obtained at baseline and after 4 weeks were measured by ELISA (both kits from R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. VEGF and ET-1 ELISA kits had a minimum sensitivity of 1.6 and 1.0 pg/ml, respectively. Although intra- and interassay coefficients of variation with all methods have been found to be <5% at our laboratory, baseline and 4-week measurements of the 4 vascular markers under study were performed using 1 ELISA kit.

Statistical analysis.

Results are expressed as the mean ± SD. Normal distribution of the parameters under study was verified using the 1-sample Kolmogorov-Smirnov test. Thus, parametric tests were used to compare continuous variables between and within the 2 groups, as appropriate. In particular, a 2-sample t-test was used to compare continuous variables between the 2 groups at the baseline. To compare the change in a given vascular parameter between the 2 groups during followup, analysis of covariance (ANCOVA) was used with the followup scores as the dependent variable and the baseline measures as a covariate.

A secondary analysis was also performed using Pearson's correlation coefficient in order to compare the number of patients in the bosentan-treated group and the control patients who reached the 5.5% level of FMD% (considered the lower normal limit of FMD% in our laboratory) after 4 weeks. Spearman's correlation coefficient was estimated in bivariate correlations between changes in FMD% and systolic pulmonary pressure. Finally, repeated-measures analysis of variance (ANOVA) was used to determine the effect of bosentan treatment on a given parameter over time, e.g., from baseline through the first (i.e., 4-week measurements) and fourth months. P values less than 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Baseline vascular measurements.

Individual baseline FMD% values in all 24 bosentan-treated patients and control patients were <5.5%, and ranged from 0.10% to 5.40% (mean ± SD 2.7 ± 1.5%). These values were indicative of endothelial dysfunction, since 5.5% is considered to be the lower limit of FMD%, derived from the study of patients free of known conditions related to endothelial dysfunction, by the same physician (CP). Thus, the FMD% values in patients with SSc were profoundly reduced compared with 52 age- and sex-matched controls (8.2 ± 2.7%; P < 0.0001). In contrast, baseline values of NTG% (13.9 ± 6.5%; n = 21), AI (30.7 ± 12.3%; n = 24), and forearm blood flow (4.2 ± 1.4%; n = 23) were not different compared with those in matched healthy controls (16.7 ± 6.8%, n = 23; 27.4 ± 11.3%, n = 33; 4.7 ± 2.7%, n = 33, respectively). The mean ± SD value of peripheral flow reserve was lower in patients with SSc compared with 30 matched healthy controls (2.0 ± 0.6% versus 2.5 ± 0.7%; P = 0.04).

As shown in Table 2, the mean values of hemodynamic parameters and serum concentrations of vascular markers did not differ between the control patients and the bosentan-treated group at baseline. Systolic pulmonary pressure at baseline exceeded 35 mm Hg, indicative of pulmonary hypertension, in 7 of 12 patients in the bosentan-treated group and in 5 of the 12 control patients (Table 1) (mean ± SD 59 ± 12 mm Hg and 48 ± 7 mm Hg, respectively). The presence of pulmonary hypertension at baseline did not correlate significantly with serum concentrations of ICAM-1, E-selectin, VEGF, or ET-1 in the patients (data not shown).

Table 2. Effects of bosentan treatment on hemodynamic parameters and serum concentrations of vascular markers*
 Bosentan-treated groupUntreated control group
Baseline4 weeksBaseline4 weeks
  • *

    Values are the mean ± SD. BP = blood pressure; NTG% = nitroglycerin-induced dilation; AI = augmentation index; ICAM-1 = intercellular adhesion molecule 1; VEGF = vascular endothelial growth factor.

Systolic BP, mm Hg121 ± 29118 ± 26124 ± 16120 ± 17
Diastolic BP, mm Hg70 ± 1564 ± 1075 ± 1069 ± 12
Reactive hyperemia, %199 ± 74214 ± 100198 ± 83186 ± 38
NTG%13.1 ± 4.618.8 ± 6.813.9 ± 8.419.2 ± 5.6
AI, %31.1 ± 15.828.6 ± 12.630.2 ± 9.530.5 ± 10.9
Forearm blood flow, ml/minute/100 ml4.2 ± 1.64.4 ± 1.24.1 ± 1.24.0 ± 1.1
Peripheral flow reserve2.2 ± 0.61.9 ± 0.51.8 ± 0.61.7 ± 0.5
ICAM-1, ng/ml1,390 ± 3951,466 ± 3681,640 ± 5311,573 ± 419
E-selectin, ng/ml63 ± 3569 ± 3077 ± 4179 ± 29
VEGF, pg/ml339 ± 201320 ± 221449 ± 244429 ± 225
Endothelin 1, pg/ml3.5 ± 0.53.5 ± 0.44.1 ± 13.7 ± 0.8

Endothelial function after 4 weeks of bosentan treatment.

Vascular studies were repeated in all patients 4 weeks after the study baseline. In the bosentan-treated (125 mg/day) patients, FMD% increased from 3.1 ± 1.3% to 8.4 ± 2.6% (mean ± SD change 5.3 ± 3.4%), with 11 of 12 patients exceeding the 5.5% value; FMD% in the other patient decreased from 4.9% to 2.6%. Conversely, only 1 of 12 control patients had an FMD% >5.5% at 4 weeks (P < 0.0001). This 2.7-fold increase was significant (P < 0.001) compared with the control patients, in which FMD% remained essentially unchanged (mean ± SD change −0.1 ± 3.0%) (Figure 1). In contrast, the values of arterial blood pressure, reactive hyperemia, NTG%, AI, forearm blood flow, and peripheral flow reserve did not change significantly from baseline values in either the bosentan-treated group or the group of control patients (Table 2).

thumbnail image

Figure 1. Effect of bosentan treatment on flow-mediated dilation (FMD%) at 4 weeks. Endothelium-dependent FMD% increased from baseline to 4 weeks in 12 bosentan-treated patients with systemic sclerosis and endothelial dysfunction compared with 12 patients with similar characteristics who did not receive bosentan (P = 0.001). Bars represent the mean; lines represent the 95% confidence interval.

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Treatment with bosentan reduced systolic pulmonary pressure in all 7 patients with pulmonary hypertension, from a mean ± SD of 59 ± 12 mm Hg to 48 ± 6.5 mm Hg (P < 0.004). Within this subgroup, bivariate correlations comparing the reduction in pulmonary pressure with the bosentan-induced increase in FMD% revealed an inverse correlation (ρ − 0.7) with marginal significance (P = 0.07). Bosentan treatment was well tolerated, and no adverse events, including elevations in transaminase values, were noted.

Sustained effect of bosentan treatment on endothelial dysfunction.

Vascular studies were repeated at the end of 4 months in 10 of 12 patients who received continuous treatment, during which the dosage of bosentan remained at 125 mg/day or was increased to 250 mg/day in the 5 patients with pulmonary hypertension, according to dosing guidelines. Repeated-measures ANOVA showed that the significantly increased FMD% values seen at the end of the 4-week treatment period were sustained at the end of 4 months in these patients (Figure 2). The patient in whom a reduction in FMD% was observed at 4 weeks after baseline improved remarkably, with an FMD% of 8.1% after 4 months. The mean values of reactive hyperemia and NTG%, AI, peripheral flow reserve, and arterial blood pressure did not change significantly after 4 months compared with baseline values. A separate analysis of patients who received 125 or 250 mg of bosentan daily did not reveal a significant dose-related effect (data not shown). Bosentan treatment was well tolerated and no adverse events were noted.

thumbnail image

Figure 2. Effects of bosentan treatment on flow-mediated dilation (FMD%) and levels of soluble adhesion molecules in 10 patients with systemic sclerosis. Bosentan had beneficial effects on endothelium-dependent FMD% after 4 weeks and 4 months of treatment (P = 0.0006) (A), but its effects on circulating levels of soluble intercellular adhesion molecule 1 (ICAM-1) (B) and E-selectin (C) were not significant. Bars represent the mean; lines represent the 95% confidence interval.

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Effects of bosentan treatment on circulating vascular markers.

Baseline serum concentrations of soluble ICAM-1 and E-selectin, both in the bosentan-treated group and the control patient group (Table 2), were increased compared with concentrations in sera from healthy controls (mean ± SD 450 ± 85 ng/ml and 30 ± 17 ng/ml, respectively, in healthy controls; P < 0.001 versus bosentan-treated and control patients). As shown in Table 2, serum levels of soluble adhesion molecules, as well as VEGF and ET-1, did not change significantly in either the bosentan-treated group or the group of control patients at 4 weeks. Repeated measurements of soluble ICAM-1 and E-selectin in serum derived from the 10 patients after 4 months of continuous treatment were no different from the respective baseline levels, and repeated-measures ANOVA again showed a nonsignificant effect of bosentan treatment (Figure 2).

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

After the vascular hypothesis of SSc pathogenesis was introduced (3), endothelial cell function became the focus of research aimed at a better understanding of the pathogenesis of SSc, leading to more effective treatments (4, 31). Indeed, drugs currently used to treat the vascular complications of SSc, including bosentan for pulmonary arterial hypertension, have increased survival rates and can truly be classified as disease-modifying compounds (2). The present study was designed specifically to examine whether endothelial dysfunction of medium-sized conduit arteries, which are readily accessible for measurement, is affected by oral bosentan treatment. Endothelium-dependent and -independent vascular function was assessed by measuring brachial artery FMD% and NTG%, i.e., the change in diameter during hand hyperemia and after sublingual glyceryl trinitrate administration, respectively. This technique is attractive because it is noninvasive and allows for repeated measurements (24, 25).

FMD% is nitric oxide (NO)–dependent in humans and it is considered to be a reliable marker of endothelial function (32). As previous studies from our laboratory have shown (33, 34), this method can be applied in SSc patients without technical difficulties related to the thickened skin, while decreased brachial artery FMD% is a common finding in these patients (33–35). Using this approach, a highly significant improvement of endothelial function to a level comparable to that in healthy individuals, which was not due to changes in the degree of reactive hyperemia, was found. Moreover, all medications had to be discontinued for 12 hours before the vascular studies, thus implying that the bosentan-induced beneficial effect was probably due to rather long-lasting changes.

In contrast, bosentan treatment had no apparent effect on NTG%, which is considered to be NO-independent (24, 25, 32). Although the mechanism of bosentan-induced effect was not directly addressed in the present study, the results suggest that the improvement in endothelial function was mediated by enhanced NO production. Since increased endothelin activity inhibits NO synthesis (36), which has been found to be impaired in SSc (37), the bosentan-induced increase in FMD% is not surprising. Accordingly, using endothelial NO synthase–deficient mice, Gonon et al (38) showed that bosentan preserves endothelial and cardiac contractile function during ischemia and reperfusion via a mechanism dependent on endothelial NO production.

Improved endothelial dysfunction in the brachial artery may thus imply that inhibition of the endothelin/endothelin receptor system promotes NO bioavailability in different organ systems. Along this line, Girgis et al (39) have found that decreased exhaled NO in patients with pulmonary arterial hypertension is reversed with bosentan treatment. An improvement in endothelial function within the lungs induced by bosentan might therefore contribute to the beneficial effects of this treatment in pulmonary hypertension (11, 12), which is consistent with the inverse correlation between pulmonary pressure and FMD% changes observed in our patients.

Treatment with bosentan at a dosage of 125 mg/day had no apparent effect on systemic vasodilation, since arterial blood pressure was not significantly affected. This is consistent with previous reports, in which daily administration of 100 mg of bosentan did not affect the sympathetic nervous system or the renin– angiotensin–aldosterone system (40). Also, venous occlusion plethysmography failed to reveal an apparent effect of bosentan on peripheral flow reserve, probably because irreversible structural changes had already occurred in the microvasculature of patients with advanced, severe SSc (5).

Alternatively, the daily dose of 125 mg may be insufficient to reach the maximal effect, particularly on structural changes. This dose is efficacious, but the maximal effect of decreasing pulmonary pressure in patients with pulmonary hypertension is achieved with a daily dose of 250 mg (11, 12). Although the gold standard noninvasive measure of arterial stiffness, i.e., the radial–femoral or carotid–femoral pulse wave velocity, was not available, the parallel measurements of AI, an indirect marker of arterial stiffness, and peripheral vascular resistance (29), were similar before and after treatment, suggesting that bosentan exerted a direct effect on endothelial function.

We also determined, by measuring soluble ICAM-1 and E-selectin concentrations, whether the effect of bosentan on NO-dependent endothelial dysfunction is related to alterations in proinflammatory processes. Other findings suggest that increased concentrations of ICAM-1 in the serum, which are commonly found in patients with SSc (41), may contribute to impaired vasomotor function in forearm microvessels (42), but may also be related to FMD% in healthy individuals, independently of cardiovascular risk factors (43). Moreover, Xu et al (44) have shown that endothelin up-regulates ICAM-1 expression in normal and SSc-derived fibroblasts. In contrast to other molecules with wider cell-type expression, soluble E-selectin is shed only by activated endothelium, and elevated circulating levels have been found in various diseases, including SSc (for review, see ref.45). We found that treatment with bosentan for 4 weeks had no apparent effect on the increased baseline levels of either soluble ICAM-1 or E-selectin, which remained elevated for as long as 4 months after treatment.

Moreover, we observed that bosentan did not significantly modify serum concentrations of either VEGF or ET-1, which are commonly elevated in SSc (6, 46), at least after 4 weeks of treatment. Increased tissue expression of both these angiogenic molecules were shown to be prevented by bosentan in an animal model of pulmonary arterial hypertension (47), but relevant studies in humans are lacking. Taken together, these results suggest that the observed improvement of endothelial function can be attributed to the endothelin receptor level, since it is independent of effects related to deactivation of the endothelium or to decreases in proinflammatory processes. Such effects are shared by cyclophosphamide and prednisolone and may explain the mechanism by which immunosuppressive regimens act favorably on endothelial cell function in patients with early diffuse SSc (48).

The bosentan-induced improvement of FMD% in these patients was observed as early as the end of the first month of treatment and continued during the following months. Again, at the end of 4 months of treatment, bosentan had no significant effect on endothelium-independent vascular function, arterial blood pressure, arterial stiffness, and peripheral flow reserve. A similarly sustained effect was observed regardless of the dose, which was increased in 5 patients according to the standard dose for pulmonary hypertension. Finally, we had the opportunity to repeat the vascular studies at 12 months from baseline in 10 patients who received bosentan. FMD% values were considered normal at this point in 6 patients who continued bosentan treatment, whereas 4 patients who had discontinued bosentan treatment prior to this point had profoundly impaired FMD%, indicative of relapsed endothelial dysfunction, further suggesting a direct role of endothelin in SSc-associated vascular injury. Nevertheless, there is an obvious need for caution in applying these noncontrolled results (data not shown).

In conclusion, a rapid and sustained improvement in endothelial function in the brachial artery was evident following the administration of bosentan to patients with SSc. This improvement was not associated with hemodynamic changes, proinflammatory processes, or activated-endothelium effects, but rather, was due to an enhancement of NO production following inhibition of endothelin action, as seen in pulmonary hypertension (4, 39). These findings suggest that the endothelin receptor system is an important molecular pathway that is directly involved in certain reversible aspects of SSc-associated vascular injury. It remains to be determined whether a bosentan-induced improvement in vascular endothelial function may possibly translate into a better long-term clinical outcome, regardless of the presence of pulmonary hypertension (49).

Although due to the specific design of this study patients with normal FMD% were excluded, it would be interesting to further examine whether bosentan may prevent deterioration in patients with no apparent endothelial dysfunction of the brachial artery. Thus, taken together with findings suggesting that endothelin is also directly involved in lung fibrosis development (50, 51), a controlled trial may be warranted to examine the potentially global clinical benefit of endothelin receptor blockade in patients with early SSc, using small doses of bosentan or similar drugs.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Dr. Sfikakis had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Sfikakis, Papamichael, Stefanadis, Mavrikakis.

Acquisition of data. Papamichael, Stamatelopoulos, Tousoulis, Fragiadaki, Katsichti, Stefanadis.

Analysis and interpretation of data. Sfikakis, Papamichael, Tousoulis, Stefanadis, Mavrikakis.

Manuscript preparation. Sfikakis, Stamatelopoulos, Tousoulis, Mavrikakis.

Statistical analysis. Sfikakis, Stamatelopoulos.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  • 1
    Denton CP, Black CM, Abraham DJ. Mechanisms and consequences of fibrosis in systemic sclerosis [review]. Nat Clin Pract Rheumatol 2006; 2: 13444.
  • 2
    Charles C, Clements P, Furst DE. Systemic sclerosis: hypothesis-driven treatment strategies [review]. Lancet 2006; 367: 168391.
  • 3
    Leroy EC. Systemic sclerosis: a vascular perspective [review]. Rheum Dis Clin North Am 1996; 22: 67594.
  • 4
    Kahaleh MB. Vascular involvement in systemic sclerosis (SSc) [review]. Clin Exp Rheumatol 2004; 22 Suppl 33: S1923.
  • 5
    Matucci-Cerinic M, Kahaleh MB, LeRoy CE. Pathogenesis: vascular involvement. In: ClementsPJ, FurstDE, editors. Systemic sclerosis. Baltimore: Williams & Wilkins; 1996. p. 15374.
  • 6
    Mayes MD. Endothelin and endothelin receptor antagonists in systemic rheumatic diseases [review]. Arthritis Rheum 2003; 48: 11909.
  • 7
    Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 4115.
  • 8
    Kim NH, Rubin LJ. Endothelin in health and disease: endothelin receptor antagonists in the management of pulmonary artery hypertension [review]. J Cardiovasc Pharmacol Ther 2002; 7: 919.
  • 9
    Clarke JG, Benjamin N, Larkin SW, Webb DJ, Davies GJ, Maseri A. Endothelin is a potent long-lasting vasoconstrictor in men. Am J Physiol 1995; 257: H20335.
  • 10
    Roux S, Breu V, Ertel SI, Clozel M. Endothelin antagonism with bosentan: a review of potential applications [review]. J Mol Med 1999; 77: 36476.
  • 11
    Channick RN, Simonneau G, Sitbon O, Robbins IM, Frost A, Tapson VF, et al. Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomized placebo-controlled study. Lancet 2001; 358: 111923.
  • 12
    Rubin LJ, Badesch DB, Barst RJ, Galie N, Black CM, Keogh A, et al. Bosentan therapy for pulmonary arterial hypertension [published erratum appears in N Engl J Med 2002;346:1258]. N Engl J Med 2002; 346: 896903.
  • 13
    Denton CP, Humbert M, Rubin L, Black CM. Bosentan treatment for pulmonary arterial hypertension related to connective tissue disease: a subgroup analysis of the pivotal trials and their open-label extensions. Ann Rheum Dis 2006; 65: 133640.
  • 14
    Girgis RE, Mathai SC, Krishnan JA, Wigley FM, Hassoun PM. Long-term outcome of bosentan treatment in idiopathic pulmonary arterial hypertension and pulmonary arterial hypertension associated with the scleroderma spectrum of diseases. J Heart Lung Transplant 2005; 24: 162631.
  • 15
    Joglekar A, Tsai FS, McCloskey DA, Wilson JE, Seibold JR, Riley DJ. Bosentan in pulmonary arterial hypertension secondary to scleroderma. J Rheumatol 2006; 33: 618.
  • 16
    Korn JH, Mayes M, Matucci Cerinic M, Ranisio M, Pope J, Hachulla E, et al. Digital ulcers in systemic sclerosis: prevention by treatment with bosentan, an oral endothelin receptor antagonist. Arthritis Rheum 2004; 50: 398593.
  • 17
    Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease [review]. Am J Hypertens 2001; 14: 8389S.
  • 18
    Stewart DJ, Levy RD, Cernacek P, Langleben D. Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease? Ann Intern Med 1991; 114: 4649.
  • 19
    Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders [review]. Blood 1998; 91: 352761.
  • 20
    Barton M, d'Uscio LV, Shaw S, Meyer P, Moreau P, Luscher TF. ETA receptor blockade prevents increased tissue endothelin-1, vascular hypertrophy, and endothelial dysfunction in salt-sensitive hypertension. Hypertension 1998; 31: 499504.
  • 21
    Spiers JP, Kelso EJ, Siah WF, Edge G, Song G, McDermott BJ, et al. Alterations in vascular matrix metalloproteinase due to ageing and chronic hypertension: effects of endothelin receptor blockade. J Hypertens 2005; 23: 171724.
  • 22
    Barton M, Haudenschild CC, d'Uscio LV, Shaw S, Munter K, Luscher TF. Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A 1998; 95: 1436772.
  • 23
    Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980; 23: 58190.
  • 24
    Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force [published erratum appears in J Am Coll Cardiol 2002;39:1082]. J Am Coll Cardiol 2002; 39: 25765.
  • 25
    Tousoulis D, Antoniades C, Stefanadis C. Evaluating endothelial function in humans: a guide to invasive and non-invasive techniques [review]. Heart 2005; 91: 5538.
  • 26
    Moyssakis I, Gialafos E, Vassiliou V, Taktikou E, Katsiari C, Papadopoulos D, et al. Aortic stiffness in systemic sclerosis is increased independently of the extent of skin involvement. Rheumatology (Oxford) 2005; 44: 2514.
  • 27
    Holling HE, Boland HC, Russ E. Investigation of arterial obstruction using a mercury-in-rubber strain gauge. Am Heart J 1961; 62: 194205.
  • 28
    Zelis R, Mason DT, Braunwald E, Levy RI. Effects of hyperlipoproteinemias and their treatment on the peripheral circulation. J Clin Invest 1970; 19: 100715.
  • 29
    O'Rourke MF, Pauca A, Jiang XJ. Pulse wave analysis [review]. Br J Clin Pharmacol 2001; 51: 50722.
  • 30
    Wilkinson IB, Fuchs SA, Jansen IM, Spratt JC, Murray GD, Cockcroft JR, et al. Reproducibility of pulse wave velocity and augmentation index measured by pulse wave analysis. J Hypertens 1998; 16: 207984.
  • 31
    Schacha L, Wigley FM. Targeting mediators of vascular injury in scleroderma [review]. Curr Opin Rheumatol 2002; 14: 68693.
  • 32
    Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, Thuillez C, et al. Nitric oxide is responsible for flow-mediated dilatation of human peripheral conduit arteries in vivo. Circulation 1995; 91: 13149.
  • 33
    Lekakis J, Mavrikakis M, Papamichael C, Papazoglou S, Economou O, Scotiniotis I, et al. Short-term estrogen administration improves abnormal endothelial function in women with systemic sclerosis and Raynaud's phenomenon. Am Heart J 1998; 136: 90512.
  • 34
    Mavrikakis ME, Lekakis JP, Papamichael CM, Stamatelopoulos KS, Kostopoulos CC, Stamatelopoulos SF. Ascorbic acid does not improve endothelium-dependent flow-mediated dilatation of the brachial artery in patients with Raynaud's phenomenon secondary to systemic sclerosis. Int J Vitam Nutr Res 2003; 73: 37.
  • 35
    Freedman RR, Girgis R, Mayes MD. Abnormal responses to endothelial agonists in Raynaud's phenomenon and scleroderma. J Rheumatol 2001; 28: 11921.
  • 36
    Ikeda U, Yamamoto K, Maeda Y, Shimpo M, Kanbe T, Shimada K. Endothelin-1 inhibits nitric oxide synthesis in vascular smooth muscle cells. Hypertension 1997; 29: 659.
  • 37
    Allanore Y, Borderie D, Hilliquin P, Hernvann A, Levacher M, Lemarechal H, et al. Low levels of nitric oxide (NO) in systemic sclerosis: inducible NO synthase production is decreased in cultured peripheral blood monocyte/macrophage cells. Rheumatology (Oxford) 2001; 40: 108996.
  • 38
    Gonon AT, Erbas D, Broijersen A, Valen G, Pernow J. Nitric oxide mediates protective effect of endothelin receptor antagonism during myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol 2004; 286: H176774.
  • 39
    Girgis RE, Champion HC, Diette GB, Johns RA, Permutt S, Sylvester JT. Decreased exhaled nitric oxide in pulmonary arterial hypertension: response to bosentan therapy. Am J Respir Crit Care Med 2005; 172: 3527.
  • 40
    Krum H, Viscoper RJ, Lacourciere Y, Budde M, Charlon V. The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. N Engl J Med 1998; 338: 78490.
  • 41
    Sfikakis PP, Tesar J, Baraf H, Lipnick R, Klipple G, Tsokos GC. Circulating intercellular adhesion molecule-1 in patients with systemic sclerosis. Clin Immunol Immunopathol 1993; 68: 8892.
  • 42
    Vita JA, Keaney JF Jr, Larson MG, Keyes MJ, Massaro JM, Lipinska I, et al. Brachial artery vasodilator function and systemic inflammation in the Framingham Offspring Study. Circulation 2004; 110: 36049.
  • 43
    Witte DR, Broekmans WM, Kardinaal AF, Klopping-Ketelaars IA, van Poppel G, Bots ML, et al. Soluble intercellular adhesion molecule 1 and flow-mediated dilatation are related to the estimated risk of coronary heart disease independently from each other. Atherosclerosis 2003; 170: 14753.
  • 44
    Xu SW, Denton CP, Dashwood MR, Abraham DJ, Black CM. Endothelin-1 regulation of intercellular adhesion molecule-1 expression in normal and sclerodermal fibroblasts. J Cardiovasc Pharmacol 1998; 31 Suppl 1: S5457.
  • 45
    Sfikakis PP, Mavrikakis M. Adhesion and lymphocyte co-stimulatory molecules in systemic rheumatic diseases [review]. Clin Rheumatol 1999; 18: 31727.
  • 46
    Kuryliszyn-Moskal A, Klimiuk PA, Sierakowski S. Soluble adhesion molecules (sVCAM-1, sE-selectin), vascular endothelial growth factor (VEGF) and endothelin-1 in patients with systemic sclerosis: relationship to organ systemic involvement. Clin Rheumatol 2005; 24: 1116.
  • 47
    Rondelet B, Kerbaul F, Motte S, van Beneden R, Remmelink M, Brimioulle S, et al. Bosentan for the prevention of overcirculation-induced experimental pulmonary arterial hypertension. Circulation 2003; 107: 132935.
  • 48
    Apras S, Ertenli I, Ozbalkan Z, Kiraz S, Ozturk MA, Haznedaroglu IC, et al. Effects of oral cyclophosphamide and prednisolone therapy on endothelial functions and clinical findings in patients with early diffuse systemic sclerosis. Arthritis Rheum 2003; 48: 225661.
  • 49
    Williams MH, Das C, Handler CE, Akram MR, Davar J, Denton CP, et al. Systemic sclerosis associated pulmonary hypertension: improved survival in the current era. Heart 2006; 92: 92632.
  • 50
    Abraham DJ, Vancheeswaran R, Dashwood MR, Rajkumar VS, Pantelides P, Xu SW, et al. Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease. Am J Pathol 1997; 151: 83141.
  • 51
    Shi-Wen X, Chen Y, Denton CP, Eastwood M, Renzoni EA, Bou-Gharios G, et al. Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts. Mol Biol Cell 2004; 15: 270719.