Improved endothelial function after endothelin receptor blockade in patients with systemic sclerosis

Abstract

Objective

Impaired endothelium-dependent vasodilator function may contribute to vascular damage in patients with systemic sclerosis (SSc). This study was undertaken to investigate whether increased activity of the endothelin 1 (ET-1) system plays a role in the occurrence of endothelial dysfunction in patients with SSc.

Methods

In 12 patients with SSc (6 with diffuse cutaneous SSc [dcSSc] and 6 with limited cutaneous SSc [lcSSc]), forearm blood flow responses to graded doses of acetylcholine (ACh) and sodium nitroprusside (SNP) given intraarterially were assessed by plethysmography, during infusion of saline and following selective blockade of ET_A receptors with BQ-123 (10 nmoles/minute).

Results

During saline infusion, the vasodilator response to ACh was blunted in patients with SSc as compared with that in healthy controls (P < 0.001), whereas the response to SNP was not different between groups (P = 0.27). The vasodilator effect of ET_A receptor antagonism was higher in patients than in controls (P < 0.001), indicating enhanced ET-1–mediated vasoconstriction in SSc. In patients, ET_A receptor blockade resulted in a potentiation of the vasodilator response to ACh (P < 0.001 versus saline), but did not affect the response to SNP (P = 0.31). Notably, both the vasodilator effect of ET_A receptor antagonism and the improvement in the responsiveness to ACh following BQ-123 infusion were higher in patients with dcSSc than in those with lcSSc (P < 0.01).

Conclusion

ET-1–dependent vasoconstrictor tone is increased predominantly in the subgroup of SSc patients with dcSSc, in whom acute blockade of ET_A receptors was able to improve impaired endothelium-dependent vasodilator function. Our results suggest novel vasculoprotective effects of ET_A receptor antagonism and support further exploration of strategies that target the ET-1 pathway in SSc.

Vascular injury and organ devascularization are among the major features of systemic sclerosis (SSc). Findings of several studies have suggested that altered function of endothelial cells plays a central role in determining the clinical manifestations of the disease and its complications (1). Thus, abnormal endothelial vasodilator function seems to be the early stage of the process that adversely impacts the microvascular bed, with a reduction in the size of microvessels leading to decreased blood flow to organs and, ultimately, to a state of chronic ischemia (1).

Endothelial control of vasomotor function results from a complex balance between endothelium-derived relaxing and contracting factors. Therefore, a disruption of the critical equilibrium between these opposing forces may predispose a patient to increased tone and decreased vasomotion (2). Principal among the vasoconstrictor substances produced by vascular endothelial cells is endothelin 1 (ET-1), which exerts its vasoactive effects through interaction with 2 subtypes of specific receptors: ET_A and ET_B. ET-1 can damage the vasculature by regulating vascular growth-promoting factors and inducing vascular remodeling, both of which are important mechanisms of organ damage.

Over the last few years, growing interest in the role of ET-1 as a factor involved in the vascular abnormalities of SSc has been sparked by the availability of antagonists of ET-1 receptors. Thus, the use of combined ET_A/B receptor antagonists has allowed researchers to ascertain the involvement of ET-1 in SSc vasculopathy, as indicated by the efficacy of bosentan in the prevention of digital ulcers (3) and improvement in symptoms of pulmonary hypertension (4). However, whether activation of the ET-1 system also plays a role in determining endothelial dysfunction in patients with SSc remains poorly understood. Therefore, in the present study, we investigated this in the blood vessels of patients with SSc, using plethysmography to assess the effects of ET_A receptor antagonism on endothelium-dependent and -independent vasodilation.

PATIENTS AND METHODS

Study subjects.

Twelve patients with SSc (2 men and 10 women; mean ± SEM age 50 ± 3 years) who attended the outpatient clinic of the Division of Rheumatology at our institution were recruited for this study. None of the patients had a history of diabetes mellitus (DM), hypertension, hypercholesterolemia, peripheral vascular disease, coagulopathy, or any other systemic condition. All patients fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for the diagnosis of SSc (5), and patients were grouped according to whether they had limited cutaneous SSc (lcSSc) (6 patients) or diffuse cutaneous SSc (dcSSc) (6 patients) (6). The mean ± SEM disease duration, calculated from the onset of the first clinical event (other than Raynaud's phenomenon) that was a clear manifestation of SSc, was 5.7 ± 4.8 years. Five patients presented with early disease (<3 years). Six patients had a history of digital ulcers, and 4 had circulating lupus anticoagulant antibodies. None of the patients had pulmonary arterial hypertension or interstitial lung disease (evaluated by pulmonary function testing and high-resolution computed tomography of the lungs). All patients had previously been treated with iloprost, nifedipine, and aspirin, but iloprost had been discontinued at least 6 months prior to recruitment, and nifedipine and aspirin at least 2 weeks prior to recruitment.

Twelve healthy volunteers (2 men and 10 women; mean ± SEM age 46 ± 3 years), with no evidence of present or past hypertension, hyperlipidemia, DM, cardiovascular disease, or any other systemic disease were selected as a control group. Each study subject was screened according to clinical history, physical examination, electrocardiography, chest radiography, and routine chemical analyses. None of the subjects was taking medications at the time of the study. The local Institutional Review Board approved the study protocol, and all participants gave written informed consent.

Assessment of vascular responses by plethysmography.

Each study in patients and controls consisted of an infusion of drugs into the brachial artery and measurement of the response of the forearm vasculature by means of strain-gauge venous occlusion plethysmography, as described previously (7). After basal measurements were obtained, dose-response curves to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, were obtained during concurrent administration of saline. At the end of the infusion of ACh and SNP and after the return of forearm blood flow to baseline, BQ-123, a selective antagonist of ET_A receptors, was infused for 60 minutes in both patients and controls, and forearm blood flow was measured every 10 minutes. In the SSc patients, infusion of BQ-123 was then continued at the same dose as before, and forearm blood flow was measured again during administration of ACh and SNP, given at the same doses and for the same duration as before.

Statistical analysis.

Within-group analyses were performed by paired t-test and repeated-measures analysis of variance (ANOVA), as appropriate. Between-group comparisons were performed using unpaired t-test and ANOVA, as appropriate. All calculated P values were 2-tailed, and P values less than 0.05 were considered significant. All group data are reported as the mean ± SEM.

RESULTS

Differences in age, sex, anthropometric variables, blood pressure, baseline forearm blood flow, and levels of plasma glucose, total cholesterol, high-density lipoprotein cholesterol, and triglyceride in patients compared with healthy controls were not significant (P > 0.05 for all comparisons). Similarly, differences in disease duration, presence of digital ulcers, and drug treatment in dcSSc patients compared with lcSSc patients were not significant (P > 0.05 for all comparisons).

During the concurrent infusion of saline, infusion of increasing doses of ACh resulted in a progressive increase in forearm blood flow compared with baseline in both SSc patients and controls. However, the vasodilator response to ACh was significantly blunted in patients compared with controls (Figure 1). Similarly, administration of SNP induced a dose-dependent vasodilator response in both groups. However, in contrast to the ACh results, the vasodilator response to SNP was not significantly different between patients and controls (Figure 1).

Figure 1. Forearm blood flow response to intraarterial infusion of increasing doses of acetylcholine and sodium nitroprusside in healthy control subjects and in patients with systemic sclerosis (SSc). Values are the mean and SEM. P values were determined by two-way analysis of variance.

As shown in Figure 2A, the degree of increase in forearm blood flow over baseline following ET_A receptor antagonism by infusion of BQ-123 was significantly higher in patients than in controls. In patients, blockade of ET_A receptors resulted in a significant increase in the vasodilator response to ACh compared with saline (Figure 2B). Infusion of BQ-123, in contrast, was not associated with significant changes in the vasodilator response to SNP (Figure 2B).

Figure 2. A, Forearm blood flow response to intraarterial infusion of BQ-123 in healthy control subjects and in patients with systemic sclerosis (SSc). B, Forearm blood flow values in response to intraarterial infusion of increasing doses of acetylcholine and sodium nitroprusside in patients with SSc during infusion of saline or following selective endothelin A receptor blockade. Values are the mean and SEM. P values were determined by two-way repeated-measures analysis of variance.

During saline administration, there was no difference in the vasodilator response to ACh between patients with lcSSc and patients with dcSSc (P = 0.22), thus indicating similar impairment of endothelium-dependent reactivity in the 2 groups. However, the vasodilator response to infusion of BQ-123 was significantly higher in patients with dcSSc than in those with lcSSc (Figure 3A). The response in the latter group was not significantly different from that in the controls (P = 0.48). Interestingly, infusion of BQ-123 resulted in a significant potentiation of the vasodilator response to ACh in patients with dcSSc (P = 0.02 versus saline), but not in those with lcSSc (P = 0.40 versus saline). As a result, reactivity to ACh during ET_A receptor antagonism was significantly higher in dcSSc patients than in lcSSc patients (Figure 3B). In contrast, there was no difference in the vasodilator response to SNP following ET_A receptor blockade in the 2 groups (Figure 3B).

Figure 3. A, Forearm blood flow response to BQ-123 in patients with limited cutaneous systemic sclerosis (lcSSc) and in patients with diffuse cutaneous SSc (dcSSc). B, Forearm blood flow values in response to intraarterial infusion of increasing doses of acetylcholine and sodium nitroprusside in patients with lcSSc and in patients with dcSSc following selective endothelin A receptor blockade. Values are the mean and SEM. P values were determined by two-way analysis of variance.

DISCUSSION

The main finding of the present study is that the blunted endothelium-dependent vasodilator responsiveness to ACh in patients with SSc is reversed following blockade of ET_A receptors. In the absence of ET_A antagonism, patients exhibited impaired vasodilator response to ACh, whereas the endothelium-independent vasorelaxing effect of SNP was not different in the SSc patients as compared with the healthy controls. Furthermore, blockade of ET_A receptors resulted in a higher vasodilator response in patients than in controls. Taken together, these findings suggest that increased ET_A-dependent vasoconstrictor tone in patients with SSc plays a role in their defective endothelium-dependent vasodilator capacity. Importantly, despite similar impairment of endothelial function in patients with lcSSc and dcSSc, activation of the ET-1 vasoconstrictor system was observed only in patients with dcSSc, and blockade of ET_A receptors resulted in improved endothelium-dependent vasodilation only in the arteries of these patients.

The results of our investigation are consistent with those of a recent study showing that nonselective ET_A/B blockade by systemic administration of bosentan was associated with improved flow-mediated, nitric oxide (NO)–dependent vasodilation in patients with SSc (8). However, the present investigation expands on those findings by also demonstrating a beneficial effect of selective ET_A blockade on endothelium-dependent vascular function in SSc patients. Theoretically, selective ET_A receptor antagonism may have an advantage over nonselective ET_A/B blockade in that it does not affect NO production through endothelial ET_B receptor stimulation (9). Therefore, it may be postulated that endothelial ET_B receptor binding of increased intravascular levels of ET-1 might have contributed to the restoration of NO bioavailability in patients with dcSSc following ET_A receptor blockade, thereby leading to the improved endothelium-dependent vasodilation observed in our study.

This hypothesis is supported by the findings of previous investigations demonstrating down-regulation of ET_B receptors in experimental models of endothelial dysfunction, as well as enhanced vascular endothelial cell NO synthase function and nitrate levels following ET_A receptor antagonism (10). Unfortunately, in our study, we could not obtain direct comparison between the effects of selective ET_A blockade and nonselective ET_A/B blockade on endothelial function in patients with SSc. Also, this postulated mechanism would be contradictory to the favorable vascular effects of nonselective ET_A/B antagonism by bosentan that was previously demonstrated in SSc (3, 4, 8). Therefore, it is reasonable to speculate that other mechanisms might have been implicated in the improved endothelial function observed in our group of patients with dcSSc following blockade of ET_A receptors.

One such mechanism could involve reduction of oxidative stress in the arteries of these patients following antagonism of the ET-1 system, leading in turn to increased NO bioavailability. Thus, enhanced activity of the ET-1 system has been related to augmented vascular release of superoxide anions (11), and an imbalance between NO and the production of superoxide anions has been previously associated with endothelial dysfunction both in experimental animals (12) and in humans (13).

Another potential mechanism for the favorable effect of ET-1 receptor antagonism on endothelial function in the blood vessels of SSc patients could be reversal of the imbalance between vasoconstrictor and vasodilator forces after removal of the vasoconstrictor effect of ET-1 on vascular smooth muscle cells. However, the absence of increased responsiveness to SNP in patients with SSc following ET-1 receptor blockade provides evidence against this hypothesis, and nonspecific improvement in vasodilator function in SSc during ET_A antagonism does not appear to be a plausible mechanism to explain our findings. This view is strengthened by the results of a previous study showing that ET-1 receptor antagonism does not affect the vascular response to ACh in healthy subjects (7). This observation further enhances the specificity of the effect of BQ-123 on the responsiveness to ACh observed in patients with dcSSc and overcomes another possible limitation of the current study, i.e., that the effect of BQ-123 on the response to ACh was not tested in controls.

Despite the limited number of patients examined in each SSc subgroup in the current study, the selective activation of the ET-1 system in patients with dcSSc and its likely involvement in endothelial dysfunction only in this subgroup clearly suggest that different pathophysiologic mechanisms underlie the vascular pathology in various forms of the disease. Since dcSSc is characterized by widespread fibrosis and more severe organ involvement, selective activation of ET-1 in this form of the disease is compatible with the known profibrotic effect of ET-1 (14). As such, our data are supported by the findings of previous studies demonstrating increased levels of ET-1 in patients with dcSSc (15) and suggest an involvement of the ET-1 system in mediating the effect of profibrotic cytokines, such as transforming growth factor β, in these patients.

In conclusion, our results demonstrate that targeting of the activated ET-1 system by blockade of ET_A receptors improves endothelium-dependent vasorelaxation in subgroups of patients with SSc. These findings further our understanding of the biologic mechanisms involved in the cascade of events that drive the occurrence and progression of vascular disease in SSc. In addition, the findings of our study suggest a vasculoprotective effect of chronic ET_A receptor blockade in these patients and support further exploration of strategies that target the ET-1 pathway to preserve vascular function in SSc.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Cardillo 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 conception and design. Cardillo, Zoli, Ferraccioli.

Acquisition of data. Cardillo, Schinzari, Mores, Bosello, Peluso.

Analysis and interpretation of data. Cardillo, Schinzari, Melina, Mores, Bosello, Peluso, Zoli, Ferraccioli.