In vitro study of the effects of endothelin-1: relevance to the pathogenesis of pulmonary arterial hypertension in systemic sclerosis.

Background: Pulmonary arterial hypertension is a devastating disease with an estimated patients’ survival of 68% at 1 year, 48% at 3 years and 34% at 5 years from diagnosis. The role of endothelin-1 has been studied in different conditions including systemic sclerosis. In systemic sclerosis patients, circulating endothelin- 1 is present at high levels, suggesting its involvement in the development of fibrotic and vascular damage. Indeed endothelin-1 is a potent pro-fibrotic peptide and inhibitors of endothelin-1 receptors (ETA and ETB) may exert an anti-fibrotic effect. The balance of endothelin-1 effects on smooth muscle cells and on endothelial cells is responsible for the homeostasis of vascular tone. Another interesting effect of endothelin-1 is the induction of aldosterone in endothelial cells that eventually results in the production of reactive oxygen species. Aldosterone is a key mediator of the pulmonary vascular injury response to hypoxia: mineralocorticoid receptor activation by aldosterone is involved in the remodeling/fibrosis response to hypoxia in endothelial cells in vitro and aldosterone antagonism attenuates these events. Methods: Reverse transcriptase-PCR, western blot and cytofluorimetric analyses were performed to evaluate the expression of the two isoforms of endothelin-1 receptors and mineralocorticoid receptor. Activation of fibroblast was evaluated measuring the amount of collagen-1, TGF-β and PDGF with ELISA kits. The detection of intracellular oxidative stress formation was measured with flow cytometry using fluorescence of CM-H2DCFDA after long or short incubations with endothelin-1 and its receptors inhibitors or with aldosterone and its antagonist. Results: Both endothelin-1 receptors are present in commercially available human pulmonary fibroblasts and normal human dermal fibroblasts, human dermal fibroblasts from healthy donors and scleroderma human dermal fibroblasts. Mineralocorticoid receptor expression was detected in human pulmonary fibroblasts, scleroderma human dermal fibroblasts and human pulmonary artery endothelial cells while in normal human dermal fibroblasts and human dermal fibroblasts from healthy donors the amount of this receptor was lower than the other cells analyzed. In human pulmonary fibroblasts, scleroderma human dermal fibroblasts and human pulmonary artery endothelial cells, endothelin-1 stimulates the production of reactive oxygen species after a long incubation. In addition, the production of oxygen radicals is inhibited by the use of ETB antagonist. Interestingly, aldosterone stimulates the production of reactive oxygen species after a short incubation in human pulmonary fibroblasts, scleroderma human dermal fibroblasts and human pulmonary artery endothelial cells, but not in normal human dermal fibroblasts and human dermal fibroblasts from healthy donors. The same results were obtained by human pulmonary fibroblasts, scleroderma human dermal fibroblasts and human pulmonary artery endothelial cells stimulated with endothelin-1 for long time with mineralocorticoid receptor inhibitor. Conclusions: These results suggest that endothelin-1 activates fibroblasts and the production of aldosterone, the true responsible for oxidative stress. The inhibition of reactive oxygen species production using the ETB antagonist indicates that the ETB receptor is implicated in this process. Moreover we found differences between fibroblasts from healthy donors and from scleroderma patients in oxidative stress production after endothelin-1 stimulation. Finally we found differences in mineralocorticoid receptor presence in the same cell types.