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A3.11 Endothelin-1 directly induces a profibrotic phenotype both in human dermal microvascular endothelial cells and skin fibroblasts
  1. S Soldano,
  2. P Montagna,
  3. R Brizzolara,
  4. A Sulli,
  5. M Cutolo
  1. Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy

Abstract

Background and objectives Endothelial cell activation and dysfunction contribute to vascular damage, which represents an early event in fibrotic diseases, including systemic sclerosis (SSc).1 These activated cells secrete several mediators including endothelin-1 (ET-1) and transforming growth factor-β1 (TGFβ1).2 In SSc, myofibroblast activation and their overproduction of profibrotic molecules, such as fibroblast specific protein-1 (S100A4), α-smooth muscle actin (α-SMA), fibronectin (FN) and type I collagen (COL1) were known to characterise the late phase of fibrotic process. To investigate the involvement of ET-1 and TGFβ1 on microvascular alteration through the directly induction of profibrotic molecules S100A4, α-SMA and FN in cultured human dermal microvascular endothelial cells (HMVECs). In addition, the effects of ET-1 in inducing SSc myofibroblasts phenotype were also investigated in cultured normal skin fibroblasts.

Materials and methods HMVECs were treated for 6 days with or without ET-1 (100nM) and TGFβ1 (10ng/ml). Cultured normal and SSc skin fibroblasts were isolated from ten SSc patients (mean age 65 ± 7 years) who fulfilled the new criteria of EULAR/ACR3 and five age matched healthy subjects after signing an Informed Consent and Ethical Committee. Cultured normal fibroblasts were treated for 48 h with or without ET-1. Untreated cultured SSc fibroblasts were used as positive controls. S100A4, α-SMA and FN protein synthesis and expression were evaluated by immunofluorescence, immunocytochemistry and quantitative real time in HMVECs. α-SMA, COL1 and FN were evaluated by immunofluorescence, immunocytochemistry, and western blotting in cultured fibroblasts. Statistical analysis was performed by non-parametric Mann-Whitney test.

Results In HMVECs, qRT-PCR showed that ET-1 and TGFβ1 were able to significantly up-regulate the gene expression of S100A4, α-SMA (p < 0.01 for both proteins) and FN (p = 0.01; p = 0.04) compared to untreated cells. Results were confirmed by immunofluorescence and immunocytochemistry.

In cultured normal skin fibroblasts, ET-1 induced the expression of α-SMA leading to the myofibroblast phenotype activation similar to that observed in SSc fibroblasts. In cultured normal fibroblasts ET-1 significantly increased both COL1 and FN synthesis compared to untreated cells (p < 0.01 for both). The synthesis of these ECM protein was similar to that observed in SSc fibroblasts. Results were confirmed by Western blotting.

Conclusion Present results seem to suggest a possible direct action of ET-1 both in the early and late phase of fibrotic process, inducing endothelial cell activation and production of a pro-fibrotic microvascular environment, as well as determining myofibroblast activation and their ECM overproduction.

References

  1. Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 2012;18:1028–40.

  2. Iglarz M, Clozel M. At the heart of tissue: endothelin system and end-organ damage. Clin Sci 2010;119:453–63.

  3. van den Hoogen F, Khanna D, Fransen J, et al. 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthrit Rheum 2013;65:2737–47.

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