Session Title: Systemic Sclerosis, Fibrosing Syndromes, and Raynaud's II
Session Type: Abstract Submissions (ACR)
The process of endothelial-to-mesenchymal transition (EndoMT) may be a crucial pathway in the generation of activated myofibroblasts, cells that play a pivotal role in the development of tissue and organ fibrosis in fibrotic diseases such as Systemic Sclerosis (SSc). It has been previously demonstrated that TGF-β1 induces EndoMT in vitro in murine lung endothelial cells (ECs). Since extensive previous studies demonstrated a potent profibrotic role for Endothelin-1 (ET-1), the role of ET-1 in the induction of EndoMT was investigated in cultured murine lung ECs.
Pulmonary EC were isolated from three C57Bl/6J mice employing trypsin/collagenase tissue digestion followed by sequential immunomagnetic selection with anti-CD31 and anti-CD102 antibodies. After in vitro culture, the purified EC were treated with ET-1 in vitro in the presence and absence of TGF-β1. EndoMT was assessed by immunofluorescence for α-smooth muscle actin (α-SMA) and by Western blot analysis for α-SMA and type I collagen. Induction of type I, type III and type IV collagens, α-SMA, fibronectin, as well as several mesenchymal specific genes, including the transcription factor Twist1 and the transcriptional repressors Snail1 and Snail2 was assessed by semi-quantitative RT-PCR triplicate assays for two replicates per cell line.
Treatment of murine pulmonary ECs with TGF-β1 induced high levels of α-SMA expression in comparison to saline-treated control ECs. ET-1 alone did not affect α-SMA levels but produced a synergistic effect with TGF-β1 by potentiating TGF-β1-induced EndoMT as indicated by increased α-SMA production. A quantitative assessment of the number of α-SMA expressing EC in TGF-β1-treated cultures was 27% compared to 52% in cultures treated with both TGF-β1 and ET-1. These results were confirmed by Western blot analysis. Semi-quantitative RT-PCR analysis demonstrated that ET-1 synergistically potentiated TGF-β1-mediated increased expression levels of types I and III collagens, α-SMA, fibronectin, Twist1, Snail1 and Snail2. Expression of EC-specific VE-cadherin (Cdh5) was downregulated in TGF-β1-treated cultures compared to saline controls and ET-1 also synergistically potentiated this TGF-β1-mediated effect.
ET-1 plays an important role in regulating EndoMT by causing a synergistic potentiation of TGF-β1-induced EndoMT-mediated generation of activated myofibroblasts and of EndoMT-mediated increased expression of extracellular matrix components including Types I and III collagens. Furthermore, ET-1 also potentiated the TGF-β1-induced increase in expression of various genes involved in the phenotypic conversion of EC into myofibroblasts including Twist1, Snail1 and Snail2. Since ET-1 plays a crucial role in the pathogenesis of SSc-associated pulmonary arterial hypertension and may play a profibrotic role in skin and lung fibrosis, the results described here identify a novel mechanism supporting the concept that ET-1 plays a key pathogenetic role in SSc-associated pulmonary fibrosis.
Supported by NIH Grant R01AM19616 to SAJ.
P. J. Wermuth,
S. A. Jimenez,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/synergistic-effects-of-endothelin-1-on-transforming-growth-factor-%ce%b21-tgf-%ce%b21-induced-endothelial-to-mesenchymal-transition-a-novel-mechanism-for-the-fibrogenic-effects-of-endothelin/