Session Information
Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's - Pathogenesis, Animal Models and Genetics
Session Type: Abstract Submissions (ACR)
Background/Purpose: Tissue hypoxia is a consequence of vascular damage and Hif-1α accumulation is a major mechanism of hypoxia response pathways. HIF-1α induces the transcriptional upregulation of expression of numerous genes encoding proteins involved in vascular repair including soluble growth factors (TGF-β and VEGF), and extracellular matrix components (type I collagen and fibronectin), rendering HIF-1α as a positive regulator of wound healing and a potential mediator of organ repair and tissue fibrosis, suggesting that HIF1-α may play an important role in the pathogenesis of fibrotic diseases such as Systemic Sclerosis (SSc). Recent studies have shown that TGF-β causes a potent increase in HIF-1α protein levels. Since endothelin 1 (ET-1) synergistically enhances TGF-β mediated endothelial-to-mesenchymal transition (EndoMT), we examined the transcriptional regulation of Hif-1α and of several of its downstream targets in response to TGF-β alone or TGF-β in combination with ET-1 in cultured murine lung microvascular endothelial cells (MVEC).
Methods: Murine pulmonary MVEC were isolated from C57Bl/6J mice employing trypsin/collagenase tissue digestion followed by sequential immunomagnetic selection with anti-CD31 and anti-CD102 antibodies. The purified EC were treated in monolayer cultures with ET-1 (100 ng/mL) in the presence and absence of TGF-β1 (10 ng/mL) and the induction of Hif-1α as well as several Hif-1α targets including lysyl oxidase (Lox), Lox-like 1 protein (Loxl1), Loxl2, Loxl3, and LoxL4 was assessed employing semi-quantitative RT-PCR.
Results: Exposure of murine pulmonary MVEC to ET-1 induced a three-fold increase in Hif-1α levels compared to non-treated controls. TGF-β1, however, induced a nearly 500-fold increase in expression whereas samples treated with TGF-β1 plus ET-1 increased Hif-1α transcript levels nearly 2500-fold compared to the saline control. This profound synergistic increase was abrogated by the dual ET-1 receptor antagonist, Bosentan, demonstrating that the observed effect was indeed mediated by ET-1. Similarly, TGF-β alone induced significant increases in the expression of Hif-1α target genes Lox, LoxL1, LoxL2, LoxL3 and LoxL4. ET-1 alone did not alter the expression of these genes whereas ET-1 in combination with TGF-β synergistically increased their levels. Bosentan abrogated the increased expression levels of these genes observed for TGF-β plus ET-1, indicating that the synergistic increases were mediated by ET-1.
Conclusion:
TGF-β potently increased Hif-1α and Hif-1α target gene expression in murine pulmonary MVEC under normoxic conditions. ET-1 synergistically enhanced these effects. Since vascular damage plays a crucial role in SSc-associated pulmonary arterial hypertension pathogenesis and participates in the development of skin and lung fibrosis, the results described here identify ET-1 as a potentially key regulator of TGF-β-mediated activation of Hif-1α in response to vascular damage causing a synergistic enhancement of HIF-1α-mediated profibrotic effects suggesting that this process may play a key pathogenetic role in SSc-associated tissue fibrosis and fibroproliferative vasculopathy.
Disclosure:
P. J. Wermuth,
None;
S. A. Jimenez,
None.
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