Background/Purpose: Transient receptor potential (Trp) cation channel, subfamily V (TRPV) members can impact a diverse range of vascular functions, including vascular tone, vascular permeability, mechanosensing, angiogenesis and endothelial cell (EC) proliferation, apoptosis, and death.  Recently, it has been shown that endothelial-to-mesenchymal transition (EndoMT) may play a role in generating myofibroblasts responsible for the uncontrolled production of extracellular matrix in many fibrotic diseases, including Systemic Sclerosis (SSc).  Thus the purpose of these studies was to investigate the effect of TRPV1 and TRPV2 inhibition on the EndoMT process.

Methods: Pulmonary EC were isolated from three C57BL/6J mice employing sequential immunomagnetic selection with anti-CD31 and anti-CD102 antibodies followed by in vitro culture and treatment with TRPV1 and TRPV2 agonists or antagonists in the presence and absence of TGF-β1.  EndoMT was assessed by α-smooth muscle actin (α-SMA) immunofluorescence 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 various mesenchymal cell specific genes, including the transcription factor Twist1, and the transcriptional repressors Snail1 and Snail2 as well as expression of EC-specific genes (Pecam1 and VE-cadherin) was assessed by semi-quantitative RT-PCR in triplicate for two replicates per cell line.

Results: Exposure of lung ECs to either the TRPV1 antagonist capsazepine or the TRPV2 antagonist tranilast caused downregulation of expression of EC-specific genes and upregulation of mesenchymal-specific genes.  These agents did not induce noticeable changes in cell morphology.  Treatment of cells with either capsazepine or tranilast in combination with TGF-β1 showed synergisitic stimulation of TGF-β1 effects on gene expression.  Capsazepine in combination with TGF-β1 produced a pronounced morphological change from EC to fibroblast morphology cells which was of greater magnitude than those induced by TGF-β1 alone.  In contrast, neither the TRPV1 agonist capsaicin nor the TRPV2 agonist probenecid alone or in combination with TGF-β1 induced EndoMT in murine lung ECs.  Capsaicin and probenecid, however, abrogated TGF-β1-induced EndoMT, and reversed TGF-β1-induced morphological changes in the lung EC.

Conclusion: Functional downregulation of TRPV1 and TRPV2 causes strong synergistic potentiation of TGF-β1-induced EndoMT and may play a pathogenetic role in pulmonary fibrosis and other fibrotic diseases, including SSc.  The abrogation of TGF-β1-induced EndoMT by capsaicin and probenecid suggests that induction or activation of TRPV1 and TRPV2 activation could represent an important and novel strategy to prevent EndoMT-mediated generation of activated myofibroblasts in fibrotic diseases.

Supported by NIH grant R01AM19616 to SAJ.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functional-activation-of-the-trpv1-and-trpv2-non-selective-cation-channels-potentiates-tgf-%ce%b21-induced-endothelial-to-mesenchymal-transition-in-murine-pulmonary-endothelial-cells-suggest-a-potenti/