Background/Purpose:

Systemic sclerosis (SSc) is a systemic autoimmune disease of unknown etiology whose pathogenesis involves the regulation of a diverse range of molecular pathways. The clinical and pathologic manifestations of SSc arise from fibroproliferative vascular lesions; excessive and progressive synthesis and deposition of collagen and other extracellular matrix (ECM) macromolecules in the skin and various internal organs; and activation of innate and adaptive immunity. Several recent studies indicate a pivotal role for Toll-like receptor (TLR) signaling in the pathogenesis of several autoimmune diseases including SSc. Endothelial-to-mesenchymal transition (EndoMT) may play a role in generating activated myofibroblasts responsible for the uncontrolled ECM production in many fibrotic diseases, including SSc.  Thus, the purpose of these studies was to compare the ability of TGF-β1 and the Tlr4 inducer lipopolysaccharide (LPS) to induce EndoMT in endothelial cells (EC) isolated from C57BL6/J or Tlr4 knockout (KO) mice.

Methods: Lung EC were isolated from three C57Bl/6J control and three TLR4 KO mice by enzymatic dissociation and were immumopurified employing sequential immunomagnetic selection with anti-CD31 and anti-CD102 antibodies followed by in vitro culture and treatment with either TGF-β1, LPS or TGF- β1 plus LPS.  EndoMT was assessed by monitoring EC morphological changes and loss of EC molecular markers.  Type I, type III and type IV collagens, α-SMA, fibronectin, and various mesenchymal cell- or EC-specific gene expression was assessed by semi-quantitative RT-PCR in triplicate for three replicates per cell line. 

Results: Treatment of lung ECs isolated from C57BL6/J control mice with TGF-β1 induced the expected loss of EC morphology causing them to assume a more spindle fibroblast-like appearance. Remarkably, LPS alone caused similar morphologic EC changes. Both agents caused downregulation of the expression of EC-specific genes and concomitant upregulation of mesenchymal-specific genes, including Col1a1, Col3a1, fibronectin 1 (Fn1) and the Fn-Eda splice variant. In contrast, the EndoMT-associated changes in gene expression induced by TGF-β1 or LPS were completely abrogated in EC from Tlr4 KO mice compared to EC from C57BL6/J control or Tlr4 KO mice and their morphology was unaffected by treatment.

Conclusion: Signaling through Tlr4 can mediate EndoMT in murine lung EC since LPS treatment induces morphologic and gene expression changes associated with the EndoMT process.  Further, Tlr4 signaling is required for TGF-β1 EndoMT in murine lung EC since genetic deletion of the Tlr4 gene results in the loss of TGF-β1-mediated EndoMT as shown by the rentention of characteristic EC morphology and failure to downregulate EC-specific genes or upregulate mesenchymal-specific genes. Thus, one mechanism by which Tlr4 signaling can assume a pathogenetic role in SSc-associated pulmonary fibrosis and other fibrotic diseases is by inducing the transdifferentiation of EC to profibrotic activated myofibroblasts. Thus, Tlr4 signaling inhibition in EC could represent an important and novel strategy to ameliorate the fibroproliferative process responsible for fibrotic diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/genetic-deletion-of-toll-like-receptor-4-tlr4-abrogates-tgf-1-induced-endothelial-to-mesenchymal-transition-endomt-in-murine-pulmonary-endothelial-cells/