Background/Purpose: Progressive tissue fibrosis and microvascular alterations are the hallmarks of Systemic Sclerosis (SSc). The mechanisms involved in SSc pathogenesis are complex and have not been fully elucidated. One notable feature of SSc is the extension of the pathologic process to normal tissues. The mechanisms involved in this topographic extension have remained elusive. Recent studies suggested that the phenotypic transition of endothelial cells (EC) into mesenchymal cells (endothelial to mesenchymal transition; EndoMT) may be involved in the progressive tissue fibrosis and fibroproliferative vasculopathy in SSc although the mechanisms involved have not been elucidated. We here suggest that one potential mechanism may be that EndoMT activated EC exert paracrine effects on normal/healthy fibroblasts and EC and induce them to express a profibrotic phenotype and transdifferentiate into activated profibrotic cells. The purpose of the study was to evaluate whether secreted molecules released by microvascular lung EC undergoing EndoMT exert paracrine effects on normal human pulmonary fibroblasts inducing them to become profibrotic.

Methods: Normal human lung fibroblasts and microvascular EC were isolated from normal lung tissues by enzymatic digestion with collagenase. The isolated cell suspension was incubated with CD45 microbeads to remove leukocytes. Fibroblasts and EC were isolated employing anti-human CD31 antibody magnetic beads. The CD31- fibroblasts and the CD31+ EC were expanded separately. For EndoMT induction the lung microvascular EC were treated with TGF-β1 or TGF-β2 for 48 h. Following a change of media to serum-free media for 24h the supernatants were collected and aliquotes added to triplicate wells of normal human lung fibroblasts. After 24h, culture supernatants were isolated for Western blot analysis. The cells were lysed and total fibroblast RNA isolated and cDNA generated. Expression of various fibrosis-associated +genes was assessed by quantitative real time PCR.  Validation of the gene expression results was performed employing Western blot analysis of secreted and intracellular proteins. 

Results: RTPCR results showed a marked increase in levels of COL1A2, COL3A1, FN1, CTGF and TGFβ1 mRNA in normal lung fibroblasts treated with supernatants from normal lung microvascular EC pretreated with TGF-β1 or TGF-β2 compared to medium from untreated EC. Increased amounts of fibronectin were found in the media of lung fibroblasts treated with supernatants from the lung microvascular EC treated with TGF-β1 or TGF-β2.

Conclusion: Treatment of normal lung fibroblasts with proteins released by cultured human lung microvascular EC induced to undergo EndoMT in vitro resulted in a profound change in the expression of numerous profibrotic genes. The results demonstrated potent paracrine effects of EndoMT-activated lung microvascular EC on normal target lung fibroblasts and suggest a novel mechanism for the progression of SSc-associated pulmonary fibrosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/paracrine-effect-of-proteins-secreted-by-normal-lung-microvascular-endothelial-cells-undergoing-endothelial-mesenchymal-transition-on-the-expression-of-genes-associated-with-tissue-fibrosis-in-normal/