Adipose tissue–derived stromal cells’ conditioned medium modulates endothelial‐mesenchymal transition induced by IL‐1β/TGF‐β2 but does not restore endothelial function

Tácia Tavares Aquinas Liguori,Luiz Felipe Pinho Moreira,Martin Conrad Harmsen,Gabriel Romero Liguori

doi:10.1111/cpr.12629

Tácia Tavares Aquinas Liguori, Luiz Felipe Pinho Moreira + Show 2 more

Open Access

https://doi.org/10.1111/cpr.12629

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Abstract

ObjectivesEndothelial cells undergo TGF‐β–driven endothelial‐mesenchymal transition (EndMT), representing up to 25% of cardiac myofibroblasts in ischaemic hearts. Previous research showed that conditioned medium of adipose tissue–derived stromal cells (ASC‐CMed) blocks the activation of fibroblasts into fibrotic myofibroblasts. We tested the hypothesis that ASC‐CMed abrogates EndMT and prevents the formation of adverse myofibroblasts.Materials and methodsHuman umbilical vein endothelial cells (HUVEC) were treated with IL‐1β and TGF‐β2 to induce EndMT, and the influence of ASC‐CMed was assessed. As controls, non‐treated HUVEC or HUVEC treated only with IL‐1β in the absence or presence of ASC‐CMed were used. Gene expression of inflammatory, endothelial, mesenchymal and extracellular matrix markers, transcription factors and cell receptors was analysed by RT‐qPCR. The protein expression of endothelial and mesenchymal markers was evaluated by immunofluorescence microscopy and immunoblotting. Endothelial cell function was measured by sprouting assay.ResultsIL‐1β/TGF‐β2 treatment induced EndMT, as evidenced by the change in HUVEC morphology and an increase in mesenchymal markers. ASC‐CMed blocked the EndMT‐related fibrotic processes, as observed by reduced expression of mesenchymal markers TAGLN (P = 0.0008) and CNN1 (P = 0.0573), as well as SM22α (P = 0.0501). The angiogenesis potential was impaired in HUVEC undergoing EndMT and could not be restored by ASC‐CMed.ConclusionsWe demonstrated that ASC‐CMed reduces IL‐1β/TGF‐β2‐induced EndMT as observed by the loss of mesenchymal markers. The present study supports the anti‐fibrotic effects of ASC‐CMed through the modulation of the EndMT process.

Highlights

Heart failure (HF) is an irreversible and potentially lethal clinical condition that affects nearly 23 million people worldwide.[1]
The aim of our investigation was to assess the impact of factors secreted by adipose tissue–derived stromal cells (ASC) on endothelial‐ mesenchymal transition (EndMT) induced by co‐stimulation with IL‐1β and TGF‐β2
The effects demonstrated with the use of ASC in epithelial‐to‐mesenchymal transition (EMT) are an important indicator that these cells would play a role in EndMT; our findings on endothelial cells are in agreement with the findings described for epithelial cells

Summary

| INTRODUCTION

Heart failure (HF) is an irreversible and potentially lethal clinical condition that affects nearly 23 million people worldwide.[1]. In EndMT‐ induced HUVEC, all intercellular adhesions were disrupted, while all cells were spindle‐shaped at day 5 (Figure 2) These changes could be seen both in the groups cultured only with IL‐1β and those. To corroborate the gene expression results, protein expression was assessed by immunoblotting of the endothelial marker VE‐cad‐ herin (CD144, CDH5 gene) and the mesenchymal marker SM22 (trans‐ gelin, TAGLN gene) (Figure 5) The expression of both proteins did not change in upon pro‐inflammatory stimulation, nor was it affected by co‐treatment with ASC‐CMed. The expression of VE‐cadherin, how‐ ever, was slightly increased by ASC‐CMed, irrespective of cytokine treatment (Figure 5A, one‐way ANOVA, P = 0.1990; Sidak's multiple comparison test, P = 0.0673). HUVEC, while control treatment of HUVEC with ASC‐CMed did not influence sprouting

| DISCUSSION

Findings

| CONCLUSION