Helium Conditioning Increases Cardiac Fibroblast Migration Which Effect Is Not Propagated via Soluble Factors or Extracellular Vesicles

Figures

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  Figure 1. HeC induces fibroblast migration, but not myofibroblast transformation or inflammation; (A) Migration of CTRL and HeC NRCFs after scratch stimuli with representative images 0, 8 and 24 h after scratch with 5× magnification; (B) qPCR analysis of markers of myofibroblast transformation (α-SMA, Col1α2, FAP and TGF-β) and inflammatory markers (IL1β and IL6) of CTRL and HeC NRCFs. Vimentin was used as a control for fibroblast phenotype; (C) WB representative bands and relative protein expression of α-SMA and Vimentin compared to DDR2 and TGF-β compared to GAPDH in CTRL and HeC NRCF cells; n = 4–7. Results are represented as mean ± SEM; * p < 0.05 vs. CTRL.

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  Figure 2. HeC tends to decrease NRCF mEV secretion: (A) EM image of mEV isolate, black arrow: membrane vesicles; white arrow: protein aggregates; (B) Qualitative analysis of Annexin A1 expression of mEVs with WB; (C) NTA concentration analysis of mEV isolates; (D) NTA size distribution analysis of mEV isolates. Results are represented as mean ± SEM.

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  Figure 3. Effect of HeC is not transferred to naïve fibroblast or endothelial cells via soluble factors; (A) Migration of naïve NRCF cells after transfer of CTRL or HeC NRCF supernatant with representative images 0, 8 and 24 h after scratch with 5× magnification; (B) Migration of HUVEC-TERT2 treated with CTRL or HeC NRCF supernatant with representative images 0 and 8 h after scratch with 5× magnification; (C) In vitro angiogenesis of HUVEC-TERT2 treated with CTRL or HeC NRCF supernatant with representative images 6 h after treatment with 5× magnification. Number of master junctions represent the number of nodes, mean mesh size represent the mean size of the meshes, Mesh index represents the mean length of the branches. n = 4–15. Results are represented as mean ± SEM.

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  Figure 4. HeC accelerates fibroblast migration without inducing myofibroblast transformation but does not affect fibrosis and angiogenesis via secreted factors. In vitro HeC protocol composed of 4 times one hour 95% helium 5% CO2 followed by one hour 95% air 5% CO2 conditions. NRCF cells migrated faster after HeC treatment, but they did not transform to myofibroblasts. mEV concentration tended to decrease in HeC NRCF supernatant. Supernatant of HeC-treated NRCFs did not induce fibrosis or angiogenesis in naïve cells.