Irradiated Human Umbilical Vein Endothelial Cells Undergo Endothelial-Mesenchymal Transition via the Snail/miR-199a-5p Axis to Promote the Differentiation of Fibroblasts into Myofibroblasts - PubMed (original) (raw)

Irradiated Human Umbilical Vein Endothelial Cells Undergo Endothelial-Mesenchymal Transition via the Snail/miR-199a-5p Axis to Promote the Differentiation of Fibroblasts into Myofibroblasts

Minxiao Yi et al. Biomed Res Int. 2018.

Abstract

Radiation induced pulmonary fibrosis (RIPF) is one of the major side effects of radiotherapy for lung cancer. Previous studies have shown that endothelial cells and activated myofibroblasts play a key role in RIPF. However, the interaction between irradiated endothelial cells and activation of myofibroblasts has not been reported. The aim of the present study was to examine whether irradiated endothelial cells would affect the differentiation of fibroblasts into myofibroblasts in the process of RIPF. In the current study, we used a coculture system that allowed direct contact between human fetal lung fibroblasts (MRC-5) and irradiated human umbilical vein endothelial cells (HUVECs). After 24 or 48 h, cells were sorted by flow cytometry. Radiation induced endothelial-mesenchymal transition (EndMT) by significantly increasing the expression of Snail and vimentin and reducing the expression of CD31 in HUVECs. In addition, irradiation of HUVECs induced the expression of collagen type I and _α_-smooth muscle actin (_α_-SMA) in MRC-5 cells. Further investigation indicated that irradiation of HUVECs induced the differentiation of fibroblasts into myofibroblasts through the Snail/miR-199a-5p axis. We conclude that irradiated endothelial cells undergo EndMT to promote differentiation of fibroblasts into myofibroblasts via the Snail/miR-199a-5p axis.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Irradiated HUVECs promote the expression of MRC-5 fibrotic markers. Panel (a): scheme illustrating the procedure to evaluate the contribution of HUVECs to MRC-5 fibrotic markers. MRC-5 cells were cocultured with irradiated HUVECs with no physical contact (noncontact). MRC-5 cells were cocultured with irradiated HUVECs with physical contact (contact). Panel (b): HUVECs were preincubated in the presence or absence of irradiation and then coincubated with MRC-5 cells (noncontact). After incubation for 24 or 48 h, the MRC-5 cells were harvested. Total protein was extracted. Western blot shows the protein expression of collagen I and _α_-SMA. Panel (c): cell sorting by flow cytometry. MRC-5 cells were labeled with fluorescent GFP (green cells) and plated with unlabeled HUVECs at a ratio of 9 : 1. Panel (d): HUVECs were preincubated in the presence or absence of radiation and then coincubated with MRC-5 cells (contact). After incubation for 24 or 48 h, the MRC-5 cells were sorted by flow cytometry and harvested. Western blot was performed to examine total protein. Panel (e): dual immunofluorescence analysis of _α_-SMA and stress fibers stained with phalloidin green. Colocalization of stress fibers and _α_-SMA in MRC-5 was evident under treatment of irradiated HUVECs.

Figure 2

Figure 2

Radiation induced EndMT contributes to the fibrotic effect in MRC-5. Panel (a): HUVECs were incubated in the presence or absence of irradiation. After incubation for 48 h, cells were harvested. The expression of Snail, vimentin, and CD31 was examined. Panels (b) and (c): Snail overexpression reduced the expression of CD31, while increasing the expression of vimentin. Panel (d): Snail overexpression in HUVECs significantly increased migration capacity. Panel (e): Snail overexpression led to impaired ability of endothelial cells to form capillary-like structures. Panel (f): Snail-overexpressed HUVECs were preincubated in the presence or absence of irradiation and then coincubated with MRC-5 (contact). After incubation for 48 h, cells were sorted by flow cytometry and MRC-5 were harvested. Total protein was examined by western blot. Error bars represent SEM from three replicates (∗ P < 0.05).

Figure 3

Figure 3

Snail-induced miR-199a-5p promotes the expression of MRC-5 fibrotic markers. Panel (a): eight fibrosis-associated microRNAs were selected. The expression levels of miR-145-5p and miR-199a-5p were increased in irradiated HUVECs compared to that in the control group. Panel (b): the expression of miR-145-5p and miR-199a-5p in Snail-overexpressed HUVECs. Panel (c): transfer of miR-199a-5p from HUVECs to cocultured MRC-5 cells. Total RNA was extracted. The miRNA expression was measured using real-time qPCR. Error bars represent SEM from three replicates (∗ P < 0.05).

Figure 4

Figure 4

Transfer of miR-199a-5p from HUVECs to cocultured MRC-5 cells promotes the expression of MRC-5 fibrotic markers. Panel (a): HUVECs were transfected with miR-199a-5p mimic, miR-199a-5p inhibitor, and control vector. miR-199a-5p was transferred from HUVECs to MRC-5. Panel (b): miR-199a-5p was transferred from HUVECs to MRC-5 cells and promoted the expression of collagen I and _α_-SMA in MRC-5 cells. Panel (c): MRC-5 cells were transfected with miR-199a-5p mimic, miR-199a-5p inhibitor, and control vector. Fibrotic markers were examined after such transfection. Error bars represent SEM from three replicates (∗ P < 0.05).

References

    1. DeSantis C. E., Lin C. C., Mariotto A. B., et al. Cancer treatment and survivorship statistics, 2014. CA: A Cancer Journal for Clinicians. 2014;64(4):252–271. doi: 10.3322/caac.21235. - DOI - PubMed
    1. Lee J.-G., Shim S., Kim M.-J., et al. Pentoxifylline Regulates Plasminogen Activator Inhibitor-1 Expression and Protein Kinase A Phosphorylation in Radiation-Induced Lung Fibrosis. BioMed Research International. 2017;2017 doi: 10.1155/2017/1279280.1279280 - DOI - PMC - PubMed
    1. Ding N.-H., Li J. J., Sun L.-Q. Molecular mechanisms and treatment of radiation-induced lung fibrosis. Current Drug Targets. 2013;14(11):1347–1356. doi: 10.2174/13894501113149990198. - DOI - PMC - PubMed
    1. Oh Y.-T., Noh O. K., Jang H., et al. The features of radiation induced lung fibrosis related with dosimetric parameters. Radiotherapy & Oncology. 2012;102(3):343–346. doi: 10.1016/j.radonc.2012.02.003. - DOI - PubMed
    1. Chung E. J., Sowers A., Thetford A., et al. Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model. International Journal of Radiation Oncology • Biology • Physics. 2016;96(4):857–866. doi: 10.1016/j.ijrobp.2016.07.026. - DOI - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources