Endothelial Mesenchymal Transition: Comparative Analysis of Different Induction Methods - PubMed (original) (raw)

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Endothelial Mesenchymal Transition: Comparative Analysis of Different Induction Methods

Mariana T Pinto et al. Biol Proced Online. 2016.

Abstract

Background: Endothelial-Mesenchymal-Transition (EndMT) plays an essential role in cardiovascular development, and recently became an attractive therapeutic target based on evidence supporting its involvement in fibrosis and cancer. Important questions that remain to be answered are related to the molecular mechanisms that control EndMT in different organs and distinct pathological conditions. The lack of a detailed protocol for induction of EndMT and the assumption that TGF-β isoforms play similar roles on different types of endothelial cells, limit progress in the field. The aim of this study was to compare the induction of EndMT by TGF-β isoforms in endothelial cells of different sources, and define a detailed protocol for EndMT assessment in vitro.

Results: We compared the dose-dependent effect of TGF-β isoforms, under normoxia and hypoxia, on the induction of EndMT in human coronary and pulmonary artery endothelial cells. Our results suggest that endothelial cells undergo spontaneous EndMT with time in culture under the conditions tested. The extent of EndMT induction by TGF-β was dependent on the dose and endothelial cell type. Furthermore, the potential of TGF-β to induce EndMT was reduced under hypoxia relative to normoxia.

Conclusions: Our work suggests that the response of endothelial cells to TGF-β is intrinsic to the dose, cell type and environment. Optimization of induction conditions may be essential, as pathways triggering EndMT may vary during development and pathological conditions. Therefore, caution is needed regarding indiscriminate use of TGF-β to induce EndMT for mechanistic studies.

Keywords: Endothelial-mesenchymal transition; Hypoxia; TGF-β.

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Figures

Fig. 1

Fig. 1

Time-dependent Change in the Expression of Endothelial and Smooth Muscle Markers in HMPAECs and HCAECs. a Representative Western blot image comparing changes in the expression of the endothelial marker CD31 and the smooth muscle markers α-SMA and SM22-α in HMPAECs and HCAECs with time in culture. b Quantification of endothelial and smooth muscle markers expression by densitometry analysis of western blots. Results were normalized to α-tubulin (loading control) and expressed as fold-change relative to baseline (day 5 versus day 2 after plating). CD31 (HMPAECs, n = 4; HCAECs, n = 3, non-significant); α-SMA (n = 3, non-significant), SM22-α (n = 3, non-significant)

Fig. 2

Fig. 2

Effect of TGF-β on the Expression of Endothelial and Smooth Muscle Markers in HCAECs. a Representative Western blot images comparing changes in the expression of the endothelial marker CD31 and the smooth muscle marker α-SMA and SM22-α in HCAECs after exposure to different doses of TGF-β1 and TGF-β2. b Quantification of endothelial and smooth muscle marker expression by densitometry analysis of western blots. Results were normalized to α-Tubulin (loading control) and expressed as fold-change relative to control without TGF-β. (n = 3, *p < 0.05, **p < 0.005)

Fig. 3

Fig. 3

Effect of TGF-β on the Expression of Endothelial and Smooth Muscle Markers in HMPAECs. a Representative Western blot images comparing changes in the expression of the endothelial marker CD31 and the smooth muscle marker α-SMA and SM22-α in HMPAECs after exposure to different doses of TGF-β1 and TGF-β2. b Quantification of endothelial and smooth muscle marker expression by densitometry analysis of western blots. Results were normalized to α-Tubulin and Gapdh (loading control) and expressed as fold-change relative to control without TGF-β. (n = 3–4, *p < 0.05)

Fig. 4

Fig. 4

Effect of Hypoxia and TGF-β on the Expression of Endothelial and Smooth Muscle Markers in HCAECs. a Representative Western blot images comparing changes in the expression of the endothelial marker CD31 and the smooth muscle marker α-SMA and SM22-α in HCAECs after exposure to different doses of TGF-β1 and TGF-β2 under Normoxia (21 % O2) and Hypoxia (1 % O2). b Quantification of endothelial and smooth muscle marker expression by densitometry analysis of western blots comparing the effect of hypoxia to baseline and normoxia control. c Quantification of endothelial and smooth muscle marker expression by densitometry analysis of western blots comparing the effect of TGF-β1 and TGF-β2 under Normoxia (21 % O2) and Hypoxia (1 % O2). Results were normalized to α-Tubulin and Gapdh (loading control) and expressed as fold-change relative to baseline (BL) in B, and relative to control without TGF-β in C. (n = 3, *p < 0.05, **p < 0.005)

Fig. 5

Fig. 5

Effect of TGF-β on the Expression of Endothelial and Smooth Muscle Markers in HMPAECs. a Representative Western blot images comparing changes in the expression of the endothelial marker CD31 and the smooth muscle marker α-SMA and SM22-α in HMPAECs after exposure to different doses of TGF-β1 and TGF-β2 under Normoxia (21 % O2) and Hypoxia (1 % O2). b Quantification of endothelial and smooth muscle marker expression by densitometry analysis of western blots comparing the effect of hypoxia to baseline and normoxia control. c Quantification of endothelial and smooth muscle marker expression by densitometry analysis of western blots comparing the effect of TGF-β1 and TGF-β2 under Normoxia (21 % O2) and Hypoxia (1 % O2). Results were normalized to Gapdh (loading control) and expressed as fold-change relative to baseline (BL) in B, and relative to control without TGF-β in C. (n = 3–4, *p < 0.05)

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