Isoform-specific effects of transforming growth factor on endothelial-to-mesenchymal transition

Endothelial-to-mesenchymal transition (EndMT) was first reported in the embryogenesis. Recent studies show that EndMT also occurs in the disease progression of atherosclerosis, cardiac and pulmonary fibrosis, pulmonary hypertension, diabetic nephropathy, and cancer. Although transforming growth factor (TGF) is crucial for EndMT, it is not clear which isoform elicits a predominant effect. The current study aims to directly compare the dose-dependent effects of TGF1, TGF2, and TGF3 on EndMT and characterize the underlying mechanisms. In our results, all three TGF isoforms induced EndMT in human microvascular endothelial cells after 72hr, as evidenced by the increased expression of mesenchymal markers N-cadherin and -smooth muscle actinas well as the decreased expression of endothelial nitric oxide synthase. Interestingly, the effect of TGF2 was the most pronounced. At 1ng/ml, only TGF2 treatment resulted in significantly increased phosphorylation (activation) of Smad2/3 and p38-MAPK and increased expression of mesenchymal transcription factorsSnail and FoxC2. Intriguingly, we observed that treatment with 1ng/ml TGF1 and TGF3, but not TGF2, resulted in an increased expression of TGF2, thus indicating that EndMT with TGF1 and TGF3 treatments was due to the secondary effects through TGF2 secretion. Furthermore, silencing TGF2 using small interfering RNA blunted the expression of EndMT markers in TGF1- and TGF3-treated cells. Together, our results indicate that TGF2 is the most potent inducer of EndMT and that TGF1- and TGF3-induced EndMT necessitates a paracrine loop involving TGF2.