Under different pathophysiological conditions, as after myocardial infarction, the growth factor TGFβ is increased in hearts and contributes to cardiac remodelling. Since there are various cell types in the heart that can serve as a source of TGFβ, we now analyzed if cardiac endothelial cells can release bioactive TGFβ under hypoxic/reoxygenated conditions, which signaling pathways are induced by TGFβ and if microRNAs are TGFβ-induced targets.

Microvascular endothelial cells were isolated from rat hearts and exposed to hypoxia (Hx) for 2 hours and reoxygenated (Rx) up to 24 hours. The TGFβ precursor protein expression was increased significantly after 15 min. and 24 h Rx (n=21, p<0.05). In order to analyse, if the increase of TGFβ precursor protein results in enhancement of bioactive TGFβ, we analysed activation of SMAD transcription factors, as classical signaling molecules of TGFβ. An increase in phosphorylation of SMAD2 to 130 ± 15% or 113 ± 4% (n=13, p<0.05) was found after 2h Hx/2h or 24h Rx. This effect was abolished by incubation of endothelial cells with TGFβ type I receptor blocker SB431542 (1µM). Addition of supernatants from hypoxic/reoxygenated endothelial cells to ventricular cardiomyocytes enhanced phosphorylation of SMAD2 also in cardiomyocytes within 1.5 h to 112 ± 1% (n=4, p<0.05). Also this phosphorylation of SMAD2 was blocked by SB431542 (1µM). Simultaneously with the induction of P-SMAD2 after 2 h Rx in endothelial cells the transcription factor GATA2 increases to 126 ± 10 % (n=11, p<0.05), whereas after 24 h Rx, enhancement of GATA2 was not present. This indicates a TGFβ induced gene regulation by GATA2/SMAD2 interaction at early time points of reoxygenation, whereas after 24h Rx SMAD2 interacts probably with different binding partners. In addition to these transcription factors, expression of miRNA-21 and -24 increased after 2 h Hx/24 h Rx to 157 ± 23 % (n=12) or 230 ± 25 % (n=13, p<0.5 vs. controls). This increase was blocked by SB431542 (1 µM).

In conclusion, under hypoxic conditions endothelial cells release TGFβ at early and late time points. This can induce SMAD2/GATA2 dependent processes of transcription in cardiomyocytes as well as in endothelial cells. After hypoxia/reoxygenation TGFβ-dependent induction of miRNA-21 and -24 is found, which are both discussed to contribute to angiogenesis and fibrosis in the heart.