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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 (Hx) and reoxygenated (Rx) conditions, and which signalling pathways are induced by TGFβ.Microvascular endothelial cells were isolated from rat hearts and exposed to hypoxia (Hx) for 2 hours and followed by reoxygenation (Rx) up to 24 hours. The TGFβ precursor protein expression increased significantly after 1h and 24 h Rx (n = 21, p < 0.05). In order to analyse, if this increase of TGFβ precursor protein results in enhancement of bioactive TGFβ, we analysed activation of SMAD transcription factors, as classical signalling molecules of TGFβ. An increase in phosphorylation of SMAD2 to 130 ± 15 % or 113 ± 4% (n = 13, p < 0.05) was found after 2 h Hx followed by 2 h or 24 h Rx, respectively. This effect was abolished by incubation of endothelial cells with TGFβ type I receptor blocker SB431542 (1μM). Simultaneously with the induction of P-SMAD2 after 2 h Rx, expression of transcription factor GATA2 increased 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 in endothelial cells via GATA2/SMAD2 interaction at early time points of reoxygenation, whereas after 24 h Rx SMAD2 interacts probably with different binding partners to regulate different target genes. To analyse, if TGFβ released from endothelial cells can also influence other cell types in the heart, supernatants of endothelial cells that underwent 2 h Hx followed by 1h Rx were added to ventricular cardiomyocytes of rat. These supernatants of hypoxia/reoxygenated endothelial cells enhanced phosphorylation of SMAD2 within 1.5 h in cardiomyocytes to 112 ± 1% (n = 4, p < 0.05).In conclusion, after hypoxia endothelial cells release TGFβ at early and late time points of reperfusion. Then TGFβ acts in an autocrine manner on endothelial cells to regulate gene expression via the transcription factors SMAD2 and GATA2. Furthermore, TGFβ acts in a paracrine manner on cardiomyocytes and activates SMAD2 signalling in theses cells as well.