Endotheliale-mesenchymale Transition in hypoxen mikrovaskulären Endothelzellen und der Einfluss auf die TGFβ1/SMAD-Signalkaskade
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Beschreibung
In this study, a single cell type endothelial cells culture from cardiac microvasculature of rats (MVECs) was established, and endothelilal mesenchymal transition of these cells under hypoxia or TGFβ1 was determined.
First, the purity of endothelial cell culture was improved. By optimising digestion of collagenase at 45 minutes and early washing after one hour of adhesion, I obtained a purity of 90% MVECs. These cells presented microscopically a cobblestone endothelial-like monolayer, whereas cells with shorter digestion and longer adhesion times appeared fibroblast like spindle-shaped and elongated. Due to cell transformations after every step of sub-culturing, we used just the first passage of cells at a confluence of 95 %.
To analyse the influence of hypoxia and reoxygenation on these cells, efficiency of oxygen deprivation was proven by detection of HIF-1α-protein.
Transforming growth factor beta (TGFβ) as growth factor is mainly involved in cardial transforming processes, termed cardial remodeling. In this study, it could be shown that hypoxia with following reoxygenation activates well-known signaling molecules of TGFβ1, named SMAD-proteins. In particular, hypoxia induced the expression of SMAD2 and SMAD1/5. Because both molecules are phosphorylated and activated at the outer membrane via the TGFß-receptor it might be argued that hypoxia induces TGFβ1 release from MVECs. A specific TGFβ-receptor-inhibitor against ALK5 could block the SMAD2 activation.
Furthermore, I could demonstrate that hypoxia provokes endothelial mesenchymal transition (EndoMT). This was detected by the increased expression of mesenchymal markers like α-SMA and FSP-1. The upregulation could be blocked by specific ALK5-inhibition, thereby showing contribution of TFGβ/SMAD2 signaling. Low concentrations of TGFβ1 (0,01 ng/ml) revealedvimilar results, whereas high TGFβ1 concentrations (1 ng/ml) triggerd an impressive transformation of endothel cells. It can therefore be concluded that a small release of TGFβ1 provokes a cardial EndoMT.
In our study, hypoxia induced a reduction in cell count, presumable by a diminished proliferation rate, which then contributes to pore formation in the monolayer. Once rhomboid-oval single cells transformed around the pores to spindle-shaped, mainly α-SMA marked cells. Equally to hypoxia, TGFβ1 triggered pore fomations.
The endothelial marker CD31 did not show any change in its expression, neither under hypoxia nor by TGFβ1. Thus, it can be concluded that the endothelial cell marker still exists after EndoMT.
In vivo hypoxic conditions, as they are found in myocardial infarction, may provoke EndoMT. This can contribute to desintegration of the cell layer and may facilitate immigration of endothelial derived mesenchymal cells and the release of TGFβ1 into cardiac tissue. These processes can contribute to cardiac fibrosis and heart failure progression after myocardial infarction. As both processes are mediated via TGFβ1/SMAD2 signaling, interference with this pathway should be a major aim for prevention of myocardial damage due to infarction.
Autor*in
Isabella Maria Sniegon