Endothelial Mesenchymal Transition in Hypoxic Microvascular Endothelial Cells and Paracrine Induction of Cardiomyocyte Apoptosis Are Mediated via TGFβ₁/SMAD Signaling.

Isabella Sniegon,Gerhild Euler,Mona Prieß,Rainer Schulz,Jacqueline Heger

doi:10.3390/ijms18112290

Isabella Sniegon, Gerhild Euler + Show 3 more

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https://doi.org/10.3390/ijms18112290

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Abstract

Cardiac remodeling plays a crucial role in the development of heart failure after mycocardial infarction. Besides cardiomyocytes, endothelial cells are recognized to contribute to cardiac remodeling. We now investigated processes of endothelial mesenchymal transition (EndoMT) in microvascular endothelial cells of rat (MVEC) under hypoxia and paracrine effects on ventricular cardiomyocytes of adult rat. Exposure of MVECs to hypoxia/reoxygenation enhanced TGFβ/SMAD signaling, since phosphorylation, and thus activation, of SMAD1/5 and SMAD2 increased. This increase was blocked by inhibitors of TGFβ receptor types ALK1 or ALK5. Exposure of ventricular cardiomyocytes to conditioned medium from hypoxic/reoxygenated MVECs enhanced SMAD2 phosphorylation and provoked apoptosis in cardiomyoyctes. Both were blocked by ALK5 inhibition. To analyze autocrine effects of hypoxic TGFβ signaling we investigated EndoMT in MVECs. After 3 days of hypoxia the mesenchymal marker protein α-smooth muscle actin (α-SMA), and the number of α-SMA- and fibroblast specific protein 1 (FSP1)-positive cells increased in MVECs cultures. This was blocked by ALK5 inhibition. Similarly, TGFβ1 provoked enhanced expression of α-SMA and FSP1 in MVECs. In conclusion, hypoxia provokes EndoMT in MVECs via TGFβ1/SMAD2 signaling. Furthermore, release of TGFβ1 from MVECs acts in a paracrine loop on cardiomyocytes and provokes apoptotic death. Thus, in myocardial infarction hypoxic endothelial cells may contribute to cardiac remodeling and heart failure progression by promotion of cardiac fibrosis and cardiomyocytes death.

Highlights

Myocardial infarction provokes cardiomyocyte death and scar formation in the infarct area, and causes diverse remodeling processes, like cardiomyocytes hypertrophy or fibrosis
This indicates that microvascular endothelial cells of rat (MVEC) adhere faster to the culture dish, so that α-smooth muscle actin (α-SMA) expressing, mesenchymal cells can be washed off
The remaining cells with greater than 90% purity could be identified as endothelial cells, since they express the typical marker CD31 and von Willebrandt factor (Figure 1D)

Summary

Introduction

Myocardial infarction provokes cardiomyocyte death and scar formation in the infarct area, and causes diverse remodeling processes, like cardiomyocytes hypertrophy or fibrosis. Cardiac fibrosis is established by resident fibroblasts, and by transition of other cell types into fibroblasts In this context, a pivotal role is attributed to the transition of endothelial to mesenchymal cells (EndoMT). Hypoxic conditions can disturb the barrier function of the endothelium, since cell–cell contacts are reduced in hypoxic rat coronary endothelial cells due to disarrangement of the actin cytoskeleton and the loss of cadherins at the cell surface. This phenomenon leads to reductions of adherence junctions [3]. Thereby, the endothelial cells can exercise substantial control over the contractility and growth of cardiomyocytes [5,6]

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