Human Cardiac Microvascular Endothelial Cells

Hypoxia Triggered EndMT in HCMECs

Endothelial-to-mesenchymal transition (EndMT) has been reported in a variety of physiological and pathological phenomena. For example, during heart embryonic development, ECs from endocardium can give rise to the valves and septa of the heart via EndMT. Under the pathological condition, EndMT contributes to many tissue fibrosis diseases such as pulmonary arterial hypertension (PAH), systemic sclerosis (SSc), and unilateral ureteral obstruction (UUO). In particular, EndMT is involved in the development of cardiac fibrosis diseases, such as acute myocardial infarction (AMI), heart failure, and hypertension. It has been shown that the elevation of exogenous and endogenous TGFβ is a potent inducer of EndMT, but accumulating evidence have also suggested that hypoxia, inflammation, and high glucose also induce EndMT.

In order to establish the model of EndMT in vitro, we exposed human cardiac microvascular endothelial cells (HCMECs) to hypoxia condition for 3 days. ECs changed gradually from a cobblestone phenotype to an elongated spindle-shaped morphology (Fig. 1A). RT-PCR analysis (Fig. 1B) showed that hypoxia decreased the expression of CD31 and E-cadherin mRNAs but increased α-SMA, FSP1, and Snail mRNA levels. Compared with the control groups, cells acquired mesenchymal makers (α-SMA, FSP1, and Snail) and lost endothelial makers (CD31 and E-cadherin) (Fig. 1C). Immunofluorescence results (Fig. 1D) further showed that CD31+α-SMA/FSP1+ cells or CD31−α-SMA/FSP1+ cells were found in the hypoxia groups, and CD31+α-SMA/FSP1− cells were found in the normoxia group. All these changes were time dependent (results not shown). Taken together, these data demonstrated that hypoxia induced EndMT.

Fig. 1. EndMT was induced by hypoxia in HCMECs (Zou, Jin, et al., 2017).

Hypoxia Enhances RUNX3 Expression in HCMECs

To investigate the expression of runt-related transcription factor 3 (RUNX3) in HCMECs during hypoxia-induced EndMT, we examined RUNX3 expression by western blotting and RT-qPCR. Both mRNA and protein levels of RUNX3 were low and increased significantly after the cells were cultured in hypoxic conditions for 4 days (Fig. 2 and 3A and B).

To examine whether RUNX3 plays a pivotal role in EndMT of HCMECs, we suppressed RUNX3 expression in HCMECs with RUNX3-RNAi lentivirus and found that RUNX3 knockdown ameliorated hypoxia-induced EndMT (Fig. 4). Suppress of RUNX3 downregulated the mRNA expression of α-SMA and FSP-1 and upregulated the expression of endothelial markers (Fig. 4A). Hypoxia induced a notable increase in the expression of α-SMA and FSP-1, while RUNX3 knockdown attenuated α-SMA and FSP-1 protein expression and increased CD31 and VE-cadherin levels in the HCMECs treated under a hypoxic condition (Fig. 2 and 4B). Double immunofluorescence staining assays were carried out to further confirm that knockdown of RUNX3 attenuated EndMT. CD31 was downregulated while α-SMA was upregulated in the hypoxia group compared with the control group (Fig. 4C), while CD31 was upregulated and α-SMA was downregulated in the RUNX3i group compared with the GFP group (Fig. 4D).

Fig. 2. Western blotting of RUNX3 and its targeting proteins in the control, hypoxia, RUNX3i and green fluorescent protein (GFP) groups (Liu, Yanhua, et al., 2017).

Fig. 3. RT-PCR data showing the mRNA and protein levels of RUNX3 in the control, hypoxia, RUNX3i and green fluorescent protein (GFP) groups (Liu, Yanhua, et al., 2017).

Fig. 4. Knockdown of RUNX3 attenuates EndMT of HCMECs (Liu, Yanhua, et al., 2017).