RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

Abstract

Endothelial-mesenchymal transition (EndMT) is an essential mechanism in the cardiovascular system, for both cardiovascular development and cardiovascular diseases (CVDs). Recent studies indicate that runt-related transcriprunt-related transcription factor 3 (RUNX3) contributes to EndMT and endothelial cell dysfunction. However, the underlying molecular mechanism remains unknown. The present study was designed to investigate the role of RUNX3 in EndMT and endothelial cell function, and to elucidate the underlying molecular mechanism. Human cardiac microvascular endothelial cells (HCMECs) were incubated in strictly controlled hypoxic conditions (1% O2). HCMECs were cultured under normoxic conditions (21% O2), and then moved to a strictly controlled hypoxic environment (1% O2). Under this hypoxic condition, the cells were transfected with the lentiviral vector containing RUNX3 or an empty lentiviral vector for 8 h. After the cells were cultured under hypoxic conditions for 4 days, CD31 and α-smooth muscle actin colocalization were assessed by immunofluorescence microscopy. Transwell migration and tube formation assays were used to examine the migration and angiogenesis ability. RT-qPCR and western blotting were used to determine the expression of molecules involved in EndMT. Hypoxia induced the transition of HCMECs to mesenchymal cells and markedly promoted tube formation and cell migration. Transforming growth factor-β (TGF-β) and Notch signaling were activated during the hypoxia-induced EndMT of HCMECs. RUNX3 knockdown attenuated EndMT of HCMECs, promoted angiogenic phenotype, and reduced endothelial cell migration. In conclusion, our results showed that RUNX3 knockdown attenuated hypoxia-induced EndMT and reversed endothelial cell functions. RUNX3 is a common downstream target of TGF-β and Notch signaling, and may be a novel therapeutic target for treating CVD mediated by EndMT.

Autophagy attenuates endothelial-to-mesenchymal transition by promoting Snail degradation in human cardiac microvascular endothelial cells

“…Immunofluorescence results ( Figure 1 D) 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), similar as shown in our previous studies [ 27 ]. Taken together, these data demonstrated that hypoxia induced EndMT.…”

“…As shown in Figure 4 A,B, hypoxia up-regulated TGF-β2, p-Smad2, and p-Smad3, which suggested that hypoxia induced EndMT in HCMECs by activating TGF-β signaling pathway, consistent with recent studies [ 27 ]. Snail is the key transcription factor in the downstream of TGF-β signaling pathway.…”

“…HCMECs were purchased from Sciencell Research Laboratories and maintained in EC medium (Sciencell, California, U.S.A.) as described recently [ 27 ]. A density of 10000 cells/cm 2 of passages 2–6 were seeded in a six-well plate.…”

“…The angiogenesis assays were performed as described recently [ 27 ]. Briefly, the matrigel matrix (BD, New Jersey, U.S.A.) was thawed at 4°C overnight and placed in 96-cell culture plate at 37°C for 1 h to allow the matrix solution to solidify.…”

“…Our recent study has reported that hypoxia induces EndMT in human cardiac microvascular ECs (HCMECs), during which TGF-β signaling pathway is activated [ 27 ]. In the present study, we confirmed the findings and demonstrated that autophagy exerts cytoprotection against EndMT in HCMECs to promote tube formation under hypoxia by mediating Snail degradation.…”