RUNX3 Modulates Hypoxia-Induced Endothelial To Mesenchymal Transition Of Human Cardiac Microvascular Endothelial Cells

Objectives:1. To observe the effects of hypoxia on endothelial to mensenchymal transition(End MT) involving human cardiac microvascular cells(HCMECs).2. To investingate the changes of transforming growth factor β and Notch signaling pathways in hypoxia-induced End MT of HCMECs.3. To investigate the role of RUNX3 in HCMECs function and the effects of knockdown of RUNX3 on End MT of HCMECs, and elucidate the underlying molecular mechanisms. Methods:1. To observe the effects of hypoxia on End MT of HCMECs:The HCMECs were cultured under normoxic conditions before they were divided into 2 groups as follows: i)Control group: the HCMECs were cultured under normoxic conditions, without any treatment; ii)Hypoxia group: the HCMECs were cultured under strictly controlled hypoxic conditions(1% O2) for 1 day,4 days, 5 days respectively. Then perform the experiments as follows: Examining the morphological changes under an inverted microscope; Using double immunofluorescence staining and observing the changes of CD31, αSMA expression; RT-PCR was used to detect the relative m RNA expressions of CD31, VE-cadherin, αSMA and FSP-1 in the HCMECs from each group.2. To investingate the changes of transforming growth factor β and Notch signaling pathways in hypoxia-induced End MT of HCMECs, the role of RUNX3 in HCMECs function and the effects of knockdown of RUNX3 on End MT of HCMECs:The HCMECs were cultured under normoxic conditions before they were divided into 4 groups as follows: i)Control group: the HCMECs were cultured under normoxic conditions for 4 days; ii)Hypoxia group: the HCMECs were cultured under strictly controlled hypoxic conditions(1% O2) for 4 days; ⅲ)RUNX3 group: the HCMECs were transfected with the lentiviral vector containing RUNX3(RUNX3-RNAi lentivirus) for 8 h and cultured under strictly controlled hypoxic conditions(1% O2) for 4 days; ⅳ) GFP group: the HCMECs were transfected with an empty lentiviral vector for 8 h and cultured under strictly controlled hypoxic conditions(1% O2) for 4 days. Then perform the experiments as follows: Using double immunofluorescence staining and observing the changes of CD31, αSMA expression; The transwell migration assay and tube formation assay was used to examine the migration and angiogenesis ability of HCMECs from each group; RT-PCR was used to detect the relative m RNA expressions of CD31, VE-cadherin, αSMA, FSP-1, RUNX3,TGF-β1, Smad2, Smad3, Notch-1, Hes1, Hey1, Slug and Snail in the HCMECs from each group; Western blot analysis was used to detect the relative protein expressions of CD31, VE-cadherin, αSMA, FSP-1, RUNX3, Smad2/3, p-Smad2/3, Notch-1, Hes1, Hey1, Slug and Snail in the HCMECs from each group. Results:1.The effects of hypoxia on End MT of HCMECs:⑴The HCMECs from the Hypoxia group acquired an elongated spindle-shaped apperance. ⑵ Double immunofluorescence staining showed that the hypoxia-treated HCMECs obtained the expression of αSMA, whereas lost the expression of CD31, the number of CD31 and αSMA double positive cells was most in H4 group(P<0.01). ⑶RT-PCR datas showed that the relative m RNA expression of endothelial markers, such as CD31 and VE-cadherin, was downregulated. At the same time, the mesenchymal markers, such as FSP1 and αSMA,was upregulated(P<0.05).2. The effects of knockdown of RUNX3 on End MT of HCMECs: ⑴ Double immunofluorescence staining showed that the expression of αSMA decreased and the expression of CD31 increased in the HCMECs from the RUNX3 group compared with the Hypoxia group(P<0.05).⑵RT-PCR and Western Blot data showed that: Compared with the Hypoxia group: the expression of CD31 and VE-Cadherin up-regulated(P<0.05), αSMA and FSP-1 down-regulated(P<0.05).3. The role of RUNX3 in HCMECs function: Compared to the control HCMECs, hypoxia promoted tube formation and induced cell migration remarkably, whereas knockdown of RUNX3 attenuated cell migration and increased angiogenesis in HCMECs treated under hypoxia condition(P<0.05).4. The changes of TGFβ signaling pathways in hypoxia-induced End MT:⑴RT-PCR results showed that: Hypoxia activated TGFβ signaling pathways, the relative m RNA expression of TGF-β1, Smad2 and Smad3 up-regulated(P<0.05). Knockdown of RUNX3 increased TGF-β1 m RNA expression, whereas reduced the expression of Smad2、Smad3(P<0.05).⑵Western Blot data showed that: Hypoxia increased the relative protein expression of Smad2/3 and P-smad2/3(P<0.05); the level of Smad2/3 and P-smad2/3 in the HCMECs from the RUNX3 group were lower than the Hypoxia group(P<0.05).5. The changes of Notch signaling pathways in hypoxia-induced End MT: RT-PCR and Western Blot results showed that: Hypoxia activated Notch signaling pathways, the expression of Notch-1, Hes1, Hey1, Slug and Snail up-regulated(P<0.05). After knockdown of RUNX3, the expression of Notch-1 remarkably up-regulated,however the principal End MT transcriptional factors Slug and Snail down-regulated(P<0.05), the expression of Hes1 and Hey1 down-regulated obviously(P<0.05). Conclusion:1. Hypoxia induced the transition of HCMECs to mesenchymal cells and promoted tube formation and cell migration remarkably.2. TGF-β and Notch signaling were activated during the Hypoxia-induced End MT of HCMECs.3. Knockdown of RUNX3 attenuated End MT of HCMECs and positively affected angiogenic phenotype and reduced endothelial cell migration, RUNX3 is likely to be a common downstream target of TGF-β and Notch signaling pathway.