Endothelial-Dependent Effect Of Rtef-1 In Pressure Overload Induced Cardiac Hypertrophy-Role Of VEGF-B

ObjectiveRelated Transcription Enhancer Factor-1 (RTEF-1) is a member of transcription enhancer family. It mainly exists in muscle cells and regulates muscle specific genes expression via binding to M-CAT sequence on the promoter area of these genes. We previously established that RTEF-1 regulates vascular endothelial growth factor A (VEGF-A) via binding to stimulating protein 1 (SP1) sequence on VEGF-A promoter in endothelial cells. However, it remains unclear whether RTEF-1 participates in the crosstalk between endothelial cell and cardiomyocytes. To determine the role of RTEF-1 in endothelial cell, especially in the conditions of cardiac hypertrophy, we developed vascular endothelial specific RTEF-1 overexpressed transgenic mice and RTEF-1 overexpressed endothelial cells. The changes and effects of these mice and cells under physiological and pathophysiological conditions provided an insight of the function of endothelial cells in the development of cardiac hypertrophy.Materials and methods 1) Endothelial specific RTEF-1 overexpressed transgenic mice2) Transverse aortic constriction (TAC) surgery and assessment3) Cardiomyocyte cross section area and vessel density assessment4) Cell culture5) Retroviral Transduction and siRNA Transfection6) Dual luciferase assay and Chromatin Immunoprecipitation (ChIP)7) Messenger ribonucleic acid (mRNA) and protein analysesResults1) VE-Cad/RTEF-1 miceGenotyping and western blotting indicated RTEF-1 gene was transferred in the mice genome, and specifically overexpressed in the endothelial cells in the VE-Cad/RTEF-1 mice heart.2) Pressure overload induced cardiac hypertrophy in VE-Cad/RTEF-1 miceVE-Cad/RTEF-1 mice developed heavier cardiac hypertrophy when compared with wild type mice (WT) after 8 weeks of TAC surgery. There were increased ratio of heart weight to tibia bong length (HW/TBL), enlarged cross section area of cardiomyocyte, thickened left ventricular wall and incrased expression of cardiac hypertrophic gene markers in VE-Cad/RTEF-1 mice after TAC surgery. Meanwhile, elevated VEGF-B and phosphorylation of extracellular regulated protein kinase 1/2 (ERK1/2) were observed.3) RTEF-1 overexpression in Human microvascular endothelial cell-1 (HMEC-1/RTEF-1) and changes of VEGF-BWestern blotting results demonstrated overexpression of RTEF-1 in the HMEC-1 cell. Both HMEC-1/RTEF-1 lysates and culture medium showed increased VEGF-B expression after normoxic or hypoxic treatment. However, decreased VEGF-B was found using RTEF-1 siRNA.4) RTEF-1 regulating VEGF-B promoter activity Dual luciferase assay and ChIP assay demonstrated that RTEF-1 regulated VEGF-B promoter activity via binding to its M-CAT elements, which could be enhanced under hypoxic conditions.5) Prohypertrophic effect of VEGF-B H9C2 showed increased expression of cardiac hypertrophic gene markers after incubating with culture medium from hypoxia treated HME-1/RTEF-1 or human recombinant VEGF-B167 (hrVEGF-B167), which could be blocked by VEGF-B siRNA. Moreover, hrVEGF-B167 induced significant enlargement of NRVMs.6) Signaling pathway in the prohypertrophic effect of VEGF-B Western blotting showed ERK1/2 was significantly phosphorylated by hrVEGF-B167 in NRVMS. Moreover, U0126, an ERK1/2 specific inhibitor, blocked hrVEGF-B167 induced ERK1/2 phosphorylation and prohypertrophic effect in cardiomyocytes.Conclusion1) VE-Cad/RTEF-1 mice exhibit an exacerbation of left ventricular hypertrophy after pressure overload.2) VEGF-B is upregulated in VE-Cad/RTEF-1 mice after TAC surgery and HMEC-1/RTEF-1 via a direct interaction between RTEF-1 and VEGF-B promoter. The upregulated VEGF-B shows a prohypertrophic effect via ERK1/2 signaling pathway.These results suggest that endothelial cells play an important role in the development of cardiac hypertrophy via RTEF-1 trancriptionally regulating VEGF-B expression. It also reveals a direct interaction between endothelial cells and cardiomyocytes in the development of cardiac hypertrophy.