Endothelial Cells Require Related Transcription Enhancer Factor-1 for Cell–Cell Connections Through the Induction of Gap Junction Proteins

Abstract

Objective-Capillary network formation represents a specialized endothelial cell function and is a prerequisite to establish a continuous vessel lumen. Formation of endothelial cell connections that form the vascular structure is regulated, at least in part, at the transcriptional level. We report here that related transcription enhancer factor-1 (RTEF-1) plays an important role in vascular structure formation. Methods and Results-Knockdown of RTEF-1 by small interfering RNA or blockage of RTEF-1 function by the transcription enhancer activators domain decreased endothelial connections in a Matrigel assay, whereas overexpression of RTEF-1 in endothelial cells resulted in a significant increase in cell connections and aggregation. In a model of oxygen-induced retinopathy, endothelial-specific RTEF-1 overexpressing mice had enhanced angiogenic sprouting and vascular structure remodeling, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. Mechanistic studies revealed that RTEF-1 induced the expression of functional gap junction proteins including connexin 43, connexin 40, and connexin 37. Blocking connexin 43 function inhibited RTEF-1-induced endothelial cell connections and aggregation. We further found that gap junction proteins, specifically connexin (Cx) 37, Cx40, and Cx43, were significantly regulated by RTEF-1 and acted as adhesion sites that enhanced endothelial cellular connections and aggregation, as well as promoted the timely formation of competent vascular networks. We selected a mouse retina model for our in vivo studies because of the retinal vasculature developing shortly after birth in a highly stereotypic manner, 9 and RTEF-1 appeared as an essential gene for retinal vascular development by enhancing angiogenic sprouting and promoting the formation of a denser primary capillary network in an oxygen-induced retinopathy (OIR) model. These observations indicate that RTEF-1 coordinated endothelial cell-to-cell connections and is essential for the assembly of vascular networks in vitro and in vivo. Materials and Methods Cell CultureHuman umbilical vein endothelial cells (Lonza, Allendale, NJ) were cultured in endothelial basal medium-2 (Lonza). Human microvascular endothelial cells-1 (HMEC-1, Center for Disease Control and Prevention, Atlanta, GA) were cultured in MCDB-131 (Invitrogen, Carlsbad, CA). HMEC-1 is an immortalized HMEC line that retains the morphological, phenotypic, and functional characteristics of normal HMEC.10 Human embryonic kidney 293 and 293T were cultured in DMEM (Invitrogen). Retroviral Transduction and Small Interfering RNA TransfectionThe coding sequence of RTEF-1 (NM_003213) was subcloned from a PXJ40/RTEF-1 construct (a gift from Dr Alexandre Stewart, University of Ottawa) into the pBMN-GFP vector (Orbigen, San Diego, CA) to produce pBMN-GFP-RTEF-1. 293T cells were transfected with pBMN-GFP (or pBMN-GFP-RTEF-1), pMD-VSVG, pJK3, and pCMV-tat using Polyethylenimine (Polysciences, Warrington, PA) as previously reported 11 to a packag...

miR‐125a‐5p impairs endothelial cell angiogenesis in aging mice via RTEF ‐1 downregulation

“…Many functions have been described for RTEF-1, including enhancement of the promoter activity of VEGF (Shie et al ., 2004) and hypoxia-inducible factor-1α (HIF-1α) (Jin, 2011) under hypoxia, increased angiogenesis in the hindlimb ischaemia model (Shie et al ., 2004; Jin, 2011), facilitation of communication between the endothelium and myocardium (Xu et al ., 2011), enhancement of endothelial-dependent microvascular relaxation (Messmer-Blust et al ., 2012a), regulation of cell-to-cell connections and aggregation via connexin 43, connexin 40, and connexin 37 (An et al ., 2012) and attenuation of blood glucose levels through insulin-like growth factor binding protein-1 (IGFBP1) regulation in ECs (Messmer-Blust et al ., 2012b). We hypothesized that RTEF-1 may be impaired during aging, as it serves as a regulatory factor during angiogenesis progression.…”

RTEF-1 protects against oxidative damage induced by H2O2 in human umbilical vein endothelial cells through Klotho activation

“…Results are expressed as fold (FI) of control (control ¼ 1 arbitrary unit). Data represent the mean AE SD from three independent experiments (n ¼ 3) [10][11][12][13][14] Thus, we hypothesize that RTEF-1 might play a role in anti-oxidation. Interestingly, we found that stable overexpression of RTEF-1 cDNA in endothelial cells led to a decrease in H 2 O 2 -treated endothelial cell growth arrest and knockdown of RTEF-1 expression by siRNA showed an increase in cell growth arrest induced by H 2 O 2 .…”

“…Downstream genes of RTEF-1 such as vascular endothelial growth factor (VEGF) and connexin (Cx43) have been suggested to be involved in RTEF-1-induced angiogenesis. 10,11 RTEF-1 also upregulates hypoxia inducible factor-1 and endothelial differentiation gene-1 transcription and accelerates endothelial tube formation and enhanced cell aggregation in Matrigel models. 12,13 In addition, RTEF-1 acts as a transcriptional stimulator of fibroblast growth factor receptor 1 and can stimulate microvascular relaxation via eNOS upregulation.…”

Endothelial Differentiation Gene-1, a New Downstream Gene Is Involved in RTEF-1 Induced Angiogenesis in Endothelial Cells

“…Downstream genes such as VEGF and Cx43 have been suggested to be involved in RTEF-1 induced angiogenesis [7], [9] and Cx43 has been revealed to involve in RTEF-1-enhanced endothelial cell connections. Quantitative real-time PCR detection showed that RTEF-1 over-experssion increased Cx43 mRNA in HMEC-1 cells at Matrigel.…”

“…In addition, RTEF-1 impacts the Fibroblast growth factor (FGF)/FGFR system through the eNOS pathway in the regulation of angiogenesis and vasodilation [8]. We have recently found that endothelial specific RTEF-1-deficient mice lack the ability to form normal capillary networks suggesting that a loss of RTEF-1 signaling leads to the disintegration of the mature vasculature and that RTEF-1 is required for endothelial connections and capillary network formation [9]. However, the direct effect of RTEF-1 on angiogenesis in endothelial cells and new target genes that might be involved in RTEF-1 driven angiogenesis has not been fully understood.…”