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

Related Transcriptional Enhancer Factor-1 (RTEF-1) has been suggested to induce angiogenesis through regulating target genes. Whether RTEF-1 has a direct role in angiogenesis and what specific genes are involved in RTEF-1 driven angiogenisis have not been elucidated. We found that over-expressing RTEF-1 in Human dermal microvascular endothelial cells-1 (HMEC-1) significantly increased endothelial cell aggregation, growth and migration while the processes were inhibited by siRNA of RTEF-1. In addition, we observed that Endothelial differentiation gene-1 (Edg-1) expression was up-regulated by RTEF-1 at the transcriptional level. RTEF-1 could bind to Edg-1 promoter and subsequently induce its activity. Edg-1 siRNA significantly blocked RTEF-1-driven increases in endothelial cell aggregation in a Matrigel assay and retarded RTEF-1-induced endothelial cell growth and migration. Pertussis Toxin (PTX), a Gi/Go protein sensitive inhibitor, was found to inhibit RTEF-1 driven endothelial cell aggregation and migration. Our data demonstrates that Edg-1 is a potential target gene of RTEF-1 and is involved in RTEF-1-induced angiogenesis in endothelial cells. Gi/Go protein coupled receptor pathway plays a role in RTEF-1 driven angiogenesis in endothelial cells

Role of A20 in cIAP-2 Protection against Tumor Necrosis Factor α (TNF-α)-Mediated Apoptosis in Endothelial Cells

Tumor necrosis factor α (TNF-α) influences endothelial cell viability by altering the regulatory molecules involved in induction or suppression of apoptosis. However, the underlying mechanisms are still not completely understood. In this study, we demonstrated that A20 (also known as TNFAIP3, tumor necrosis factor α-induced protein 3, and an anti-apoptotic protein) regulates the inhibitor of apoptosis protein-2 (cIAP-2) expression upon TNF-α induction in endothelial cells. Inhibition of A20 expression by its siRNA resulted in attenuating expression of TNF-α-induced cIAP-2, yet not cIAP-1 or XIAP. A20-induced cIAP-2 expression can be blocked by the inhibition of phosphatidyl inositol-3 kinase (PI3-K), but not nuclear factor (NF)-κB, while concomitantly increasing the number of endothelial apoptotic cells and caspase 3 activation. Moreover, TNF-α-mediated induction of apoptosis was enhanced by A20 inhibition, which could be rescued by cIAP-2. Taken together, these results identify A20 as a cytoprotective factor involved in cIAP-2 inhibitory pathway of TNF-α-induced apoptosis. This is consistent with the idea that endothelial cell viability is dependent on interactions between inducers and suppressors of apoptosis, susceptible to modulation by TNF-α

PTX inhibits RTEF-1 driven endothelial cell aggregation and migration.

<p><b>A.</b> PTX decreased RTEF-1 driven endothelial cell tube formation and aggregation. (Δ = <i>p</i><0.05, <i>vs</i> control HMEC-1 at 6 h Matrigel; * = <i>p</i><0.05, <i>vs</i> RTEF-1 o/e HMEC-1 at 6 h Matrigel; ** = <i>p</i><0.01, <i>vs</i> RTEF-1 o/e HMEC-1 at 6 h Matrigel; § = <i>p</i><0.05, <i>vs</i> control HMEC-1 at 24 h Matrigel; §§<i> = p</i><0.01, <i>vs</i> control HMEC-1 at 24 h Matrigel; ##<i> = p</i><0.01, <i>vs</i> RTEF-1 o/e HMEC-1 at 24 h Matrigel; Triangle: aggregation area; Arrow: branch) <b>B.</b> PTX retarded RTEF-1 driven endothelial cell migration (# = <i>p</i><0.05, <i>vs</i> control HMEC-1 without PTX treatment; ** = <i>p</i><0.01, <i>vs</i> RTEF-1 o/e HMEC-1 without PTX treatment). The results were quantified based on three experiments and are presented as mean±S.D.</p

RTEF-1 induces angiogenesis in endothelial cells.

<p><b>A.</b> RTEF-1 was over-expressed in HMEC-1. RTEF-1 mRNA (left) and protein (right) were detected in control HMEC-1 and RTEF-1 o/e HMEC-1. (** = <i>p</i><0.01, <i>vs</i> control HMEC-1) <b>B.</b> RTEF-1 expression was knocked down by RTEF-1 siRNA in control HMEC-1. (** = <i>p</i><0.01, <i>vs</i> control HMEC-1; left: mRNA; right: protein) <b>C.</b> RTEF-1 enhanced endothelial cell connections. The tubule length (left) and branch number (middle) were increased by RTEF-1 at 6 h in Matrigel compared to control. Notably, tube formation was more apparent at 24 h than at 6 h in Matrigel. The aggregation area (right) were increased by RTEF-1 both at 6 h and 24 h in Matrigel and pronounced at 24 h in Matrigel. (* = <i>p</i><0.05, <i>vs</i> control 6 h HMEC-1; ** = <i>p</i><0.01, <i>vs</i> control 6 h HMEC-1; ΔΔ = <i>p</i><0.01, <i>vs</i> RTEF-1 o/e 6 h Matrigel; ##<i> = p</i><0.01, <i>vs</i> control 24 h HMEC-1; Triangle: aggregation area; Arrow: branch) <b>D.</b> RTEF-1 siRNA decreased endothelial cell connections. The tubule length (left), branch number (middle) and aggregation area (right) in Matrigel assay were significantly lowered by RTEF-1 siRNA. (* = <i>p</i><0.05, <i>vs</i> negative control siRNA; Triangle: aggregation area; Arrow: branch) <b>E.</b> RTEF-1 increased endothelial cell migration. (** = <i>p</i><0.01, <i>vs</i> control HMEC-1) <b>F.</b> RTEF-1 siRNA retarded endothelial cell migration. (* = <i>p</i><0.05, <i>vs</i> control HMEC-1) <b>G.</b> RTEF-1 increased endothelial cell growth. (* = <i>p</i><0.05, <i>vs</i> control HMEC-1; ** = <i>p</i><0.01, <i>vs</i> control HMEC-1) <b>H.</b> RTEF-1 siRNA inhibited endothelial cell growth (** = <i>p</i><0.01, <i>vs</i> control HMEC-1). The results were quantified based on three independent experiments and are presented as mean±S.D.</p

Edg-1 is involved in RTEF-1 driven angiogenesis in endothelial cells.

<p><b>A.</b> Edg-1 expression was knocked down by Edg-1 siRNA. (** = <i>p</i><0.01, <i>vs</i> control siRNA treated RTEF-1 o/e HMEC-1) <b>B.</b> Edg-1 siRNA inhibited RTEF-1 driven endothelial cell aggregation. (# = <i>p</i><0.05, <i>vs</i> negative control siRNA treated control HMEC-1; ** = <i>p</i><0.01, <i>vs</i> negative control siRNA treated RTEF-1 o/e HMEC-1; ## = <i>p</i><0.01, <i>vs</i> negative control siRNA treated control HMEC-1; Triangle: aggregation area; Arrow: branch) <b>C.</b> Edg-1 siRNA inhibited RTEF-1 driven endothelial cell migration. (# = <i>p</i><0.05, <i>vs</i> negative control siRNA treated control HMEC-1; * = <i>p</i><0.05, <i>vs</i> negative control siRNA treated RTEF-1 o/e HMEC-1) <b>D.</b> Treatment with Edg-1 siRNA attenuated cell growth in RTEF-1 o/e HMEC-1 (* = <i>p</i><0.05; ** = <i>p</i><0.01, <i>vs</i> negative control siRNA). The results were quantified based on three experiments and are presented as mean±S.D.</p

Edg-1 is a potential target gene of RTEF-1 <i>in vitro</i> and <i>in vivo</i>.

<p><b>A.</b> Edg-1 mRNA levels were significantly increased in RTEF-1 o/e HMEC-1 and decreased in RTEF-1 knockdown control HMEC-1. (* = <i>p</i><0.05; ** = <i>p</i><0.01, <i>vs</i> control HMEC-1) <b>B.</b> Edg-1 protein levels showed similar results to mRNA levels when RTEF-1 was over-expressed (left panel) or knocked down (right panel). <b>C.</b> Edg-1 mRNA expression level in the lung was increased in VE-Cad: RTEF-1 mice and decreased in RTEF-1^−/− mice (* = <i>p</i><0.05; ** = <i>p</i><0.01, <i>vs</i> control mice). <b>D.</b> Immunoblot analysis of lungs from both VE- Cad: RTEF-1 mice and RTEF-1^−/− mice. VE-Cad: RTEF-1 mice showed an increase in Edg-1 expression, while RTEF-1^−/− mice showed a decrease in Edg-1 expression when compared to their littermate controls. <b>E.</b> Lung tissues from VE-Cad: RTEF-1 mice, RTEF-1^−/− mice and their littermate controls were stained with anti-Edg-1 and anti-CD31 antibodies. <b>F.</b> Edg-1 full length promoter was transiently co-transfected with different concentrations of RTEF-1, and luciferase activity was examined. Edg-1 promoter activity was shown to be up-regulated in an RTEF-1 dose-dependent manner. (* = <i>p</i><0.05; ** = <i>p</i><0.01). <b>G.</b> ChIP assays were performed by immunoprecipitating chromatin from HMEC-1 cells with control IgG or anti-RTEF-1 antibody and followed by RT-PCR. The results were quantified from three independent experiments and are presented as mean±S.D.</p

Endothelial cells require related transcription enhancer factor-1 for cell-cell connections through the induction of gap junction proteins.

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. CONCLUSIONS: These findings provide novel insights into the transcriptional control of endothelial function in the coordination of cell-cell connections