Vascular Endothelial Growth Factor Promoter Activity Research Articles

Expanded Hemodialysis Therapy Ameliorates Uremia-Induced Systemic Microinflammation and Endothelial Dysfunction by Modulating VEGF, TNF-α and AP-1 Signaling.

Systemic chronic microinflammation and altered cytokine signaling, with adjunct cardiovascular disease (CVD), endothelial maladaptation and dysfunction is common in dialysis patients suffering from end-stage renal disease and associated with increased morbidity and mortality. New hemodialysis filters might offer improvements. We here studied the impact of novel improved molecular cut-off hemodialysis filters on systemic microinflammation, uremia and endothelial dysfunction. Human endothelial cells (ECs) were incubated with uremic serum obtained from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation (PERCI-II) crossover clinical trial, comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes, and then assessed for their vascular endothelial growth factor (VEGF) production and angiogenesis. Compared to HF membranes, dialysis with MCO membranes lead to a reduction in proinflammatory mediators and reduced endothelial VEGF production and angiogenesis. Cytokine multiplex screening identified tumor necrosis factor (TNF) superfamily members as promising targets. The influence of TNF-α and its soluble receptors (sTNF-R1 and sTNF-R2) on endothelial VEGF promoter activation, protein release, and the involved signaling pathways was analyzed, revealing that this detrimental signaling was indeed induced by TNF-α and mediated by AP-1/c-FOS signaling. In conclusion, uremic toxins, in particular TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel MCO membranes.Translational Perspective and Graphical Systemic microinflammation, altered cytokine signaling, cardiovascular disease, and endothelial maladaptation/dysfunction are common clinical complications in dialysis patients suffering from end-stage renal disease. We studied the impact of novel improved medium-cut-off hemodialysis filters on uremia and endothelial dysfunction. We can show that uremic toxins, especially TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel improved medium-cut-off membranes.

Cigarette smoke modulates binding of the transcription factor MZF1 to the VEGF promoter and regulates VEGF expression in dependence of genetic variation SNP 405.

Vascular endothelial growth factor (VEGF) affects carcinogenesis of the upper aerodigestive tract. Cigarette smoke (CSE) influences VEGF-gene regulation. The single nucleotide polymorphism +405 G/C (SNP +405 G/C) and the transcriptional factor (TF) myeloid zinc finger 1 (MZF1) are endogenic regulators of the VEGFpromoter as the polymorphism 405 potentially affects binding of the transcription factor MZF1. Therefore, this in vitro study analysed cancer cells of the upper aerodigestive tract after CSE incubation concerning MZF1-binding specificity and VEGF expression in dependency of VEGF polymorphism +405 G/C compared to wild type (wt). In human alveolar epithelial-like type-II cells (A549) and oral squamous cell cancer cells (HNSCCUM-02T) SNP +405 G/C- and MZF1-dependent VEGF promoter activity and VEGF expression were analysed by qRT-PCR and Western blot after incubation with 10% CSE. Temporary knock-down of MZF1 was performed using siRNA. MZF1 binding was analysed by Co-Chromatin-Immunoprecipitation (Co-ChiP) (each test n=3). We found a stronger MZF1 binding to VEGF polymorphism 405 in A549 cells (P<.05) compared to HNSCCUM-02T cells (P=.02), where MZF1 binding was reduced. MZF1 knock out reduced VEGF promoter activity in HNSCCUM-02T cells, showing the relevance of the factor for transcriptional activation of the VEGF promoter. Finally, we found that CSE increases promoter activity in both cell lines and no significant differences between the two analysed polymorphisms concerning their activating capacity. In summary, both VEGF promoter polymorphisms are similar effective in terms of transcriptional activity, and MZF1 is a transcriptional activator of VEGF promoter. Moreover, cigarette smoke increases MZF1 binding of VEGF-promoter and directly affects VEGF-gene regulation.

Trichostatin A resistance is facilitated by HIF-1α acetylation in HeLa human cervical cancer cells under normoxic conditions.

Trichostatin A (TSA) is an anticancer drug that inhibits histone deacetylases (HDACs). Hypoxia-inducible factor 1 (HIF-1) participates in tumor angiogenesis by upregulating target genes, such as vascular endothelial growth factor (VEGF). In the present study, we investigated whether TSA treatment increases HIF-1α stabilization via acetylation under normoxic conditions, which would lead to VEGF upregulation and resistance to anticancer drugs. TSA enhanced total HIF-1α and VEGF-HRE reporter activity under normoxic conditions. When cells were transfected with GFP-HIF-1α, treatment with TSA increased the number of green fluorescence protein (GFP)-positive cells. TSA also enhanced the nuclear translocation of HIF-1α protein, as assessed by immunoblotting and as evidenced by increased nuclear localization of GFP-HIF-1α. An increase in the interaction between HIF-1α and the VEGF promoter, which was assessed by a chromatin immunoprecipitation (ChIP) assay, led to activation of the VEGF promoter. TSA acetylated HIF-1α at lysine (K) 674, which led to an increase in TSA-induced VEGF-HRE reporter activity. In addition, TSA-mediated cell death was reduced by the overexpression of HIF-1α but it was rescued by transfection with a HIF-1α mutant (K674R). These data demonstrate that HIF-1α may be stabilized and translocated into the nucleus for the activation of VEGF promoter by TSA-mediated acetylation at K674 under normoxic conditions. These findings suggest that HIF-1α acetylation may lead to resistance to anticancer therapeutics, such as HDAC inhibitors, including TSA.

Interleukin-17 upregulates vascular endothelial growth factor by activating the JAK/STAT pathway in nucleus pulposus cells

ObjectivesIntervertebral disc (IVD) related diseases and age-related IVD degeneration are responsible for significant morbidity. Inflammatory mediators and pro-inflammatory cytokines, including interleukin (IL)-17, show elevated expression in degenerated disc tissue. IL-17 is reported to transduce signals across the cell membrane predominantly via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction pathway, leading to transcriptional activation of target genes. MethodsIn this study, we investigated whether the JAK/STAT pathway plays a role in IL-17-mediated signaling in the nucleus pulposus (NP) cells of IVDs. Vascular endothelial growth factor (VEGF) and IL-17 were found to be highly expressed in human degenerated NP tissue. In isolated rat NP cells, IL-17-induced VEGF expression in a time- and dose-dependent manner. Rat NP cells were co-transfected with VEGF promoter plasmid along with constitutively active STAT1, STAT3 or JAK2 plasmid. VEGF promoter activity was found to be increased by STAT1, STAT3 and JAK2 in IL-17-treated cells. Transfection of cultured rat NP cells with STAT1 or STAT3 lentiviral short hairpin RNAs or treatment with the JAK2 inhibitor AG490 significantly reduced IL-17-stimulated VEGF expression. ConclusionsIL-17 upregulated VEGF expression in rat NP cells mediated by the JAK/STAT pathway, and elevated levels of IL-17 and VEGF are present in human degenerated NP tissue. These findings provide new insight into the pathology of IVD degeneration.

Intracellular autocrine VEGF signaling promotes EBDC cell proliferation, which can be inhibited by Apatinib

Tumor cells produce vascular endothelial growth factor (VEGF) which can interact with membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth. We aimed to investigate the role of extracellular/intracellular autocrine VEGF signaling and Apatinib, a highly selective VEGFR2 inhibitor, in extrahepatic bile duct cancer (EBDC). We found conditioned medium or recombinant human VEGF treatment promoted EBDC cell proliferation through a phospholipase C-γ1-dependent pathway. This pro-proliferative effect was diminished by VEGF, VEGFR1 or VEGFR2 neutralizing antibodies, but more significantly suppressed by intracellular VEGFR inhibitor. The rhVEGF induced intracellular VEGF signaling by promoting nuclear accumulation of pVEGFR1/2 and enhancing VEGF promoter activity, mRNA and protein expression. Internal VEGFR2 inhibitor Apatinib significantly inhibited intracellular VEGF signaling, suppressed cell proliferation in vitro and delayed xenograft tumor growth in vivo, while anti-VEGF antibody Bevacizumab showed no effect. Clinically, overexpression of pVEGFR1 and pVEGFR2 was significantly correlated with poorer overall survival (P = .007 and P = .020, respectively). In conclusion, the intracellular autocrine VEGF loop plays a predominant role in VEGF-induced cell proliferation. Apatinib is an effective intracellular VEGF pathway blocker that presents a great therapeutic potential in EBDC.

Critical role of CDK11(p58) in human breast cancer growth and angiogenesis.

BackgroundA capillary network is needed in cancer growth and metastasis. Induction of angiogenesis represents one of the major hallmarks of cancer. CDK11p58, a Ser/Thr kinase that belongs to the Cell Division Cycle 2-like 1 (CDC2L1) subfamily is associated with cell cycle progression, tumorigenesis, sister chromatid cohesion and apoptotic signaling. However, its role in breast cancer proliferation and angiogenesis remains unclear.MethodsTumorigenicity assays and blood vessel assessment in athymic mice were used to assess the function of CDK11p58 in tumor proliferation and angiogenesis. CCK-8 assay was used to detect breast cancer cell growth. Immunohistochemistry was used to detect the expression of vascular endothelial growth factor (VEGF), CD31 and CD34 in CDK11 positive patient breast cancer tissues. Dual-Luciferase array was used to analyze the function of CDK11p58 in the regulation of VEGF promoter activity. Western blot was used to detect related protein expression levels.ResultsCDK11p58 inhibited breast cancer growth and angiogenesis in breast cancer cells and in nude mice transplanted with tumors. Immunohistochemistry confirmed that CDK11p58 was negatively associated with angiogenesis-related proteins such as VEGF, CD31 and CD34 in breast cancer patients. Real-time PCR and dual-luciferase assay showed CDK11p58 inhibited the mRNA levels of VEGF and the promoter activity of VEGF. As CDK11p58 is a Ser/Thr kinase, the kinase-dead mutant failed to inhibit VEGF mRNA and promoter activity. Western blot analysis showed the same pattern of related protein expression. The data suggested angiogenesis inhibition was dependent on CDK11p58 kinase activity.ConclusionThis study indicates that CDK11p58 inhibits the growth and angiogenesis of breast cancer dependent on its kinase activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1698-7) contains supplementary material, which is available to authorized users.

Induction of VEGF expression by alpha-tocopherol and alpha-tocopheryl phosphate via PI3Kγ/PKB and hTAP1/SEC14L2-mediated lipid exchange.

In several studies, vitamin E has been observed to influence angiogenesis and vasculogenesis. We recently showed that the phosphorylated form of α-tocopherol (αT), α-tocopheryl phosphate (αTP), increases the expression of the vascular endothelial growth factor (VEGF). Thus, αTP may act as an active lipid mediator increasing VEGF expression, angiogenesis, and vasculogenesis. Here, we investigated the molecular signaling mechanisms by which αTP induces VEGF expression using cultured HEK293 cells as model system. αT and more so αTP increased VEGF-promoter activity in a phosphatidylinositol-3-kinase gamma (PI3Kγ)-dependent manner. In contrast, after overexpression of PI3Kγ and/or protein kinase B (PKB), VEGF promoter activity was inhibited by αT and more so by αTP. Inhibition by αT and αTP was dependent on the lipid kinase activity of PI3Kγ, whereas an induction was seen with the protein kinase activity, consistent with a model in which PKB inhibition by αT or αTP occurs only when activated at the plasma membrane and possibly involves a phosphatase such as PHLPP1. PI3Kγ-induced VEGF expression was reduced when the human tocopherol-associated protein 1 (hTAP1/SEC14L2) was overexpressed suggesting formation of an inactive PI3Kγ/hTAP1 heterodimer, that could be reactivated by αT and more so by αTP. We suggest a novel signaling mechanism by which αTP stimulates PI3Kγ activity by stimulating hTAP-mediated phosphatidylinositol exchange and presentation to the enzyme and/or dissociation of an inactive heterodimer. At cellular level, hTAP may act as sensor for intracellular lipid information (location, type, and amount of lipid) and translate it into responses of PI3K-mediated signaling and gene expression.

Molecular imaging of expression of vascular endothelial growth factor a (VEGF a) in femoral bone grafts transplanted into living mice.

The biology of cells transplanted with bone grafts is incompletely understood. Focusing on the early angiogenic response postgrafting, we report a mouse femur graft model in which grafts were derived from mice transgenic for a firefly luciferase (FLuc) bioluminescence reporter gene driven by a promoter for the angiogenic signaling molecule vascular endothelial growth factor (VEGF). Upon transplantation into wild-type (wt) mice, in vivo bioluminescence imaging (BLI) permitted longitudinal visualization and measurements of VEGF promoter activity in the transplanted graft cells and demonstrated a lag period of 7 days posttransplantation prior to robust induction of the promoter. To determine cellular mediators of VEGF induction in graft bone, primary graft-derived osteoblastic cells (GDOsts) were characterized. In vitro BLI on GDOsts showed hypoxia-induced VEGF expression and that this induction depended on PI3K signaling and, to a lesser degree, on the MEK pathway. This transcriptional regulation correlated with VEGF protein production and was validated in GDOsts seeded on demineralized bone matrix (DBM), a bone graft substitute material. Together, combined imaging of VEGF expression in living animals and in live cells provided clues about the regulation of VEGF in cells post-bone grafting. These data are particularly significant toward the development of future smart bone graft substitutes.

Induction of VEGF expression in monocytes by alpha‐tocopherol and alpha‐tocopheryl phosphate via PI3Kgamma/Akt and hTAP1/SEC14L2‐mediated lipid exchange (1000.1)

In several studies, vitamin E has been observed to influence angiogenesis and vasculogenesis. We recently observed that the phosphorylated form of α‐tocopherol (αT), α‐tocopheryl phosphate (αTP), increases the expression of the vascular endothelial growth factor (VEGF), which may stimulate angiogenesis and vasculogenesis and enhance the survival of cells. Here we investigated the molecular signaling mechanisms by which αTP induces VEGF expression using cultured THP‐1 monocytes and HEK293 cells as model systems. αT and more so αTP increased VEGF‐promoter activity in a phosphatidylinositol‐3‐kinase gamma (PI3Kγ) ‐dependent manner. In contrast, after over‐expression of PI3Kγ and/or Akt, VEGF promoter activity is inhibited by αT and more so by αTP. PI3Kγ‐induced VEGF expression was reduced when the human tocopherol associated protein 1 (hTAP1/SEC14L2) was over‐expressed, that could be reverted by αT and more so by αTP. Recombinant hTAP1/hSEC14L2 binds both αT and αTP and reduces the in vitro activity of the PI3Kγ most likely by forming a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition αT, βT, γT, δT or αTP differentially stimulates PI3Kγ. We suggest a novel signaling mechanism by which αT or αTP stimulate PI3Kγ activity by facilitating egress and presentation of sequestered PI from hTAP1 to the enzyme and/or dissociating the inactive heterodimer. Supported by USDA Contract #58‐1950‐0‐014.Grant Funding Source: Supported by USDA Contract #58‐1950‐0‐014

The proto-oncogene c-Fos transcriptionally regulates VEGF production during peritoneal inflammation

Vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) are key mediators of adverse peritoneal membrane remodeling in peritoneal dialysis eventually leading to ultrafiltration failure. Both are pleiotropic growth factors with cell type-dependent regulation of expression and biological effects. Here we studied regulation of TGF-β1-induced VEGF expression in human peritoneal mesothelial cells in the absence or presence of proinflammatory stimuli, tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Quiescent human peritoneal mesothelial cells secreted only trace amounts of VEGF. Stimulation with TGF-β1 resulted in time- and dose-dependent increases in VEGF mRNA expression and protein release. TNF-α and IL-1β alone had minimal effects but acted in synergy with TGF-β1. Combined stimulation led to induction of transcription factor c-Fos and activation of the VEGF promoter region with high-affinity binding sites for c-Fos. Inhibition of c-Fos by small interfering RNA interference or by pharmacological blockade with SR-11302 decreased VEGF promoter activity and downregulated its expression and release. Exposure of human peritoneal mesothelial cells to dialysate effluent containing increased levels of TGF-β1, TNF-α, and IL-1β obtained during peritonitis resulted in a dose-dependent VEGF induction that was significantly attenuated by SR-11302. Thus, dialysate TGF-β1, IL-1β, and TNF-α act through c-Fos to synergistically upregulate VEGF production in peritoneal mesothelium and may represent an important regulatory link between inflammation and angiogenesis in the peritoneal membrane.

FHL1 and Smad4 synergistically inhibit vascular endothelial growth factor expression

Vascular endothelial growth factor (VEGF) plays an important role in many disease states, including ischemia, chronic and acute inflammation, and pathologies associated with angiogenesis such as tumors and wounds. A number of factors regulate VEGF promoter activity and VEGF expression such as four and a half LIM domains 1 (FHL1) and Smad4. FHL1 belongs to a family of LIM-only proteins that regulate gene transcription, cell proliferation, differentiation and apoptosis. Smad4 is a tumor suppressor gene, initially identified as deleted in pancreatic carcinoma locus 4 (DPC4). The aim of this study was to determine whether FHL1 and Smad4 inhibited VEGF signaling. HepG2 cells were transfected with the VEGF-Luc reporter, Smad4 and FHL1 or Smad4 and FHL1 siRNA. Results showed that the overexpression of FHL1 and Smad4 synergistically inhibited the promoter activity, mRNA expression and secretion of VEGF, whereas knockdown of endogenous Smad4 and FHL1 had opposite effects. Moreover, the reduction of endogenous Smad4 eliminated FHL1-mediated inhibition of the VEGF promoter activity. In conclusion, a cooperative regulation of VEGF signaling by FHL1 and Smad4 was evidenced, which may provide a novel regulation mechanism underlying cancer development and progression.

Sanguinarine is a novel VEGF inhibitor involved in the suppression of angiogenesis and cell migration

Vascular endothelial growth factor (VEGF) is a main angiogenic factor which is known to be upregulated in lung cancer. In the present study, it was demonstrated that sanguinarine, an alkaloid obtained from the bloodroot plant, markedly repressed the VEGF-induced tube formation of human microvascular endothelial cells (HMVECs) and the migration of human A549 lung cancer cells. Furthermore, sanguinarine decreased VEGF secretion and expression in HMVECs and A549 lung cancer cells in a dose- and time-dependent manner. Additionally, sanguinarine inhibited the activation of serum starvation- and hypoxia-induced VEGF promoter activity. Sanguinarine also inhibited the VEGF-mediated Akt and p38 activation, as well as VE-cadherin protein phosphorylation. To the best of our knowledge, this is the first study demonstrating that VEGF inhibition appears to be an important mechanism involved in the antiangiogenic and anti-invasive activities of sanguinarine in lung cancer treatment.

Thymosin β4 stabilizes hypoxia‐inducible factor‐1α protein in an oxygen‐independent manner

The small actin-binding protein thymosin β4 (Tβ4) is understood to stimulate angiogenesis. Previously, we reported that Tβ4 induces angiogenesis by increasing vascular endothelial growth factor (VEGF) expression, but the mechanism underlying how Tβ4 upregulates VEGF expression remain unknown. To identify the mechanism of VEGF induction by Tβ4, we measured VEGF promoter activity and analyzed the effect of Tβ4 on VEGF RNA stability. The Tβ4 peptide had no effect on either VEGF promoter activity or VEGF RNA stability. We focused on the possibility that Tβ4 may indirectly induce VEGF expression via hypoxia-inducible factor (HIF)-1α. We determined that Tβ4 increased the stability of HIF-1α protein under normoxic conditions. These data suggest that Tβ4 indirectly induces VEGF expression by increasing the protein stability of HIF-1α in an oxygen-independent manner.

Elevated SP-1 Transcription Factor Expression and Activity Drives Basal and Hypoxia-induced Vascular Endothelial Growth Factor (VEGF) Expression in Non-Small Cell Lung Cancer

VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.

Osteoblast-specific transcription factor Osterix (Osx) and HIF-1α cooperatively regulate gene expression of vascular endothelial growth factor (VEGF)

Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Angiogenesis and osteogenesis are tightly coupled during bone formation. Vascular endothelial growth factor (VEGF) is involved in both processes. Relatively little is known about VEGF gene regulation in osteoblasts. Osterix (Osx) is a bone morphogenetic protein 2 (BMP-2) inducible osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Our recent study has demonstrated that Osx controls VEGF expression in osteoblasts. Here, we further characterized Osx regulation of VEGF. To address which domain of Osx is responsible for VEGF regulation, the deletion mutant analysis and transfection assay were carried out to show that proline-rich region (PRR) is required for Osx activation of VEGF promoter activity. Hypoxia-inducible factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis, and to upregulate VEGF. Effect of Osx on HIF-1α expression was examined in this study. Quantitative RT-PCR results revealed that HIF-1α remained unchanged between wild type and Osx knockout calvaria at E18.5 in mouse embryos. Overexpression of Osx in stable C2C12 mesenchymal cells using Tet-off system did not affect HIF-1α expression. HIF-1α level did not change after Osx inhibition by siRNA in osteoblasts. Moreover, BMP-2 stimulation led to upregulation of Osx and VEGF, but not HIF-1α. These results demonstrate that HIF-1α is not a downstream target of Osx in osteoblasts, suggesting that Osx regulation of VEGF is independent of HIF-1α expression level. Interestingly, synergistic interplays were observed between Osx and HIF-1α in VEGF promoter activation in transfection assay. Our findings indicate that Osx and HIF-1α cooperatively regulate VEGF expression.

The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.

A cell-autonomous role of Cited2 in controlling myocardial and coronary vascular development

AimsMyocardial development is dependent on concomitant growth of cardiomyocytes and a supporting vascular network. The coupling of myocardial and coronary vascular development is partly mediated by vascular endothelial growth factor (VEGFA) signalling and additional unknown mechanisms. We examined the cardiomyocyte specific role of the transcriptional co-activator Cited2 on myocardial microstructure and vessel growth, in relation to Vegfa expression.Methods and resultsA cardiomyocyte-specific knockout of mouse Cited2 (Cited2Nkx) was analysed using magnetic resonance imaging and histology. Ventricular septal defects and significant compact layer thinning (P< 0.02 at right ventricular apex, P< 0.009 at the left ventricular apex in Cited2Nkx vs. controls, n= 11 vs. n= 7, respectively) were found. This was associated with a significant decrease in the number of capillaries to larger vessels (ratio 1.56 ± 0.56 vs. 3.25 ± 1.63, P= 2.7 × 10−6 Cited2Nkx vs. controls, n= 11 vs. n= 7, respectively) concomitant with a 1.5-fold reduction in Vegfa expression (P< 0.02, Cited2Nkx vs. controls, n= 12 vs. n= 12, respectively). CITED2 was subsequently found at the Vegfa promoter in mouse embryonic hearts using chromatin immunoprecipitation, and moreover found to stimulate human VEGFA promoter activity in cooperation with TFAP2 transcription factors in transient transfection assays. There was no change in the myocardial expression of the left-right patterning gene Pitx2c, a previously known target of CITED2.ConclusionsThis study delineates a novel cell-autonomous role of Cited2 in regulating VEGFA transcription and the development of myocardium and coronary vasculature in the mouse. We suggest that coupling of myocardial and coronary growth in the developing heart may occur in part through a Cited2→Vegfa pathway.

TRAF6 inhibits proangiogenic signals in endothelial cells and regulates the expression of vascular endothelial growth factor

TNF-family molecules induce the expression Vascular Endothelial Growth Factor (VEGF) in endothelial cells (EC) and elicit signaling responses that result in angiogenesis. However, the role of TNF-receptor associated factors (TRAFs) as upstream regulators of VEGF expression or as mediators of angiogenesis is not known. In this study, HUVEC were cotransfected with a full-length VEGF promoter-luciferase construct and siRNAs to TRAF 1, -2, -3, -5, -6, and promoter activity was measured. Paradoxically, rather than inhibiting VEGF expression, we found that knockdown of TRAF6 resulted in a 4–6-fold increase in basal VEGF promoter activity compared to control siRNA-transfected EC (P<0.0001). In addition, knockdown of TRAF 1, -2, -3 or -5 resulted in a slight increase or no change in VEGF promoter activation. Using [3H]thymidine incorporation assays as well as the in vitro wound healing assay, we also found that basal rates of EC proliferation and migration were increased following TRAF6 knockdown; and this response was inhibited by the addition of a blocking anti-VEGF antibody into cell cultures. Using a limited protein array to gain insight into TRAF6-dependent intermediary signaling responses, we observed that TRAF6 knockdown resulted in an increase in the activity of Src family kinases. In addition, we found that treatment with AZD-0530, a pharmacological Src inhibitor, reduced the regulatory effect of TRAF6 knockdown on VEGF promoter activity. Collectively, these findings define a novel pro-angiogenic signaling response in EC that is regulated by TRAF6.

Transcriptional Regulation of Vascular Endothelial Growth Factor (VEGF) by Osteoblast-specific Transcription Factor Osterix (Osx) in Osteoblasts

Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.

Calcineurin Inhibitor-Induced and Ras-Mediated Overexpression of VEGF in Renal Cancer Cells Involves mTOR through the Regulation of PRAS40

Malignancy is a major problem in patients treated with immunosuppressive agents. We have demonstrated that treatment with calcineurin inhibitors (CNIs) can induce the activation of proto-oncogenic Ras, and may promote a rapid progression of human renal cancer through the overexpression of vascular endothelial growth factor (VEGF). Interestingly, we found that CNI-induced VEGF overexpression and cancer cell proliferation was inhibited by rapamycin treatment, indicating potential involvement of the mammalian target of rapamycin (mTOR) pathway in this tumorigenic process. Here, we examined the role of mTOR pathway in mediating CNI- and Ras-induced overexpression of VEGF in human renal cancer cells (786-0 and Caki-1). We found that the knockdown of raptor (using siRNA) significantly decreased CNI-induced VEGF promoter activity as observed by promoter-luciferase assay, suggesting the role of mTOR complex1 (mTORC1) in CNI-induced VEGF transcription. It is known that mTOR becomes activated following phosphorylation of its negative regulator PRAS40, which is a part of mTORC1. We observed that CNI treatment and activation of H-Ras (through transfection of an active H-Ras plasmid) markedly increased the phosphorylation of PRAS40, and the transfection of cells using a dominant-negative plasmid of Ras, significantly decreased PRAS40 phosphorylation. Protein kinase C (PKC)-ζ and PKC-δ, which are critical intermediary signaling molecules for CNI-induced tumorigenic pathway, formed complex with PRAS40; and we found that the CNI treatment increased the complex formation between PRAS40 and PKC, particularly (PKC)-ζ. Inhibition of PKC activity using pharmacological inhibitor markedly decreased H-Ras-induced phosphorylation of PRAS40. The overexpression of PRAS40 in renal cancer cells significantly down-regulated CNI- and H-Ras-induced VEGF transcriptional activation. Finally, it was observed that CNI treatment increased the expression of phosho-PRAS40 in renal tumor tissues in vivo. Together, the phosphorylation of PRAS40 is critical for the activation of mTOR in CNI-induced VEGF overexpression and renal cancer progression.