Vascular Endothelial Growth Factor Promoter Region Research Articles

VEGF Gene Polymorphism Among Diabetes Mellitus and Diabetic Retinopathy

Vascular endothelial growth factor (VEGF) is a major angiogenic factor and a prime regulator of endothelial cell proliferation. VEGF gene is located on chromosome 6 (6p21.3). Objectives: To determine whether deletion at -2549 position of promoter region of the VEGF gene exert influence on the development of diabetic retinopathy. Methods: Diseased and control subjects were selected for blood sampling. The blood samples (n=50) was taken from diabetic retinopathy patients and blood samples(n=50) was taken from type 2 diabetes mellitus patients as control group. After DNA extraction Polymerase Chain Reaction was performed to amplify the VEGF gene and sequencing was commercially done for molecular analysis of VEGF gene. Results: The molecular analysis confirmed that deletion at -2549 position of the promoter region of VEGF significantly greater in DR group. 2-11 % deletion was examined at -2549 position of promoter region of VEGF. The DD genotype was responsible for the development of DR. Conclusions: This study indicates that DD gene mutation and D allele is an autonomous hazard aspect for the advancement and progression of retinopathy in people with type 2 diabetes, additionally different issues for example diabetic age and family ancestry of diabetes assumes key function in the advancement of retinopathy in diabetic patients.

Study of the Association between VEGF Polymorphisms and the Risk of Coronary Artery Disease in Koreans.

Coronary artery disease (CAD), a leading cause of death worldwide, has a complex etiology comprising both traditional risk factors (type 2 diabetes, dyslipidemia, arterial hypertension, and cigarette smoking) and genetic factors. Vascular endothelial growth factor (VEGF) notably contributes to angiogenesis and endothelial homeostasis. However, little is known about the relationship between CAD and VEGF polymorphisms in Koreans. The aim of this study is to investigate the associations of 2 VEGF promoter region polymorphisms (−1154G>A [rs1570360], −1498T>C [rs833061]) and 4 VEGF 3′-UTR polymorphisms (+936C>T [rs3025039], +1451C>T [rs3025040], +1612G>A [rs10434], and +1725G>A [rs3025053]) with CAD susceptibility in Koreans. We studied 885 subjects: 463 CAD patients and 422 controls. Genotyping was conducted with polymerase chain reaction-restriction fragment length polymorphism analysis and TaqMan allelic discrimination assays, and the genotype frequencies were calculated. We then performed haplotype and genotype combination analyses and measured the associations between VEGF polymorphisms and clinical variables in both the CAD patients and control subjects. We detected statistically significant associations between CAD and certain VEGF allele combinations. In the haplotypes of 5 single-nucleotide polymorphisms, the VEGF allele combination −1154A/+936T was associated with a decreased prevalence of CAD (A-T-T-G-G of VEGF −1154G>A/−1498T>C/+936C>T/+1612G>A/+1725G>A, AOR = 0.077, p = 0.021). In contrast, the VEGF allele combinations −1498T/+1725A and −1498T/+1612A/+1725A were associated with an increased prevalence of CAD (G-T-C-C-A of VEGF −1154G>A/−1498T>C/+936C>T/+1451C>T/+1725G>A, AOR = 1.602, p = 0.047; T-C-C-A-A of VEGF −1498T>C/+936C>T/+1451C>T/+1612G>A/+1725G>A, AOR = 1.582, p = 0.045). Gene–environment combinatorial analysis showed that the combination of the VEGF +1725AA genotype and several clinical factors (e.g., body mass index, hemoglobin A1c, and low-density lipoprotein cholesterol) increased the risk of CAD. Therefore, we suggest that VEGF polymorphisms and clinical factors may impact CAD prevalence.

Molecular dynamics simulations of the human VEGF promoter G-quadruplex

Cancerous tumors rely on increased formation of blood vessels to obtain nutrients and oxygen for proliferation and metastasis. Blood vessels are formed via a process known as angiogenesis, which in humans requires signaling by the vascular endothelial growth factor (VEGF), and overexpression of VEGF is implicated in various types of cancers. A 22-nucleotide segment of the VEGF promoter region (Pu22) is known to form a G-quadruplex (GQ) under experimental conditions. Due to the enrichment of GQ structures within promoter regions, it is suggested that they may serve as regulators of transcription.

Ribavirin inhibits the growth and ascites formation of hepatocellular carcinoma through downregulation of type I CARM1 and type II PRMT5

Type I co-activator-associated arginine methyltransferase 1 (CARM1) and type II protein arginine methyltransferase 5 (PRMT5) are highly expressed in multiple cancers including liver cancer and their overexpression contributes to poor prognosis, thus making them promising therapeutic targets. Here, we evaluated anti-tumor activity of ribavirin in hepatocellular carcinoma (HCC). We found that ribavirin significantly inhibited the proliferation of HCC cells in a time- and dose-dependent manner. Furthermore, ribavirin suppressed the growth of subcutaneous and orthotopic xenograft of HCC in mice, decreased vascular endothelial growth factor (VEGF) and peritoneal permeability to reduce ascites production, and prolonged the survival of mice in HCC ascites tumor models. Mechanistically, ribavirin potently down-regulated global protein expression of CARM1 and PRMT5, and concurrently decreased accumulation of H3R17me2a and H3R8me2s/H4R3me2s. However, ribavirin did not affect the activity and mRNA levels of both CARM1 and PRMT5 in vivo and in vitro HCC cells. In addition, our ChIP results shown that ribavirin inhibited CARM1 which in turn decreased the H3R17me2a, binds to the eukaryotic translation initiation factor 4E (eIF4E) and VEGF promoter region, and reduced the relative mRNA expression level of eIF4E and VEGF in HCC cells. Our findings suggested a potential therapeutic strategy for patients with HCC through inhibition of the abnormal activation/expression of both CARM1 and PRMT5.

The -2549 -2567 del18 Polymorphism in VEGF and Irreversible Bronchoconstriction in Asthmatics.

While the importance of vascular endothelial growth factor (VEGF) in the pathogenesis of several diseases (eg, neoplasms) has been proven, its role in asthma, especially in terms of the potential associations between genetic variants of VEGF and airway remodeling, has received relatively little attention. Objectives: This study aimed to evaluate the possible connection between a genetic factor, ie, the polymorphism del/ins in the VEGF promoter region, and airway remodeling potential in asthmatics with and without irreversible bronchoconstriction. The study population comprised 82 patients with asthma (of whom 42 had irreversible bronchoconstriction) and a group of 40 controls. DNA was isolated from peripheral blood leukocytes. Polymerase chain reaction was used to type the VEGF (18-bp deletion/insertion) gene polymorphism at loci -2549 -2567. Other factors (ie, smoking, disease duration) were also taken into consideration. The del/del genotype was found in 74.39% of patients with asthma (P=.031; OR=2.38), 80.95% of patients with irreversible bronchoconstriction (P=.012; OR=3.48), and 67.5% patients with reversible bronchoconstriction (P=.251; OR=1.70). The proportion of smokers to nonsmokers was higher (P=.032) and disease duration was longer (P=.041) in patients with irreversible bronchoconstriction than in those with reversible bronchoconstriction. Our results showed that the risk of irreversible bronchoconstriction in asthmatics was associated with the presence of the del18 genotype at the -2549 -2567 position in the promoter region of the VEGF gene, as were disease duration and other factors such as smoking.

Distinct APE1 Activities Affect the Regulation of VEGF Transcription Under Hypoxic Conditions

Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF), a crucial factor in tumor angiogenesis, has been reported to be transcriptionally regulated by hypoxia-inducible factor-1 (HIF-1). An 8-oxo-G or apurinic/apyrimidinic (AP) site, which is frequently associated with DNA damage, has been identified in the promoter region of VEGF. However, the detailed molecular mechanisms by which AP sites regulate VEGF gene transcription are largely unknown. The dual functional protein apurinic/apyrimidinic endonuclease 1 (APE1) is both the key enzyme in DNA base excision repair and the redox factor shown to regulate HIF-1 DNA-binding activity. In the present study, we tested the involvement of both the AP endonuclease and redox activity of APE1 in regulating HIF-1 DNA binding and VEGF transcription in HUVECs. By employing two APE1 activity-specific inhibitors and AP-site-containing reporter constructs, we confirmed that both activities of APE1 were involved in regulating VEGF expression under hypoxic conditions. Furthermore, we found that the interaction between APE1 and its downstream repair enzyme, DNA polymerase β, was compromised when the N-terminal structure of APE1 was distorted under oxidative conditions. Our data suggest that the DNA repair and redox activity of APE1 can play a collaborative role in regulating the transcriptional initiation of the AP-site-containing promoter.

The -2549 insertion/deletion polymorphism in the promoter region of VEGF is associated with the risk of recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) is a common health problem affecting women of reproductive age. Altered expression of vascular endothelial growth factor (VEGF) has been associated with spontaneous abortion. The present case-control study aimed to evaluate the impact of the 18-bp insertion/deletion (ins/del) polymorphism (rs35569394) in the promoter region of the VEGF gene on idiopathic RSA. Genomic DNA from 93 patients with RSA and 93 healthy fertile women of southeastern Iran was isolated using the salting-out method. Genotyping of the rs35569394 variant was performed by a polymerase chain reaction (PCR) method. The findings indicated that the VEGF 18-bp ins/del variant significantly increased the risk of RSA under codominant (ins/ins vs. del/del; OR=2.85, 95% CI=1.31-6.22, P=0.019), dominant (del/ins+ins/ins vs. del/del; OR=2.19, 95% CI=1.20-4.01, P=0.015) and allelic (ins vs. del; OR=1.90, 95% CI=1.25-2.88, P=0.003) inheritance models. In summary, the findings propose a significant association between the VEGF 18-bp ins/del polymorphism and risk of RSA in a sample of the southeast Iranian population. Further studies on larger sample sizes and different ethnicities are required to validate the present findings.

Effects of 5-Hydroxymethylcytosine Epigenetic Modifications within the VEGF Promoter Region on G-Quadruplex and I-Motif DNA Structure and Stability

Epigenetic modifications to DNA base sequences may regulate gene expression. CpG islands can contain methylated (5mC) or hydroxymethylated (5hmC) cytosine. Most CpG islands are found primarily in promoter regions that may also contain a high number of repeated cytosines and/or guanines. G-quadraplexes (G4) and i-motifs (iM) are two unique DNA secondary structures that can form in repeating sequences of either guanine or cytosine, respectively. Both G4 and iM sequences may contain CpG sequences that can be methylated or hydroxymethylated. The effects of CpG islands on DNA secondary structures were determined by incorporating a single 5hmC at varying positions in the Vascular Endothelial Growth Factor (VEGF) G4 and iM sequences. An Olis DSM-20 spectropolarimeter and a Cary 100 UV-visible spectrometer were used to monitor the effect of 5hmC on G4 and iM thermal stability. Two of the three 5hmC-containing loops showed a notable decrease in stability for G4's and increased intermolecular structure formation. Contrastingly, the iM stability increased when 5hmC was incorporated into its sequence. Additionally, there was little change in the iM pka. In summary, our results suggest the 5hmC has little effect on iM structures, but can destabilize the G4's.

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.

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K+ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K+ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins.LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.

TLR-4 and VEGF Polymorphisms in Chronic Periaortitis

ObjectiveChronic periaortitis (CP) is a rare disease that is characterised by fibro-inflammatory tissue surrounding the abdominal aorta and has both non-aneurysmal (idiopathic retroperitoneal fibrosis [IRF]) and aneurysmal forms (inflammatory abdominal aortic aneurysm [IAAA]). We investigated whether toll-like receptor 4 (TLR-4) and vascular endothelial growth factor (VEGF) polymorphisms were associated with susceptibility to, and the clinical features of CP.MethodsOne hundred and two CP patients and 200 healthy controls were molecularly genotyped for TLR-4 gene polymorphism (+896 A/G) (rs4986790), VEGF mutations +936 C/T (rs3025039) and −634 C/G (rs2010963), and an 18 base pair (bp) insertion/deletion (I/D) polymorphism at −2549 of the VEGF promoter region. The patients were grouped on the basis of the type of CP (IRF or IAAA), and the presence or absence of established atherosclerotic disease (ischemic heart disease, cerebrovascular disease, and peripheral arterial disease).ResultsThere were no significant differences in the distribution of the studied polymorphisms between the patients and controls. However, carriage of the +936 T allele was significantly more frequent in the patients with IRF than in those with IAAA (26.5% vs 5.3%; p = 0.046; OR 6.49 [95% CI 0.82–51.54]). There were significantly more carriers of the I allele among the patients with ureteral obstruction (83.8% vs 58.8%; p = 0.006; OR 3.63 [95% CI 1.42–9.28]) and those who received conservative treatment (48.5% vs 23.5%; p = 0.015; OR 3.06 [95% CI 1.22–7.721]) than among those without, and II homozygosity was significantly more frequent in the patients with deep vein thrombosis than in those without (30.4% vs 11.7%, p = 0.031; OR 3.31 [95% CI 1.07–10.21]).ConclusionThe VEGF +936 C/T polymorphism may be associated with an increased risk of developing the non-aneurysmal IRF form of CP. Carriers of the I allele and II homozygosity are respectively at increased risk of developing ureteral obstruction and deep vein thrombosis.

The Vascular Endothelial Growth Factor-2549 Insertion/Deletion Polymorphism Is Not Associated with Susceptibility to Hepatocellular Carcinoma in Chinese

Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, which is crucial for development and metastasis of tumors including hepatocellular carcinoma (HCC), and elevated VEGF levels in serum and tissues have been known to be related with poor prognosis in patients with HCC. Polymorphisms in VEGF may alter VEGF protein concentrations, influence the process of angiogenesis, and may relate to interindividual variation in tumorigenesis. In this study, we carried out a case-control study in a Chinese population (206 cases and 302 controls) to estimate the susceptibility to HCC associated with an 18-bp insertion/deletion polymorphism (rs35569394) in the promoter region of VEGF. After adjusting the data by gender, age, smoking status, drinking status, and hepatitis B virus (HBV) infection using logistic regression model, we found that rs35569394 was not associated with HCC, at both the allele and genotype levels. Thus, rs35569394 should not be viewed as a major contributor to the development of HCC in Chinese.

Vascular endothelial growth factor gene polymorphisms and serum levels in Behçet’s disease

Vascular endothelial growth factor (VEGF) is important for angiogenesis and inflammation, both of which are codependent and contribute to the pathophysiology of Behçet's disease (BD). In this report, we sought to investigate whether the selected VEGF polymorphisms [-634 C/G, +936 C/T and an 18 bp insertion/deletion (I/D) at -2549 of the VEGF promoter region] are associated with susceptibility and severity of BD in the Tunisian population. One hundred and thirty-five Tunisian BD patients and 157 healthy controls were recruited. The VEGF gene was genotyped by polymerase chain reaction followed by digestion with restriction endonucleases. VEGF serum levels of BD patients and healthy controls were measured by enzyme-linked immunosorbent assay. We found no association between the VEGF polymorphisms and the susceptibility to BD. However, when data were analysed according to the presence of each symptom, we found a positive association between VEGF 18 bp I/D polymorphism and ocular involvement as well as BD severity. Indeed, among the BD patients, the frequency of the 18 bp I/I genotype was less in patients with ocular inflammation (6.1% vs 24.6%, P = 0.007) and in patients with severe BD (9.2% vs 21.4%, P = 0.0014). Moreover, the mean serum VEGF level was considerably higher in BD patients (P < 0.01) than in healthy controls. We suggest that VEGF gene polymorphisms may be involved in the development of the ocular BD as well as the severity of the disease.

The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex–interactive agents

Previous studies on the functional analysis of the human vascular endothelial growth factor (VEGF) promoter using the full-length VEGF promoter reporter revealed that the proximal 36-bp region (-85 to -50 relative to transcription initiation site) is essential for basal or inducible VEGF promoter activity in several human cancer cells. This region consists of a polypurine (guanine) tract that contains four runs of at least three contiguous guanines separated by one or more bases, thus conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that the G-rich strand in this region is able to form an intramolecular propeller-type parallel-stranded G-quadruplex structure in vitro by using the electrophoretic mobility shift assay, dimethyl sulfate footprinting technique, the DNA polymerase stop assay, circular dichroism spectroscopy, and computer-aided molecular modeling. Two well-known G-quadruplex-interactive agents, TMPyP4 and Se2SAP, stabilize G-quadruplex structures formed by this sequence in the presence of a potassium ion, although Se2SAP is at least 10-fold more effective in binding to the G-quadruplex than TMPyP4. Between these two agents, Se2SAP better suppresses VEGF transcription in different cancer cell lines, including HEC1A and MDA-MB-231. Collectively, our results provide evidence that specific G-quadruplex structures can be formed in the VEGF promoter region, and that the transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. Our results also provide further support for the idea that G-quadruplex structures may play structural roles in vivo and therefore might provide insight into novel methodologies for rational drug design.

Association of Vascular Endothelial Growth Factor Polymorphisms with Asthma in Tunisian Children

BackgroundPrevious studies demonstrated that the vascular endothelial growth factor (VEGF) was being implicated in the airways inflammation and remodeling process in patients with asthma.AimsWe explored the relationship of three polymorphisms in the VEGF gene with asthma in both case control and family studies.MethodsWe Genotyped a total of 210 children with asthma, 224 unrelated controls and 160 parents for the +936 C &gt; T (rs3025039), –634 G &gt; C (rs2010963) and –2549 –2567 del 18 of the VEGF promoter region. The Mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis for the +936 C &gt; T, and –634 G &gt; C polymorphisms.ResultsOf the three polymorphisms studied, a borderline association with asthma was found for the G allele in the –634 G &gt; C polymorphism (p = 0.059). No Statistically significant differences were observed for both +936 C &gt; T, and –2549 –2567 del 18 polymorphisms between asthmatic patients and controls, considering either allelic or genotypic frequencies. The distribution of genotypes according to the severity status revealed a significant differences for the +936 C &gt; T, and –2549 –2567 del 18 polymorphisms. In addition, association was found with the haplotypes inferred by the three polymorphisms and asthma susceptibility.ConclusionWe suggest that VEGF Gene polymorphisms can be implicated in asthma.

Sympathetic Control of VEGF Angiogenic Signaling

See related article, pages 682–691 It is now accepted that vascular endothelial growth factor (VEGF), a dimeric 46-kDa heparin-binding glycoprotein, is one of the most important vasculogenic and angiogenic factors reported to date. VEGF acts as a potent and specific mitogen for vascular endothelial cells in vitro and possesses a signal sequence which allows it to be secreted.1 Furthermore, numerous studies in vivo have shown that expression of VEGF and its receptors is upregulated during angiogenesis under physiological and pathological processes, such as development of the embryo,2–4 estrous cycle,5 tumor growth,6,7 and wound healing.8 Thus, VEGF plays an important role in the formation of new vessels from existing ones and the microvascular remodeling which involves structural alterations—usually enlargement—of arterioles, capillaries, or venules, in inflammatory or neoplastic diseases. Different mechanisms appear to participate in the regulation of VEGF mRNA expression. It is well known that low oxygen tension is a strong inducer of VEGF mRNA expression in a variety of cells.6,9 Besides hypoxia-sensitive elements, the VEGF promoter region contains potential binding sites for the transcription factors AP-1, AP-2, and SP-1.10 Consistent with this fact, cAMP analogues and phorbol esters have been shown to …

Rat strain-dependent susceptibility to ischemia-induced retinopathy associated with retinal vascular endothelial growth factor regulation

Vascular endothelial growth factor (VEGF) is a potent inflammation, vascular permeability, and angiogenic factor. Variations of the VEGF gene are implicated in the pathogenesis of diabetic retinopathy. Previous studies have shown that Brown Norway (BN) rats have higher retinal VEGF levels and more severe retinal vascular leakage than Sprague-Dawley (SD) rats in response to ischemia and diabetes. To investigate the molecular mechanism of vascular leakage in this animal model, F2 progeny were generated by crossbreeding BN and SD rats. Neonatal rats were exposed to hyperoxia to induce oxygen-induced retinopathy (OIR) models. The F2 rats in response to ischemia have shown a linear distribution of retinal VEGF levels, which is significantly and positively correlated to retinal vascular leakage. We identified a single nucleotide polymorphism (SNP) at upstream stimulating factor-binding site in the VEGF promoter region between BN and SD rats. No differences were found in retinal vascular permeability or VEGF levels between F2 rats with BN, SD, and BN/SD alleles of VEGF SNP. The increased retinal VEGF levels are correlated to ischemia-induced retinal vascular leakage in the OIR rat model. The VEGF mRNA and promoter are not responsible for increased retinal VEGF level and vascular permeability. The up-regulation of VEGF expression activated by a yet to be identified upstream factor or mediator affecting VEGF stability may be associated with a high susceptibility to retinal vascular leakage in BN rats.

Effects of Polycyclic Aromatic Hydrocarbons (PAHs) on Vascular Endothelial Growth Factor Induction through Phosphatidylinositol 3-Kinase/AP-1-dependent, HIF-1α-independent Pathway

Previous studies have demonstrated that exposure to polycyclic aromatic hydrocarbons (PAHs) and its derivatives is associated with an increased risk of skin cancers, and the carcinogenic effect of PAHs is thought to involve both tumor initiation and promotion. Whereas PAH tumor initiation is well characterized, the mechanisms involved in the tumor promotion of PAHs remain elusive. In the present study, we investigated the effects of PAHs on vascular endothelial growth factor (VEGF) expression by comparison of its induction between the active metabolite and its parent compound (B[a]PDE versus B[a]P) or between active compound and its relatively inactive analog (5-MCDE versus CDE). We found that exposure of cells to (+/-)-anti-benzo-[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) or (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) led to marked induction of VEGF in Cl41 cells, whereas benzo[a]pyrene (B[a]P) or chrysene-1,2-diol-3,4-epoxide (CDE) did not exhibit significant inductive effects. Exposure of cells to B[a]PDE and 5-MCDE did not induce HIF-1alpha activation, whereas AP-1 was significantly activated. Moreover, overexpression of TAM67 (a dominant-negative mutant c-Jun) dramatically blocked that VEGF induction. Electrophoretic mobility shift assay showed that AP-1 was only able to specifically recognize and bind to its AP-1 potential binding site within -1136 and -1115 of the VEGF promoter region. Site-directed mutation of this AP-1 binding site eliminated the VEGF transcriptional activity induced by B[a]PDE, suggesting that the AP-1 binding site between -1136 and -1115 in the VEGF promoter region is critical for VEGF induction by B[a]PDE. In addition, overexpression of Deltap85 (a dominant-negative mutant PI-3K) impaired B[a]PDE- and 5-MCDE-induced VEGF induction. Considering our previous findings that PI-3K is an upstream mediator for c-Jun/AP-1 activation, we conclude that the VEGF induction by B[a]PDE and 5-MCDE is through PI-3K/AP-1-dependent and HIF-1alpha-independent pathways. These findings may help us to understand the mechanisms involved in PAH carcinogenic effects.

VEGF gene variability and type 1 diabetes: evidence for a protective role

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine originally described as an angiogenic factor. A number of reports have recently demonstrated that VEGF increases pancreatic islet survival after islet transplantation by stimulating angiogenesis and improving islet revascularization. Whether VEGF can protect from the autoimmune destruction of insulin-producing beta-cells that characterizes the development of type 1 diabetes is presently unknown. To clarify this issue, we studied the association of three polymorphisms of the promoter region of VEGF with type 1 diabetes in the Italian and the Finnish populations. The polymorphisms considered [C(-2578)A, G(-1190)A, and G(-1154)A] are known to modulate in vitro and in vivo VEGF expression. We found that VEGF promoter genotypes are associated with type 1 diabetes in both populations, but with different combinations. In Italian individuals, the -2578AA and -1190AA genotypes are associated with type 1 diabetes and accelerate its onset, while in Finnish individuals, -1154GG and -1190GG protect from type 1 diabetes and delay its onset. In conclusion, because the expected functional consequence of both genotype combinations is a reduced VEGF expression in diabetic patients, we propose a protective role of VEGF in the development of type 1 diabetes.

Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents

The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3′-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents.