Human Vascular Endothelial Growth factor-A Research Articles

In vivo antiangiogenic effect of nimbolide, trans-chalcone and piperine for use against glioblastoma

BackgroundAngiogenesis is an important hallmark of Glioblastoma (GBM) marked by elevated vascular endothelial growth factor-A (VEGF-A) and its receptor 2 (VEGFR-2). As previously reported nimbolide (NBL), trans-chalcone (TC) and piperine (PPR) possess promising antiangiogenic activity in several cancers however, their comparative efficacy and mechanism of antiangiogenic activity in GBM against VEGFR-2 has not been elucidated.Methods2D and 3D spheroids cultures of U87 (Uppsala 87 Malignant Glioma) were used for evaluation of non-cytotxoic dose for anti-angiogenic activity. The antiangiogenic effect was investigated by the GBM U87 cell line bearing chick CAM model. Excised U87 xenografts were histologically examined for blood vascular density by histochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of avian and human VEGF-A and VEGFR-2 mRNA transcripts.ResultsUsing 2D and 3D spheroid models, the non-cytotoxic dose of NBL, TC and PPR was ≤ 11 µM. We found NBL, TC and PPR inhibit U87-induced neoangiogenesis in a dose-dependent manner in the CAM stand-alone model as well as in CAM U87 xenograft model. The results also indicate that these natural compounds inhibit the expression of notable angiogenic factors, VEGF-A and VEGFR-2. A positive correlation was found between blood vascular density and VEGF-A as well as VEGFR-2 transcripts.ConclusionTaken together, NBL, TC and PPR can suppress U87-induced neoangiogenesis via a reduction in VEGF-A and its receptor VEGFR-2 transcript expression at noncytotoxic concentrations. These phytochemicals showed their utility as adjuvants to GBM therapy, with Piperine demonstrating superior effectiveness among them all.

VEGFA promotes odonto/osteoblastic differentiation in dental pulp stem cells via ERK/p38 signaling

Background/purpose: Vascular endothelial growth factor A (VEGFA) is a potent angiogenic factor and an essential growth factor for vascular endothelial cells, but its effects on dental pulp stem cells, such as stem cells from human exfoliated deciduous teeth (SHEDs), have not been fully evaluated. The aim of this study was to explore the effects and underlying mechanisms of VEGFA on odonto/osteoblastic differentiation in SHEDs in vitro. This study also aimed to examine the mineralized tissue-forming and pro-angiogenic potentials of VEGFA in rat dental pulp in vivo. Materials and methodsProliferation, migration, odonto/osteoblastic gene expression, and mineralized nodule formation were evaluated in SHEDs after stimulation with recombinant human VEGFA (rhVEGFA). Expression patterns of extracellular signal-regulated kinase (ERK) and p38/mitogen-activated protein kinase (MAPK) were analysed by western blotting. Rat molar pulp was histologically and immunohistochemically examined after 10 days of rhVEGFA-soaked agarose bead exposure. ResultsrhVEGFA stimulation promoted migration, mRNA expression of odonto/osteoblastic markers RUNX family transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), and mineralized nodule formation in SHEDs; these effects were reduced by ERK and p38/MAPK inhibitors. RhVEGFA-treated rat molar pulp tissues exhibited a reparative dentin-like mineralized tissue with surrounding nestin-positive cells and densely distributed CD146+ vascular vessels. ConclusionrhVEGFA can promote migration, odonto/osteoblastic differentiation, and mineralized nodule formation via ERK/p38 signaling in SHEDs in vitro; it promotes mineralized tissue formation and neovascularization in pulp tissue in vivo.

Increased VEGF-A Expression of Human Dental Pulp Stem Cells (hDPSCs) Cultured with Advanced Platelet Rich Fibrin (A-PRF)

Background: VEGF-A expression of human dental pulp stem cells (hDPSCs) can induce the angiogenesis process of dental pulp regeneration. This in vitro study aimed to analyze the effect of various concentrations of Advanced Platelet Rich Fibrin (A-PRF) conditioned media (CM) on the increased expression of vascular endothelial growth factor-A (VEGF-A) of hDPSCs. Methods: hDPSCs were collected from ten third molars extracted from nine healthy donors, cultured, and then harvested at the end of the third passage. The hDPSCs were seeded in four different CM (control group: hDPSCs + DMEM; 1% A-PRF CM group: hDPSCs + 1% A-PRF CM; 5% A-PRF CM group: hDPSCs + 5% A-PRF CM; 10% A-PRF CM group: hDPSCs + 10% A-PRF CM). All of the groups were cultured in biological triplicates (Triplo) and observed for 5, 12, and 24 hours. The VEGF-A protein expression of hDPSCs was measured using human VEGF-A ELISA at a wavelength of 405 nm. Data was analyzed with Kruskal Wallis and post hoc Mann Whitney test with p<0.05. Results: The VEGF-A expression rate of hDPSCs among all groups was statistically significantly different at 5, 12 and 24 hours of observations (p<0.05). Post hoc analysis test showed a statistically significant difference of hDPSCs’s VEGF-A expression between 5% A-PRF groups compared to other groups at 5 and 12 hours of observation (p<0.05). However, there were no statistically significant differences observed of hDPSCs’ VEGF-A expression at 24 hours of observation between 1%, 5% and 10% A-PRF groups (p>0.05). Conclusion: 5% A-PRF CM was superior in increasing VEGF-A expression of hDPSCs at 5, 12 and 24 hours of observations.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris.

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

The role of recovered thiols in venous endothel adaptation in case of autovenous reconstruction of lower limb arteries

Purpose. To evaluate the role of thiol status and its correlative relationship with the level of nitric oxide metabolites and vascular endothelial growth factor A (VEGF-A) in the blood plasma of patients with critical lower limb ischemia (CLI) after autovenous reconstructions of femoropopliteal segment’s arteries in the setting of venous endothelium of the arterial bed. Materials and methods . 54 patients with critical lower limb ischemia had been examined and divided into groups: synthetic prosthesis, in situ autovenous bypass procedure and reversed vein autovenous bypass procedure. Peripheral venous blood was taken on the 1 st and 10th days, and in 1, 3 and 6 months after the operation. Nitric oxide metabolites level was examined with photocolorimetric method by reaction with Griess reagent on a microplate analyzer (Awareness Technology, USA). VEGF-A number estimation was done by ELISA test (PersonalLab, Italy) with the use of Human VEGF-A Platinum ELISA. The level of thiol (SH-) groups was estimated with the use of Ellman’s reagent (SERVA, Germany) on spectrophotometer (Saint-Petersburg, Russian Federation). Results. The concentration of VEGF-A and the level of SH-groups increase on the 10th day and after 1 month in the group of patients operated on using a synthetic prosthesis. The level of NO metabolites, the concentration of VEGF-A, and the content of SH-groups increase statistically significantly and then decrease to the initial values in the group of patients operated on by reversed vein autovenous bypass procedure. In the group of patients operated on by the in situ method, the level of nitric oxide metabolites increases, the concentration of VEGF-A increases on the 10th day, the level of SH-groups increases, and a positive correlation was found between the content of SHgroups and the concentration of VEGF-A. Conclusion . NO metabolites contribute to the build-up of SH groups and VEGF-A in patients operated by the “reversed vein” method, and in patients in the in situ group, the concentration of VEGF-A and the level of SH groups are not affected, which may be of clinical importance when prescribing NO donors. The revealed patterns of change in the level of recovered thiols, nitrogen oxide metabolites, VEGF-A in combination with analysis of early and late postoperative complications make it possible to conclude the advantage of autovenous reconstruction of the femoropopliteal segment due to functional adaptation of the venous endothelium compared to the group of patients, operated on by the method of “synthetic prosthesis”. Method in situ on biochemical and angiological indicators proved to be more favorable in terms of the clinical course, than the “reversed vein” method.

Abstract 783: miR-375 downregulates VEGFA via YAP1-EBRR2 pathway in esophageal cancer

Abstract Esophageal cancer (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), is one of the deadliest cancers with extremely aggressive nature and poor survival rate. Diagnostic methods for EC mainly include endoscopy, barium swallow and ultrasound, which are either invasive or lack of sensitivity and specificity at early stages. Therefore, the development of a non-invasive and reliable diagnostic method with high sensitivity and specificity is crucial in improving early detection, diagnosis and treatment. microRNAs (miRNAs) are small RNA molecules that regulate the expression of protein-coding genes by directly binding to target mRNAs in a sequence-specific manner. The aim of this study is to identify novel miRNA biomarkers for EC diagnosis. By cross-referencing our data with others’ from NCBI, we identified a list of dysregulated miRNAs in EC, including miR-375. We assessed the expression of miR-375 in EC cell lines and tissues using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). We found that miR-375 expression is significantly decreased in human EC cell lines (KEYS-70, KEYS-180, FLO-1 and JHU-ad1) compared to the normal esophageal squamous cell line, Het-1A. Using microdissection technique, we isolated normal epithelium, dysplasia, and invasive EC components from formalin-fixed, paraffin-embedded (FFPE) tissue followed by qRT-PCR analysis. Decreased expression of miR-375 was detected in 12 of 14 EC tissue samples (86%) compared to the matched normal tissues. MTT and Matrigel invasion assay demonstrated that forced expression of miR-375 suppressed the proliferation and invasion in EC cell lines. TargetScanS and miRanda were used to identify potential target genes of miR-375, which includes epidermal growth factor receptor 2 (HER2) and YES-associated protein (YAP1), both of them act as oncogenes in EC development. Interestingly, forced expression of miR-375 resulted in significantly reduced expression of human vascular endothelial growth factor-A (VEGF-A) gene, which is a downstream target gene of YAP1/ERBB2 pathway involving in tumor angiogenesis in EC cell lines. More functional analysis is under way. These findings suggest that miR-375 may act as a tumor suppressor, and could serve as a marker for EC diagnosis and management. Citation Format: Shuchang Ren, Xiaohui Tan, Xiaoling Wu, Tao Chen, Patricia Latham, Sidney W. Fu. miR-375 downregulates VEGFA via YAP1-EBRR2 pathway in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 783.

Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.

Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development.

Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear sub-population of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3′ sites. In FDLE cultured at the PO2 of the fetal lung, FGF-10 relieved the Spry2 interaction at the HRE region by promoting clearance of a 39 kDa form and this was accompanied by histone-3 S10K14 phosphoacetylation, promoter de-methylation, hypoxia inducible factor-1α activation and VEGF-A expression. This repressive characteristic of nuclear Spry2 was relieved in 16HBE14o- by shRNA knockdown, and stable expression of mutants (C218A; C221A) that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung.

Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A)

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

A potential role of endoplasmic reticulum stress in development of ovarian hyperstimulation syndrome

Vascular endothelial growth factor A (VEGFA) is crucial for ovarian angiogenesis, but its excess production induces ovarian hyperstimulation syndrome (OHSS). The aim of this study was to determine whether endoplasmic reticulum (ER) stress regulates VEGFA expression in granulosa cells, and whether its activation is involved in OHSS development. The expression of the spliced form of X-box-binding protein 1 [XBP1(S)], induced by ER stress, in cumulus cells from OHSS patients was higher than that in cumulus cells from non-OHSS patients. The ER stress inducer tunicamycin increased human chorionic gonadotropin-induced VEGFA production in human granulosa cells through the induction of XBP1(S), and pretreatment with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) abrogated the effect of tunicamycin. In OHSS model rats, TUDCA administration prevented the OHSS development, reducing ovarian VEGFA production. Our findings suggest ER stress upregulates hCG-induced VEGFA production in granulosa cells, indicating that ER stress might be involved in OHSS development.

Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions.

In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an astrocyte-derived permeability factor, and suggest TYMP and VEGFA together promote blood-brain barrier breakdown.

Upregulation of non-β cell-derived vascular endothelial growth factor A increases small clusters of insulin-producing cells in the pancreas.

Pancreatic β cell-derived vascular endothelial growth factor A (VEGF-A) contributes to normal β cell function. We therefore hypothesized that non-β cell-derived VEGF-A may affect its properties in adult mice.We generated transgenic mice expressing human VEGF-A (hVEGF-A) in a visceral smooth muscle cell (SMC)-dominant manner under the control of the transgelin (Tagln/SM22α) promoter via a tamoxifen-induced Cre/loxP recombination system (SM-CreER(T2)/hVEGF mice).SM-CreER(T2)/hVEGF mice received tamoxifen orally followed by microscopic examination of their pancreas 4 weeks after the hVEGF-A induction. The number of clusters of insulin-producing cells (IPCs) in islets, pancreatic ducts, and individual IPCs were counted.The number of small IPC clusters (100-215 μm(2)) in the pancreas increased significantly in SM-CreER(T2)/hVEGF mice compared with SM-CreER(T2)(Ki) mice (473 out of 1 992 counts vs. 199 out of 976 counts, p<0.05), although total IPC area and the number of pancreatic duct IPCs, in proportion to exocrine area, were similar between the 2 groups. Although most small IPC clusters observed in SM-CreER(T2)/hVEGF mice were not accompanied by α and/or δ cells, some were attached to a single or a few α cells. An STZ-induced diabetic state in SM-CreER(T2)/hVEGF mice was slightly ameliorated, with only one point of significance 12 weeks after STZ administration, compared with SM-CreER(T2)(Ki) mice.Upregulation of non-β cell-derived VEGF-A may alter the composition of pancreatic IPCs by increasing the number of small IPC clusters. These findings provide new information on the role of non-β cell-derived VEGF-A to IPC regeneration and insulin production.

TRPS1 expression promotes angiogenesis and affects VEGFA expression in breast cancer

Angiogenesis is a hallmark of the malignant process in breast cancer in which vascular endothelial growth factor A (VEGFA) plays an important role. Trichorhinophalangeal syndrome type 1 (TRPS1) is a GATA-type transcription factor and is involved in trichorhinophalangeal syndrome type 1. To investigate the role of TRPS1 in breast cancer angiogenesis, we analyzed the expression of TRPS1 and microvessel density (MVD) marker CD31 by immunohistochemistry in 117 paraffin-embedded breast tissues. TRPS1 expression was positively correlated with CD31. We further investigated whether TRPS1 induces human umbilical vein endothelial cell (HUVEC) migration and VEGFA expression of breast cancer cells. The over-expression of TRPS1 induced a significant increase in HUVEC migration accompanied by VEGFA up-regulation in transfected cells. In contrast, knockdown of TRPS1 decreased the induction of HUVEC migration and significantly down-regulated VEGFA expression. Furthermore, endogenous TRPS1 was present in the VEGFA promoter, as determined by chromatin immunoprecipitation assay. Taken together, this study showed that TRPS1 promotes angiogenesis and affects VEGFA expression in breast cancer.

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in Vitro

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.

Transcriptional Regulation of VEGFA by the Endoplasmic Reticulum Stress Transducer OASIS in ARPE-19 Cells

BackgroundVascular endothelial growth factor-A (VEGFA) is the main mediator of angiogenesis. Angiogenesis plays important roles not only in many physiological processes, but also in the pathophysiology of many diseases. VEGFA is one of the therapeutic targets of treatment for ocular diseases with neovascularization. Therefore, elucidation of the regulatory mechanisms for VEGFA expression is important for the development of pharmaceutical drugs. Recent studies have demonstrated that the unfolded protein response is involved in the transcriptional regulation of VEGFA. However, the precise regulation of VEGFA in the human retina is not fully understood.Principal FindingsWhen human retinal pigment epithelial cells, ARPE-19, were exposed to endoplasmic reticulum stressors, VEGFA mRNA was significantly upregulated. The unfolded protein response-related transcription factors XBP1, ATF4, ATF6, and OASIS were expressed in ARPE-19 cells. To determine which transcription factors preferentially contribute to the induction of VEGFA expression after endoplasmic reticulum stress, we carried out reporter assays using an approximately 6-kbp 5′-upstream region of the human VEGFA gene. Among these transcription factors, OASIS acted most effectively on the VEGFA promoter in ARPE-19 cells. Based on data obtained for certain deleted and mutated reporter constructs, we determined that OASIS promoted VEGFA expression by acting on a cyclic AMP-responsive element-like site located at around –500 bp relative to the VEGFA transcription start site. Furthermore, we confirmed that OASIS directly bound to the promoter region containing this site by chromatin immunoprecipitation assays.Conclusions and SignificanceWe have demonstrated a novel regulatory mechanism for VEGFA transcription by OASIS in human retinal pigment epithelial cells. Chemical compounds that regulate the binding of OASIS to the promoter region of the VEGFA gene may have potential as therapeutic agents for ocular diseases with neovascularization.

Regulation of endothelial lipase and systemic HDL cholesterol levels by SREBPs and VEGF-A

ObjectiveEndothelial lipase (EL) regulates HDL cholesterol levels and in inflammatory states, like atherosclerosis, EL expression is increased contributing to low HDL cholesterol. The regulation of EL expression is poorly understood and has mainly been attributed to inflammatory stimuli. As sterol regulatory element binding proteins (SREBPs) are regulators of genes involved in lipid metabolism, we hypothesized that EL is regulated by SREBPs and that EL expression is modified by the SREBP activator vascular endothelial growth factor A (VEGF-A). Methodsand results: Quantitative PCR and Western blot results demonstrated that starvation increased EL expression in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). Also, 25-hydroxycholesterol (25HC), an inhibitor of SREBP activation inhibited EL expression. With siRNA-mediated inhibition of SREBPs the effect of starvation was shown to be SREBP-2 dependent. VEGF-A decreased EL expression in both endothelial cell lines used, most likely via inhibition of SREBP-2 binding determined by chromatin immunoprecipitation (ChIP). Furthermore, in atherosclerosis prone LDLR−/−ApoB100/100 mice, systemic adenoviral gene transfer with human VEGF-A decreased EL mRNA in peripheral tissues and increased plasma HDL cholesterol. ConclusionsThese results identify SREBPs as novel regulators of EL expression. VEGF-A as an endogenous EL inhibitor could be therapeutically relevant in atherosclerosis by increasing systemic HDL cholesterol levels.

Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues.

The Herpes Simplex Virus-1 Transactivator Infected Cell Protein-4 Drives VEGF-A Dependent Neovascularization

Herpes simplex virus-1 (HSV-1) causes lifelong infection affecting between 50 and 90% of the global population. In addition to causing dermal lesions, HSV-1 is a leading cause of blindness resulting from recurrent corneal infection. Corneal disease is characterized by loss of corneal immunologic privilege and extensive neovascularization driven by vascular endothelial growth factor-A (VEGF-A). In the current study, we identify HSV-1 infected cells as the dominant source of VEGF-A during acute infection, and VEGF-A transcription did not require TLR signaling or MAP kinase activation. Rather than being an innate response to the pathogen, VEGF-A transcription was directly activated by the HSV-1 encoded immediate early transcription factor, ICP4. ICP4 bound the proximal human VEGF-A promoter and was sufficient to promote transcription. Transcriptional activation also required cis GC-box elements common to the VEGF-A promoter and HSV-1 early genes. Our results suggest that the neovascularization characteristic of ocular HSV-1 disease is a direct result of HSV-1's major transcriptional regulator, ICP4, and similarities between the VEGF-A promoter and those of HSV-1 early genes.

Upregulation of VEGF-A and CD24 gene expression by the tGLI1 transcription factor contributes to the aggressive behavior of breast cancer cells

The Hedgehog signaling pathway is one of the most dysregulated pathways in human cancers. The glioma-associated oncogene homolog 1 (GLI1) transcription factor is the terminal effector of the Hedgehog pathway, frequently activated in human breast cancers and an emerging target of breast cancer therapy. While somatic mutations in the human GLI1 gene have never been reported in any cell or tumor type, we recently uncovered the existence of a novel alternatively spliced, truncated GLI1 (tGLI1) that has an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene. Using glioblastoma models, we showed that tGLI1 has gained the ability to promote glioblastoma migration and invasion via its gain-of-function transcriptional activity. However, the pathological impact of tGLI1 on breast cancer remains undefined. Here, we report that tGLI1 is frequently expressed in human breast cancer cell lines and primary specimens we have examined to date, but is undetectable in normal breast tissues. We found for the first time that tGLI1, but not GLI1, binds to and enhances the human vascular endothelial growth factor-A (VEGF-A) gene promoter, leading to its upregulation. Consequently, tGLI1-expressing MDA-MB-231 breast cancer cells secret higher levels of VEGF-A and contain a higher propensity, than the isogenic cells with control vector and GLI1, to stimulate in vitro angiogenesis of human vascular endothelial cells. We further showed that tGLI1 has gained the ability to enhance the motility and invasiveness of breast cancer cells in a proliferation-independent fashion and that this functional gain is associated with increased expression of migration/invasion-associated genes, CD24, MMP-2 and MMP-9. tGLI1 has also acquired the property to facilitate anchorage-independent growth of breast cancer cells. Collectively, our results define tGLI1 as a gain-of-function GLI1 transcription factor and a novel mediator of the behavior of clinically more aggressive breast cancer.