Role Of Vascular Endothelial Growth Factor A Research Articles

Treatment with the vascular endothelial growth factor-A antibody, bevacizumab, has sex-specific effects in a rat model of mild traumatic brain injury

Mild traumatic brain injury (mTBI) involves damage to the cerebrovascular system. Vascular endothelial growth factor-A (VEGF-A) is an important modulator of vascular health and VEGF-A promotes the brain’s ability to recover after more severe forms of brain injury; however, the role of VEGF-A in mTBI remains poorly understood. Bevacizumab (BEV) is a monoclonal antibody that binds to VEGF-A and neutralises its actions. To better understand the role of VEGF-A in mTBI recovery, this study examined how BEV treatment affected outcomes in rats given a mTBI. Adult Sprague-Dawley rats were assigned to sham-injury + vehicle treatment (VEH), sham-injury + BEV treatment, mTBI + VEH treatment, mTBI + BEV treatment groups. Treatment was administered intracerebroventricularly via a cannula beginning at the time of injury and continuing until the end of the study. Rats underwent behavioral testing after injury and were euthanized on day 11. In both females and males, BEV had a negative impact on cognitive function. mTBI and BEV treatment increased the expression of inflammatory markers in females. In males, BEV treatment altered markers related to hypoxia and vascular health. These novel findings of sex-specific responses to BEV and mTBI provide important insights into the role of VEGF-A in mTBI.

Vascular Endothelial Growth Factor as Molecular Target for Bronchopulmonary Dysplasia Prevention in Very Low Birth Weight Infants.

Bronchopulmonary dysplasia (BPD) still represents an important burden of neonatal care. The definition of the disease is currently undergoing several revisions, and, to date, BPD is actually defined by its treatment rather than diagnostic or clinic criteria. BPD is associated with many prenatal and postnatal risk factors, such as maternal smoking, chorioamnionitis, intrauterine growth restriction (IUGR), patent ductus arteriosus (PDA), parenteral nutrition, sepsis, and mechanical ventilation. Various experimental models have shown how these factors cause distorted alveolar and vascular growth, as well as alterations in the composition and differentiation of the mesenchymal cells of a newborn's lungs, demonstrating a multifactorial pathogenesis of the disease. In addition, inflammation and oxidative stress are the common denominators of the mechanisms that contribute to BPD development. Vascular endothelial growth factor-A (VEGFA) constitutes the most prominent and best studied candidate for vascular development. Animal models have confirmed the important regulatory roles of epithelial-expressed VEGF in lung development and function. This educational review aims to discuss the inflammatory pathways in BPD onset for preterm newborns, focusing on the role of VEGFA and providing a summary of current and emerging evidence.

VEGFA promotes the occurrence of PLA2R-associated idiopathic membranous nephropathy by angiogenesis via the PI3K/AKT signalling pathway

BackgroundThe M-type phospholipase A2 receptor (PLA2R)-associated idiopathic membranous nephropathy (IMN) is a common immune-related disease in adults. Vascular endothelial growth factor A (VEGFA) is the key mediator of angiogenesis, which leads to numerous kidney diseases. However, the role of VEGFA in IMN is poorly understood.MethodsIn the present study, we downloaded the microarray data GSE115857 from Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) were identified with R software. The cytoHubba plug-in were used to identify hub genes from the protein–protein interaction network. Gene set enrichment analysis (GSEA) was used to identify signalling pathway in IMN. CCK8 was performed to assess the cell viability in human vascular endothelial cells (HVECs). Then, passive Heymann nephritis (PHN) was induced in rats by a single tail vein injection of anti-Fx1A antiserum. Animals treated with VEGFA inhibitor bevacizumab (BV), with saline as a positive control. Proteinuria was evaluated by biochemical measurements. Immunohistochemistry and immunofluorescence was used to evaluate relative proteins expression. Electron microscopy was performed to observe the thickness of the glomerular basement membrane (GBM).ResultsWe revealed 3 hub genes, including one up-regulated gene VEGFA and two down-regulated genes JUN and FOS, which are closely related to the development of PLA2R-associated IMN. Pathway enrichment analysis found that the biological process induced by VEGFA is associated with PI3K/Akt signalling. GSEA showed that the signalling pathway of DEGs in GSE115857 was focused on angiogenesis, in which VEGFA acts as a core gene. We confirmed the high expression of VEGFA, PI3K, and AKT in IMN renal biopsy samples with immunohistochemistry. In HVECs, we found that BV suppresses cell viability in a time and dose dependent manner. In vivo, we found low dose of BV attenuates proteinuria via inhibiting VEGFA/PI3K/AKT signalling. Meanwhile, low dose of BV alleviates the thickening of the GBM.ConclusionVEGFA/PI3K/AKT signalling may play significant roles in the pathogenesis of IMN, which may provide new targets for the treatment of IMN.

Placental Decidual Arteriopathy and Vascular Endothelial Growth Factor A Expression Among Women With or Without Human Immunodeficiency Virus.

Women with human immunodeficiency virus (HIV) (WHIV) are at higher risk of adverse birth outcomes. Proposed mechanisms for the increased risk include placental arteriopathy (vasculopathy) and maternal vascular malperfusion (MVM) due to antiretroviral therapy and medical comorbid conditions. However, these features and their underlying pathophysiologic mechanisms have not been well characterized in WHIV. We performed gross and histologic examination and immunohistochemistry staining for vascular endothelial growth factor A (VEGF-A), a key angiogenic factor, on placentas from women with ≥1 MVM risk factors including: weight below the fifth percentile, histologic infarct or distal villous hypoplasia, nevirapine-based antiretroviral therapy, hypertension, and preeclampsia/eclampsia during pregnancy. We compared pathologic characteristics by maternal HIV serostatus. Twenty-seven of 41 (placentas 66%) assessed for VEGF-A were from WHIV. Mean maternal age was 27 years. Among WHIV, median CD4 T-cell count was 440/µL, and the HIV viral load was undetectable in 74%. Of VEGF-A-stained placentas, both decidua and villous endothelium tissue layers were present in 36 (88%). VEGF-A was detected in 31 of 36 (86%) with decidua present, and 39 of 40 (98%) with villous endothelium present. There were no differences in VEGF-A presence in any tissue type by maternal HIV serostatus (P = .28 to >.99). MVM was more common in placentas selected for VEGF-A staining (51 vs 8%; P < .001). VEGF-A immunostaining was highly prevalent, and staining patterns did not differ by maternal HIV serostatus among those with MVM risk factors, indicating that the role of VEGF-A in placental vasculopathy may not differ by maternal HIV serostatus.

Hypoxic Preconditioning Enhances Cellular Viability and Pro-angiogenic Paracrine Activity: The Roles of VEGF-A and SDF-1a in Rat Adipose Stem Cells.

To achieve the full therapeutic potential of implanted adipose stem cells (ASCs) in vivo, it is crucial to improve the viability and pro-angiogenic properties of the stem cells. Here, we first simulated the conditions of ischemia and hypoxia using the in vitro oxygen-glucose deprivation (OGD) model and confirmed that hypoxic preconditioning of ASCs could provide improved protection against OGD and enhance ASC viability. Second, we assessed the effect of hypoxic preconditioning on pro-angiogenic potential of ASCs, with a particular focus on the role of vascular endothelial growth factor-A (VEGF-A) and stromal derived factor-1a (SDF-1a) paracrine activity in mediating angiogenesis. We found that the conditioned medium of ASCs (ASCCM) with hypoxic preconditioning enhanced angiogenesis by a series of angiogenesis assay models in vivo and in vitro through the upregulation of and a synergistic effect between VEGF-A and SDF-1a. Finally, to investigate the possible downstream mechanisms of VEGF/VEGFR2 and SDF-1a/CXCR4 axes-driven angiogenesis, we evaluated relevant protein kinases involved the signal transduction pathway of angiogenesis and showed that VEGF/VEGFR2 and SDF-1a/CXCR4 axes may synergistically promote angiogenesis by activating Akt. Collectively, our findings demonstrate that hypoxic preconditioning may constitute a promising strategy to enhance cellular viability and angiogenesis of transplanted ASCs, therein improving the success rate of stem cell-based therapies in tissue engineering.

Role of VEGF-A and LRG1 in Abnormal Angiogenesis Associated With Diabetic Nephropathy.

Diabetic nephropathy (DN) is an important public health concern of increasing proportions and the leading cause of end-stage renal disease (ESRD) in diabetic patients. It is one of the most common long-term microvascular complications of diabetes mellitus that is characterized by proteinuria and glomerular structural changes. Angiogenesis has long been considered to contribute to the pathogenesis of DN, whereas the molecular mechanisms of which are barely known. Angiogenic factors associated with angiogenesis are the major candidates to explain the microvascular and pathologic finds of DN. Vascular endothelial growth factor A (VEGF-A), leucine-rich α-2-glycoprotein 1, angiopoietins and vasohibin family signal between the podocytes, endothelium, and mesangium have important roles in the maintenance of renal functions. An appropriate amount of VEGF-A is beneficial to maintaining glomerular structure, while excessive VEGF-A can lead to abnormal angiogenesis. LRG1 is a novel pro-angiogenic factors involved in the abnormal angiogenesis and renal fibrosis in DN. The imbalance of Ang1/Ang2 ratio has a role in leading to glomerular disease. Vasohibin-2 is recently shown to be in diabetes-induced glomerular alterations. This review will focus on current understanding of these angiogenic factors in angiogenesis and pathogenesis associated with the development of DN, with the aim of evaluating the potential of anti-angiogenesis therapy in patients with DN.

Vascular endothelial growth factor-A as a promising therapeutic target for the management of psoriasis.

Vascular endothelial growth factor-A (VEGF-A), the main angiogenic mediator, plays a critical role in the pathogenesis of several inflammatory immune-mediated diseases, including psoriasis. Even though anti-angiogenic therapies, such as VEGF inhibitors, are licensed for the treatment of various cancers and eye disease, VEGF-targeting interventions are not part of current psoriasis therapy. In this viewpoint essay, we argue that the existing preclinical research evidence on the role of VEGF-A in the pathogenesis of psoriasis as well as clinical observations in patients who have experienced psoriasis remission during oncological anti-VEGF-A therapy strongly suggests to systematically explore angiogenesis targeting also in the management of psoriasis. We also point out that some psoriasis therapies decrease circulating levels of VEGF-A and normalise the psoriasis-associated vascular pathology in the papillary dermis of plaques of psoriasis and that a subset of patients with constitutionally high levels of VEGF-A may benefit most from the anti-angiogenic therapy we advocate here. Given that novel, well-targeted personalised medicine therapies for the development of psoriasis need to be developed, we explore the hypothesis that VEGF-A and signalling through its receptors constitute a promising target for therapeutic intervention in the future management of psoriasis.

Vascular Endothelial Growth Factor A and VEGFR-1 Change during Preimplantation in Heifers.

Vascular endothelial growth factor A (VEGFA) plays a critical angiogenic role in the endometrium of placentalia during preimplantation. The role of VEGFA and its receptors is not fully characterised in bovine reproduction. We analysed the mRNA expression of VEGFA isoforms 121, 165 and 189, and VEGF receptors 1 and 2 in three experimental settings (A, B and C). We compared intercaruncular endometrium of cyclic to pregnant heifers at Days 12, 15 and 18 post insemination (Day 0), and between Day 15 and Day 18 conceptuses (A). We further compared caruncular versus intercaruncular endometrium at Day 15 (B), and endometrium of heifers carrying embryos originating from somatic cell nuclear transfer (SCNT) versus in vitro fertilisation (IVF) at Day 18 (C). Endometrial VEGFA protein was localised and quantified. Pregnant heifers displayed lower intercaruncular endometrial mRNA expression of VEGFA-121 (p = 0.045) and VEGFA-189 (p = 0.009) as well as lower VEGFA protein abundance (p < 0.001) at Day 15. The VEGFA protein was localised in intercaruncular luminal, glandular epithelium and in tunica muscularis of blood vessels. At Day 15, caruncular endometrium displayed higher VEGFA mRNA expression than intercaruncular endometrium (p < 0.05). Intercaruncular endometrial VEGFA protein at Day 18 was higher in abundance in SCNT than in IVF (p = 0.038). Therefore, during preimplantation in cattle, there may be a need for timely physiological reduction in intercaruncular endometrial VEGFA expression in favour of the caruncular area to facilitate a gradient towards the implantation sites. A higher expression of VEGFA in SCNT may predispose for later placentation abnormalities frequently observed following SCNT.

The distribution and expression of vascular endothelial growth factor A (VEGFA) during follicular development and atresia in the pig.

The involvement of vascular endothelial growth factor A (VEGFA) in ovarian physiological processes has been widely reported, but the location and role of VEGFA during follicular atresia remain unknown. This study investigated the distribution and expression of VEGFA during porcine follicular development and atresia. Pig ovaries were obtained, individual medium-sized (3-5mm in diameter) antral follicles were separated and classified into healthy, early atretic or progressively atretic groups. Immunobiology and quantitative techniques were used to investigate the varied follicular distribution of VEGFA at both the morphological and molecular level. The results indicated that VEGFA protein expression peaked in tertiary follicles, mostly distributed in the thecal and inner granulosa layers, during follicular development while VEGFA mRNA was mainly expressed in the inner granulosa layers. Additionally, healthy antral follicles showed a significantly higher expression of VEGFA than atretic follicles in both theca and granulosa cells. Knockdown of VEGFA using siRNA revealed an antiapoptosis effect of VEGFA in cultured pig granulosa cells. Our results increase the knowledge of VEGFA functions in follicles.

VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells.

Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca2+ release and subsequent activation of intermediate conductance Ca2+ -activated K+ (IKCa ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca2+ responses and the EDH-vasodilatation mediated by IKCa activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IKCa channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries. These results indicate a novel role for VEGF-A in resistance arteries and suggest a new avenue for investigation into the role of VEGF-A in cardiovascular diseases. Vascular endothelial growth factor A (VEGF-A) is a potent permeability and angiogenic factor that is also associated with the remodelling of the microvasculature. Elevated VEGF-A levels are linked to a significant increase in the risk of cardiovascular dysfunction, although it is unclear how VEGF-A has a detrimental, disease-related effect. Small resistance arteries are central determinants of peripheral resistance and endothelium-dependent hyperpolarization (EDH) is the predominant mechanism by which these arteries vasodilate. Using isolated, pressurized resistance arteries, we demonstrate that VEGF-A acts via VEGF receptor-2 (R2) to inhibit both endothelial cell (EC) Ca2+ release and the associated EDH vasodilatation mediated by intermediate conductance Ca2+ -activated K+ (IKCa ) channels. Importantly, VEGF-A had no direct effect against IKCa channels. Instead, the inhibition was crucially reliant on the downstream activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). The distribution of EC inositol 1,4,5-trisphosphate (IP3 ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP3 R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca2+ via VEGFR2 has profound implications for EDH-mediated dilatation of resistance arteries and could provide a mechanism by which elevated VEGF-A contributes towards cardiovascular dysfunction.

Expression and role of VEGFA and miR-381 in portal vein tumor thrombi in patients with hepatocellular carcinoma.

The aim of the present study was to examine the expression and role of vascular endothelial growth factor A (VEGFA) and microRNA (miRNA or miR)-381 in tumor thrombi from patients with hepatocellular carcinoma and portal vein tumor thrombus (PVTT). Tumor thrombi and adjacent paired tissues were collected from 39 patients with hepatocellular carcinoma with PVTT. VEGFA expression levels were assessed using reverse transcription-quantitative polymerase chain reaction and western blotting. miRNAs that may regulate VEGFA expression were predicted using bioinformatics analysis and confirmed via a dual luciferase reporter assay. The effects of VEGFA and its upstream miRNA on proliferation of the proliferation of EAhy926 human venous endothelial cells were analyzed using an MTT assay. Compared with the paired adjacent tissues, VEGFA was significantly upregulated at both the mRNA and protein level in tumor thrombi (P<0.05). VEGFA was predicted to be a target of miR-381 and this was confirmed experimentally. miR-381 expression was significantly downregulated in tumor thrombi from patients with PVTT compared with paired adjacent tissues (P<0.05). In addition, transfection with antagomirs against miR-381 or short interfering RNA against VEGFA significantly inhibited EAhy926 cell proliferation (P<0.05). In conclusion, the results of the present study indicate that VEGFA is upregulated in tumor thrombi whereas miR-381 is downregulated. VEGFA is regulated by miR-381 and both may be associated with the development of PVTT.

Abstract 4592: Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy

Abstract Background: Resistance to anti-cancer therapies results in relapsed/refractory disease of Glioblastoma (GBM) and Ewing’s Sarcoma. Up-regulation of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59 can enable solid tumors to confer resistance to antibody-mediated immunotherapy by preventing complement and antibody-dependent cytotoxicity. mCRPs’ inhibitory role in monoclonal antibody treatments for liquid tumors have been reported, but their role and regulation in solid tumors has not been explored. In the context of refractory tumors, others have reported that vascular endothelial growth factor-A (VEGF-A) can induce mCRP expression in endothelial cells. Notably, p53 mutational status induces VEGF-A and its receptor (VEGFR2) in breast cancer cell lines. To investigate potential links among p53 status, VEGF-A, and mCRP, we screened wildtype (wt-p53) and mutant p53 solid tumor cell lines for mCRP expression and VEGF-A secretion. Our data suggest that p53 mutational status is associated with expression of CD55 and VEGF-A secretion. These studies provide foundation for potentially recognizing mCRPs as immune biomarkers in solid tumors, ultimately, resulting in development of novel immunotherapies for improved clinical outcomes. Methods: Pediatric Ewing’s sarcoma (CHLA9 and CHLA10) and adult GBM (GBM10 and GBM43) cell lines differing in p53 status were selected for in vitro studies. GBM43 originates from a primary GBM, while GBM10 is from a recurrent GBM patient. Ewing’s Sarcoma cell lines, CHLA9 and CHLA10, were generated from the same patient at primary diagnosis and at relapse respectively. Western blot, and sequencing confirmed the expression and p53 mutational status. mCRP expression was evaluated using RT-PCR and flow cytometry. Milliplex platform assessed VEGF-A expression in cell supernatants. Results: Whole genome sequencing data confirmed p53 mutations in all cell lines. CHLA9 and GBM10 harbor wt-p53. CHLA10 cells have p53 deletion and GBM43 cells have a F270C p53 mutation in both alleles CD55 transcripts were undetected in wt-p53 lines (CHLA9 and GBM10), but CD55 transcripts were increased in mutant/deleted p53 lines (CHLA10 and GBM43). Flow cytometry data show increased CD55 expression in mutant p53 glioblastoma (GBM43) versus wt-p53 (GBM10) cells (p&amp;lt;0.001). Transcript and flow cytometric analysis of CD46 and CD59 in Ewing’s sarcoma and GBM cell lines are in progress. Mutant p53 Ewing’s sarcoma and GBM cell lines secreted more VEGF-A compared to wt-p53 cell lines (p&amp;lt;0.05 and p&amp;lt;0.001, respectively). Conclusion: These findings highlight the importance of further investigating role of VEGF-A in regulating mCRPs in wt-p53 versus mutant p53 solid tumor cell lines. Elucidating mechanisms for mCRP regulation is critical for immune biomarker development and in facilitating the use of antibody-based therapeutic approaches for solid tumors. Citation Format: Pankita Hemant Pandya, M. R. Saadatzadeh, Jixin Ding, Barbara Bailey, Sydney Ross, Khadijeh Bijangi-Vishehsaraei, Mary E. Murray, Karen E. Pollok, Jamie L. Renbarger. Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4592. doi:10.1158/1538-7445.AM2017-4592

Neuroprotective effects of vascular endothelial growth factor A in the experimental autoimmune encephalomyelitis model of multiple sclerosis

Neuroprotective effects of vascular endothelial growth factor A in the experimental autoimmune encephalomyelitis model of multiple sclerosis

Abstract B170: Blockade of checkpoint programmed death 1 (PD1) delays tumor growth and induces early transferable protective immunity in a murine orthotopic pancreatic ductal adenocarcinoma model

Abstract Despite the growing understanding of how PD1 signaling contributes to the immunosuppressed tumor microenvironment (TME), the mechanisms underlying tumor regression by antibody blockade remain unclear. In this study, we sought to identify the early events involved in mediating the anti-tumor effects of anti-PD1 monoclonal antibody (α-PD1 mAb) using an orthotopic mutant KrasG12D/p53R172H Pdx-Cre (KPC) pancreatic ductal adenocarcinoma (PDA) model. This model is clinically relevant because in addition to KPC cells constitutively expressing the programmed death 1 ligand 1 (PD-L1), it represents advanced tumors due to its fast growth and drug resistant phenotype. When compared to hamster IgG isotype, single dose treatment with α-PD1 mAb (clone J43, 250µg/mouse) prolonged survival in C57BL6 mice bearing well established tumors (32 vs 76 days median survival time, respectively, p=0.0003). Mice that received α-PD1 mAb therapy presented transient moderate splenomegaly with increased spleen cellularity 72 hours following mAb administration. To determine if the transient splenomegaly represented a correlate of immune protection against tumor challenge, splenocytes from tumor bearing mice treated with IgG isotype or α-PD1 mAb for 72 hours, 7 days or 4 weeks were transferred into recipient tumor bearing mice that were pre-conditioned with cyclophosphamide (100mg/kg). Surprisingly, transferable protective immunity was only observed in the group that received splenocytes from donor mice treated with α-PD1 mAb for 72 hours (39.5 vs. 63 days median survival time for the IgG isotype vs. α-PD1 mAb treated group, p=0.042). Analysis by real time qPCR of tumor-infiltrating lymphocytes (TIL) obtained by selection of CD45+ cells using magnetic beads showed an increase in mRNA expression of IL-6 (4.6 fold), IL-8 (2.7 fold), IL-10 (6.2 fold), IFN-γ (3.8 fold) and TNF-α (4 fold) at 7 days, but not at 72 hours after PD1 blockade, suggesting that alterations in the splenic microenvironment influenced subsequent immune activation in the TME. Furthermore, individual depletion of immune cell subsets generally regarded as involved with the innate (Gr1+ cells, NK cells) or adaptive arms of the immune response (CD4 or CD8 T cells) were all capable of blocking the effect of α-PD1 mAb therapy in donor mice (p≤0.001). These results corroborate the importance of tumor recognition by the innate immune system in order to activate T cell-mediated killing, showing that stimulation of innate and/or adaptive immunity may play a direct role in controlling tumor progression. Alternatively, gene expression analysis using the non-TIL enriched fraction (remaining CD45- cells) extracted from tumors 7 days after PD1 inhibition showed downregulation of vascular endothelial growth factor A (VEGF-A) and G1/S related cyclin mRNA, such as cyclin-A, -D2 and E, suggesting that inhibition of PD1 induced tumor cell cycle arrest occurred concomitantly with upregulation of cytokine expression. Downregulation of VEGF-A was confirmed by ELISA using whole tumor extracts (p=0.03). Even though IL-10 is an inhibitor of VEGF-A, in vitro experiments using KPC cells failed to demonstrate a direct connection. However, TME conditions are complex, and often non-reproducible in vitro. Based on our results, we challenge the notion that PD1 blockade inhibits tumor progression mainly as a result of activating canonical adaptive immune responses. In addition, induction of G1/S tumor cell cycle arrest could increase sensitivity to cytotoxic therapies, thus providing a rationale for drug combinations. Given the broad role of VEGF-A in fostering an immunosuppressive TME and as an endothelial/epithelial growth factor, further studies are underway to investigate the role of VEGF-A inhibition in α-PD1 mAb-mediated tumor regression. Citation Format: Marcela d'Alincourt Salazar, Edwin Manuel, Weimin Tsai, Massimo D'Apuzzo, Leanne Goldstein, Bruce R. Blazar, Don J. Diamond. Blockade of checkpoint programmed death 1 (PD1) delays tumor growth and induces early transferable protective immunity in a murine orthotopic pancreatic ductal adenocarcinoma model. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B170.

Epigenetic regulation of vascular endothelial growth factor a dynamic expression in transitional cell carcinoma

Vascular endothelial growth factor A (VEGF-A) is a key mediator in the neovascularization of cancers. We have found that VEGF-A was expressed at significantly higher levels in high-grade transitional cell carcinoma (TCC) cells than low-grade TCC cells in our previous study. In the present study, promoter methylation pattern was assessed and quantified by bisulfite genomic sequencing (BGS) and specific VEGF-A CpG sites in low-grade, but not in high-grade, TCC cells were observed. Reporter assays indicated that hypermethylation of nine CpG sites can inhibit the transcriptional activity of the VEGF-A gene. Subsequent chromatin immunoprecipitation (ChIP) assay revealed down-regulation of transcription activity of VEGF-A with increasing binding of methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in low-grade TCC cells during hypermethylation. Furthermore, treatment of low-grade TCC cells with DNA methyltransferase inhibitor and histone deacetylase inhibitor can restore the expression of VEGF-A and promote the invasive ability of low-grade TCC cells. Hypermethylation with lower expression levels of VEGF-A in low-grade TCC tumors than high-grade TCC tumors was also confirmed in clinical specimens by reverse transcriptase-PCR and pyrosequencing analyses. Our findings are the first results indicating that VEGF-A expression is suppressed in low-grade TCC tumors by promoter hypermethylation. This offers a new perspective on the role of VEGF-A in TCC tumor behavior.

The expression of VEGF-A is down regulated in peripheral blood mononuclear cells of patients with secondary progressive multiple sclerosis.

BackgroundMost patients with relapsing-remitting multiple sclerosis (RRMS) eventually enter a secondary progressive (SPMS) phase, characterized by increasing neurological disability. The mechanisms underlying transition to SPMS are unknown and effective treatments and biomarkers are lacking. Vascular endothelial growth factor-A (VEGF-A) is an angiogenic factor with neuroprotective effects that has been associated with neurodegenerative diseases. SPMS has a prominent neurodegenerative facet and we investigated a possible role for VEGF-A during transition from RRMS to SPMS.Methodology/Principal FindingsVEGF-A mRNA expression in peripheral blood mononuclear (PBMC) and cerebrospinal fluid (CSF) cells from RRMS (n = 128), SPMS (n = 55) and controls (n = 116) were analyzed using real time PCR. We demonstrate reduced expression of VEGF-A mRNA in MS CSF cells compared to controls (p<0.001) irrespective of disease course and expression levels are restored by natalizumab treatment(p<0.001). VEGF-A was primarily expressed in monocytes and our CSF findings in part may be explained by effects on relative monocyte proportions. However, VEGF-A mRNA expression was also down regulated in the peripheral compartment of SPMS (p<0.001), despite unchanged monocyte counts, demonstrating a particular phenotype differentiating SPMS from RRMS and controls. A possible association of allelic variability in the VEGF-A gene to risk of MS was also studied by genotyping for six single nucleotide polymorphisms (SNPs) in MS (n = 1114) and controls (n = 1234), which, however, did not demonstrate any significant association between VEGF-A alleles and risk of MS.Conclusions/SignificanceExpression of VEGF-A in CSF cells is reduced in MS patients compared to controls irrespective of disease course. In addition, SPMS patients display reduced VEGF-A mRNA expression in PBMC, which distinguish them from RRMS and controls. This indicates a possible role for VEGF-A in the mechanisms regulating transition to SPMS. Decreased levels of PBMC VEGF-A mRNA expression should be further evaluated as a biomarker for SPMS.

Urinary albumin excretion is elevated in sepsis, but does not correlate with circulating VEGF-A levels

In critically ill patients admitted with SIRS, endothelial dysfunction leads to increased capillary permeability. In the glomerulus, it manifests as increased albumin excretion. Microalbuminuria is a common finding in various acute conditions like sepsis, trauma and surgery. Studies have shown increased levels of vascular endothelial growth factor-A (VEGF-A), a potent vascular permeability inducing agent, in LPS-induced endotoxemia, severe sepsis and septic shock. The pathogenic mechanism of the glomerular leakage of albumin in acute inflammatory conditions remains to be clarified. We wished to investigate the causative role of VEGF-A, in this regard which might have therapeutic implications.

Spatiotemporal Expression of flk-1 in Pulmonary Epithelial Cells during Lung Development

Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.

Increased expression of Vascular Endothelial Growth Factor-A in seasonal allergic rhinitis.

Increased vascular dilatation and permeability characterize allergic rhinitis. In this study oligonucleotide microarrays (Affymetrix HuGe95A) were used to identify differentially expressed vasoactive genes in nasal biopsies from 23 patients with symptomatic seasonal allergic rhinitis (SAR) and 12 healthy controls. RNA was extracted from the biopsies and pooled in three patient and three control pools. Out of 12,626 analysed transcripts, 39 were higher and 81 lower in the patients. Of these transcripts two have vasoactive effects: Vascular Endothelial Growth Factor-A (VEGF-A) and the Beta-1-Adrenergic Receptor. Both were higher in patients than in controls. The mean +/- SEM expression levels in arbitrary units of VEGF-A were 130 +/- 123 in the patients and 59 +/- 53 in the controls. The fold ratio in expression levels between patients/controls was 2.2. The corresponding values for the beta-1-adrenergic receptor were 129 +/- 123 in the patients and 40 +/- 31 in the controls. The fold ratio between patient/controls was 3.2. The role of VEGF-A was assessed by determining VEGF-A concentrations in nasal fluids from another 30 patients with SAR before and after allergen provocation. VEGF-A increased from 124.3 +/- 30.2 to 163.2 +/- 37.8 pg/ml after challenge, P < 0.05. In summary, oligonucleotide microarray analysis of nasal biopsies and protein analyses of nasal fluids indicate that VEGF-A may be an important mediator in SAR.

Distinct Requirements for Zebrafish Angiogenesis Revealed by a VEGF-A Morphant

Angiogenesis is a fundamental vertebrate developmental process that requires signalling by the secreted protein vascular endothelial growth factor-A (VEGF-A). VEGF-A functions in the development of embryonic structures, during tissue remodelling and for the growth of tumour-induced vasculature. The study of the role of VEGF-A during normal development has been significantly complicated by the dominant, haplo-insufficient nature of VEGF-A-targeted mutations in mice. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish VEGF-A morphant loss of function model. Zebrafish VEGF-A morphant embryos develop with an enlarged pericardium and with major blood vessel deficiencies. Morphological assessment at 2 days of development indicates a nearly complete absence of both axial and intersegmental vasculature, with no or reduced numbers of circulating red blood cells. Molecular analysis using the endothelial markers fli-1 and flk-1 at 1 day of development demonstrates a fundamental distinction between VEGF-A requirements for axial and intersegmental vascular structure specification. VEGF-A is not required for the initial establishment of axial vasculature patterning, whereas all development of intersegmental vasculature is dependent on VEGF-A signalling. The zebrafish thus serves as a quality model for the study of conserved vertebrate angiogenesis processes during embryonic development.