Plasma Vascular Endothelial Growth factor-A Research Articles

Prognostic Significance of Plasma VEGFA and VEGFR2 in Acute Ischemic Stroke-a Prospective Cohort Study.

Enhancement of vascular remodeling in affected brain tissue is a novel therapy for acute ischemic stroke (AIS). However, conclusions regarding angiogenesis after AIS remain ambiguous. Vascular endothelial growth factor A (VEGFA) and VEGF receptor 2 (VEGFR2) are potent regulators of angiogenesis and vascular permeability. We aimed to investigate the association between VEGFA/VEGFR2 expression in the acute stage of stroke and prognosis of patients with AIS. We enrolled 120 patients with AIS within 24h of stroke onset and 26 healthy controls. Plasma levels of VEGFA and VEGFR2 were measured by enzyme-linked immunosorbent assay (ELISA). The primary endpoint was an unfavorable outcome defined as a modified Rankin Scale (mRS) score > 2 at 3months after AIS. Univariate and multivariate logistic regression analyses were used to identify risk factors affecting prognosis. Plasma VEGFA and VEGFR2 were significantly higher in patients with AIS than in health controls, and also significantly higher in patients with unfavorable than those with favorable outcomes. Moreover, both VEGFA and VEGFR2 showed a significantly positive correlation with mRS at 3months. Univariate and multivariate analyses showed VEGFA and VEGFR2 remained associated with unfavorable outcomes, and adding VEGFA and VEGFR2 to the clinical model significantly improved risk reclassification (continuous net reclassification improvement, 105.71%; integrated discrimination improvement, 23.45%). The new risk model curve exhibited a good fit with an area under the receiver operating characteristic curve (ROC) curve of 0.9166 (0.8658-0.9674). Plasma VEGFA and VEGFR2 are potential markers for predicting prognosis; thus these two plasma biomarkers may improve risk stratification in patients with AIS.

Biomarker analysis of the phase II JO25567 study comparing erlotinib with or without bevacizumab in first-line advanced EGFR+non-small-cell lung cancer.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), such as erlotinib, are standard-of-care for patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC), but most patients progress within 1 year. Previously, we demonstrated that erlotinib plus bevacizumab (EB) improved progression-free survival (PFS) in patients with EGFR-positive non-squamous NSCLC in the randomized JO25567 study. To understand this effect, we conducted comprehensive exploratory biomarker analyses. Using blood and tissue specimens from patients enrolled in the JO25567 study, angiogenesis-related serum factors, plasma vascular endothelial growth factor-A (pVEGFA), angiogenesis-related gene polymorphisms, and messenger RNAs (mRNAs) in tumor tissue were analyzed. Interactions between potential predictors and treatment effect on PFS were analyzed in a Cox model. Continuous variable predictors were evaluated by multivariate fractional polynomial interaction methodology and subpopulation treatment effect pattern plotting (STEPP). Overall, 152 patients treated with EB or erlotinib alone (E) were included in the analysis. Among 26 factors analyzed in 134 baseline serum samples, high follistatin and low leptin were identified as potential biomarkers for worse and better outcomes with EB, with interaction P values of 0.0168 and 0.0049, respectively. Serum concentrations of 12 angiogenic factors were significantly higher in patients with high follistatin. Low pVEGFA levels related to better outcomes with EB, interaction P=0.033. VEGF-A165a was the only predictive tissue mRNA, showing a similar trend to pVEGFA. No valid results were obtained in 13 polymorphisms of eight genes. EB treatment showed better treatment outcomes in patients with low pVEGFA and serum leptin, and limited efficacy in patients with high serum follistatin.

A phase II study to explore biomarkers for the use of mFOLFOX6/XELOX plus bevacizumab as a first-line chemotherapy in patients with metastatic colorectal cancer (WJOG7612GTR)

The purpose of this prospective study was to assess the ability of plasma vascular endothelial growth factor-A short isoforms (pVEGF-Asi) to predict bevacizumab (BV) efficacy and to explore other circulating biomarkers in metastatic colorectal cancer (mCRC) patients treated with modified FOLFOX6/XELOX plus BV (mFOLFOX6/XELOX+ BV). Pre-treatment plasma samples were collected from 100 mCRC patients receiving first-line chemotherapy with mFOLFOX6/XELOX+ BV. The plasma levels of 11 angiogenesis-associated molecules, including pVEGF-Asi and 22 cancer-associated gene mutations in circulating tumor DNA, were analyzed. For the primary endpoint, we assumed that the hazard ratio (HR) for progression-free survival (PFS) calculated using a Cox proportional hazards model was <1.15, comparing patients with a high versus those with a low pVEGF-Asi level divided according to the median pVEGF-Asi value. The median value of pVEGF-Asi was 37 (range 6.5-262) pg/ml. The HR for PFS between the high and low pVEGF-Asi patient groups was 1.3 [95% confidence interval (CI) 0.8-2.1; log rank, P= 0.25], which was larger than the predefined threshold of 1.15. The multivariate analysis demonstrated that PFS was significantly associated with plasma intercellular adhesion molecule-1 (pICAM-1) (≥190.0 versus <190.0 ng/ml; HR 2.1; 95% CI 1.3-3.5), RAS (mutant versus wild; HR 2.5; 95% CI 1.5-4.3), and FBXW7 (mutant versus wild; HR 2.8; 95% CI 1.2-6.8), whereas overall survival was significantly associated with pICAM-1 (HR 2.0; 95% CI 1.1-3.7) and RAS (HR 2.6; 95% CI 1.5-4.6). The addition of BV was unable to compensate for the poor PFS associated with a high pVEGF-Asi level, suggesting that pVEGF-Asi is unlikely to be a good predictive biomarker of the efficacy of mFOLFOX6/XELOX+ BV therapy. The clinical significance of circulating ICAM-1, mutant RAS, and mutant FBXW7 levels should be studied further.

Association between Common Variants in VEGFA Gene and the Susceptibility of Primary Knee Osteoarthritis.

This study aimed to analyze the relationship between vascular endothelial growth factor A (VEGFA) gene polymorphisms, plasma VEGFA, and the susceptibility of knee osteoarthritis (OA). A total of 404 subjects, 202 knee OA subjects and 202 healthy volunteers, were enrolled into the study. Four distinct polymorphisms of the VEGFA gene were evaluated using polymerase chain reaction-restriction fragment length polymorphism: -2578C/A (rs699947), -1154G/A (rs1570360), -634C/G (rs2010963), and +936C/T (rs3025039). Plasma VEGFA levels were analyzed using enzyme-linked immunosorbent assay. The most common nucleotides in both knee OA subjects and healthy controls were CC for -2578C/A, GG for -1154G/A, CG for -634C/G, and CC for +936C/T in the VEGFA gene. Genotype distribution and allele frequencies of VEGFA -2578C/A, -1154G/A, -634C/G, and +936C/T single nucleotide polymorphisms did not differ between OA patients and the controls. Plasma VEGFA levels showed no difference between OA patients and the controls. In contrast, plasma VEGFA levels of -634C/C genotype were significantly higher in OA patients than in the controls (P = 0.035). According to the -2578A/A genotype, patients with early stage OA had a higher odds ratio than those with advanced stage OA (P = 0.023). VEGFA -2578C/A (rs699947), -1154G/A (rs1570360), -634C/G (rs2010963), and +936C/T (rs3025039) polymorphisms may not be responsible for OA susceptibility in the Thai population. However, the OA patients with A/A genotype at the -2578C/A seemed to have a lower potential risk of developing severe OA than those with the C/A and C/C genotypes. These findings would help elucidate and facilitate a better understanding of the genetic fundamentals of OA.

The blunted vascular endothelial growth factor-A (VEGF-A) response to high-altitude hypoxia and genetic variants in the promoter region of the VEGFA gene in Sherpa highlanders.

BackgroundSherpa highlanders demonstrate extraordinary tolerance to hypoxia at high altitudes, which may be achieved by mechanisms promoting microcirculatory blood flow and capillary density at high altitudes for restoring oxygen supply to tissues. Vascular endothelial growth factors (VEGFs) are important signaling proteins involved in vasculogenesis and angiogenesis which are stimulated by hypoxia. We hypothesize that the VEGF-A, the major member of the VEGF family, and the gene encoding VEGF-A (VEGFA) play a part in the adaptation to high-altitude hypoxia in Sherpa highlanders.MethodsFifty-one Sherpa highlanders in Namche Bazaar village at a high altitude of 3,440 meters (m) above sea level and 76 non-Sherpa lowlanders in Kathmandu city at 1,300 m in Nepal were recruited for the study. Venous blood was sampled to obtain plasma and extract DNA from each subject. The plasma VEGF-A concentrations were measured and five single-nucleotide polymorphisms (SNPs, rs699947, rs833061, rs1570360, rs2010963, and rs3025039) in the VEGFA were genotyped. The VEGF-A levels and allelic frequencies of the SNPs were compared between the two populations.ResultsA significant difference in oxygen saturation (SpO2) was observed between the two ethnic groups locating at different elevations (93.7 ± 0.2% in Sherpas at 3,440 m vs. 96.7 ± 0.2% in non-Sherpas at 1,300 m, P < 0.05). The plasma VEGF-A concentration in the Sherpas at high altitude was on the same level as that in the non-Sherpas at low altitude (262.8 ± 17.9 pg/ml vs. 266.8 ± 21.8 pg/ml, P = 0.88). This result suggested that the plasma VEGF-A concentration in Sherpa highlanders was stable despite a high-altitude hypoxic stimulus and that therefore the Sherpas exhibited a phenotype of blunted response to hypoxic stress. Moreover, the allele frequencies of the SNPs rs699947, rs833061, and rs2010963 in the promoter region of the VEGFA were different between the Sherpa highlanders and non-Sherpa lowlanders (corrected P values = 3.30 ×10−5, 4.95 ×10−4, and 1.19 ×10−7, respectively).ConclusionsSherpa highlanders exhibited a blunted VEGF-A response to hypoxia at high altitudes, which was speculated to be associated with the distinctive genetic variations of the SNPs and haplotype in the promoter region of VEGFA in Sherpa highlanders.

Fatty acid metabolism is involved in both retinal physiology and the pathology of retinal vascular diseases

To study the pathophysiological roles of the fatty acid-binding proteins (FABPs) within the retina, we performed; (1) immunolabeling of human retinas, wild type (WT) rat and mouse retinas, rat models for diabetic retinopathy (DR) and retinitis pigmentosa (RP) with anti-FABP3, FABP4, FABP5, FABP7, FABP8 and FABP12, (2) electroretinogram (ERG) measurements of WT and FABP4-deficient (Fabp4-/-) mice, (3) ELISA or gas chromatography measurements of plasma (P-) and vitreous (V-) levels of FABP4 and vascular endothelial growth factor A (VEGFA), and fatty acids (FAs) from patients with retinal vascular disease (RVD) including proliferative DR (PDR, n = 30) and retinal vein occlusion (RVO, n = 18) and non-RVD (n = 18). Within the human retina, diverse expressions of FABP3, FABP4, FABP7 and FABP8 were identified. In contrast, positive immunoreactivities toward only FABP4 and FABP12 were detected in the cases of rat and mouse retinas, and interestingly, the FABP4 labeling patterns for the WT, DR and RP rat retinas were different. The ERG amplitudes of Fabp4-/- mice were enhanced compared with those of WT mice. The concentrations of V-FABP4, V-VEGFA and total FAs were significantly higher in RVD patients than in non-PDR patients (P < 0.05). The V-FAs levels of each were significantly and positively correlated with V-FABP4 and V-VEGFA, although no significant correlation between vitreous (V-) and plasma (P-) FABP4, VEGFA and FAs were detected. The current study reveals that V-FAs appear to have significant roles in both retinal physiology as well as the pathogenesis of RVD with FABP4, which is commonly expressed within the retina in most species.

Clinical evidence for elevated levels of caveolin-1 in circulation of patients with diabetic foot ulcers.

Caveolin-1 directly interacts vascular endothelial growth factor receptor-2 (VEGFR2) and therefore prevents VEGF-induced angiogenesis. In addition, the production of nitric oxide (NO), which is effective in reducing ischemia in diabetic foot ulcers (DFU), is suppressed by caveolin-1 in endothelial cells. The present study was designed to investigate the change of caveolin-1 concentrations in DFU patients. A total of 150 participants were consecutively enrolled, including 40 DFU patients (DFU group), 40 diabetes patients without DFU (type 2 diabetes mellitus [T2DM] group), and 70 participants without diabetes (control group). Significant increased levels of plasma caveolin-1, accompanied with decreased concentration of plasma VEGF-A (vascular endothelial growth factor-A) and NO, were detected in DFU patients. Moreover, Pearson's correlation analysis revealed a negative correlation between plasma caveolin-1 and VEGF-A as well as NO levels in DFU patients. Furthermore, DFU patients had higher expression of caveolin-1 in the popliteal artery, compared to those in control and T2DM groups. Simultaneously, the amounts of eNOS (an enzyme responsible for the production of NO) and VEGFR2 were attenuated in the popliteal artery of DFU patients. Taken together, our study provided clinical evidence for the possible association of elevated caveolin-1 levels and the development of DFU. This may be induced by the suppressed VEGF-A/VEGFR2 and eNOS/NO signalling axis.

Evaluation of Plasma MMP-9 and VEGF-A mRNAs as Non-invasive Diagnostic Biomarkers in Women with Endometriosis

Background: Endometriosis is one of the common gynecological diseases and can lead to pelvic pain, dysmenorrhea, dyspareunia, and infertility in women. Thus, accurate and early diagnosis is a pivotal issue and an essential need for managing this disorder. At the present, the gold standard diagnostic method for endometriosis is laparoscopic surgery that is an invasive method and can lead to delay in diagnosis. Thus, there is an immediate necessity to search for non-invasive diagnostic biomarkers, such as blood-based ones. Objectives: Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A) have essential roles in the pathogenesis of endometriosis. Therefore, in this study, we evaluated the plasma mRNA levels of MMP-9 and VEGF-A, as potential non-invasive diagnostic biomarkers for endometriosis. Methods: This study included 48 women (24 cases and 24 controls) who underwent laparoscopy for suspected endometriosis. Preoperative plasma samples were collected, and after RNA extraction, the levels of MMP-9 and VEGF-A mRNAs were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results: Plasma MMP-9 mRNA level was statistically higher in endometriosis patients compared with the control group (P value = 0.01). However, plasma VEGF-A mRNA level did not show a significant difference between the two groups (P value =0.5). Conclusions: It seems that the plasma level of MMP-9 mRNA in endometriosis patients is significantly higher than in non-endometriosis women. This finding can provide new insights regarding this mRNA’s applicability as a non-invasive diagnostic biomarker for discovering new cases of endometriosis (newly diagnosed). According to our results, despite the suggested role of VEGF-A in endometriosis pathogenesis, it seems that the plasma level of VEGF-A mRNA does not have the potential to be used as a non-invasive diagnostic biomarker.

Chronic elevation of plasma vascular endothelial growth factor-A (VEGF-A) is associated with a history of blast exposure

Mounting evidence points to the significance of neurovascular-related dysfunction in veterans with blast-related mTBI, which is also associated with reduced [18F]-fluorodeoxyglucose (FDG) uptake.The goal of this study was to determine whether plasma VEGF-A is altered in veterans with blast-related mTBI and address whether VEGF-A levels correlate with FDG uptake in the cerebellum, a brain region that is vulnerable to blast-related injury 72 veterans with blast-related mTBI (mTBI) and 24 deployed control (DC) veterans with no lifetime history of TBI were studied. Plasma VEGF-A was significantly elevated in mTBIs compared to DCs. Plasma VEGF-A levels in mTBIs were significantly negatively correlated with FDG uptake in cerebellum. In addition, performance on a Stroop color/word interference task was inversely correlated with plasma VEGF-A levels in blast mTBI veterans. Finally, we observed aberrant perivascular VEGF-A immunoreactivity in postmortem cerebellar tissue and not cortical or hippocampal tissues from blast mTBI veterans.These findings add to the limited number of plasma proteins that are chronically elevated in veterans with a history of blast exposure associated with mTBI. It is likely the elevated VEGF-A levels are from peripheral sources. Nonetheless, increasing plasma VEGF-A concentrations correlated with chronically decreased cerebellar glucose metabolism and poorer performance on tasks involving cognitive inhibition and set shifting. These results strengthen an emerging view that cognitive complaints and functional brain deficits caused by blast exposure are associated with chronic blood-brain barrier injury and prolonged recovery in affected regions.

Plasma and Urinary FGF-2 and VEGF-A Levels Identify Children at Risk for Severe Bleeding after Pediatric Cardiopulmonary Bypass: A Pilot Study.

Severe bleeding after cardiothoracic surgery with cardiopulmonary bypass (CPB) is associated with increased morbidity and mortality in adults and children. Fibroblast Growth Factor-2 (FGF-2) and Vascular Endothelial Growth Factor-A (VEGF-A) induce hemorrhage in murine models with heparin exposure. We aim to determine if plasma and urine levels of FGF-2 and VEGF-A in the immediate perioperative period can identify children with severe bleeding after CPB. We performed a prospective, observational biomarker study in 64 children undergoing CPB for congenital heart disease repair from June 2015 - January 2017 in a tertiary pediatric referral center. Primary outcome was severe bleeding defined as ≥ 20% estimated blood volume loss within 24-hours. Independent variables included perioperative plasma and urinary FGF-2 and VEGF-A levels. Analyses included comparative (Wilcoxon rank sum, Fisher's exact, and Student's t tests) and discriminative (receiver operator characteristic [ROC] curve) analyses. Forty-eight (75%) children developed severe bleeding. Median plasma and urinary FGF-2 and VEGF-A levels were elevated in children with severe bleeding compared to without bleeding (preoperative: plasma FGF-2 = 16[10-35] vs. 9[2-13] pg/ml; urine FGF-2= 28[15-76] vs. 14.5[1.5-22] pg/mg; postoperative: plasma VEGF-A = 146[34-379] vs. 53 [0-134] pg/ml; urine VEGF-A = 132 [52-257] vs. 45[0.1-144] pg/mg; all p < 0.05). ROC curve analyses of combined plasma and urinary FGF-2 and VEGF-A levels discriminated severe postoperative bleeding (AUC: 0.73-0.77) with mean sensitivity and specificity above 80%. We conclude that the perioperative plasma and urinary levels of FGF-2 and VEGF-A discriminate risk of severe bleeding after pediatric CPB.

Beneficial Impact of Moderate to Vigorous Physical Activity Program on Circulating Number and Functional Capacity of Endothelial Progenitor Cells in Children: The Crucial Role of Nitric Oxide and VEGF-A.

Endothelial progenitor cells (EPCs) appear to interact with physical training. This study aimed to provide a comprehensive assessment of the relationship of moderate to vigorous physical activity (MVPA) with both angiogenic factors and EPC function in healthy children. Forty children (22 boys and 18 girls) aged 7 to 11 years participated in a 10-week MVPA program (duration: 45min; intensity: 75%-85% of heart rate reserve; frequency: 4sessions/wk). The anthropometric data, biochemical profile, EPCs number, EPCs colony-forming units, and vascular endothelial growth factor-A (VEGF-A) and nitric oxide (NO) plasma levels were evaluated before and after the MVPA program. After a 10-week MVPA program, a significant increase was detected in circulating/functional capacity of EPCs, NO, and VEGF-A levels, associated with improvement of waist circumference and estimated maximum rate of oxygen consumption (VO2max). A strong positive correlation was found between delta of EPCs number and variation of both NO level (r = .677, P < .001) and VEGF-A level (r = .588, P < .001). Furthermore, a significant correlation between NO level variation and delta of VEGF-A level was observed (r = .708, P < .001). Our findings suggest that lifestyle intervention implemented by MVPA program can contribute meaningfully to improve circulating/functional capacity of EPCs in healthy children, possibly due to the increase of plasma NO and VEGF-A levels.

Abstract P5-12-17: Prognostic and predictive value of serum level of vascular endothelial growth factor-A in metastatic breast cancer patients treated with bevacizumab plus paclitaxel

Abstract Background Several studies showed that first-line bevacizumab plus chemotherapy for HER2-negative metastatic breast cancer improves progression-free survival and tumor response rate but not overall survival. MERiDiAN trial evaluated plasma vascular endothelial growth factor-A (VEGF-A) prospectively as a predictive biomarker for bevacizumab efficacy in metastatic breast cancer. However, results of this trial do not support using baseline plasma VEGF-A to identify patients benefitting most from bevacizumab. We measured baseline serum VEGF-A level from stored blood samples of metastatic breast cancer patient with treated bevacizumab plus paclitaxel as fist-line and later line therapy, and evaluated a correlation between serum VEGF-A level and efficacy of bevacizumab and prognosis of breast cancer patients tread with bevacizumab, retrospectively. Patients and methods We examined blood samples from 57 metastatic breast cancer patients treated with bevacizumab and paclitaxel, after obtaining written informed consent. And, we evaluated a correlation between baseline serum VEGF-A level and time to treatment failure (TTF) and overall survival (OS). We also compared the serum VEGF-A level of response group (CR and PR) and that of non-response group (SD and PD). Results Baseline serum level of VEGF-A ranged from 80 to 2079 pg/ml. Cases of treatment line were as follows: first-line, 22 cases (38.6%); second line, 11 cases (19.3%) and third-line and the later line, 24 cases (42.1%). The cutoff identified by ROC curve analysis that was able to differentiate response group and non-response group in first-line setting was 360pg/ml for serum VEGF-A. And, we separated high serum VGEF-A group and low serum VEGF-A group of patients treated with bevacizumab plus paclitaxel. In patients treated as first line therapy, median TTF was 4.0 months with high serum VGEF-A group versus 5.0 months with low serum VEGF-A group, and median OS was 12 months with high serum VGEF-A group versus 11months with low serum VEGF-A group. There were no significant differences in both TTF and OS in first line setting. In patients treated as second line and later line therapy, median TTF was 2.8 months with high serum VGEF-A group versus 7.1 months with low serum VEGF-A group, and median OS was 6.4 months with high serum VGEF-A group versus 12.7 months with low serum VEGF-A group. The prognosis of high serum VEGF-A group was significantly worse than that of low serum group in both TTF and OS. The serum VEGF-A level of response group was tend to be higher than that of non-response group in first line setting, and was lower in second and later line setting. However, there were no significant differences. Conclusion In this study, serum VEGF-A cannot be a predictor for efficacy of bevacizumab plus paclitaxel as first line therapy for metastatic breast cancer patients. On the other hand, there was a possibility that high serum level of VEGF-A can be a poor prognostic factor in late line therapy setting of bevacizumab. Citation Format: Tozuka K, Nagai SE, Matsumoto H, Hayashi Y, Kubo K, Tsuboi M, Sato A, Takai K, Wang X, Yamada Y, Inoue K. Prognostic and predictive value of serum level of vascular endothelial growth factor-A in metastatic breast cancer patients treated with bevacizumab plus paclitaxel [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-17.

Junctional Adhesion Molecule-a Regulates Vascular Endothelial Growth Factor-a-Dependent Permeability

Introduction: Vascular barrier function is important for maintaining vascular integrity and is regulated by endothelial tight junctions. Breaches in the tight junction result in increased endothelial cell (EC) permeability, which leads to a variety of pathological conditions such as edema, vascular degeneration, diabetic retinopathy, and ischemia reperfusion injury. Junctional adhesion molecule A (JAM-A), is an integral part of the tight junctions of ECs. It has been shown that a lack of Jam-A affects neutrophil extravasation and FGF-2-induced angiogenesis. Role of JAM-A in regulation of vascular permeability is not known.Methods: Jam-A knockout (KO) and age-matched wild-type (WT) control mice of both sexes were used in this study. In vivo vascular permeability was assessed using Miles assay and quantified colorimetrically by extracting Evans blue in formamide at 56oC. The circulating plasma level of vascular endothelial growth factor A (VEGF-A) in WT and Jam-A KO mice was measured by ELISA assay. Surface expression of VEGF receptor 2 (VEGFR2) was analyzed by flow cytometry. Quantitative real- time PCR was used to determine gene transcription. Protein expression was determined by western blotting. VEGF-induced WT and Jam-A KO EC migration was analyzed by both scratch and trans-well assays.Results: Although Jam-A resides at the endothelial tight junctions, genetic ablation of Jam-A does not affect blood vessel structure. However, basal vascular permeability was significantly enhanced (P<0.05) in Jam-A KO mice compared to the WT controls, suggesting that a lack of Jam-A in EC compromises vascular integrity. This was further supported by the fact that there was increased edema in the lungs of the Jam-A KO mice compared to WT controls. Since VEGF is also known as vascular permeability factor, we evaluated the effect of VEGF-A on vascular permeability in Jam-A KO mice. Upon intravenous injection of VEGF-A (100ng/ml), EC permeability was significantly increased in both WT and Jam-A KO mice. In order to determine the cause of this enhanced basal permeability, we evaluated the levels of VEGF-A in WT and Jam-A KO mice. Jam-A deficient mice showed a three-fold (P<0.004) increased level of plasma VEGF-A compared to WT mice. Additionally, VEGF-A protein level was also increased in the ECs from Jam-A KO mice compared to WT controls. Furthermore, mRNA levels of VEGFR2, but not sFlt1, were increased in the Jam-A KO ECs compared to WT ECs. Protein levels as well as surface expression of VEGFR2 were significantly increased (P<0.001) in Jam-A KO ECs compared to WT ECs, suggesting that lack of Jam-A results in increased levels of VEGF-A/VEGFR2. We next evaluated the protein and mRNA levels of both hypoxia-inducible factor-1 α (HIF-1α), a transcription factor known to regulate expression of VEGF-A, and inhibitor of DNA binding 1 (Id1), a transcriptional factor known to regulate expression of VEGFR2. We found that bothHIF-1α and Id1 mRNA as well as protein levels were significantly (P<0.001 and P<0.05 respectively) augmented in Jam-A KO ECs compared to WT ECs. Additionally, overexpression of JAM-A in human umbilical vein endothelial cells (HUVECs) resulted in significantly reduced expression of bothHIF-1α and Id1 proteins. Furthermore, ECs isolated from Jam-A KO mouse showed significantly enhanced (P<0.05) cell migration in response to VEGF-A as compared to WT ECs, suggesting that Jam-A ECs respond favorably to VEGF-A due to increased VEGFR2 on their surface. To confirm that the observed augmented permeability in Jam-A KO mice was primarily due to enhanced VEGFR2 signaling, we inhibited VEGFR2 in mice using DC101 (200μg/kg), a function-blocking antibody. Isotype specific IgG (200μg/kg) was used as control. Inhibition of VEGFR2 completely abrogated basal as well as VEGF-A-induced vascular permeability in both Jam-A KO and WT mice, suggesting that the augmented EC permeability in Jam-A KO mice is not due to structural defect in tight junctions, but due to enhanced VEGF-A/VEGFR2 signaling.Conclusion: Our results suggest that JAM-A positively regulates EC tight junction integrity by suppressing levels of VEGF-A and VEGFR2 through downregulation ofHIF-1α and Id1 transcription factors. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Circulating Plasma VEGF-A, ET-1 and Magnesium Levels as the Predictive Markers for Proliferative Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common cause for preventable blindness in India. The onset of micro and macrovascular complications in T2DM is multifactorial and difficult to predict. The status of micronutrients, several inflammatory cytokines, elevated triacylglycerols, oxidative stress etc., are being studied extensively. Hypomagnesemia plays a pivotal role in worsening of insulin resistance. Although, Vascular Endothelial Growth Factor-A (VEGF-A) and Endothelin-1 (ET-1) are known to be elevated in DR, yet few reports cite their role, especially in Indian population. In this study, we included thirty subjects with T2DM in each of the three groupsnamely, T2DM cases without retinopathy, Non Proliferative DR (NPDR) and Proliferative DR (PDR) retinopathy. The glycemic status, circulating plasma VEGF-A, ET-1 levels, serum magnesium and lipids were estimated and compared among the groups. An ROC was drawn to evaluate VEGF-A, ET-1 and serum magnesium levels as the predictive markers for PDR. On comparison VEGF-A, ET-1 and serum magnesium levels showed a significant difference among the three groups. PDR cases had higher circulating levels of VEGF-A, ET-1 and low serum magnesium levels when compared to others. ROC for VEGF-A and ET-1 showed an optimum cut-off of 1521ng/ml (AUC 0.975) and 16pg/ml (AUC 0.96) respectively. A negative ROC was drawn to check the lower cut-off limit for serum magnesium; we documented an optimum cut off of 1.7mg/dl (AUC 0.837). ET-1, VEGF-A and serum Magnesium levels are significantlyaltered in PDR and can be used as the predictive markers of PDR.

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

A new angiogenesis prognostic index with VEGFA, PlGF, and angiopoietin1 predicts survival in patients with advanced gastric cancer.

The role of angiogenic factors in gastric cancer is not clear. We aimed to assess the role of vascular endothelial growth factor A (VEGFA), angiopoietin 1 (Ang-1), and placental growth factor (PlGF) in the prognosis of patients with advanced gastric cancer. Thirty consecutive patients treated with a modified DCF (docetaxel, cisplatin, and fluorouracil) regimen were included in the study. The plasma VEGFA, Ang-1, and PlGF levels of the patients before treatment and following two cycles of chemotherapy were measured and evaluated as prognostic factors. Poor performance status and lower Ang-1 levels were correlated with poor overall survival (OS). No significant correlation between VEGFA or PlGF and OS was found. An angiogenesis prognostic index (API) based on the levels of VEGFA, Ang-1, and PlGF was found to be highly correlated with OS. Performance status and API were found as independent prognostic factors for OS. Furthermore, a decrease in VEGFA by 25% from the pretreatment level was also found as a prognostic factor for OS independent of response to DCF regimen. Our results support the use of the new API including VEGFA, Ang-1, and PlGF levels in patients with advanced gastric cancer as a predictor of survival.

Bevacizumab plus paclitaxel versus placebo plus paclitaxel as first-line therapy for HER2-negative metastatic breast cancer (MERiDiAN): A double-blind placebo-controlled randomised phase III trial with prospective biomarker evaluation

AimMERiDiAN evaluated plasma vascular endothelial growth factor-A (pVEGF-A) prospectively as a predictive biomarker for bevacizumab efficacy in metastatic breast cancer (mBC). MethodsIn this double-blind placebo-controlled randomised phase III trial, eligible patients had HER2-negative mBC previously untreated with chemotherapy. pVEGF-A was measured before randomisation to paclitaxel 90 mg/m2 on days 1, 8 and 15 with either placebo or bevacizumab 10 mg/kg on days 1 and 15, repeated every 4 weeks until disease progression, unacceptable toxicity or consent withdrawal. Stratification factors were baseline pVEGF-A, prior adjuvant chemotherapy, hormone receptor status and geographic region. Co-primary end-points were investigator-assessed progression-free survival (PFS) in the intent-to-treat and pVEGF-Ahigh populations. ResultsOf 481 patients randomised (242 placebo–paclitaxel; 239 bevacizumab–paclitaxel), 471 received study treatment. The stratified PFS hazard ratio was 0.68 (99% confidence interval, 0.51–0.91; log-rank p = 0.0007) in the intent-to-treat population (median 8.8 months with placebo–paclitaxel versus 11.0 months with bevacizumab–paclitaxel) and 0.64 (96% confidence interval, 0.47–0.88; log-rank p = 0.0038) in the pVEGF-Ahigh subgroup. The PFS treatment-by-VEGF-A interaction p value (secondary end-point) was 0.4619. Bevacizumab was associated with increased incidences of bleeding (all grades: 45% versus 27% with placebo), neutropenia (all grades: 39% versus 29%; grade ≥3: 25% versus 13%) and hypertension (all grades: 31% versus 13%; grade ≥3: 11% versus 4%). ConclusionThe significant PFS improvement with bevacizumab is consistent with previous placebo-controlled first-line trials in mBC. Results do not support using baseline pVEGF-A to identify patients benefitting most from bevacizumab. Clinical trials registrationClinicalTrials.gov NCT01663727.

In vivo localisation and vascular effects of nano-vesicles derived from normal first trimester human placentae

In vivo localisation and vascular effects of nano-vesicles derived from normal first trimester human placentae

89Zr-bevacizumab PET visualizes heterogeneous tracer accumulation in tumor lesions of renal cell carcinoma patients and differential effects of antiangiogenic treatment.

No validated predictive biomarkers for antiangiogenic treatment of metastatic renal cell carcinoma (mRCC) exist. Tumor vascular endothelial growth factor A (VEGF-A) level may be useful. We determined tumor uptake of (89)Zr-bevacizumab, a VEGF-A-binding PET tracer, in mRCC patients before and during antiangiogenic treatment in a pilot study. Patients underwent (89)Zr-bevacizumab PET scans at baseline and 2 and 6 wk after initiating either bevacizumab (10 mg/kg every 2 wk) with interferon-α (3-9 million IU 3 times/wk) (n = 11) or sunitinib (50 mg daily, 4 of every 6 wk) (n = 11). Standardized uptake values were compared with plasma VEGF-A and time to disease progression. (89)Zr-bevacizumab PET scans visualized 125 evaluable tumor lesions in 22 patients, with a median SUV(max) (maximum standardized uptake value) of 6.9 (range, 2.3-46.9). Bevacizumab/interferon-α induced a mean change in tumor SUV(max) of -47.0% (range, -84.7 to +20.0%; P < 0.0001) at 2 wk and an additional -9.7% (range, -44.8 to +38.9%; P = 0.015) at 6 wk. In the sunitinib group, the mean change in tumor SUV(max) was -14.3% at 2 wk (range, -80.4 to +269.9; P = 0.006), but at 6 wk the mean change in tumor SUV(max) was +72.6% (range, -46.4 to +236%; P < 0.0001) above baseline. SUV(max) was not related to plasma VEGF-A at all scan moments. A baseline mean tumor SUV(max) greater than 10.0 in the 3 most intense lesions corresponded with longer time to disease progression (89.7 vs. 23.0 wk; hazard ratio, 0.22; 95% confidence interval, 0.05-1.00). Tumor uptake of (89)Zr-bevacizumab is high in mRCC, with remarkable interpatient and intrapatient heterogeneity. Bevacizumab/interferon-α strongly decreases tumor uptake whereas sunitinib results in a modest reduction with an overshoot after 2 drug-free weeks. High baseline tumor SUV(max) was associated with longer time to progression.

Effect of laser photocoagulation on plasma levels of VEGF-A, VEGFR-2, and Tie2 in infants with retinopathy of prematurity

To report the baseline plasma levels of vascular endothelial growth factor-A (VEGF-A), soluble vascular endothelial growth factor receptor-2 (sVEGFR-2) and soluble Tie2 in infants with treatment-requiring retinopathy of prematurity (ROP) and to investigate the effect of laser treatment on the plasma levels. Blood samples were collected from infants with prethreshold type 1 ROP before, 1 day after, and 1 week after confluent laser photocoagulation. Enzyme-linked immunosorbent assay procedure was used to determine plasma VEGF-A, sVEGFR-2, and sTie2 levels. A total of 48 eyes of 30 infants were included. The mean postconceptional age at which laser photocoagulation was performed was 37.5 ± 2.9 weeks. The mean number of laser spots applied to each eye was 1,480 ± 420. Regression of active retinopathy occurred within 1 week of treatment in all cases. Baseline plasma VEGF-A, sVEGFR-2, and sTie2 levels did not differ between unilateral and bilateral and also zone I and zone II disease. A significantly progressive decrease was observed in plasma VEGF-A, sVEGFR-2, and sTie2 levels at 1 day and 1 week after laser photocoagulation. Plasma levels of VEGF-A, sVEGFR-2, and sTie2 decreased significantly in our patients after laser treatment; however, because of the variability in the measurements, further studies evaluating the clinical value of these angiogenic factors in patients with treatment-requiring ROP are necessary.