Elevated Vascular Endothelial Growth factor-A Research Articles

Serum VEGF-A levels on admission in COVID-19 patients correlate with SP-D and neutrophils, reflecting disease severity: A prospective study

Background and objectiveThe coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global morbidity and mortality. This study aimed to investigate the clinical significance of serum vascular endothelial growth factor A (VEGF-A) in COVID-19 patients and its association with disease severity and pulmonary injury. MethodsWe prospectively collected data from 71 hospitalized COVID-19 patients between June 2020 and January 2021. Patients were classified as either mild or severe based on their oxygen requirements during hospitalization. Serum VEGF-A levels were measured using an ELISA kit. ResultsIn comparison to mild cases, significantly elevated serum VEGF-A levels were observed in severe COVID-19 patients. Furthermore, VEGF-A levels exhibited a positive correlation with white blood cell count, neutrophil count, and lymphocyte count. Notably, serum surfactant protein-D (SP-D), an indicator of alveolar epithelial cell damage, was significantly higher in patients with elevated VEGF-A levels. ConclusionThese results suggest that elevated serum VEGF-A levels could serve as a prognostic biomarker for COVID-19 as it is indicative of alveolar epithelial cell injury caused by SARS-CoV-2 infection. Additionally, we observed a correlation between VEGF-A and neutrophil activation, which plays a role in the immune response during endothelial cell injury, indicating a potential involvement of angiogenesis in disease progression. Further research is needed to elucidate the underlying mechanisms of VEGF-A elevation in COVID-19.

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.

Abstract 11433: Elevated Vascular Endothelial Growth Factor A is Associated With Disruption of Default Network Connectivity

Introduction: Vascular Endothelial Growth Factor A (VEGF-A) is an angiogenic signaling protein involved in the maintenance of the cerebral vasculature. VEGF-A may play a role in the pathogenesis of Alzheimer’s disease and vascular dementia; however, whether elevated VEGF-A levels indicate a pathophysiolocal response or protective mechanism remains unclear. No prior study has explored whether VEGF-A levels may be associated with changes in functional connectivity among brain regions prior to the development of cognitive decline. Hypothesis: Disruption of the default network often precedes the development of cognitive changes. In a sample of older adults, we hypothesized that VEGF-A levels may be associated with default network connectivity, indicating early involvement of VEGF-A in disease processes. Methods: Forty-six independently living older adults (mean age = 72.0 years; SD = 6.4; 34.8% male) free of dementia or clinical stroke underwent venipuncture and brain MRI. Plasma was assayed for VEGF-A. Using resting state functional MRI, blood-oxygen level dependent (BOLD) time series from all voxels in each region of interest (ROI) was averaged to determine mean ROI activity. Medial prefrontal cortex, lateral parietal cortex and precuneus cortex ROIs comprised the default network. Results: Accounting for age and sex, multiple linear regression analysis revealed a significant negative association between VEGF-A levels and default mode network connectivity (B =-.0007, 95% CI (-.0012, -.0002), p = .0096). This association persisted after adjusting for APOE4 carrier status and Framingham score. A significant association was present in both APOE4 carriers (p = .019) and non-carriers (p = .029). Conclusions: Prior studies evaluating the association between VEGF-A levels and cognition have been conflicted. This study is the first to examine whether VEGF-A may be linked to early changes in brain network connectivity which precede cognitive decline. These findings suggest that VEGF-A may be elevated early in the progression of neurocognitive disorders. Whether higher levels of VEGF-A contribute to the pathogenesis of neurocognitive disorders or play a protective role in persevering cognitive function warrants further investigation.

Elevated Urinary VEGF-A, Transferrin, and Angiotensinogen Levels in Normoalbuminuric Children and Adolescents with Type 1 Diabetes: Can They Be Early Markers of Diabetic Kidney Disease?

Objective: We hypothesized that diabetic kidney disease (DKD) begins early, before albuminuria occurs. We therefore aimed to assess potential early urinary biomarkers of DKD in normoalbuminuric and normotensive children and adolescents with type 1 diabetes (T1D) to evaluate the relationship between these markers and clinical and laboratory risk factors for DKD. Methods: This cross-sectional study included 75 children and adolescents with T1D (62% females, mean age 13.9 ± 3.2 years) with normoalbuminuria (an albumin/creatinine ratio [ACR] below 30 mg/g creatinine). Fifty-five age- and sex-matched healthy children and adolescents served as controls. For the assessment of early DKD, urinary levels of angiotensinogen (AGT), transferrin, nephrin, vascular endothelial growth factor-A (VEGF-A), and kidney injury molecule-1 (KIM-1) were measured in adequately collected 24-h urine samples using enzyme-linked immunoassays. Results: The mean disease duration was 7.3 ± 3.2 (range 2.1–15.7) years, and the mean HbA1c level was 8.8 ± 1.4%. The median levels of urine VEGF-A/Cr, AGT/Cr, and transferrin/Cr were significantly higher in normoalbuminuric patients with T1D, compared with those of controls (p < 0.001, p = 0.02, and p = 0.001, respectively), but there was no difference in nephrin/Cr and KIM-1/Cr between the 2 groups. Although none of the patients had albuminuria, the median level of urine ACR was significantly higher in the patient group than the control group (p = 0.003). The ACR was positively correlated with glomerular filtration rate (GFR). Urinary transferrin/Cr, AGT/Cr, and VEGF-A/Cr were significantly correlated with ACR, but not with either GFR or diabetic risk factors including HbA1c or disease duration. Conclusion: Normoalbuminuric and normotensive children and adolescents with T1D have elevated urinary VEGF, AGT, and transferrin levels, which may indicate the development of DKD before albuminuria occurs.

Serum VEGF-A and VEGFR-1 levels in patients with adult immunoglobulin A vasculitis.

Immunoglobulin A vasculitis (IgAV) is classified as a leukocytoclastic vasculitis characterized by immune deposits in endothelial walls of small vessels causing vascular endothelial injury. The aim of the present study is to evaluate levels of vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-1 (VEGFR-1) levels in adult IgAV patients. Thirty-seven adult IgAV patients admitted to the Rheumatology Clinic meeting the IgAV American College of Rheumatology (ACR) criteria and 32 control subjects were enrolled in the study. Disease activity was categorized as "remission" or "active" according to Birmingham Vasculitis Activity Score (BVAS). Serum VEGF-A, VEGFR-1 levels and VEGFR-1/VEGF-A ratio were evaluated in patient and control groups. Serum median VEGF-A, VEGFR-1 and VEGFR-1/VEGF-A ratios were significantly higher in the patient group when compared to controls (235.9 [155-308.4] pg/mL vs. 78.8 [29.7-210.3] pg/mL, 400 [277.2-724.3] pg/mL vs. 31.5 [12.5-214.4] pg/mL and 1.85 [0.57-2.97] vs. 0.46 [0.38-0.63] respectively, all P values <.001). VEGFR-1 had the strongest predictive value with a cut-off value of 0.6 with 75% sensitivity and 73% specificity (P<.001). This study is the first report indicating elevated serum VEGF-A, VEGFR-1, and more importantly VEGFR-1/VEGF-A ratio can be good representatives of the inflammatory processes together with vascular endothelial injury in adult IgAV patients. VEGFR-1 seems to be a more important indicator of the ongoing inflammation.

Melanoma Associated Chitinase 3-Like 1 Promoted Endothelial Cell Activation and Immune Cell Recruitment.

Chitinase 3-like 1 (CHI3L1) is an enzymatically inactive mammalian chitinase that is associated with tumor inflammation. Previous research indicated that CHI3L1 is able to interact with different extracellular matrix components, such as heparan sulfate. In the present work, we investigated whether the interaction of CHI3L1 with the extracellular matrix of melanoma cells can trigger an inflammatory activation of endothelial cells. The analysis of the melanoma cell secretome indicated that CHI3L1 increases the abundance of various cytokines, such as CC-chemokine ligand 2 (CCL2), and growth factors, such as vascular endothelial growth factor A (VEGF-A). Using a solid-phase binding assay, we found that heparan sulfate-bound VEGF-A and CCL2 were displaced by recombinant CHI3L1 in a dose-dependent manner. Microfluidic experiments indicated that the CHI3L1 altered melanoma cell secretome promoted immune cell recruitment to the vascular endothelium. In line with the elevated VEGF-A levels, CHI3L1 was also able to promote angiogenesis through the release of extracellular matrix-bound pro-angiogenic factors. In conclusion, we showed that CHI3L1 is able to affect the tumor cell secretome, which in turn can regulate immune cell recruitment and blood vessel formation. Accordingly, our data suggest that the molecular targeting of CHI3L1 in the course of cancer immunotherapies can tune patients’ response and antitumoral inflammation.

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.

Elevated VEGF-A &amp; PLGF concentration in aqueous humor of patients with uveal melanoma following Iodine-125 plaque radiotherapy

To measure the concentration of vascular endothelial growth factor-A (VEGF-A), and placental growth factor (PLGF) in aqueous humor of uveal melanoma patients before and after Iodine-125 plaque therapy (IPT), determine the postoperative fluctuation and evaluate associated factors in vivo. Participants were 18 Chinese patients with uveal melanoma who were elected to IPT. Undiluted aqueous humor samples were collected at Iodine plaque implant and removal time, then stored immediately at -80°C until assayed. The concentration of VEGF-A, PLGF and other 7 cytokines comprising interleukin-2 (IL-2), IL-8, IL-10, interferon (IFN)-γ, programmed death (PD)-1, transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-1 in aqueous humor was measured using Raybiotech immunoassay kit, a high throughput strategy. The VEGF-A and PLGF levels were compared across preoperation and postoperation subgroups, as well as those of other 7 interleukins. Correlation and grouped analyses were conducted to determine the independent effects of clinical parameters and other cytokines on VEGF-A and PLGF concentration or fluctuation. This study set a self-control design. VEGF-A (P=0.038) and PLGF (P=0.026) were the only two increased cytokines after IPT. Preoperative and postoperative level of VEGF-A and PLGF (r=0.575, P=0.013; r=0.987, P<0.001) correlated with each other significantly. Level of VEGF-A (r=0.626, P=0.005; r=0.588, P=0.01) and PLGF (r=0.616, P=0.007; r=0.588, P=0.01) had positive correlation with tumor thickness consistently. Elevated VEGF-A or PLGF level were strong predictive factors of each other (P=0.007, OR=60.0). The elevated VEGF-A group showed a higher postoperative level of IFN-γ (P=0.005), IL-2 (P<0.001) and IL-10 (P=0.004) in aqueous humor. When the elevated PLGF group got similar results that a higher postoperative level of IFN-γ (P=0.007), IL-2 (P<0.001) and IL-10 (P=0.013) in aqueous humor. This study reveals that VEGF-A and PLGF in aqueous humor significantly increased with tumor thickness and radiation process in uveal melanoma patients. VEGF-A and PLGF may be crucial in uveal melanoma genesis and radiotherapy reactions. Immune mediators comprised IFN-γ, IL-2 and IL-10 could play roles in the link between inflammation and angiogenesis in uveal melanoma when exposed to radiotherapy.

Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes

Chemically modified mRNA is an efficient, biocompatible modality for therapeutic protein expression. We report a first-time-in-human study of this modality, aiming to evaluate safety and potential therapeutic effects. Men with type 2 diabetes mellitus (T2DM) received intradermal injections of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) or buffered saline placebo (ethical obligations precluded use of a non-translatable mRNA control) at randomized sites on the forearm. The only causally treatment-related adverse events were mild injection-site reactions. Skin microdialysis revealed elevated VEGF-A protein levels at mRNA-treated sites versus placebo-treated sites from about 4–24 hours post-administration. Enhancements in basal skin blood flow at 4 hours and 7 days post-administration were detected using laser Doppler fluximetry and imaging. Intradermal VEGF-A mRNA was well tolerated and led to local functional VEGF-A protein expression and transient skin blood flow enhancement in men with T2DM. VEGF-A mRNA may have therapeutic potential for regenerative angiogenesis.

Association of SNPs in the Promoter Regions of VEGF (rs699947 и rs2010963), ICAM1 (rs281437) and ET-1 (rs1800541) with Serum Levels of Related Proteins and Alcoholic Liver Cirrhosis Risk

Background: Uncontrolled use of alcohol can lead to the development of cirrhosis of the liver, which is manifested by fibrosis with the formation of regenerative nodes, an increase in pressure in the portal vein system and impaired liver function. Hepatic endothelium dysfunction during the formation of portal hypertension is accompanied by an increase in the level of protein molecules involved in the functioning of the endothelium: vascular endothelial growth factor A (VEGF-A), a soluble form of the intercellular adhesion molecule (s-ICAM-1) and endothelin-1 (ET -one). It is assumed that elevated levels of VEGF-A, s-ICAM-1 and ET-1 in alcoholic liver cirrhosis (AHC) may be interconnected with the structure of polymorphic loci, the promoter regions of the respective genes, which in turn may be a genetic risk factor for developing cirrhosis.Aims: Investigate the relationship of carriage of variant forms of polymorphic loci located in the promoter regions of VEGF-A, ICAM-1 and ET-1 with the level of the corresponding proteins in the blood serum and the risk of AHC.Materials and methods: The main group consisted of patients with pathological dependence on alcohol, aggravated by cirrhosis of the liver (AHC, n=60). The control group consisted of persons suffering from alcohol abuse, without liver pathology (AA, n=24). The observation period was the period of hospitalization. The serum levels of VEGF-A, s-ICAM-1 and ET-1 were evaluated by enzyme immunoassay. The distribution of variant forms of polymorphic loci located in the promoter regions of the VEGF-A genes (rs699947 and rs2010963), ICAM1 (rs281437) and ET-1 (rs1800541) in the studied sample was performed by real-time PCR.Results: The development of alcoholic cirrhosis was accompanied by a significant increase in the concentration of VEGF-A, s-ICAM-1 and ET-1 in serum. At the same time, direct correlations between the concentrations of VEGF-A, s-ICAM-1 and ET-1 in serum and the diameter of the portal vein in persons with liver cirrhosis were revealed. Patients with AHC are often carriers of the G allele of rs1800541 locus, located in the promoter of the ET-1 gene, compared with individuals suffering from control without liver pathology, which is associated with an increased risk of developing cirrhosis in alcohol dependence. The carriage of the C allele rs699947, as well as the C allele rs2010963 located in the promoter of the VEGF gene was associated with an increased level of VEGF-A in the AHC compared to carriers of this allele in the AA group. In addition, in the group of patients with AHC, carriers of allele C, homozygous CC genotype and heterozygous GC genotype of rs2010963 locus compared with carriers of G allele or homozygous GG genotype, respectively, were characterized by elevated serum VEGF-A levels.Conclusion: Carrier allele G of the rs1800541 locus (ET-1) is a risk factor for liver cirrhosis with alcohol abuse. The carriage of the C allele rs699947, as well as the C allele rs2010963 located in the promoter of the VEGF gene, can determine the elevated serum VEGF-A level in the AHC.

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.

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.

Blocking elevated VEGF-A attenuates non-vasculature Fragile X syndrome abnormalities.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation. In exploring abnormalities associated with the syndrome, we have recently demonstrated abnormal vascular density in a FXS mouse model (Galvan and Galvez, ). One of the most prominent regulators of vascular growth is VEGF-A (Vascular Endothelial Growth Factor A), suggesting that FXS is associated with abnormal VEGF-A expression. In addition to its role in vascular regulation, VEGF-A also induces cellular changes such as increasing cell proliferation, and axonal and neurite outgrowth independent of its effects on vasculature. These VEGF-A induced cellular changes are consistent with FXS abnormalities such as increased axonal material, dendritic spine density, and cell proliferation. In support of these findings, the following study demonstrated that FXS mice exhibit increased expression of VEGF-A in brain. These studies suggest that increased VEGF-A expression in FXS is contributing to non-vascular FXS abnormalities. To explore the role of VEGF-A in mediating non-vascular FXS abnormalities, the monoclonal antibody Bevacizumab was used to block free VEGF-A. Bevacizumab treatment was found to decrease FXS Synapsin-1 expression, a presynaptic marker for synapse density, and reduce FXS testicle weight to control levels. Blocking VEGF-A also alleviated FXS abnormalities on novel object recognition, a test of cognitive performance. These findings demonstrate that VEGF-A is elevated in FXS brain and suggest that its expression promotes non-vascular FXS abnormalities. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 14-25, 2017.

Phase I study of every 2- or 3-week dosing of ramucirumab, a human immunoglobulin G1 monoclonal antibody targeting the vascular endothelial growth factor receptor-2 in patients with advanced solid tumors

Ramucirumab is a fully human immunoglobulin G1 monoclonal antibody receptor antagonist designed to block the ligand-binding site of vascular endothelial growth factor receptor-2 (VEGFR-2). An initial phase I study evaluated ramucirumab administered weekly in advanced cancer patients. This phase I study of ramucirumab [administered every 2 or 3 weeks (Q2W or Q3W)] examined safety, maximum tolerated dose, pharmacokinetics, immunogenicity, antitumor activity, and pharmacodynamics. Patients with advanced solid malignancies were treated with escalating doses of ramucirumab i.v. over 1 h. Blood was sampled for pharmacokinetics studies throughout treatment; levels of circulating vascular endothelial growth factor-A (VEGF-A) and soluble VEGF receptors (R)-1 and -2 were assessed. Twenty-five patients were treated with ramucirumab: 13 with 6, 8, or 10 mg/kg Q2W, and 12 with 15 or 20 mg/kg Q3W. The median treatment duration was 12 weeks (range 2-81). No dose-limiting toxicities were observed. The most frequently reported adverse events (AEs) included proteinuria and hypertension (n = 6 each), and diarrhea, fatigue and headache (n = 4 each). Treatment-related grade 3/4 AEs were: two grade 3 hypertension (10 and 20 mg/kg), one each grade 3 vomiting, fatigue (20 mg/kg), atrial flutter (15 mg/kg), and one each grade 4 duodenal ulcer hemorrhage (6 mg/kg) and grade 4 pneumothorax (20 mg/kg). Pharmacokinetic analysis revealed low clearance and half-life of ∼110-160 h. Analysis of serum biomarkers indicated considerable patient-to-patient variability, but trends toward elevated VEGF-A and a transient decline in soluble VEGFR-2. Fifteen patients (60%) had best response of stable disease, with a median duration of 13 months (range 2-18 months) in tumor types including colorectal, renal, liver, and neuroendocrine cancers. Ramucirumab was well tolerated. Study results led to recommended phase II doses of 8 mg/kg Q2W and 10 mg/kg Q3W. Prolonged stable disease was observed, suggesting ramucirumab efficacy in various solid tumors. NCT00786383.

Vascular endothelial growth factor-A and changes in a tumor-bearing mouse model with Lewis lung cancer

Vascular endothelial growth factor-A (VEGF-A) affects tumor growth and metastasis through stimulation of angiogenesis. The purpose of this study was to describe features of Lewis lung cancer (LLC) in mice and compare the serum VEGF-A levels with those of normal control mice. Two groups of mice were compared: one was subcutaneously injected with LLC cells (n=16) and the other served as the normal control (n=6). The serum VEGF-A levels were measured by ELISA prior to inoculation, and at 7, 21 and 35 days post-inoculation. The tumor weight and the metastatic condition were evaluated on day 35. Changes in body weight and serum VEGF-A concentration over a period of time were compared between the groups using generalized estimating equations. The relationship between the primary tumor and the metastatic condition was analyzed using the Spearman's rank correlation test. The survival rate was 56.3% on day 35 post-tumor inoculation. No difference was found between the groups with regard to gastrocnemius muscle weight on day 35 post-inoculation [0.1315±0.0066 g vs. 0.1308±0.0069 g (normal control)]. In tumor-bearing mice, the weight gain at sacrifice was less (0.24±0.45 vs. 1.93±0.47 g, P=0.01), the final mean tumor volume and weight were 4264.69±1038.32 mm(3) and 3.70±0.83 g, the number of nodules in the lungs and livers was 6.33 (range 0-20) and 2.22 (range 0-11), respectively, and the serum VEGF-A levels were significantly higher than those of control mice. In conclusion, lower body weight gain, metastasis in the liver and lungs, and elevated VEGF-A levels are features of LLC in mice.

Abstract 5142: VEGFA gene amplification and protein expression in breast cancer

Abstract Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression. Methods: VEGFA gene amplification was evaluated by fluorescence in situ hybridization (FISH) on a multitumor tissue microarray (MTMA) comprising 3417 tissue samples from more than 100 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence immunoassay. In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers. Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 70) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p&amp;lt;0.0001). Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5142. doi:10.1158/1538-7445.AM2011-5142

Overexpression of VEGF 165 b, an inhibitory splice variant of vascular endothelial growth factor, leads to insufficient angiogenesis in patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic connective tissue disorder characterized by widespread microangiopathy, fibrosis, and autoimmunity that affects the skin and internal organs. Although in SSc there is a lack of sufficient angiogenic response to chronic tissue ischemia culminating in the loss of capillary vessels, the expression of vascular endothelial growth factor-A (VEGF) has paradoxically been shown to be upregulated in SSc skin and circulation. However, previous studies in the field did not distinguish between the proangiogenic VEGF 165 and antiangiogenic VEGF 165 b isoforms that are generated by alternative splicing in the terminal exon of VEGF pre-RNA. In the present study, we investigated whether VEGF isoform expression could be altered in skin and circulation of SSc patients. Using RT-PCR, quantitative real-time PCR, Western blotting, immunohistochemistry and confocal microscopy, we could show that the VEGF 165 b splice variant was selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF 165 b expression correlated with increased expression of profibrotic transforming growth factor-β1 (TGF-β1) and serine/arginine protein 55 (SRp55) splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. ELISA on plasma samples revealed that circulating levels of VEGF 165 b were significantly higher in SSc patients than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF 165 b than healthy MVECs (H-MVECs). TGF-β1 upregulated the expression of VEGF 165 b and SRp55 in both SSc- and H-MVECs. In SSc-MVECs, VEGF receptor-2 (VEGFR-2) was overexpressed, but its phosphorylation and ERK1/2 downstream signaling were impaired. Recombinant human VEGF 165 b and SSc-MVEC–conditioned medium inhibited VEGF 165 -mediated VEGFR-2 phosphorylation, ERK1/2 activation and capillary morphogenesis on Matrigel in H-MVECs. The addition of anti-VEGF 165 b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC–conditioned medium. Capillary morphogenesis was severely impaired in SSc-MVECs and could be ameliorated by treatment with recombinant VEGF 165 and anti-VEGF 165 b blocking antibodies. In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia. The combination of proangiogenic VEGF 165 administration and VEGF 165 b neutralization might represent a potential therapeutic strategy to promote effective angiogenesis and capillary regeneration in SSc.

Engineered Zinc Finger Protein–Mediated VEGF-A Activation Restores Deficient VEGF-A in Sensory Neurons in Experimental Diabetes

OBJECTIVEThe objectives of the study were to evaluate retrograde axonal transport of vascular endothelial growth factor A (VEGF-A) protein to sensory neurons after intramuscular administration of an engineered zinc finger protein activator of endogenous VEGF-A (VZ+434) in an experimental model of diabetes, and to characterize the VEGF-A target neurons.RESEARCH DESIGN AND METHODSWe compared the expression of VEGF-A in lumbar (L)4/5 dorsal root ganglia (DRG) of control rats and VZ+434-treated and untreated streptozotocin (STZ)-induced diabetic rats. In addition, axonal transport of VEGF-A, activation of signal transduction pathways in the DRG, and mechanical sensitivity were assessed.RESULTSVEGF-A immunoreactivity (IR) was detected in small- to medium-diameter neurons in DRG of control rats. Fewer VEGF-A-IR neurons were observed in DRG from STZ-induced diabetic rats; this decrease was confirmed and quantified by Western blotting. VZ+434 administration resulted in a significant increase in VEGF-A protein expression in ipsilateral DRG, 24 h after injection. VEGF-A was axonally transported to the DRG via the sciatic nerve. VZ+434 administration resulted in significant activation of AKT in the ipsilateral DRG by 48 h that was sustained for 1 week after injection. VZ+434 protected against mechanical allodynia 8 weeks after STZ injection.CONCLUSIONSIntramuscular administration of VZ+434 increases VEGF-A protein levels in L4/5 DRG, correcting the deficit observed after induction of diabetes, and protects against mechanical allodynia. Elevated VEGF-A levels result from retrograde axonal transport and are associated with altered signal transduction, via the phosphatidylinositol 3′-kinase pathway. These data support a neuroprotective role for VEGF-A in the therapeutic actions of VZ+434 and suggest a mechanism by which VEGF-A exerts this activity.

Elevation of Vascular Endothelial Growth Factor-A Serum Levels Following Acute Myocardial Infarction. Evidence for its Origin and Functional Significance

A. Kranz, C. Rau, M. Kochs and J. Waltenberger. Elevation of Vascular Endothelial Growth Factor-A Serum Levels Following Acute Myocardial Infarction. Evidence for its Origin and Functional Significance. Journal of Molecular and Cellular Cardiology (2000) 32, 65–72. Following the onset of acute myocardial infarction (AMI), a number of serum parameters show well-defined changes reflecting myocardial injury. During the consecutive repair phase, compensatory processes are initiated including the formation of a collateral circulation on the basis of angiogenesis and arteriogenesis. An important angiogenic factor is vascular endothelial growth factor-A (VEGF-A), shown to be upregulated in the ischemic myocardium. It is unclear, however, whether acute myocardial ischemia leads to a detectable elevation of VEGF-A serum concentrations. With the use of an immunoradiometric assay, we measured the levels of VEGF-A in the serum of patients after AMI at defined time intervals, of patients with unstable angina pectoris (UAP) and of healthy individuals. In addition, in a small group of patients with subacute myocardial infarction VEGF-A concentrations were measured in coronary sinus blood. The data are given as median followed by the 25th and 75th percentiles. In the group with AMI serum VEGF-A measured 105 [78; 176] pg/ml on day 1 and 114 pg/ml [72; 163] pg/ml on day 3 after onset of AMI. Serum levels of VEGF-A significantly increased on day 7 after AMI to 189 [119; 373] pg/ml (P=0.0103) and on day 10 to 255 [162; 371] pg/ml (P=0.0007). The VEGF-A serum level in healthy controls and in patients with UAP measured 98 [75; 137] pg/ml and 116 [57; 140] pg/ml, respectively. Serum at day 10 after AMI contained VEGF-A at a biologically relevant concentration capable of stimulating proliferation of endothelial cells. Surprisingly, VEGF-A serum levels were similar in samples taken from the coronary sinus with 61 [43; 83] pg/ml. Therefore the main source for VEGF-A in the blood stream is not the infarcted myocardium. However, the number of platelets, a rich source of VEGF-A, is significantly increased after myocardial infarction, i.e. 284 [252; 363]×109/litre v 220 [177; 250]×109/litre. In conclusion, the time course of VEGF-A elevation following AMI strongly suggests that VEGF-A plays a role as an endogenous activator of coronary collateral formation in the human heart. The most likely source of the elevated VEGF-A are platelets, rather than the infarcted myocardium.