Vascular Endothelial Growth Factor Receptors VEGFR-1 Research Articles

Endothelial Regulation of Microvascular Growth and Stability by Ang‐Tie and VEGF Signaling Pathways: A Mechanistic Computational Systems Biology Model

The Angiopoietin‐Tie (Ang‐Tie) pathway is a key signaling pathway regulating vascular stability and permeability, and it significantly intersects and crosstalk with the vascular endothelial growth factor (VEGF) signaling pathway, a major signaling pathway regulating angiogenesis and vascular permeability. Disrupted Ang‐Tie and VEGF signaling is linked to vascular dysfunction related to cancer, systemic inflammation, cardiovascular disease, and diabetic macular edema. Ang‐Tie pathway is regulated by various molecular mechanisms, including ligand‐receptor interactions, receptor multimerization, binding of co‐receptor Tie1, inhibition by vascular endothelial protein tyrosine phosphatase (VE‐PTP), receptor extracellular domain shedding, junctional localization, trafficking, and turnover. VEGF pathway is regulated by ligand binding, homo‐ and heterodimerization of VEGF receptors VEGFR1 and VEGFR2, interaction with neuropilin, thrombospondin‐1/CD47, receptor internalization, recycling, and degradation. Ang‐Tie and VEGF signaling pathways also share crosstalk mechanisms and integrative downstream signaling through phosphoinositide 3‐kinases (PI3K)/Akt, sphingosine‐dependent Erk activation, Src sequestration via RhoA/mDia, and calcium cycling, forming a complex reaction network that requires an integrative model to study the signaling outcomes on a systems level. The present study uses a mechanistic computational model of the Ang‐Tie and VEGF signaling pathways, their regulatory mechanisms, crosstalk, and integrative downstream signaling to quantitatively characterize the molecular control of vascular growth, permeability, and stability by endothelial cells. The model captures and reproduces key experimental observations of the Ang‐Tie/VEGF signaling network with detailed molecular mechanisms. The model significantly expands our previous computational models of the Ang‐Tie signaling pathway [1,2] and integrates our systems biology understanding of the VEGF pathway and their crosstalk. The model serves as a platform for quantitatively predicting the signaling outcome of varying ligand concentrations and receptor expressions in physiological and pathological conditions, identifying molecular mechanisms and targets for therapeutic interventions targeting the signaling network, and simulating the effects of drugs and their combinations on the signaling outcome on a network level.

Angiopoietin-Tie Signaling Pathway in Endothelial Cells: A Computational Model.

SummaryThe angiopoietin-Tie signaling pathway is an important vascular signaling pathway involved in angiogenesis, vascular stability, and quiescence. Dysregulation in the pathway is linked to the impairments in vascular function associated with many diseases, including cancer, ocular diseases, systemic inflammation, and cardiovascular diseases. The present study uses a computational signaling pathway model validated against experimental data to quantitatively study various mechanistic aspects of the angiopoietin-Tie signaling pathway, including receptor activation, trafficking, turnover, and molecular mechanisms of its regulation. The model provides mechanistic insights into the controversial role of Ang2 and its regulators vascular endothelial protein tyrosine phosphatase (VE-PTP) and Tie1 and predicts synergistic effects of inhibition of VE-PTP, Tie1, and Tie2 cleavage on enhancing the vascular protective actions of Tie2.

Plasma levels of soluble VEGF receptor isoforms, circulating pterins and VEGF system SNPs as prognostic biomarkers in patients with acute coronary syndromes

BackgroundDevelopment of collateral circulation in coronary artery disease is cardio-protective. A key process in forming new blood vessels is attraction to occluded arteries of monocytes with their subsequent activation as macrophages. In patients from a prospectively recruited post-acute coronary syndromes cohort we investigated the prognostic performance of three products of activated macrophages, soluble vascular endothelial growth factor (VEGF) receptors (sFlt-1 and sKDR) and pterins, alongside genetic variants in VEGF receptor genes, VEGFR-1 and VEGFR-2.MethodsBaseline levels of sFlt-1 (VEGFR1), sKDR (VEGFR2) and pterins were measured in plasma samples from subgroups (n = 513; 211; 144, respectively) of the Coronary Disease Cohort Study (CDCS, n = 2067). DNA samples from the cohort were genotyped for polymorphisms from the VEGFR-1 gene SNPs (rs748252 n = 2027, rs9513070 n = 2048) and VEGFR-2 gene SNPs (rs2071559 n = 2050, rs2305948 n = 2066, rs1870377 n = 2042).ResultsAt baseline, levels of sFlt-1 were significantly correlated with age, alcohol consumption, NTproBNP, BNP and other covariates relevant to cardiovascular pathophysiology. Total neopterin levels were associated with alcohol consumption at baseline. 7,8 dihydroneopterin was associated with BMI. The A allele of VEGFR-2 variant rs1870377 was associated with higher plasma sFlt-1 and lower levels of sKDR at baseline. Baseline plasma sFlt-1 was univariately associated with all cause mortality with (p < 0.001) and in a Cox’s proportional hazards regression model sFlt-1 and pterins were both associated with mortality independent of established predictors (p < 0.027).ConclusionssFlt-1 and pterins may have potential as prognostic biomarkers in acute coronary syndromes patients. Genetic markers from VEGF system genes warrant further investigation as markers of levels of VEGF system components in these patients.Trial registrationAustralian New Zealand Clinical Trials Registry. ACTRN12605000431628. 16 September 2005, Retrospectively registered.

A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P.

Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.

Polymorphisms in Vascular Endothelial Growth Factor Receptor 2 Are Associated with Better Response Rates to Ranibizumab Treatment in Age-related Macular Degeneration

Intravitreal anti-vascular endothelial growth factor (VEGF) injections are currently the standard treatment for neovascular age-related macular degeneration (AMD), but a broad range of response rates has been observed. We evaluated the association of single nucleotide polymorphisms (SNPs) in VEGF genes and their receptors (VEGFR) with the response rate to ranibizumab in 366 patients with neovascular AMD. Case series study. A total of 366 eyes of 366 patients with neovascular AMD. Visual acuity (VA) was determined at baseline, after 3 monthly ranibizumab injections, and after 1 year of treatment. Genotyping of 126 SNPs in the genes encoding VEGF family members VEGFA, VEGFB, VEGFC, VEGFD (FIGF), and placental growth factor (PGF); VEGF receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4); and the gene encoding pigment epithelium-derived factor (PEDF) (SERPINF1) was performed. The changes in VA after 3 injections and after 1 year of treatment and their association with VEGF and VEGFR genotypes. Univariate analyses of variance (ANOVAs) revealed a significant effect of SNP rs4576072 in the VEGFR2 gene on VA change after 12 months (F[1,235]= 14.05; P= 0.02). A stepwise linear regression analysis returned a model (P= 0.01) with SNPs rs4576072 and rs6828477 in the VEGFR2 gene as independent predictors for VA change after 12 months, with a mean increase in VA of 0.26 on the logarithm of the minimum angle of resolution (logMAR) scale in patients with 3 contributing minor alleles compared with a loss of 0.03 logMAR in patients with no minor allele. Polymorphisms in the VEGFR2/KDR gene significantly influence visual outcome in patients receiving ranibizumab treatment for neovascular AMD. This study shows that genetic variation partially explains the wide range of response to ranibizumab treatment, which in the future might help clinicians tailoring medicalinterventions to individual needs.

LDL attenuates VEGF-induced angiogenesis via mechanisms involving VEGFR2 internalization and degradation following endosome-trans-Golgi network trafficking

Considerable efforts have been made to amplify angiogenesis under conditions of hypoxia and ischemia by vascular endothelial growth factor (VEGF) delivery, so far with limited success. Ischemic vascular diseases are often associated with hypercholesterolemia. To elucidate whether the exposure to blood lipids influences VEGF responses of microvessels, we characterized effects of low density lipoprotein (LDL) exposure on the proliferation, migration and tube formation of human umbilical vein endothelial cells. By examining the expression, phosphorylation and downstream signals of VEGF's receptor VEGFR2, we characterized mechanisms controlling angiogenic responses following LDL exposure. LDL attenuated endothelial proliferation, migration and tube formation in a dose-dependent way. Reduced abundance of VEGFR2 and VEGFR1 were noticed in LDL-exposed endothelial cells. In subcellular localization studies that we combined with pharmacological experiments, we showed that the loss of VEGFR2 resulted from its internalization and degradation, the latter of which required syntaxin-16-dependent endosome-trans-Golgi network trafficking. As a consequence, VEGFR2 phosphorylation and downstream signals -specifically Akt and ERK1/2 phosphorylation- were attenuated in response to VEGF treatment. VEGF only partly reversed the effects of LDL on angiogenesis under conditions of normoxia and hypoxia. Our results suggest that angiogenic responses to VEGF are compromised in hypercholesterolemia as a consequence of endosomal VEGFR2 degradation.

P.3.c.027 The effect of antipsychotic drug treatment on serum levels of vascular endothelial growth factor (VEGF), sVEGFR-1, and sVEGFR-2 in schizophrenia

Vascular endothelial growth factor-A (VEGF-A) is a crucial mediator of vascular hyperpermeability, angiogenesis, and inflammation, processes intimately involved in tissue repair. Although much emphasis has been placed on understanding the synthesis and stability of VEGF-A mRNA, relatively little attention has been given to the study of the stability and processing of VEGF-A proteins themselves. In recent years, several studies indicated that VEGF-A protein activity is highly controlled through interaction with angiogenic or non-angiogenic mediators. We analyzed mechanisms that might control extracellular VEGF-A processing during wound repair. First, our studies provide evidence that VEGF-A protein is a target of proteases present in the microenvironment of human chronic non-healing wounds. Serine proteases, in particular plasmin cleave VEGF165 and digested VEGF fragments, showed decreased mitogenic activity. Inactivation of the plasmin cleavage site Arg110/Ala111 preserved the structural integrity and increased the angiogenic potency of VEGF165 when tested in an impaired healing mouse model. Secondly, we identified significantly increased levels of the potent VEGF-A inhibitor, the soluble form of the VEGF receptor VEGFR-1 (sVEGFR-1) in non-healing wounds when compared to healing wounds. Wound closure in healing and non-healing wounds correlated significantly with a decline in sVEGFR-1 levels. These observations support the concept that VEGF-A and VEGF-A receptors are important mediators in tissue repair. Further, our data provide mechanisms how VEGF-A-mediated interactions are disturbed during impaired healing.

Expression and Localization of Angiogenic Growth Factors in Developing Porcine Mesonephric Glomeruli

The development and growth of renal glomeruli is regulated by specific angiogenic growth factors, including vascular endothelial growth factor (VEGF) and the angiopoietins (ANGPT1 and ANGPT2). The expression of these factors has already been studied during metanephric glomerulogenesis, but it remains to be elucidated during the development of the embryonic mesonephros, which can function as an interesting model for glomerular development and senescence. In this study, the presence of the angiogenic growth factors was studied in developing porcine mesonephroi, using IHC and real-time RT-qPCR on laser capture microdissected glomeruli. In addition, mesonephric glomerular growth was measured by using stereological methods. ANGPT2 remained upregulated during maturation of glomeruli, which may be explained by the continuous growth of the glomeruli, as observed by stereological examination. The mRNA for VEGFA was expressed in early developing and in maturing glomeruli. The VEGF receptor VEGFR1 was stably expressed during the whole lifespan of mesonephric glomeruli, whereas VEGFR2 mRNA was only upregulated in early glomerulogenesis, suggesting that VEGFR2 is important for the vascular growth but that VEGFR1 is important for the maintenance of endothelial fenestrations.

Monoclonal Antibodies Targeting Vascular Endothelial Growth Factor

The use of monoclonal antibodies targeting the vascular endothelial growth factor (VEGF) pathway has been a significant addition to cancer therapy. One of the VEGF family members, VEGF-A (commonly referred to as VEGF), has been demonstrated to be important in angiogenesis. Although the mechanism of action of these antibodies is still under study, the anti-VEGF antibody bevacizumab has been approved for treatment of various solid cancers including colorectal, lung, and breast cancers as well as glioblastoma and renal cell carcinoma. Addition of bevacizumab to chemotherapy as adjuvant therapy in colorectal cancer did not improve disease-free survival. Bevacizumab is being tested in other clinical settings such as adjuvant therapy, maintenance therapy, and in combination with both chemotherapy and other targeted agents such as the epidermal growth factor receptor kinase inhibitor erlotinib. In addition to bevacizumab, other antibody-based therapies targeting the VEGF pathway are being tested. Ramucirumab and IMC-18F1 are monoclonal antibodies that target the VEGF receptors VEGFR-2 and VEGFR-1, respectively. Aflibercept (VEGF-Trap), a peptide-antibody fusion targeting VEGF ligand, is being tested in clinical trials. Much research is focused on identifying biomarkers to predict which patients will benefit from anti-VEGF therapy. Recent results suggest that VEGF single nucleotide polymorphisms may be predictive of patient response to bevacizumab. Improved imaging modalities such as dynamic contrast-enhanced MRI (DCE-MRI) can better characterize the efficacy of anti-angiogenic agents. As anti-VEGF treatments such as bevacizumab have been integrated into the treatment of many different types of cancers, the development of bevacizumab-resistant tumors has become more common. Recent studies show that targeting other angiogenesis signaling pathways such as platelet-derived growth factor-C (PDGF-C), Bombina variagata peptide 8 (Bv8, also known as prokineticin-2), and VEGFR-3 may lead to enhanced response in anti-VEGF resistant tumors. In the future, tailored treatments consisting of combinations of chemotherapy, other targeted therapies, and anti-angiogenesis agents will hopefully result in better patient outcomes.

VEGF‐Functionalized Polyelectrolyte Multilayers as Proangiogenic Prosthetic Coatings

AbstractStimulation of transprosthetic vascularization represents an interesting strategy in implantology to allow rapid tissue integration and finally to avoid prosthetic rejection. To achieve this goal, we modified the surface of porous titanium implants with polyelectrolyte multilayer (PEM) films functionalized with vascular endothelial growth factor (VEGF). Among the two PEM systems investigated, poly(L‐lysine)/poly(L‐glutamic acid) (PLL/PGA) and poly(allylamine hydrochloride)/poly(sodium 4‐styrenesulfonate) (PAH/PSS), the (PAH/PSS)4 architecture was selected to functionalize porous titanium, both for its high efficiency to adsorb VEGF and for its biocompatibility toward endothelial cells. In an original way, we unambiguously demonstrated that VEGF adsorbed on (PAH/PSS)4 maintains its bioactivity in vitro and stimulates endothelial cells proliferation. This effect was correlated with specific activation of intracellular signaling pathways induced by successive phosphorylation of the endothelial VEGF receptor VEGFR2 and mitogen‐activated protein kinases (MAPK) ERK1/2. By clearly demonstrating the proangiogenic activity of the VEGF‐PEM coating in vitro, the present study constitutes a first step toward in vivo application.

Hypoxia-induced epithelial VEGF-C/VEGFR-3 upregulation in carcinoma cell lines

Adaptation to hypoxia, a universal hallmark of carcinomas, is a critical step for tumor cell survival and growth. One of the principal regulators of hypoxia-responsive pathways is the transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha). Currently, it is known that tumoral production of members of the vascular endothelial growth factor (VEGF)-family (VEGFs) may promote tumor growth and progression by acting on carcinoma cells that express the cognate receptors (VEGFRs). However, the influence of hypoxia in the formation of such a tumoral VEGF/VEGFR loop is not completely understood. In the present study we examined the potential existence of a HIF-1 alpha/VEGF/VEGFR autocrine loop on commonly occurring carcinomas. The experiments were performed on five colorectal carcinoma cell lines, one breast (MCF7) and one lung (A549) adenocarcinoma cell line under normoxic and oxygen stress conditions using HIF-1 alpha-EIA, VEGFs-ELISA as well as RT-PCR and immunofluorescence for VEGFRs. HIF-1 alpha overexpression was found already after 2 h of exposure to hypoxia in all above mentioned cell lines, thus documenting that activation of the transcription factor HIF-1 alpha is an early cellular event. Under hypoxic conditions a significant upregulation and activation of HIF-1 alpha accompanied by an increased production of VEGF in MCF7 and A549 was observed. The well-differentiated colorectal carcinoma cell lines were 'hypoxia-resistant' showing unchanged levels of HIF-1 alpha and VEGF under hypoxia. None of the cell lines used in this study expressed the VEGF receptors VEGFR-1 and VEGFR-2 under normoxia and hypoxia. Additionally, all colorectal carcinoma cell lines were negative for VEGFR-3 transcripts in both conditions. However, VEGFR-3 mRNA and protein were expressed and under hypoxia overexpressed in MCF7 and A549. Hypoxic cultures of both cell lines secreted in elevated levels the VEGFR-3 ligand VEGF-C but not VEGF-D. Our findings suggest that under hypoxic conditions an autocrine loop between VEGF-C/VEGFR-3 and HIF-1 alpha is possible in breast carcinoma and lung carcinoma but not in colorectal carcinoma cell lines.

Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1

BackgroundWhile vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial.Methods and FindingsWe investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope.ConclusionsThis study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.

Dimerization of VEGF receptors and implications for signal transduction: A computational study

Vascular endothelial growth factor (VEGF) is a potent cytokine involved in the induction of neovascularization. Secreted as a cysteine-linked dimer, it has two binding sites at opposite poles through which it may bind VEGF receptors (VEGFRs), receptor tyrosine kinases found on the surface of endothelial and other cells. The binding of a VEGF molecule to two VEGFR molecules induces transphosphorylation of the intracellular domains of the receptors, leading to signal transduction. The dominant mechanism of receptor dimerization is not clear: the receptors may be present in an inactive pre-dimerized form, VEGF binding first to one of the receptors, the second receptor then ideally located for dimerization; or VEGF may bind receptor monomers on the cell surface, which then diffuse and bind to available unligated receptor monomers to complete the activation. Both processes take place and one or other may dominate on different cell types. We demonstrate the impact of dimerization mechanism on the binding of VEGF to the cell surface and on the formation of active signaling receptor complexes. We describe two methods to determine which process dominates, based on binding and phosphorylation assays. The presence of two VEGF receptor populations, VEGFR1 and VEGFR2, can result in receptor heterodimer formation. Our simulations predict that heterodimers will comprise 10–50% of the active, signaling VEGF receptor complexes, and that heterodimers will form at the expense of homodimers of VEGFR1 when VEGFR2 populations are larger. These results have significant implications for VEGF signal transduction and interpretation of experimental studies. These results may be applicable to other ligand–receptor pairs, in particular PDGF.

Bevacizumab-Induced Nasal Septum Perforation

A 53-year-old white male presented for evaluation and treatment after transverse colectomy for colon cancer. Postoperative computerized tomography and positron emission tomography scans confirmed synchronous liver metastases. The patient was started on 5-fluorouracil (5FU), leucovorin, and oxaliplatin (Eloxatin ® ; Sanofi-Synthelabo Inc., New York, http://www.sanofi-synthelabo.us) (mFOLFOX) in combination with bevacizumab (Avastin ® ; Genentech, Inc., South San Francisco, CA, http://www. gene.com). His concurrent medications included metoprolol (Lopressor ® ; Novartis Pharmaceuticals Corporation, East Hanover, NJ, http://www.pharma.us.novartis.com) and irbesartan (Avapro ® ; Bristol-Myers Squibb, Princeton, NJ, http://www.bms.com) for hypertension and clopidogrel (Plavix ® ; Bristol-Myers Squibb), atorvastatin (Lipitor ® ; Pfizer Pharmaceuticals, New York, http://www.pfizer. com), and aspirin for a history of single vessel coronary artery disease status post angioplasty. After three cycles of treatment with mFOLFOX/bevacizumab, the patient noted scabbing and irritation in the inferior part of the nasal septum associated with occasional bleeding. Physical examination revealed a small mucosal break. He denied any nasal instrumentation or manipulation, any history of cocaine abuse, or use of intranasal medications. After six cycles of chemotherapy, he complained of a “hole in the nose” in association with scant bloody discharge. Physical examination revealed a nasal septum perforation without any masses. A consultation with a head and neck specialist confirmed these findings on rhinoscopy. The mucosa was noted to be dry and scaly and slightly erythematous around the edges of the perforation. There were no visible masses or other abnormalities noted in the nasal vestibules. Figure 1 shows the nasal septal defect. Bevacizumab is a recombinant monoclonal immunoglobulin G1 antibody that selectively binds to vascular endothelial growth factor (VEGF), thus preventing its binding to the VEGF receptors VEGFR-1 and VEGFR-2 and inhibiting angiogenesis [1]. The combination of bevacizumab with irinotecan (Camptosar ® ; Pfizer Pharmaceuticals), 5-FU, and leucovorin has been shown to produce a superior response rate, time to progression, and overall survival in the first-line treatment of metastatic colon cancer [2]. Similarly, the combination of bevacizumab with FOLFOX has resulted in superior response rates, time to progression, and overall survival in the second-line treatment of metastatic colorectal cancer [3]. Bevacizumab has been associated with a 1% risk for bowel perforation when given as a single agent or in combination with FOLFOX [3]. Furthermore, a higher risk for wound healing complications and bleeding has been documented in patients who started treatment within 60 days from surgery [4]. Nasal

Angiogenic factors may have a different prognostic role in adult acute lymphoblastic leukemia

Angiogenesis plays an important role in solid tumors and hematologic malignancies. The prognostic significance of angiogenic factors in adult acute lymphoblastic leukemia (ALL) remains ambiguous. We therefore analyzed the impact of angiogenic factor levels on overall survival of newly diagnosed adult ALL patients. Plasma levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-1 receptor alpha (IL-1Ralpha), IL-6, IL-8, VEGF receptors VEGFR1 and VEGFR2, and thrombopoietin (TPO) were measured in plasma samples of 95 patients by enzyme-linked immunosorbent assay (ELISA). In a univariate Cox proportional hazards model, higher levels of IL-1Ralpha, IL-8, VEGFR1, and VEGFR2 were predictive of poor survival. In contrast, higher levels of VEGF were predictive of longer survival, and higher levels of bFGF suggested a similar trend (P = .09). The multivariate model simultaneously included VEGF (relative risk [RR] for death, 8.01; P = .001 for levels less than or equal to 19.5 pg/mL), IL-1Ralpha (RR, 5.12; P = .007 for levels greater than 373 pg/mL), and VEGFR2 (RR, 4.01; P = .04 for levels greater than 8222 pg/mL) as independent factors for survival. Of interest is the association of high levels of VEGF with good prognosis and higher levels of VEGF receptors with poor outcome. These data reflect the complexity by which angiogenic factors may affect the clinical behavior of patients with ALL, and this complexity should be considered in any therapeutic strategy incorporating antiangiogenic agents.

Cooperative Antitumor Effect of Multitargeted Kinase Inhibitor ZD6474 and Ionizing Radiation in Glioblastoma

Glioblastoma multiforme is an aggressive disease in which vascular endothelial growth factor (VEGF) and the EGF receptor (EGFR) are implicated in tumor growth, relapse, and resistance to radiotherapy and chemotherapy. The VEGF receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR), typically present on endothelial cells, have also been identified in human glioblastoma tissues and cell lines. In addition, EGFR is dysregulated in the majority of human glioblastomas and EGFR overexpression correlates with shorter survival. We have investigated the antitumor and antiangiogenic effect of ZD6474, an inhibitor of both VEGFR and EGFR signaling as a single agent and in combination with ionizing radiation. We have used ZD6474 and/or ionizing radiation in human glioblastoma cell lines D54 and U251 in vitro and in nude mice bearing established xenografts. The effects of treatment on tumor blood vessels and protein expression were evaluated by Western blot and immunohistochemistry. As single agents, ionizing radiation and ZD6474 caused a dose-dependent inhibition of soft agar growth in D54 and U251 cell lines, whereas a cooperative effect was obtained in combination. Treatment of mice bearing D54 xenografts with either ZD6474 or radiotherapy alone caused tumor growth inhibition that was reversible upon treatment cessation. A cooperative and long-lasting inhibition of tumor growth was obtained with ZD6474 in combination with concomitant radiotherapy. The antiproliferative effect was accompanied by inhibition of VEGF protein expression and inhibition of angiogenesis as measured by vessel counting. This study shows the antitumor activity of ZD6474 in combination with ionizing radiation in glioblastoma both in vitro and in vivo, and provides a scientific rationale to evaluate ZD6474 alone or in combination with radiotherapy in patients affected by this disease.

VEGF and VEGF Receptor Expression after Experimental Brain Contusion in Rat

Angiogenesis following traumatic brain injury (TBI) may be of importance not only for post-traumatic reparative processes but also for the development of secondary injuries. Vascular endothelial growth factor (VEGF) is a major regulator of endothelial cell proliferation, angiogenesis, and vascular permeability, though its possible involvement in secondary injuries after TBI is largely unknown. This study was undertaken to analyze the expression of VEGF and the VEGF receptors in experimental brain contusion in rat. Twenty-three adult female Sprague-Dawley rats were subjected to a focal cerebral contusion injury by use of a weight-drop model. Four additional rats underwent craniotomy only. The animals were sacrificed 6 h, or 1, 2, 4, 6, 8, or 16 days post-injury. Expression of VEGF and the VEGF receptors VEGFR1 (Flt-1) and VEGFR2 (Flk-1) were studied by in situ hybridization and immunohistochemistry. VEGF messenger (m)RNA and protein expression were detected in astrocytes, neutrophils, and macrophages in or adjacent to the injury from 1 day after injury, with a peak expression after 4-6 days. Flt-1 and Flk-1 mRNA and protein were detected in vessels adjacent to the lesion from 1 day after injury throughout day 6 after injury. It was also noted that Flt-1/Flk-1 and VEGF-positive vessels often were negative for SMI-71, a marker for vessels in areas with blood-brain barrier (BBB). In conclusion, we have demonstrated that TBI leads to an upregulation of VEGF, Flt-1, and Flk-1 mRNA and protein in and around the lesion. The data provide a foundation for future pharmacological intervention studies focusing on posttraumatic angiogenesis and possible injury repair effects of the VEGF system in TBI.

Vessels and Nerves: Marching to the Same Tune

The patterning of the nervous system is achieved through coordinated action of a variety of repulsive or attractive neuronal guidance factors that direct the growth of growing axons to specific pathways. Recent studies suggest that the same or similar sets of factors also guide migration of endothelial cells, helping to direct the stereotypical embryonic patterning of the developing vertebrate vascular system.

VEGF is required for the maintenance of dorsal root ganglia blood vessels but not neurons during development.

Vascular endothelial growth factor (VEGF) is a potent regulator of vascular function through its control of multiple endothelial cell functions. In addition to its key role in vascularization, VEGF has recently been shown to have neurotrophic activity during hypoxic stress. In the central and peripheral motor nervous system, VEGF treatment increased neuronal vascularization and perfusion, as well as having direct trophic effects on neurons and Schwann cells. However, the role of VEGF in the sensory nervous system remains unclear. To characterize the differential effects of VEGF on endothelial cells and neurons in sensory ganglia, we used explanted mouse dorsal root ganglia (DRG), a culture system containing neurons and endothelial cells in close apposition. We show that VEGF is expressed by neurons and satellite cells, but not by endothelial cells or pericytes. On the other hand, the tyrosine kinase VEGF receptor VEGFR-2 was robustly expressed by endothelial cells throughout the extensive DRG capillary network, but not found at either the transcript or protein level in sensory neurons or other nonendothelial cells of the DRG. Both soluble receptor sequestration of VEGF and small molecule kinase inhibition of VEGFR-2 signaling rapidly disrupted the connectivity, branching, and structural integrity of the capillary network of embryonic DRG; this effect was no longer evident postnatally. However, VEGF inhibition showed no detectable effect on neuronal health at any stage analyzed. These data suggest that endogenous VEGF is a strict requirement for vascular, but not neuronal, maintenance in developing sensory ganglia.

Phase I and pharmacokinetic study of VEGF Trap administered subcutaneously (sc) to patients (pts) with advanced solid malignancies

3009 Background: Vascular Endothelial Growth Factor (VEGF) Trap is a potent angiogenesis inhibitor that binds VEGF-A 100- to 1000- fold more tightly than monoclonal antibodies (Kd <1pM) and inactivates all circulating and tissue VEGF-A isoforms plus placental growth factor. VEGF Trap is comprised of portions of the human VEGF receptor VEGFR1 (Flt-1) and VEGFR2 (KDR) extracellular domains fused to human IgG1 Fc. Methods: In this phase I trial, successive cohorts of pts with relapsed or refractory solid tumors received a single sc dose of VEGF Trap, followed 4 weeks later by 6 weekly doses. Pts without disease progression subsequently entered a long-term extension study. Study endpoints included safety, pharmacokinetics, and immunogenicity. Anti-tumor activity was assessed by CT scan. Results: A total of 30 pts have been treated across 6 dose levels to date: 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg/kg. Drug-related grade 3 AEs have included hypertension (HTN; n=4), proteinuria (n=1), and afebrile neutropenia (...