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Purpose: Investigate the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) in vasculogenic mimicry (VM) formation in ocular melanoma, as well as whether or not VEGFR1-targeted contrast-enhanced ultrasound (CEUS) can evaluate and quantify VM perfusion and function in the ocular melanoma model., Methods: The expression of VEGFR1 was examined using immunofluorescence, western blot, and quantitative polymerase chain reaction. VM networks were analyzed with tube formation and periodic acid Schiff staining. Targeted microbubbles (MBs) were constructed and used for targeted CEUS imaging in vivo. Comparisons were made in perfusion parameters of tumors between targeted and non-targeted CEUS imaging., Results: VEGFR1 was highly expressed, and knockdown of VEGFR1 significantly decreased VM protein expression and disrupted VM formation in MUM-2B melanoma. VEGFR1-targeted MBs specifically bind to MUM-2B cell surfaces. CEUS with VEGFR1-targeted MBs showed significant imaging enhancement throughout the entire perfusion phase compared with CEUS with IgG MBs. VEGFR1-targeted imaging was able to detect a decrease in maximum intensity and mean transit time in VEGFR1 knockdown melanoma compared with control melanoma. The pathological VM patterns were consistent with VEGFR1-targeted CEUS findings., Conclusions: VEGFR1 was responsible for VM network formation and was required for efficient choroidal melanoma tumor growth. This study shows that VEGFR1-targeted CEUS can track VM levels in animal models of ocular melanoma at morphological levels in vivo. This experiment is noninvasive and reproducible and indicates the possibility of real-time in vivo imaging technology for VM evaluation., Translational Relevance: Based on our study results, VEGFR1 could prove to be a promising treatment that targets VM formation in choroidal melanoma. Our findings also suggest the potential use of VEGFR1-targeted CEUS for quantitative monitoring of VM processes at the molecular level in the future., Competing Interests: Disclosure: H. Liu, None; M. Gao, None; J. Gu, None; X. Wan, None; H. Wang, None; Q. Gu, None; Y. Zhou, None; X. Sun, None, (Copyright 2020 The Authors.)

After a peripheral nerve lesion, distal ends of injured axons disintegrate into small fragments that are subsequently cleared by Schwann cells and later by macrophages. Axonal debris clearing is an early step of the repair process that facilitates regeneration. We show here that Schwann cells promote distal cut axon disintegration for timely clearing. By combining cell-based and in vivo models of nerve lesion with mouse genetics, we show that this mechanism is induced by distal cut axons, which signal to Schwann cells through PlGF mediating the activation and upregulation of VEGFR1 in Schwann cells. In turn, VEGFR1 activates Pak1, leading to the formation of constricting actomyosin spheres along unfragmented distal cut axons to mediate their disintegration. Interestingly, oligodendrocytes can acquire a similar behavior as Schwann cells by enforced expression of VEGFR1. These results thus identify controllable molecular cues of a neuron-glia crosstalk essential for timely clearing of damaged axons., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: During degeneration of the intervertebral disc ingrowth of blood vessels and nerves into the disc are associated with back pain. Vascular endothelial growth factors promote vasculogenesis by binding to the membrane vascular endothelial growth factor receptor 1, while shorter soluble forms of this receptor can inhibit vascularization. We hypothesized that membrane and soluble receptor forms might change between stages of intervertebral disc degeneration. Results: Expression of soluble and membrane forms of vascular endothelial growth factor receptor 1 in human degenerated intervertebral discs and healthy bovine caudal discs was assessed by qRT-PCR and immunoblot. Comparative microarray meta-analysis across disc degeneration grades showed that membrane and soluble forms of this receptor, together with other components of classic vascularization pathways, are constitutively expressed across human disc degeneration stages. Contrary to our hypothesis, we observed that expression of the classic vascularization pathway is stable across degeneration stages and we assume that soluble vascular endothelial growth factor receptor 1 does not contribute to prevent disc degeneration. However, we observed increased expression levels of genes involved in alternative vascularization signalling pathways in severely degenerated discs, suggesting that abnormal vascularization is part of the pathological progression of disc degeneration. [ABSTRACT FROM AUTHOR]

Background: Vascular endothelial growth factor (VEGF) A and B are endothelial cell mitogens whose ligation to VEGFR1/VEGFR2 drives tumor angiogenesis and metastasis, and epithelial-mesenchymal transition (EMT). Blockade of these signaling axes could be obtained by disturbing the interactions between VEGFA and/or VEGFB with VEGFR1 and/or VEGFR2., Methods: A 14-mer peptide (VGB) that recognizes both VEGFR1 and VEGFR2 were investigated for its inhibitory effects on the VEGF-induced proliferation and migration using MTT and scratch assay, respectively. Downstream signaling pathways were also assessed by quantitative estimation of gene and protein expression using real-time PCR and immunohistochemistry (IHC)., Results: We investigated the inhibitory effects of VGB on downstream mediators of metastasis, including epithelial-cadherin (E-cadherin), matrix metalloprotease-9 (MMP-9), cancer myelocytomatosis (c-Myc), and nuclear factor-κβ (NF-κβ), and migration, comprising focal adhesion kinase (FAK) and its substrate Paxilin. VGB inhibited the VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs), 4T1 and U87 cells in a time- and dose-dependent manner and migration of HUVECs. Based on IHC analyses, treatment of 4T1 mammary carcinoma tumor with VGB led to the suppression of p-AKT, p-ERK 1/2 , MMP-9, NF-κβ, and activation of E-cadherin compared with PBS-treated controls. Moreover, quantitative real-time PCR analyses of VGB-treated tumors revealed the reduced expression level of FAK, Paxilin, NF-κβ, MMP-9, c-Myc, and increased expression level of E-cadherin compared to PBS-treated controls., Conclusions: Our results demonstrated that simultaneous blockade of VEGFR1/VEGFR2 is an effective strategy to fight solid tumors by targeting a wider range of mediators involved in tumor angiogenesis, growth, and metastasis.

Angiogenesis is required for tumor growth. Dihydroartemisinin (DHA), a the effective anti-malarial derivative of artemisinin, demonstrated potent anti-angiogenic activities that closely related to the regulation of vascular endothelial growth factor (VEGF) signaling cascade. VEGF receptor 1 (VEGFR1), a receptor in endothelial cells (ECs), coordinately regulate angiogenic activity triggered by ligand-receptor binding. Here we aimed to explore the effects of DHA on VEGFR1 expression in ECs. We found that DHA significantly increases VEGFR1 expression in human umbilical vein endothelial cells (HUVECs). In addition, DHA significantly upregulates the level of V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1 (ETS-1), a transcriptional factor which binds to the human VEGFR1 promoter. ChIP assay showed that DHA increases ETS-1 binding to the -52 ETS motif on the VEGFR1 promoter. Knockdown of ETS-1 by RNA interference abolished DHA-induced increase of VEGFR1 expression. Taken together, we demonstrated that DHA elevates VEGFR1 expression via up-regulation of ETS-1 transcription in HUVECs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Angiogenesis plays a critical role in tumor aggressiveness, and a lot of anti-angiogenic agents have been used in clinical therapy. The therapeutic efficacy of peptides are generally restricted by the short in vivo life-time, thus, we were interested in developing a novel albumin-based and maleimidopropionic acid-conjugated peptide to prolong the half-life and improve the anti-tumor effect. Methods: We developed a peptide F56 with a maleimidopropionic acid (MPA) at the C-terminal (denoted as F56-CM), which allows immediate and irreversible conjugation with serum albumin. Biological property and anti-tumor activity of F56-CM were evaluated in vitro and in vivo . Results: We showed that F56-CM reduced migration and tube formation of endothelial cells in vitro and inhibited the generation of subintestinal vessels (SIV) in zebrafish embryos in vivo . F56-CM inhibited vascular endothelial growth factor (VEGF) induced phosphorylation of VEGFR1 and activation of the PI3K-AKT axis. Furthermore, F56-CM rapidly conjugated with albumin upon intravenous injection and extended the biological half-life of F56 from 0.4249 h to 6.967 h in rats. Compared with F56, F56-CM exhibited stronger anti-tumor activity on both BGC-823 gastric cancer and HT-29 colon cancer xenografts in nude mice, and the statistical difference was remarkable. More significantly, the efficacy of F56-CM inhibiting lung metastasis of BGC-823 cells was also better than that of F56. The inhibition rates were 62.1% and 78.9% for F56 and F56-CM respectively when administrated every day, and 43.8% and 63.1% when administrated every four days at equal dose. Conclusions: Taken together, our results demonstrated that F56-CM has considerable potential for cancer therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

While a number of post-translational modifications (PTM), such as phosphorylation and ubiquitination, have been extensively studied, lysine methylation is emerging as an important PTM with implications in a growing number of diverse cellular processes. To date, there are approximately 5000 identified methylation sites on non-histone proteins, and as the methyllysine proteome expands it becomes important to identify the lysine methyltransferase enzymes responsible for each methylation event. The use of peptide SPOT methylation assay has proven to be a useful in the identification and validation of novel substrates for lysine methyltransferase enzymes as it uses a weak beta emitter coupled with fluorography to detect methylation events. The method described in this paper provides improvements to the typical protocol for this assay, as a highly sensitive tritium assay can be developed with less radioactivity than previously described. This protocol provides an inexpensive alternative to weak beta signal enhancer sprays and washes for use in lysine methylation peptide SPOT arrays, and a simple open-source method for array quantification.

Although it is well established that tumor-associated macrophages take part in each step of cancer progression, less is known about the distinct role of the so-called metastasis-associated macrophages (MAMs) at the metastatic site. Previous studies reported that Caveolin-1 (Cav1) has both tumor-promoting and tumor-suppressive functions. However, the role of Cav1 in bone-marrow-derived cells is unknown. Here, we describe Cav1 as an anti-metastatic regulator in mouse models of lung and breast cancer pulmonary metastasis. Among all the recruited inflammatory cell populations, we show that MAMs uniquely express abundant levels of Cav1. Using clodronate depletion of macrophages, we demonstrate that macrophage Cav1 signaling is critical for metastasis and not for primary tumor growth. In particular, Cav1 inhibition does not affect MAM recruitment to the metastatic site but, in turn, favors angiogenesis. We describe a mechanism by which Cav1 in MAMs specifically restrains vascular endothelial growth factor A/vascular endothelial growth factor receptor 1 (VEGF-A/VEGFR1) signaling and its downstream effectors, matrix metallopeptidase 9 (MMP9) and colony-stimulating factor 1 (CSF1)., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Natural antibodies have been found to have anti-tumorigenic function. This study was designed to investigate whether natural IgG antibodies against vascular endothelial growth factor receptor 1 (VEGFR1) could suppress the growth of hepatocellular carcinoma (HCC) cells. Three HCC cell lines and A549 lung cancer cells were used for this study. They were grown, respectively, with human plasma positive or negative for anti-VEGFR1 IgG. Cell viability, apoptosis and VEGFR1 gene expression were examined. Three patients with HCC were recruited for a case study. The results showed that plasma anti-VEGFR1 IgG significantly inhibited the proliferation of all three HCC cell lines but not A549 cell line; the proportions of apoptotic cells were significantly higher in HCC cells treated with anti-VEGFR1 IgG positive plasma than those treated with IgG negative plasma. The expression of the VEGFR1 gene was significantly higher in HCC cells than A549 cells. Of three HCC patients who received transfusion of anti-VEGFR1 IgG positive plasma, two cases with stage B showed a good response to the treatment but one with distant metastasis did not. Human plasma IgG against VEGFR1 may be a promising agent for anti-HCC therapy.

VEGFR1 rs9582036 and rs9554320 were previously reported the association with sunitinib progression-free survival (PFS) and overall survival (OS) in patients with metastatic renal cell carcinoma (mRCC). Hereafter, the association of both single nucleotide polymorphisms (SNPs) with PFS/OS was confirmed in two independent mRCC cohorts. The aim of the current study was to validate the associations of both SNPs with sunitinib outcome in three independent well-characterized cohorts (SUTOX, CCF and SOGUG) including 286 sunitinib-treated mRCC patients, as well as to perform a meta-analysis of current and published data combined. We found that rs9582036 and rs9554320 showed a significant association with sunitinib PFS in the CCF cohort (HR: 0.254, 95%CI: 0.092-0.703; P=0.008 and HR: 0.430, 95%CI: 0.200-0.927; P=0.031, respectively). Patients with the variant genotype of rs9582036 and rs9554320 had a shorter median PFS. No significant association of both SNPs with sunitinib PFS or OS was detected in either the SUTOX or SOGUG cohort. After the combination of all available data into a meta-analysis, the association of both SNPs with sunitinib PFS or OS did not achieve the threshold for statistical significance. Our findings suggest that, although VEGFR1 rs9582036 and rs9554320 are involved in sunitinib therapy outcome, its clinical use as biomarkers for prediction of sunitinib outcome in mRCC patients is limited, due to inconsistent findings when analyzing all existing studies together.

The progress in treatment of kidney cancer is related to application of anti-angiogenic medications. Therefore, investigation and searching for new molecular markers characterizing its angiogenic activity is actual still. The purpose of study is to compare VEGF, VEGFR1 and VEGFR2 in blood serum of healthy people, patients with cancer and benign tumors of kidney and to analyze their relationship with main clinical morphological characteristics of neoplasms. The study sampling consisted of 94 patients with cancer and 10 patients with benign tumors of kidney. The control group included 80 individuals. The concentration of analyzed proteins was determined using QuantikineТ (R&D Systems, USA), a reagents' kit for direct immune enzyme analysis. The content of VEGF, VEGFR1 and VEGFR2 in blood serum of patients with cancer of kidney was reliably higher than in control group. The level of VEGF also was increased in patients with benign tumors. Under threshold level of VEGF of 365 pg/ml the diagnostic sensitivity of detection of primary cancer of kidney amounted to 67% and specificity - 70%. The stage of disease and T and N indices were correlated only with VEGFR1 level. The relationship between level of markers and histological structure and degree of differentiation of cancer of kidney was not established. Therefore, VEGF and its receptors have a limited diagnostic value under cancer of kidney but they can be applied for monitoring and prognosis of efficiency of anti-angiogenic therapy., Competing Interests: The authors declare no conflict of interest.

Background: the vascular endothelial growth factor (VEGF) pathway plays a prominent role in the growth and progression of human cancer, including non-small cell lung carcinoma (NSCLC). The key mediators of VEGF signaling are a family of related receptor tyrosine kinases that include VEGFR1, VEGFR2, and VEGFR3. The relative expression levels, activity, and cross-talk among these receptors may contribute to response of NSCLC to anti-angiogenic therapies, and a better systematic, translatable approach to categorizing tumors is needed., Materials and Methods: We comparatively evaluated immunohistochemical expression of the three VEGFRs in archival primary NSCLC tissues (n=96)., Results: VEGFR1 and VEGFR2 were localized both in vessels and tumor cells, while VEGFR3 was only localized in tumor vessels. A set of eight VEGFR staining subclasses were identified: Triple VEGFR positive (n=11, 11.5%), VEGFR1 predominant (n=22, 22.9%), VEGFR2 predominant (n=9, 9.4%), VEGFR3 predominant (n=3, 3.1%), VEGFR1/2 predominant (13, 13.5%), VEGFR1/3 predominant (2, 2.1%), VEGFR2/3 predominant (n=8, 8.3%), and triple VEGFR negative (n=28, 29.2%). An objective categorization based on K-means clustering revealed four clusters, three of which showed high VEGFR2 compared to VEGFR3 (30.7% of cases), cases high in both VEGFR2 and VEGFR3 (18.2%), and cases that were negative/low for both VEGFR2 and VEGFR3 (45.4%). A positive association between VEGFR2 and VEGFR3 was found, however no associations were observed between VEGFR1 and VEGFR2, nor VEGFR1 and VEGFR3., Conclusion: The proposed subclasses of NSCLC are an approach for complementing lines of investigation of anti-angiogenic therapies beginning with systematic characterization of the disease., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: The vascular endothelial growth factor (VEGF) pathway plays an important role in growth and progression of human cancer, including colorectal carcinomas (CRC). The key mediators of VEGF signaling are VEGFR1, VEGFR2, and VEGFR3, part of a family of related receptor tyrosine kinases. The relative expression, activity, or interplay among these receptors may determine the response of CRC patients to anti-angiogenic therapies., Materials and Methods: We developed technically sound immunohistochemical (IHC) assays to quantify VEGFR1, 2 and 3, and using a well-annotated CRC tissue microarray (TMA), we carried out comprehensive comparative evaluation of the three VEGFRs in archival primary CRC tissues (n=84). For each TMA core, tumor cell VEGFR1 expression was reported as H-score (range=0-300); vascular VEGFR2/VEGFR3 expression was manually scored as the number of receptor-positive tumor stromal vessels. Each case was defined as VEGFR1/ VEGFR2/VEGFR3-negative, low, medium or high., Results: Based on the differential expression of the three VEGFRs, eight VEGFR staining profiles were observed: Triple VEGFR positive (n=12, 14%), VEGFR1 predominant (n=17, 20%), VEGFR2 predominant (n=7, 8%), VEGFR3 predominant (n=1, 1%), VEGFR1/2 predominant (n=39, 46%), VEGFR1/3 predominant (n=2, 2%), VEGFR2/3 predominant (n=3, 4%), and triple-VEGFR-negative (n=3, 4%)., Conclusion: Herein we demonstrated heterogeneity of expression of VEGFRs in human CRC stromal vessels and tumor cells. The observed VEGFR expression-based subsets of human CRCs may reflect differences in biology of pathologic angiogenesis in primary CRC tissues. Furthermore, the heterogeneity of expression of VEGFRs unraveled in this analysis merits independent validation in larger cohorts of primary and metastatic human CRC tissues and in pertinent experimental models treated with various anti-angiogenic therapies., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Angiogenesis is central to the growth of cancers and VEGFR-1/Flt-1 plays an important role during the neovascularization under pathological conditions. We previously founded a VEGFR1 antagonistic peptide, F56, by screening the phage peptide library. We showed that DHFR-F56 chimeric protein displayed anti-tumor activity and inhibited angiogenesis, however the anti-tumor activity of monomeric F56 and the mechanism remain unclear. In this study, we found that the F56 didn't affect VEGF-A induced endothelial cell proliferation, but reduced migration and tube formation of endothelial cells. F56 also inhibited the sprout of rat aortic endothelial cells, the angiogenesis of chicken embryo chorioallantoic membrane as well as the generation of subintestinal vein vessels (SIV) in zebrafish embryos. We found that F56 inhibited VEGF-induced phosphorylation of VEGFR1, as well as the phosphorylation of the downstream of PI3K-AKT axis. However, F56 had no effect on the phosphorylation of VEGFR2. Correlating with these effects, F56 inhibited xenograft growth of HT-29 and MGC-823 cells in BALB/c nude mice, and significantly suppressed the lung metastasis of B16 cells in C57BL/6 mice. Our study demonstrated that monomeric peptide F56 had a significant anti-tumor activity by inhibiting angiogenesis, and laid the foundation for its clinical application.

The biological functions of VEGF-B in cancer progression remain poorly understood. Here, we report that VEGF-B promotes cancer metastasis through the remodeling of tumor microvasculature. Knockdown of VEGF-B in tumors resulted in increased perivascular cell coverage and impaired pulmonary metastasis of human melanomas. In contrast, the gain of VEGF-B function in tumors led to pseudonormalized tumor vasculatures that were highly leaky and poorly perfused. Tumors expressing high levels of VEGF-B were more metastatic, although primary tumor growth was largely impaired. Similarly, VEGF-B in a VEGF-A-null tumor resulted in attenuated primary tumor growth but substantial pulmonary metastases. VEGF-B also led to highly metastatic phenotypes in Vegfr1 tk(-/-) mice and mice treated with anti-VEGF-A. These data indicate that VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism. High expression levels of VEGF-B in two large-cohort studies of human patients with lung squamous cell carcinoma and melanoma correlated with poor survival. Taken together, our findings demonstrate that VEGF-B is a vascular remodeling factor promoting cancer metastasis and that targeting VEGF-B may be an important therapeutic approach for cancer metastasis.

The soluble vascular endothelial growth factor receptor-1 (VEGFR1) or sFLT-1 has important role in antiangiogenesis. In this study, the increase expression and production of sFLT-1 fragment by newly designed ChPL-NPs nanoparticles (chitosan-protein lipid) using Chinese hamster ovary cell line (CHO) was evaluated. The assessment and purification of sFLT-1 were carried out by western blotting and fast protein liquid chromatography (FPLC). Thereafter, the angiostatic effect of gene transfer of sFLT-1 in Human umbilical vein endothelial cell line (HUVEC) was evaluated. Our results showed a significance rate of transfection with ChPL-NPs (80-85%) in comparison to standard lipofectamine(2000) (65-70%) (P < 0.05). The anti-angiogenic action of sFLT-1 was observed by in-vitro culture of recombinant protein (sFLT-1; 50 ng/ml) with HUVEC cell lines (5 × 10(6)). The ChPL-NPs nanoparticles can consider a potential carrier system for large scale production of sFLT-1, which ultimately may be use as therapeutic agent in targeting solid tumor tissues.

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

Little is known about the molecular pathways regulating poor differentiation and invasion of head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to determine the role of MDA-9/Syntenin, a metastasis associated molecule in HNSCC tumorigenesis. Elevated MDA-9/Syntenin expression was evident in 67% (54/81) primary HNSCC tumors (p=0.001-0.002) and 69% (9/13) pre-neoplastic tissues (p=0.02-0.03). MDA-9/Syntenin overexpression was associated with the stage (p=0.001), grade (p=0.001) and lymph node metastasis (p=0.0001). Silencing of MDA-9/Syntenin in 3 poorly differentiated HNSCC cell lines induced squamous epithelial cell differentiation, disrupted angiogenesis and reduced tumor growth in vitro and in vivo. We confirmed SPRR1B and VEGFR1 as the key molecular targets of MDA-9/Syntenin on influencing HNSCC differentiation and angiogenesis respectively. MDA-9/Syntenin disrupted SPRR1B expression interacting through its PDZ1 domain and altered VEGFR1 expression in vitro and in vivo. VEGFR1 co-localized with MDA-9/Syntenin in HNSCC cell lines and primary tumor. Downregulation of growth regulatory molecules CyclinD1, CDK4, STAT3, PI3K and CTNNB1 was also evident in the MDA-9/Syntenin depleted cells, which was reversed following over-expression of MDA-9/Syntenin in immortalized oral epithelial cells. Our results suggest that early induction of MDA-9/Syntenin expression influences HNSCC progression and should be further evaluated for potential biomarker development.

Activation of vascular endothelial growth factor receptor 1 (VEGFR1/FLT1) and 2 (VEGFR2/KDR) involves receptor dimerization. Formation of VEGFR dimer has so far not been visualized in single intact cells. In the present study we describe different optical assays which can be used to observe dimerization of VEGFR1 and VEGFR2. Bimolecular fluorescence complementation (BIFC) assays confirmed homo,- and heterodimerization of transfected receptors. Fluorescence resonance energy transfer (FRET) techniques in living and fixed CHO-K1 cells allowed observation of VEGFR1 homodimer,- and VEGFR1 and VEGFR2 heterodimer formation after ligand stimulation. After inhibition of ligand binding by the VEGFA JH121 antibody VEGFR1 homodimerization was completely abolished. Under the same conditions, cells transfected by VEGFR1 and VEGFR2 maintained relevant receptor heterodimerization. These techniques to monitor VEGFR1 and VEGFR2 homo- and heterodimerization in living and fixed cells may help in the search for new angiogenesis-directed inhibitors of VEGFR dimerization.

We have previously reported that the major isoform of Flt1/VEGFR-1 expressed in MDA-MB-231 breast cancer cells was a truncated intracellular isoform transcribed from intron 21 (i21 Flt1). This isoform upregulated the active form of Src and increased breast cancer cell invasiveness. Since expression of the transmembrane and soluble Flt1 isoforms of HUVEC is activated by Notch signaling, we wondered whether the expression of the intracellular isoform i21 Flt1 was also dependent on Notch activation. We report here that the expression of i21 Flt1 in HUVEC and MDA-MB-231 cells is downregulated by the γ-secretase inhibitor DAPT. In addition, treatment of MDA-MB-231 cells with siRNA specific for Notch-1 and Notch-3 downregulates the expression of i21 Flt1. In agreement with these findings, HUVEC and MDA-MB-231 breast cancer cells, cultured on dishes coated with recombinant human Dll4 extracellular domain, express higher levels of i21 Flt1. In cancer cells, Flt1 is a target of the micro RNA family miR-200. In MDA-MB-231 breast cancer cells, the truncated intracellular isoform i21 Flt1 is also negatively regulated by miR-200c. Retinoic acid interferes i21 Flt1 expression by downregulating Notch-3 and upregulating miR-200 expression. Treatment of MDA-MB-231 breast cancer cells with both a γ-secretase inhibitor and retinoic acid suppresses the expression of i21 Flt1, providing a new mechanism to explain the effectiveness of this therapeutic approach., (© 2013 Wiley Periodicals, Inc.)

VEGF/VEGFR signal axis has been proven to be an important target for development of novel cancer therapies. One challenging aspect in small molecular VEGFR inhibitors is to achieve sustained target inhibition at tolerable doses previously seen only with the long-acting biologics. It would require high potency (low effective drug concentrations) and sufficient drug exposures at tolerated doses so that the drug concentration can be maintained above effective drug concentration for target inhibition within the dosing period. Fruquintinib (HMPL-013) is a small molecule inhibitor with strong potency and high selectivity against VEGFR family currently in Phase II clinical studies. Analysis of Phase I pharmacokinetic data revealed that at the maximum tolerated dose of once daily oral administration fruquintinib achieved complete VEGFR2 suppression (drug concentrations were maintained above that required to produce >85% inhibition of VEGFR2 phosphorylation in mouse) for 24 hours/day. In this article, the preclinical data for fruquintinib will be described, including kinase enzyme activity and selectivity, cellular VEGFR inhibition and VEGFR-driven functional activity, in vivo VEGFR phosphorylation inhibition in the lung tissue in mouse and tumor growth inhibition in a panel of tumor xenograft and patient derive xenograft models in mouse. Pharmacokinetic and target inhibition data are also presented to provide a correlation between target inhibition and tumor growth inhibition.

Elevated permeability of retinal endothelial cells (REC), as observed in diabetic retinopathy (DR), is induced by extended exposure to ≥25 ng/ml vascular endothelial growth factor A165 (VEGF165) for up to 3 d and this effect is more pronounced when equimolar amounts of basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-1) are present. Down-regulation of the tight-junction protein claudin-1 and its loss from the plasma membrane is associated with induced higher permeability, whereas other tight-junction proteins (e.g. claudin-3, claudin-5, ZO-1) show only subtle changes in our experimental setting. Using immortalized bovine REC (iBREC) as a well-established model, we investigated effects of other members of the VEGF family, i.e. VEGF121, placental growth factor (PlGF-1 and PlGF-2) and viral VEGF-E which activate different sets of VEGF receptors, on barrier function after extended treatment: iBREC were incubated with 1-100 ng/ml of the growth factors for up to 2 days before barrier function was assessed by measuring transendothelial resistance (TER). Presence of TJ-proteins was determined by western blot analyses and immunofluorescence staining. Similar experiments were performed to evaluate whether the primary actions of PlGF-1, PlGF-2 or VEGF121 are modulated by bFGF or IGF-1 when all growth factors (each at 25 ng/ml, but 10 ng/ml IGF-1) act simultaneously at equimolar concentrations. We also studied the potential normalization of the barrier disturbed with combinations of growth factors by addition of the VEGF-specific Fab fragment ranibizumab or the recombinant protein aflibercept which binds VEGF and PlGF. Whereas 1 ng/ml VEGF-E were sufficient to impair the iBREC barrier, a higher concentration of 100 ng/ml VEGF121 was needed to reduce TER and expression of claudin-1 over 2 days. By PlGF-1 or PlGF-2, the barrier was not affected even at the highest concentration tested (100 ng/ml) and these factors also did not modulate the effect of VEGF165. The weak barrier derangement caused by VEGF121 was slightly enhanced by bFGF and IGF-1. After induction of the barrier breakdown with various combinations of all growth factors included in the study, normal TER and claudin-1 expression was re-established by ranibizumab. Both VEGF inhibitors ranibizumab and aflibercept similarly reinstated lost claudin-1, even when applied at a small fraction of the clinically relevant concentrations. These results show that VEGF-A, but not PlGF impairs the barrier function of iBREC and that the longer isoform VEGF165 is more potent than VEGF121. To induce barrier dysfunction in iBREC, activation of VEGF receptor 2 - probably in concert with neuropilin-1 - seems to be sufficient because VEGF-E and VEGF165, but not PlGF-1/-2 reduced TER or claudin-1 expression., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aim: Vascular endothelial growth factor (VEGF) signaling is frequently altered in invasive tumor cells and is associated with patient outcome. In the present study, we examined VEGF, VEGFR1, and VEGFR2 expression in non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC), and evaluated the association between VEGF and its receptors with disease characteristics and bladder cancer recurrence., Materials and Methods: Tissue microarrays containing bladder cancer specimens (n=212) and adjacent normal bladder mucosa (n=131) were immunostained using antibodies against VEGF, VEGFR1, and VEGFR2. The association between the expression of these proteins and clinical parameters including stage, lymph node metastasis, and recurrence-free survival were statistically evaluated. VEGF mRNA expression data were extracted from the public Oncomine database., Results: VEGF and VEGFR1 mRNA levels were significantly higher in bladder cancer specimens than that of normal mucosa (for VEGF, p<0.001; for VEGFR1, p=0.02). Analysis of their expression at protein levels showed that levels of VEGF and VEGFR1 were significantly higher in NMIBC than in MIBC (p<0.001), while that of VEGFR2 was significantly higher in all cancer specimens compared to benign urothelial mucosa (p=0.001). Further-more, the expression of VEGFR2 was significantly higher in MIBC, as compared to NMIBC (p<0.001). Patients with higher levels of VEGF, VEGFR1, and VEGFR2 tended to have poorer recurrence-free survival than those with lower levels, but this was not statistically significant., Conclusion: Our results suggest that alterations in the expression of VEGF and VEGF receptors are associated with disease stage and recurrence.

Perinatal estrogen exposure elicits a wide range of abnormalities in the female genital tract. Since angiogenesis is essential for morphogenesis, we investigated the vascular density, integrity of vasculatures, and expression of angiogenic factors and their receptors in the uteri of mice treated with diethylstilbestrol (DES) neonatally (DES-mice); the uteri were collected from Day 4 to Day 20. DES treatment reduced the number and density of vasculatures immunostained with PECAM1 (platelet and endothelial cell adhesion molecule 1) in the stroma. Horseradish peroxidase injected into the left ventricle leaked into the endometrium and myometrium on Day 10 in the DES-mice but not in the controls. Electron microscopy confirmed the immaturity of the capillaries, which had an incomplete basal lamina and fewer pericytes. Immunohistochemical studies demonstrated that VEGFA (vascular endothelial growth factor A) expression and ANGPT1 (angiopoietin 1) expression were down-regulated in the stromal cells until Days 20 and 10, respectively. The number of vasculatures with ANGPT2 immunoreaction was reduced in the DES-mice. DES treatment suppressed the expression of VEGFR2 (VEGF receptor 2) and the co-receptor NRP1 (neuropilin 1) as well as TEI2 in the vasculatures. The results of RT-PCR and Western blotting supported the down-regulation of the expression of angiogenic factors and their receptors in DES-mice, whereas the VEGFR1 protein expression was up-regulated. These results suggested that the low concentration of angiogenic factors in the stroma was primarily responsible for the low vascular density in the stroma of the DES-mice, and that the low vascular density and immature vasculatures resulted in uterine malformations.

Prostate cancer can usually be treated at a clinically localized stage by radical prostatectomy. Unfortunately, within 10 years following surgery, 30% of patients experience local or distant relapse. Few data exist on the association of markers of angiogenesis and distant relapse after radical prostatectomy. By immunohistochemistry in tissue microarray, we compared the expression pattern of hypoxia inducible factor 1, alpha subunit (HIF1α) and vascular endothelial growth factor (VEGF) and its receptors in 45 patients with distant relapse and 68 patients without relapse after radical prostatectomy. Expressions of HIF1α and VEGF were assessed in prostate tumor cells and those of VEGFR1, VEGFR2 and neuropilin 1 in tumor and endothelial cells. The five molecules studied were expressed by all tumors, with the exception of neuropilin 1 in endothelial cells for one tumor. Strong endothelial expression of VEGFR1 appeared to be an independent predictor of distant relapse. A moderate to strong endothelial expression of neuropilin 1 was in turn an independent predictor of absence of distant relapse. No significant difference was found for HIF1α, VEGF, VEGFR1, VEGFR2 and neuropilin 1 expression in tumor cells, nor for VEGFR2 in endothelial cells, between the two groups. To our knowledge, this is the first study to evaluate the prognostic value of VEGFR1, VEGFR2 and neuropilin 1 in endothelial cells in prostate cancer after radical prostatectomy. The evaluation by immunohistochemistry of endothelial expression of neuropilin 1 and VEGFR1 could be an additional tool in the assessment of tumor aggressiveness of clinically localized prostate cancer to better identify patients at high risk of distant relapse.

Upper tract urothelial carcinoma (UTUC) associated with Balkan endemic nephropathy (BEN) is characterized by a number of aberrations in cell-cycle regulation and apoptosis. The aim of this study was to detect angiogenesis-related marker(s) specific for BEN UTUC, and to examine the influence of HIF 1α upon angiogenesis and apoptosis in UTUC. Present investigation included 110 patients with UTUC, 50 from BEN region and 60 control tumors. Altered expression of VEGFR1 was more often present in control UTUC than in BEN tumors (p<0.005). It was associated with high grade, low and high stage, solid growth, and metaplastic change of control UTUC. Microvessel density assessed by CD31 (MVD CD31) was significantly higher in UTUC with lymphovascular invasion (p<0.05), and in BEN tumors with papillary growth (p<0.05). Discriminant analysis indicated that BEN and control tumors do not differ significantly in expression of angiogenesis related markers. The most important discriminant variable that determined control UTUC was expression of VEGFR1 (p=0.002). HIF 1α in UTUC significantly correlated with the low stage, papillary growth and expression of Bcl-2, Caspase-3 index, and MVD CD34 (p<0.001; 0.0005; 0.01; 0.005; 0.01, respectively). HIF-1α may be helpful marker in evaluation of UTUC, especially when combined with angiogenesis and apoptosis.

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.