43,990 research outputs found
Clinical procedure for colon carcinoma tissue sampling directly affects the cancer marker-capacity of VEGF family members
Background: mRNA levels of members of the Vascular Endothelial Growth Factor family (VEGF-A, -B, -C, -D, Placental Growth Factor/PlGF) have been investigated as tissue-based markers of colon cancer. These studies, which used specimens obtained by surgical resection or colonoscopic biopsy, yielded contradictory results. We studied the effect of the sampling method on the marker accuracy of VEGF family members.
Methods: Comparative RT-qPCR analysis was performed on healthy colon and colon carcinoma samples obtained by biopsy (n = 38) or resection (n = 39) to measure mRNA expression levels of individual VEGF family members. mRNA levels of genes encoding the eicosanoid enzymes cyclooxygenase 2 (COX2) and 5-lipoxygenase (5-LOX) and of genes encoding the hypoxia markers glucose transporter 1 (GLUT-1) and carbonic anhydrase IX (CAIX) were included as markers for cellular stress and hypoxia.
Results: Expression levels of COX2, 5-LOX, GLUT-1 and CAIX revealed the occurrence in healthy colon resection samples of hypoxic cellular stress and a concurrent increment of basal expression levels of VEGF family members. This increment abolished differential expression of VEGF-B and VEGF-C in matched carcinoma resection samples and created a surgery-induced underexpression of VEGF-D. VEGF-A and PlGF showed strong overexpression in carcinoma samples regardless of the sampling method.
Conclusions: Sampling-induced hypoxia in resection samples but not in biopsy samples affects the marker-reliability of VEGF family members. Therefore, biopsy samples provide a more accurate report on VEGF family mRNA levels. Furthermore, this limited expression analysis proposes VEGF-A and PlGF as reliable, sampling procedure insensitive mRNA-markers for molecular diagnosis of colon cancer
Clinical significance of VEGF-A, -C and -D expression in esophageal malignancies
Vascular endothelial growth factors ( VEGF)- A, - C and - D are members of the proangiogenic VEGF family of glycoproteins. VEGF-A is known to be the most important angiogenic factor under physiological and pathological conditions, while VEGF-C and VEGF-D are implicated in the development and sprouting of lymphatic vessels, so called lymphangiogenesis. Local tumor progression, lymph node metastases and hematogenous tumor spread are important prognostic factors for esophageal carcinoma ( EC), one of the most lethal malignancies throughout the world. We found solid evidence in the literature that VEGF expression contributes to tumor angiogenesis, tumor progression and lymph node metastasis in esophageal squamous cell carcinoma ( SCC), and many authors could show a prognostic value for VEGF-assessment. In adenocarcinoma (AC) of the esophagus angiogenic properties are acquired in early stages, particularly in precancerous lesions like Barrett's dysplasia. However, VEGF expression fails to give prognostic information in AC of the esophagus. VEGF-C and VEGF-D were detected in SCC and dysplastic lesions, but not in normal mucosa of the esophagus. VEGF-C expression might be associated with lymphatic tumor invasion, lymph node metastases and advanced disease in esophageal SCC and AC. Therapeutic interference with VEGF signaling may prove to be a promising way of anti-angiogenic co-treatment in esophageal carcinoma. However, concrete clinical data are still pending
The lymphangiogenic growth factors VEGF-C and VEGF-D : Part 1: Basic principles and embryonic development
This journal is listed by Scopus and EMBASE/Excerpta Medica, but not by Web of Science...VEGF-C and VEGF-D are the two central signaling molecules that stimulate the development and growth of the lymphatic system. Both belong to the vascular endothelial growth factor (VEGF) protein family, which plays important roles in the growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). In mammals, the VEGF family comprises five members: VEGF-A, PlGF, VEGF-B, VEGF-C and VEGF-D. The family was named after VEGF-A, the first member to be discovered. VEGF-C and VEGF-D form a subgroup within this family in terms of function and structure. Their distinctive biosynthesis differentiates them from the other VEGFs: they are produced as inactive precursors and need to be activated by proteolytic removal of their long N- and C-terminal propeptides. Unlike the other VEGFs, VEGF-C and VEGF-D are direct stimulators of lymphatic vessel growth. They exert their lymphangiogenic function via VEGF receptor 3, which is expressed in the adult organism almost exclusively on lymphatic endothelial cells. In this review, we provide an overview of the VEGF protein family and their receptors. We focus on the lymphangiogenic VEGF-C and VEGF-D, discussing their biosynthesis and their role in embryonic lymphangiogenesis.Peer reviewe
Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells
Abstract
Background
Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells.
Methods
To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system.
Results
Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines.
Conclusions
Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.http://deepblue.lib.umich.edu/bitstream/2027.42/112625/1/12885_2012_Article_3866.pd
VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy- Terminal NRP1 Fragments That Regulate Angiogenic Signaling
OBJECTIVE:
NRP1(neuropilin-1) acts as a coreceptor for VEGF (vascular endothelial growth factor) with an essential role in angiogenesis. Recent findings suggest that posttranslational proteolytic cleavage of VEGF receptors may be an important mechanism for regulating angiogenesis, but the role of NRP1 proteolysis and the NRP1 species generated by cleavage in endothelial cells is not known. To characterize NRP1 proteolytic cleavage in endothelial cells, determine the mechanism, and investigate the role of NRP1 cleavage in regulation of endothelial cell function.
APPROACH AND RESULTS:
NRP1 species comprising the carboxy (C)-terminal and transmembrane NRP1 domains but lacking the ligand-binding A and B regions are constitutively expressed in endothelial cells. Generation of these C-terminal domain NRP1 proteins is upregulated by phorbol ester and Ca2+ ionophore, and reduced by pharmacological inhibition of metalloproteinases, by small interfering RNA-mediated knockdown of 2 members of ADAM (a disintegrin and metalloproteinase) family, ADAMs 9 and 10, and by a specific ADAM10 inhibitor. Furthermore, VEGF upregulates expression of these NRP1 species in an ADAM9/10-dependent manner. Transduction of endothelial cells with adenoviral constructs expressing NRP1 C-terminal domain fragments inhibited VEGF-induced phosphorylation of VEGFR2 (VEGF receptor tyrosine kinase)/KDR and decreased VEGF-stimulated endothelial cell motility and angiogenesis in coculture and aortic ring sprouting assays.
CONCLUSIONS:
These findings identify novel NRP1 species in endothelial cells and demonstrate that regulation of NRP1 proteolysis via ADAMs 9 and 10 is a new regulatory pathway able to modulate VEGF angiogenic signaling
VEGF family members : modulators of tumor angiogenesis and lymphangiogenesis
Members of the vascular endothelial growth factor (VEGF) family and their receptors
(VEGFR) play an essential role in the development and maintenance of the blood and
lymphatic vasculature. To date, five VEGFs have been identified in the mammalian genome,
VEGF-A, -B, -C, -D, and placental growth factor (PlGF), which display distinct binding
affinities for VEGFR-1, -2, and -3. In addition to their central function in physiological
angiogenesis and lymphangiogenesis, VEGFs and VEGFRs are upregulated during
carcinogenesis and are involved in the remodeling of the tumoral blood and lymphatic
vasculature. By activating VEGFR-1 and –2, which are both expressed on blood endothelial
cells, VEGF-A promotes the formation of new tumoral blood vessels and thereby accelerates
tumor growth. In contrast, upregulation of VEGF-C, a ligand for lymphatic endothelial
VEGFR-3 as well as for VEGFR-2, induces the formation of tumor-associated lymphatic
vessels and thus promotes the passive metastatic dissemination of tumor cells to regional
lymph nodes. Much less is known about the functional consequences of tumor-expressed
VEGF-B and PlGF, two selective ligands for VEGFR-1, as well as VEGF-D, the second
VEGFR-3- and -2-binding lymphangiogenic VEGF family member. Also, the biological
effects of selective VEGFR-1, -2 or -3 signaling on tumor angiogenesis and tumor growth as
well as tumor lymphangiogenesis and metastasis are incompletely studied. Only recently, the
identification of VEGF-E, a selective ligand for VEGFR-2, as well as the generation of
VEGF-C156S, a specific ligand for VEGFR-3, has enabled the study of the distinct roles of
these receptors.
To investigate the function of lymphangiogenic VEGF-D under physiological
conditions, I analyzed transgenic mice, in which expression of VEGF-D is specifically
targeted to β-cells of pancreatic islets of Langerhans (Rip1VEGF-D mice). In these mice,
expression of VEGF-D induces the formation of large lymphatic lacunae surrounding most
islets. A few of these lymphatic vessels may be dysfunctional, which causes intra-lymphatic
accumulations of immune cells. Moreover, lymphatic lacunae often contain erythrocytes,
which may result from blood-lymphatic vessel shunts found in the vicinity of some islets.
However, the fact that erythrocytes are drained to regional lymph nodes demonstrates the
draining capacity of the de novo formed lymphatic vessels. To address the impact of VEGF-D
on tumorigenesis and metastasis, I crossed Rip1VEGF-D with Rip1Tag2 mice, a wellcharacterized
transgenic model of poorly metastatic multistage β-cell carcinogenesis.
Tumoral expression of VEGF-D in Rip1Tag2 mice promotes the growth of peri-tumoral
lymphatic vessels that frequently contain leucocyte clusters and hemorrhages. Concomitantly,
these double-transgenic mice exhibit a high incidence of regional lymph node and distant
lung metastases. Since expression of VEGF-D does not significantly affect the invasiveness
of tumors and all metastases are well differentiated, these data indicate that VEGF-D
promotes lymphogenous metastasis by upregulating tumor-associated lymphangiogenesis.
Interestingly, the presence of VEGF-D significantly represses tumor angiogenesis and tumor
growth, yet the mechanisms of this inhibition are thus far uncharacterized. Notably, syngenic
and allogenic subcutaneous transplantation of VEGF-D-producing Rip1Tag2 tumor cell lines
results in the formation of tumors exhibiting a dense intra-tumoral lymphatic network but
lacking peri-tumoral lymphatic vessels. In these transplanted tumors, no immune cell clusters
or hemorrhages are formed in tumor-associated lymphatic vessels and tumor angiogenesis is
unaffected by the expression of VEGF-D. These results demonstrate that the tumor
microenvironment critically modulates VEGF-D-elicited effects. It has been recently shown
that transgenic expression of VEGF-C during Rip1Tag2 tumorigenesis promotes metastasis to
regional lymph nodes but not to the lungs by inducing peri-tumoral lymphangiogenesis.
Tumor-associated lymphatic vessels of these mice neither contain immune cell accumulations
nor hemorrhages, and tumor angiogenesis and tumor growth are not affected by the
production of VEGF-C. Thus, by employing the Rip1Tag2 tumor model, I was able to
identify not only similarities but also significant differences between VEGF-D and –C
function.
Since VEGF-C and –D can bind both VEGFR-3 and –2, it is not fully established
whether selective activation of VEGFR-3 is sufficient to induce tumoral lymphangiogenesis
and to promote lymphogenous metastasis. Therefore, I established transgenic mice expressing
VEGF-C156S in the endocrine pancreas and crossed these mice with Rip1Tag2 animals. The
analysis of single and double transgenic mice revealed that VEGF-C156S phenocopies
VEGF-C in all investigated aspects. These results indicate that VEGFR-3 may be the
predominant receptor mediating VEGF-C-elicited effects in Rip1Tag2 mice and that selective
activation of VEGFR-3 is sufficient to promote tumor-associated lymphangiogenesis and
metastasis. Hence, VEGFR-3 might represent a valuable target for future anti-metastatic
strategies.
To further understand the specific roles of VEGFR-1 and –2 signaling in physiological
angiogenesis as well as in tumorigenesis, I established transgenic mouselines, which express
the VEGFR-1-specific ligands VEGF-B167 and PlGF-1 as well as the selective VEGFR-2
ligand VEGF-ED1701 in β-cells of pancreatic islets (Rip1VEGF-B167, Rip1PlGF-1, and
Rip1VEGF-ED1701 mice). These single transgenic mice were analyzed with regard to islet
blood vessel morphology and density. In a second set of experiments, I crossed singletransgenic
animals with Rip1Tag2 mice. These double-transgenic mice expressing either
VEGF-B167, PlGF-1 or VEGF-ED1701 in tumor cells, were analyzed for changes in tumor
angiogenesis, tumor growth, and tumor progression. The preliminary data provide evidence
that β-cell-specific upregulation of VEGF-B167 does not critically affect physiological
angiogenesis of single-transgenic mice but results in a significant increase in the tumor
microvessel density of double-transgenic animals. However, tumor growth and tumor
progression are not promoted by the stimulation of tumor angiogenesis. In contrast,
overexpression of PlGF-1 in single-transgenic mice leads to a prominent dilation of blood
capillaries, which may at least in part be caused by a significant reduction of stabilizing blood
vessel-associated pericytes. Furthermore, tumoral expression of PlGF-1 significantly inhibits
tumor angiogenesis and tumor growth, suggesting that this growth factor might be a natural
inhibitor of pathological angiogenesis. Hence, although binding to the same receptor, VEGFB167
and PlGF-1 elicit opposing effects on the tumor blood vasculature. These results suggest
that the two growth factors induce distinct signaling pathways via VEGFR-1, which might be
considered when designing inhibitors of angiogenesis involving VEGFR-1. Importantly, the
phenotype of VEGF-B167- and PlGF-1- expressing Rip1Tag2 mice is different from the
recently described VEGF-A165 transgenic Rip1Tag2 mice, which exhibited accelerated tumor
growth and early death. The analysis of VEGF-ED1701-expressing mice and effects induced by
selective activation of VEGFR-2 signaling is currently underway
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Molecular regulation of vascular endothelial growth factor expression in the retinal pigment epithelium
Purpose: Vascular endothelial growth factor (VEGF) plays an important role in homeostasis and diseases of the retinal pigment epithelium (RPE), choriocapillaris, and, most notably, age-related macular degeneration (AMD). Although much is known about VEGF regulation in pathologies, little is known about the control of VEGF expression under normal conditions. VEGF expression has been previously shown to be regulated in coordination with cell differentiation in the muscle and kidney. We therefore tested the hypothesis that VEGF in the adult RPE would similarly be regulated in conjunction with differentiation. Methods: A human retinal pigment epithelium cell line (ARPE-19), a line of immortalized human RPE cells, was used for all experiments. RPE cells were polarized in culture for 4 weeks on laminin-coated Transwells. Levels of VEGF mRNA and protein were determined with real-time PCR and enzyme-linked immunosorbent assay, respectively. VEGF-luciferase reporter constructs were used to identify regions of the VEGF promoter that control VEGF expression in the RPE. Microphthalmia-associated transcription factor (MITF)-Tfe transcription factors were blocked using either a pan MITF-Tfe dominant negative or specific small interfering RNA (siRNA). Results: VEGF mRNA and protein secretion increased over time in the RPE cells cultured on Transwells, with protein secretion occurring in a polarized fashion primarily toward the basolateral side. Overexpression of a dominant negative that targets the MITF-Tfe family resulted in a 50% reduction in VEGF expression. The role of the MITF-Tfe family in VEGF regulation in the RPE was corroborated in studies with the VEGF-luciferase reporter constructs, where deletion of the distal VEGF promoter region containing putative binding sites for the MITF-Tfe family resulted in a 50% reduction in VEGF promoter activity. siRNA knockdown of the MITF-Tfe family individually, and in combination, revealed that downregulation of Tfe3 resulted in reduced VEGF expression. Conclusions: Our results indicate that Tfe3, in conjunction with other MITF-Tfe members, regulates VEGF expression in the RPE and are consistent with the hypothesis that VEGF expression in RPE cells is regulated as part of their differentiation
Mechanism of Selective VEGF-A Binding by Neuropilin-1 Reveals a Basis for Specific Ligand Inhibition
Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A164/5 isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3
Reprogramming energy metabolism and inducing angiogenesis : co-expression of monocarboxylate transporters with VEGF family members in cervical adenocarcinomas
Background: Deregulation of cellular energetic metabolism was recently pointed out as a hallmark of cancer cells. This deregulation involves a metabolic reprogramming that leads to a high production of lactate. Lactate efflux, besides contributing for the glycolytic flux, also acts in the extracellular matrix, contributing for cancer malignancy, by, among other effects, induction of angiogenesis. However, studies on the interplay between cancer metabolism and angiogenesis are scarce. Therefore, the aim of the present study was to evaluate the metabolic and vascular molecular profiles of cervical adenocarcinomas, their co-expression, and their relation to the clinical and pathological behavior.
Methods: The immunohistochemical expression of metabolism-related proteins (MCT1, MCT4, CD147, GLUT1 and CAIX) as well as VEGF family members (VEGF-A, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2 and VEGFR-3) was assessed in a series of 232 cervical adenocarcinomas. The co-expression among proteins was assessed and the expression profiles were associated with patients’ clinicopathological parameters.
Results: Among the metabolism-related proteins, MCT4 and CAIX were the most frequently expressed in cervical adenocarcinomas while CD147 was the less frequently expressed protein. Overall, VEGF family members showed a strong and extended expression with VEGF-C and VEGFR-2 as the most frequently expressed and VEGFR-1 as the less expressed member. Co-expression of MCT isoforms with VEGF family members was demonstrated. Finally, MCT4 was associated with parametrial invasion and HPV18 infection, CD147 and GLUT1 with distant metastasis, CAIX with tumor size and HPV18 infection, and VEGFR-1 with local and lymphnode metastasis.
Conclusions: The results herein presented provide additional evidence for a crosstalk between deregulating cellular energetics and inducing angiogenesis. Also, the metabolic remodeling and angiogenic switch are relevant to cancer progression and aggressiveness in adenocarcinomas.CP received a post-doctoral fellowship (SFRH/BPD/69479/2010) and FM-S received a doctoral fellowship (SFRH/BD/87139/2012) from FCT (Portuguese Foundation for Science and Technology). This work was supported by the FCT grant ref. PTDC/SAU-FCF/104347/2008, under the scope of "Programa Operacional Tematico Factores de Competitividade" (COMPETE) of "Quadro Comunitario de Apoio III" and co-financed by Fundo Comunitario Europeu FEDER, and also by FAPESP 2008/03232-1
Vascular Endothelial Growth Factor Receptor-3 Directly Interacts with Phosphatidylinositol 3-Kinase to Regulate Lymphangiogenesis
Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCc1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis
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