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

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

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

Brain homeostasis : VEGF receptor 1 and 2 ; two unequal brothers in mind

Vascular endothelial growth factors (VEGFs), initially thought to act specifically on the vascular system, exert trophic effects on neural cells during development and adulthood. Therefore, the VEGF system serves as a promising therapeutic target for brain pathologies, but its simultaneous action on vascular cells paves the way for harmful side effects. To circumvent these deleterious effects, many studies have aimed to clarify whether VEGFs directly affect neural cells or if the effects are mediated secondarily via other cell types, like vascular cells. A great number of reports have shown the expression and function of VEGF receptors (VEGFRs), mainly VEGFR-1 and -2, in neural cells, where VEGFR-2 has been described as the major mediator of VEGF-A signals. This review aims to summarize and compare the divergent roles of VEGFR-1 and -2 during CNS development and homeostasis

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

Roles of tumor suppressors in regulating tumor-associated inflammation.

Loss or silencing of tumor suppressors (TSs) promotes neoplastic transformation and malignant progression. To date, most work on TS has focused on their cell autonomous effects. Recent evidence, however, demonstrates an important noncell autonomous role for TS in the control of tumor-associated inflammation. We review evidence from clinical data sets and mouse model studies demonstrating enhanced inflammation and altered tumor microenvironment (TME) upon TS inactivation. We discuss clinical correlations between tumor-associated inflammation and inactivation of TS, and their therapeutic implications. This review sets forth the concept that TS can also suppress tumor-associated inflammation, a concept that provides new insights into tumor-host interactions. We also propose that in some cases the loss of TS function in cancer can be overcome through inhibition of the resulting inflammatory response, regardless whether it is a direct or an indirect consequence of TS loss

The placental problem: Linking abnormal cytotrophoblast differentiation to the maternal symptoms of preeclampsia

The placenta is a remarkable organ. In normal pregnancy its specialized cells (termed cytotrophoblasts) differentiate into various specialized subpopulations that play pivotal roles in governing fetal growth and development. One cytotrophoblast subset acquires tumor-like properties that allow the cells to invade the decidua and myometrium, a process that attaches the placenta to the uterus. The same subset also adopts a vascular phenotype that allows these fetal cells to breach and subsequently line uterine blood vessels, a process that channels maternal blood to the rest of the placenta. In the pregnancy complication preeclampsia, which is characterized by the sudden onset of maternal hypertension, proteinuria and edema, cytotrophoblast invasion is shallow and vascular transformation incomplete. These findings, together with very recent evidence from animal models, suggest that preeclampsia is associated with abnormal placental production of vasculogenic/angiogenic substances that reach the maternal circulation with the potential to produce at least a subset of the clinical signs of this syndrome. The current challenge is to build on this knowledge to design clinically useful tests for predicting, diagnosing and treating this dangerous disorder

Whole blood transcript and protein abundance of the vascular endothelial growth factor family relate to cognitive performance

The vascular endothelial growth factor (VEGF) family of genes has been implicated in the clinical development of Alzheimer's Disease (AD). A previous study identified associations between gene expression of VEGF family members in the prefrontal cortex and cognitive performance and AD pathology. This study explored if those associations were also observed in the blood. Consistent with previous observations in brain tissue, higher blood gene expression of placental growth factor (PGF) was associated with a faster rate of memory decline (p=0.04). Higher protein abundance of FMS-related receptor tyrosine kinase 4 (FLT4) in blood was associated with biomarker levels indicative of lower amyloid and tau pathology, opposite the direction observed in brain. Also, higher gene expression of VEGFB in blood was associated with better baseline memory (p=0.008). Notably, we observed that higher gene expression of VEGFB in blood was associated with lower expression of VEGFB in the brain (r=-0.19, p=0.02). Together, these results suggest that the VEGFB, FLT4, and PGF alterations in the AD brain may be detectable in the blood compartment