Differential Expression of Genes Involved in Metabolism and Immune Response in Diffuse and Intestinal Gastric Cancers, a Pilot Ptudy.

Gastric cancer (GC) is one of the major causes of cancer-related mortality worldwide. The vast majority of GC cases are adenocarcinomas including intestinal and diffuse GC. The incidence of diffuse GCs, often associated with poor overall survival, has constantly increased in USA and Europe The molecular basis of diffuse GC aggressivity remains unclear. Using mRNA from diffuse and intestinal GC tumor samples of a Western cohort, this study reports the expression level of the immunomodulatory aryl-hydrocarbon receptor (AhR), and genes involved in immune suppression (PD1, PD-L1, PD-L2) and the early steps of tryptophan metabolism (IDO1, IDO2, TDO2). Strongly increased expression of IDO1 (p < 0.001) and PD1 (p < 0.003) was observed in the intestinal sub-type. The highest expression of IDO1 and PDL1 correlated with early clinical stage and absence of lymphatic invasion (×25 p = 0.004, ×3 p = 0.04, respectively). Our results suggest that kynurenine, produced by tryptophan catabolism, and AhR activation play a central role in creating an immunosuppressive environment. Correspondingly, as compared to intestinal GCs, expression levels of IDO1-TDO2 and PD-L1 were less prominent in diffuse GCs which also had less infiltration of immune cells, suggesting an inactive immune response in the advanced diffuse GC. Confirmation of these patterns of gene expression will require a larger cohort of early and advanced stages of diffuse GC samples.

Detection of Persistent Organic Pollutants in Omental Adipose Tissue from Patients with Diffuse-Gastric Cancer: A Pilot Study.

The greater omentum represents a specific adipose tissue resected with gastric surgery for cancer. Diffuse gastric adenocarcinoma (diffuse-GC) is of major relevance among gastric cancers due to its unknown origin, aggressiveness, and metastasis in the peritoneal cavity. We postulated that persistent organic pollutants (POPs) could be detected in the greater omentum. Great omentum from patients with (i) diffuse-GC, or (ii) with other peritoneal metastatic cancer, and (iii) control group without cancer disease were analyzed for the distribution of a large panel of 96 POPs. POPs include polychlorinated dioxins/furans (PCDD/Fs), polychlorobiphenyls (PCBs), polybrominated diphenyl ethers (PBDE), polybrominated biphenyls (PBB), hexabromocyclododecanes, organochlorine pesticides, and polycyclic aromatic hydrocarbons (PAHs). The widespread presence of a substantial list of POPs (PCDDs/Fs, PCBs, and brominated flame retardants) was found in the omentum from patients with aggressive diffuse-GC, with minor presence of some organochlorine pesticides and PAHs at the low analyzed levels. Some chemicals appeared in larger concentrations in diffuse-GC or other cancer groups, including some PCDDs, PCB105, 123, 138, PBDE209, and PBB153. Overall, the present pilot study provides novel information regarding POPs levels in the omental fat, which is an understudied fat depot in terms of POPs load, and diffuse-GC association.

Differential gene expression in growth factors, epithelial mesenchymal transition and chemotaxis in the diffuse type compared with the intestinal type of gastric cancer.

Gastric cancer (GC) is a highly heterogeneous disease and one of the major causes of cancer-related mortality worldwide. Diffuse-type gastric adenocarcinoma (or poorly cohesive- with independent cells) is characterized by aggressive behavior (rapid invasion, chemoresistance and peritoneal metastasis), as compared with intestinal-subtype adenocarcinoma. Diffuse subtype GC additionally has a substantially increasing incidence rate in Europe and the USA, and was often associated with younger age. Our objective was to analyze the expression and clinical significance of genes involved in several signaling pathways in diffuse-type GC. Tumors samples and non-malignant gastric tissues were obtained from patients with GC (diffuse-type and intestinal-subtype adenocarcinoma). The expression of 33 genes coding for proteins involved in four categories, growth factors and receptors, epithelial-mesenchymal transition, cell proliferation and migration, and angiogenesis was determined by reverse transcription-quantitative polymerase chain reaction. The expression of 22 genes was significantly upregulated in diffuse-type GC and two were downregulated (including CDH1) compared with normal tissues. Among these genes, acompared with intestinal-subtype adenocarcinoma, diffuse-type GC revealed elevated levels of IGF1 and IGF1R, FGF7 and FGFR1, ZEB2, CXCR4, CXCL12 and RHOA, and decreased levels of CDH1, MMP9 and MKI67. The expression of selected genes was compared with other genes and according to clinical parameters. Furthermore, TGF-ß expression was significantly increased in linitis, a sub-population of diffusely infiltrating type associated with extensive fibrosis and tumor invasion. Our study identified new target genes (IGF1, FGF7, CXCR4, TG-ß and ZEB2) whose expression is associated with aggressive phenotype of diffuse-type GC.

Alteration of mammary gland development by bisphenol a and evidence of a mode of action mediated through endocrine disruption.

The development and function of the mammary gland are endocrine-dependent processes, depending on the stage of development. Foetal and/or postnatal exposure to low doses of BPA alters tissue organisation through epithelial proliferation and stroma-epithelial interactions. BPA also alters the expression of E2-dependent epithelial and stroma transcriptomes. Several signalling pathways are consistent with the observed phenotype: proliferation and apoptosis, a focal adhesion pathway indicating changes in biomechanical properties of the extracellular matrix, and immune function. Some of BPA's effects are reversed by oestrogen and/or GPER inhibitors. BPA also alters the expression of epigenetic marks (EZH2, HOTAIR), which would explain the delayed effect of foetal BPA exposure. In conclusion, experimental evidence shows that pre- or postnatal BPA exposure consistently causes endocrine modifications in the mammary tissue of different animal species, disrupting stromal-epithelial interactions and ultimately increasing its susceptibility to carcinogens. An interspecies comparison highlights why and how these effects apply to humans.

Regulatory identification of BPA as an endocrine disruptor: Context and methodology.

BPA is one of the most investigated substances for its endocrine disruptor (ED) properties and it is at the same time in the center of many ED-related controversies. The analysis on how BPA fits to the regulatory identification as an ED is a challenge in terms of methodology. It is also a great opportunity to test the regulatory framework with a uniquely data-rich substance and learn valuable lessons for future cases. From this extensive database, it was considered important to engage in a detailed analysis so as to provide specific and strong evidences of ED while reflecting accurately the complexity of the response as well the multiplicity of adverse effects. An appropriate delineation of the scope of the analysis was therefore critical. Four effects namely, alterations of estrous cyclicity, mammary gland development, brain development and memory function, and metabolism, were considered to provide solid evidence of ED-mediated effects of BPA.

High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism.

Increasing epidemiological and animal experimental data provide substantial support for the role of aryl hydrocarbon receptor (AhR) in mammary tumorigenesis. The effects of AhR have been clearly demonstrated in rodent models of breast carcinogenesis and in several established human breast cancer cell lines following exposure to AhR ligands or AhR overexpression. However, relatively little is known about the role of AhR in human breast cancers. AhR has always been considered to be a regulator of toxic and carcinogenic responses to environmental contaminants such as TCDD (dioxin) and benzo[a]pyrene (BaP). The aim of this study was to identify the type of breast tumors (ERα-positive or ERα-negative) that express AHR and how AhR affects human tumorigenesis. The levels of AHR, AHR nuclear translocator (ARNT) and AHR repressor (AHRR) mRNA expression were analyzed in a cohort of 439 breast tumors, demonstrating a weak association between high AHR expression and age greater than fifty years and ERα-negative status, and HR-/ERBB2 breast cancer subtypes. AHRR mRNA expression was associated with metastasis-free survival, while AHR mRNA expression was not. Immunohistochemistry revealed the presence of AhR protein in both tumor cells (nucleus and/or cytoplasm) and the tumor microenvironment (including endothelial cells and lymphocytes). High AHR expression was correlated with high expression of several genes involved in signaling pathways related to inflammation (IL1B, IL6, TNF, IL8 and CXCR4), metabolism (IDO1 and TDO2 from the kynurenine pathway), invasion (MMP1, MMP2 and PLAU), and IGF signaling (IGF2R, IGF1R and TGFB1). Two well-known ligands for AHR (TCDD and BaP) induced mRNA expression of IL1B and IL6 in an ERα-negative breast tumor cell line. The breast cancer ER status likely influences AhR activity involved in these signaling pathways. The mechanisms involved in AhR activation and target gene expression in breast cancers are also discussed.

MDA-MB-231 breast cancer cells overexpressing single VEGF isoforms display distinct colonisation characteristics.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has important roles in angiogenesis. Our knowledge of the significance of VEGF isoforms in human cancer remains incomplete. METHODS: Bioluminescence imaging and transcriptomic analysis were used to study the colonisation capacity of the human breast cancer cells MDA-MB-231 controlling or overexpressing the VEGF165 or VEGF189 isoform (named cV-B, V165-B and V189-B, respectively) in nude mice. RESULTS: When injected into the bloodstream, V189-B cells induced less metastasis in the lungs and bone than V165-B and cV-B control cells, consistent with longer survival of these mice and delay in tumour uptake in the mice injected with a V189-B clone. Histological analysis confirmed that there were less αSMA-positive cells in the lungs of the mice injected with V189-B. In vitro V189-B cells decreased both cell invasion and survival. Using transcriptomic analysis, we identified a subset of 18 genes expressed differentially between V189 and V165 cell lines and in 120 human breast tumours. V165 was associated with poor prognosis, whereas V189 was not, suggesting a complex regulation by VEGF isoforms. Our results showed a negative correlation between the expression pattern of VEGF189 and the levels of expression of seven genes that influence metastasis. CONCLUSION: Our findings provide the first evidence that VEGF isoforms have different effects on breast cancer cell line colonisation in vivo.

Autocrine functions of VEGF in breast tumor cells: adhesion, survival, migration and invasion.

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions.

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.

Similar NF-κB gene signatures in TNF-α treated human endothelial cells and breast tumor biopsies.

BACKGROUND: Endothelial dysfunction has been implicated in the pathogenesis of diverse pathologies ranging from vascular and immune diseases to cancer. TNF-α is one of the mediators of endothelial dysfunction through the activation of transcription factors, including NF-κB. While HUVEC (macrovascular cells) have been largely used in the past, here, we documented an NF-κB gene signature in TNFα-stimulated microvascular endothelial cells HMEC often used in tumor angiogenesis studies. METHODOLOGY/PRINCIPAL FINDINGS: We measured mRNA expression of 55 NF-κB related genes using quantitative RT-PCR in HUVEC and HMEC. Our study identified twenty genes markedly up-regulated in response to TNFα, including adhesion molecules, cytokines, chemokines, and apoptosis regulators, some of them being identified as TNF-α-inducible genes for the first time in endothelial cells (two apoptosis regulators, TNFAIP3 and TNFRSF10B/Trail R2 (DR5), the chemokines GM-CSF/CSF2 and MCF/CSF1, and CD40 and TNF-α itself, as well as NF-κB components (RELB, NFKB1 or 50/p105 and NFKB2 or p52/p100). For eight genes, the fold induction was much higher in HMEC, as compared to HUVEC. Most importantly, our study described for the first time a connection between NF-κB activation and the induction of most, if not all, of these genes in HMEC as evaluated by pharmacological inhibition and RelA expression knock-down by RNA interference. Moreover, since TNF-α is highly expressed in tumors, we further applied the NF-κB gene signature documented in TNFα-stimulated endothelial cells to human breast tumors. We found a significant positive correlation between TNF and the majority (85 %) of the identified endothelial TNF-induced genes in a well-defined series of 96 (48 ERα positive and 48 ERα negative) breast tumors. CONCLUSION/SIGNIFICANCE: Taken together these data suggest the potential use of this NF-κB gene signature in analyzing the role of TNF-α in the endothelial dysfunction, as well as in breast tumors independently of the presence of ERα.

Administration of bisphenol A to dams during perinatal period modifies molecular and morphological reproductive parameters of the offspring.

Bisphenol A (BPA) is an estrogenic agonist compound that induces changes in diverse reproductive parameters in rats. The aim of the present study was to determine the effects of BPA given in drinking water containing 10mg/L (approximate dose 1.2mg/kg BW/day), administered chronically to rats during pregnancy and lactation, on reproductive tract parameters of the offspring. 79.2% of the female offspring from BPA-treated mothers presented irregular estrous cycles. As compared to the control group, a significant increase in the thickness of the uterine epithelia and stroma was observed in the BPA group. Additionally, 60% of the female offspring from BPA mothers did not undergo abundant uterine epithelial apoptosis during the estrus phase of the cycle while control animals did. In addition, a down regulation of ERα expression was observed in epithelial cells on estrus day. The results indicate that BPA, when administered chronically in water beverages to dams, modifies the reproductive cycle of the offspring during young adulthood.

Vascular endothelial growth factor is a target gene for estrogen receptor and contributes to breast cancer progression.

Tumor growth requires the development and remodeling of the vascular system, involving paracrine signaling between various growth factors and endothelial receptors. Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological and pathological neovascularization, especially involved in tumor growth. Recent studies indicate that 17beta-estradiol (E2) modulates VEGF expression in breast cancer cells through transcriptional activation. We have investigated both the molecular mechanisms of E2-induction of VEGF expression and of VEGF control of breast cancer angiogenesis. In transient transfection assays using the VEGF promoter-luciferase construct, E2 increased VEGF transcriptional activity in MCF-7 cells and in MDA-MB-231 cotransfected with estrogen receptor (ERalpha or ERbeta). The positive effect was abolished when MCF-7 cells were treated with the pure antiestrogen ICI 182,780 or the agonist/antagonist tamoxifen. We further identified an imperfect estrogen responsive element (ERE1520) in the VEGF promoter, which formed a complex with ERalpha or ERbeta proteins in gel shift assay using MCF-7 or MDA-MB-231 nuclear extracts; the ERE sequence is involved in the transcriptional regulation of VEGF in our experimental conditions. These results demonstrate that in breast cancer (BC) cells VEGF is a target gene for ERalpha or ERbeta. To determine the role of VEGF in the progression of human breast carcinoma, we generated stable human breast carcinoma cells (MCF-7) overexpressing VEGF165 (V165 clones). Cells or control vector clones were implanted subcutaneously in athymic mice. Our in vivo findings show that overexpression of VEGF significantly decreased tumor uptake and increased tumor growth and angiogenesis in a murine model of BC.

Xenoestrogens modulate vascular endothelial growth factor secretion in breast cancer cells through an estrogen receptor-dependent mechanism.

Environmental chemicals may affect human health by disrupting endocrine function. Their possible role in the mammary gland and breast tumors is still unknown. Previous studies have demonstrated that vascular endothelial growth factor (VEGF), a key factor in angiogenesis and tumor progression, is an estrogen-regulated gene. We analyzed whether VEGF expression is regulated by different xenoestrogens in several breast cancer cells, MELN (derived from MCF-7) and MELP (derived from MDA-MB-231) and stably expressing estrogen receptor alpha (ERalpha); these cell lines stably express estrogen response element (beta-globin)-luciferase. Genistein, bisphenol A (BPA), 4-(tert-octyl)phenol (OP), dieldrin, and several phthalates, including benzyl butyl phthalate (BBP) and di-ethyl-2-hexyle phthalate (DEHP), were first shown to be estrogenic. These compounds induced a dose-dependent increase of VEGF secretion in MELN and MCF-7 cells; maximal effect was observed at 1-10 microM non-cytotoxic concentrations and was inhibited by the antiestrogen ICI 182 780. VEGF increase was not observed in ERalpha-negative MDA-MB-231 cells. Most substances increased VEGF transcript levels in MELN cells. In contrast, gamma-hexachlorocyclohexane, vinclozolin, and the phthalates (mono-n-butyl ester phthalic acid, di-isononyle phthalate, and di-isodecyle phthalate) were ineffective on both VEGF secretion and estrogenic luciferase induction in these cell lines. Specific kinase inhibitors PD98059, SB203580, or LY294002 suppressed the xenoestrogen-induced VEGF response, suggesting activation of MEK, p38 kinase, and phosphatidylinositol-3-kinase pathways. Our in vitro results show for the first time that genistein and xenoestrogens (BPA, OP, dieldrin, BBP, and DEHP at high concentrations) up-regulate VEGF expression in MELN cells by an ER-dependent mechanism. Since VEGF increases capillary permeability and breast tumor angiogenesis in vivo, the physiological relevance of these findings is discussed.

Overexpression of vascular endothelial growth factor 189 in breast cancer cells leads to delayed tumor uptake with dilated intratumoral vessels.

Vascular endothelial growth factor (VEGF) is essential for breast cancer progression and is a relevant target in anti-angiogenesis. Although VEGF121 and VEGF165, the fully or partially secreted isoforms, respectively, have been the focus of intense studies, the role of the cell-associated VEGF189 isoform is not understood. To clarify the contribution of VEGF189 to human mammary carcinogenesis, we established several clones of MDA-MB-231 cells stably overexpressing VEGF189 (V189) and VEGF165 (V165). V189 and V165 clones increased tumor growth and angiogenesis in vivo. Remarkably, V165 induced the most rapid tumor uptake, whereas V189 increased vasodilation. In vitro overexpression of VEGF165 and VEGF189 increases the proliferation and chemokinesis of these cancer cells. Interestingly, overexpression of VEGF189 increased cell adhesion on fibronectin (1.9-fold) and vitronectin (1.6-fold), as compared to VEGF165, through alpha5beta1 and alphavbeta5 integrins. Using the BIACore system we demonstrated for the first time that VEGF189 binds directly to neuropilin-1, which is strongly expressed in MDA-MB-231 cells. In contrast, VEGF-R2 was not significantly expressed and VEGF-R1 was expressed at low level. Our in vitro results suggest an autocrine effect of VEGF189 on breast cancer cells, probably through neuropilin-1. In conclusion, our data indicate that VEGF189 participates in mammary tumor growth through both angiogenesis and nonangiogenic functions. Whether VEGF189 overexpression is correlated to prognosis in human breast tumors remains to be established.

Angiogenic activity of human chorionic gonadotropin through LH receptor activation on endothelial and epithelial cells of the endometrium.

Successful embryo development requires an extensive endometrial angiogenesis in proximity of implantation site. The glycoprotein hCG is produced even before implantation by trophoblast in normal pregnancy. In this manuscript, we demonstrate an angiogenic effect of hCG in several in vivo (chick chorioallantoïc membrane, matrigel plug assay, aortic ring assay) and in vitro experimental models. In contrast, human placental lactogen (hPL) did not display angiogenic properties. LH/hCG receptor was detected in endothelial cells by reverse-transcriptase polymerase chain reaction (RT-PCR) and by Western blotting. In mice aortic ring assay, angiostimulation by hCG was abrogated by deletion of LH/hCG receptor (LuRKO mice). Use of recombinant hCG and anti-hCG antibody (Ab) further confirmed the specificity of this angiogenic activity. By using dibutyryl cAMP, adenylate cyclase, or protein kinase A inhibitors, we demonstrate that hCG-mediated angiogenesis involves adenylyl-cyclase-protein kinase A activation. Addition of hCG to endometrial epithelial epithelial cells, but not to cultured endothelial cells, stimulated vascular endothelial growth factor (VEGF). VEGF and hCG also displayed additive activities. Altogether, these data demonstrate that peritrophoblastic angiostimulation may result from a paracrine dialogue between trophoblast, epithelial, and endothelial cells through hCG and VEGF.

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA.

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.

Vascular endothelial growth factor VEGF189 induces human neutrophil chemotaxis in extravascular tissue via an autocrine amplification mechanism.

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell mitogen involved in normal and pathological angiogenesis. Our group recently reported that, among the several VEGF isoforms, VEGF189 (V189) is selectively induced in decidual endometrial cells during the mid-late phase of the menstrual cycle, together with polymorphonuclear neutrophil (PMN) influx. We thus compared the effects of various VEGF isoforms on PMN migration in vitro, and the mechanisms involved. In transmigration and under-agarose assays, V189 was both chemotactic and chemokinetic for PMN, while VEGF165 (V165) was only chemokinetic. The chemokinetic effect of V189 for PMN was blocked by neutralizing anti-VEGF antibodies, but not by neutralizing anti-KDR antibodies, suggesting that the Flt-1 VEGF receptor that is expressed in PMN mediates these effects. Flow cytometric analysis of several adhesion molecules at the PMN surface showed that all VEGF isoforms slightly upregulated beta1- and beta2-integrins and PECAM, and downregulated L-selectin; all these molecules are activation markers. The involvement of beta1-integrins was further supported by the ability of blocking antibodies to reduce VEGF-induced PMN migration. As human PMN can secrete several cytokines and growth factors, the selective secretion of VEGF isoforms was also further examined. RT-PCR analysis showed that V165 mRNA was more strongly expressed than V189 mRNA. Conversely, the major protein isoform secreted after optimal PMN degranulation was V189, which was located in both azurophilic and specific granules. PMN-derived VEGF can thus modulate PMN migration. This autocrine amplification mechanism would allow sustained VEGF release to occur at inflammatory sites, and may contribute to both normal and pathological angiogenesis.

Transcriptional regulation of vascular endothelial growth factor by estradiol and tamoxifen in breast cancer cells: a complex interplay between estrogen receptors alpha and beta.

Vascular endothelial growth factor (VEGF) is a potent angiogenic and prognostic factor for many tumors, including those of endocrine-responsive tissues such as the breast and uterus. Recent studies indicate that 17beta-estradiol (E(2)) modulates VEGF expression in breast and uterine cells, involving transcriptional activation through estrogen receptor (ER) alpha. However, molecular mechanisms of VEGF regulation mediated by the two ER subtypes and the potential role of ERbeta in the control of breast cancer angiogenesis have not yet been investigated. In transient transfection assays using the VEGF(-2275/+54) promoter-luciferase construct, E(2) (1 nM) increased transcription activity in MCF-7 cells (either untransfected or cotransfected with ERalpha) and it increased transcription activity in MDA-MB-231 cells cotransfected with ERalpha or ERbeta (1.8- and 2-fold induction, respectively). The positive effect was abolished when MCF-7 cells were treated with pure antiestrogen ICI 182,780 or the agonist/antagonist tamoxifen (1 micro M). To identify response elements involved in this transcriptional regulation, MCF-7 or MDA-MB-231 cells were transfected with several deletion constructs of the VEGF promoter. Deletion of 1.2-2.3 kb upstream to the transcription start in the VEGF promoter abrogated E(2)-dependent transcription in these cells. This region contains an imperfect estrogen-responsive element (ERE), ERE1520, and one activator protein 1 site. Transfection of MCF-7 cells (ERalpha) with the ERE1520-luciferase construct conferred transcriptional activity with 1 nM E(2) (1.9-fold induction). Also, the imperfect ERE formed a complex with ERalpha or ERbeta proteins in gel shift assay using MCF-7 or MDA-MB-231 nuclear extracts. In contrast to ERalpha, ERbeta could transactivate VEGF reporter construct in MDA-MB-231 cells, in the presence of E(2) or tamoxifen, suggesting different transactivational mechanisms between ERalpha and ERbeta in the presence of tamoxifen. Interestingly, E(2) inhibited VEGF transcription in MCF-7 cells transfected with ERbeta or MDA-MB-231 cells cotransfected with ERalpha and ERbeta, suggesting that heterodimerization of ERalpha/ERbeta has the ability to inhibit E(2)-induced VEGF expression in breast cancer cells. These results demonstrate that VEGF is a target gene for ERalpha and ERbeta in breast cancer cells; it remains to be determined whether ERalpha and ERbeta expression in breast biopsies correlates with VEGF expression and vascular density.

A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus.

A key mechanism underlying physiological angiogenesis of the human endometrium is its ability to regenerate the vascular capillary network and to perform vascular remodeling (i.e., development of spiral arteries). Vascular endothelial growth factor (VEGF) is associated with angiogenesis and capillary permeability in this tissue. VEGF is expressed as several spliced variants, its main human isoforms contain 121 and 165 aa; 17beta-estradiol (E(2)) increases endometrial VEGF, possibly in all isoforms. Here we show that progesterone (P) selectively increases the expression of the VEGF(189) (V(189)) isoform in the human uterus. V(189) is identified in the conditioned medium of stromal cells treated with E(2) + P; its presence in this in vitro model of decidual stromal cells is detected after 6-8 days, using ELISA, and after 8-10 days, using Western blot analysis with different antibodies, including one specific for V(189). The secretion pattern of V(189) parallels that of the decidual protein IGFBP-1. V(189) is secreted as a native isoform, as compared with the migration of recombinant V(189) by SDS/PAGE. In situ hybridization and immunocytochemistry(,) performed on the same biopsies, suggest that decidual cells express V(189) during the mid-late secretory phase of the menstrual cycle and early gestation. Finally, using an in vivo permeability assay, we show that native V(189) increases capillary permeability. These observations demonstrate that P regulates V(189) expression in decidual cells, which could have important implications for understanding uterine vascular remodeling and implantation, and may be relevant in a range of disease states such as edema and irregular bleeding.