VEGF expression by epithelial and stromal cell compartments: resolving a controversy.
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ABSTRACT:
SUBMITTER: Senger DR
PROVIDER: S-EPMC1850188 | biostudies-literature | 2000 Jul
REPOSITORIES: biostudies-literature
Publications
VEGF expression by epithelial and stromal cell compartments: resolving a controversy.
Senger D R DR Van De Water L L
The American journal of pathology 20000701 1
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Project description:The β-cell mitogenic effects of ANGPTL8 have been subjected to substantial debate. The original findings suggested that ANGPTL8 overexpression in mice induced a 17-fold increase in β-cell proliferation. Subsequent studies in mice contested this claim, but a more recent report in rats supported the original observations. These conflicting results might be explained by variable ANGPTL8 expression and differing methods of β-cell quantification. To resolve the controversy, three independent labs collaborated on a blinded study to test the effects of ANGPTL8 upon β-cell proliferation. Recombinant human betatrophin (hBT) fused to maltose binding protein (MBP) was delivered to mice by intravenous injection. The results demonstrate that ANGPTL8 does not stimulate significant β-cell proliferation. Each lab employed different methods for β-cell identification, resulting in variable quantification of β-cell proliferation and suggests a need for standardizing practices for β-cell quantification. We also observed a new action of ANGPTL8 in stimulating CD45+ hematopoietic-derived cell proliferation which may explain, in part, published discrepancies. Overall, the hypothesis that ANGPTL8 induces dramatic and specific β-cell proliferation can no longer be supported. However, while ANGPTL8 does not stimulate robust β-cell proliferation, the original experimental model using drug-induced (S961) insulin resistance was validated in subsequent studies, and thus still represents a robust system for studying signals that are either necessary or sufficient for β-cell expansion. As an added note, we would like to commend collaborative group efforts, with repetition of results and procedures in multiple laboratories, as an effective method to resolve discrepancies in the literature.
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Project description:BACKGROUND:B7-H3 and B7-H4 are highly expressed by many human malignancies making them attractive immunotherapeutic targets. However, their expression patterns and immune contexts in epithelial ovarian cancer have not been well characterized. METHODS:We used flow cytometry, immunohistochemistry, and genomic analyses to determine the patterns of B7-H3, B7-H4, and PD-L1 expression by tumor, stromal, and immune cells in the ovarian tumor microenvironment (TME). We analyzed immune cell frequency and expression of PD-1, TIM3, LAG3, ICOS, TIA-1, granzyme B, 2B4, CD107a, and GITR on T cells; CD20, CD22, IgD, BTLA, and CD27 on B cells; CD16 on monocytes; and B7-H3, B7-H4, PD-L1, PD-L2, ICOSL, CD40, CD86, and CLEC9a on antigen-presenting cells by flow cytometry. We determined intratumoral cellular location of immune cells using immunohistochemistry. We compared differences in immune infiltration in tumors with low or high tumor-to-stroma ratio and in tumors from the same or unrelated patients. RESULTS:On non-immune cells, B7-H4 expression was restricted to tumor cells whereas B7-H3 was expressed by both tumor and stromal cells. Stromal cells of the ovarian TME expressed high levels of B7-H3 compared to tumor cells. We used this differential expression to assess the tumor-to-stroma ratio of ovarian tumors and found that high tumor-to-stroma ratio was associated with increased expression of CD16 by monocytes, increased frequencies of PD-1high CD8+ T cells, increased PD-L1 expression by APCs, and decreased CLEC9a expression by APCs. We found that expression of PD-L1 or CD86 on APCs and the proportion of PD-1high CD4+ T cells were strongly correlated on immune cells from tumors within the same patient, whereas expression of CD40 and ICOSL on APCs and the proportion of PD-1high CD8+ T cells were not. CONCLUSIONS:This study provides insight into the expression patterns of B7-H3 and B7-H4 in the ovarian TME. Further, we demonstrate an association between the tumor-to-stroma ratio and the phenotype of tumor-infiltrating immune cells. We also find that some but not all immune parameters show consistency between peritoneal metastatic sites. These data have implications for the design of immunotherapies targeting these B7 molecules in epithelial ovarian cancer.
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Project description:Depletion of Brca1 leads to defects in mouse mammary gland development and mammary tumors in humans and mice. To explore the role of microRNAs (miRNAs) in this process, we examined the mammary glands of MMTV-Cre Brca1(Co/Co) mice for differential miRNA expression using a candidate approach. Several miRNAs were differentially expressed in mammary tissue at day 1 of lactation and in mammary epithelial cell lines in which Brca1 messenger RNA (mRNA) levels have been reduced. Functional studies revealed that several of these miRNAs regulate mammary epithelial cell function in vitro, including miR-206. Creation and analysis of MMTV-miR-206 transgenic mice showed no effect on lactational mammary development and no tumors, but indicates a role in mammary tissue remodeling in mature mice, potentially involving Igf-1 and Sfrp1. These results indicate the potential of miRNAs to mediate the consequences of Brca1 loss and suggest a novel function for miR-206.
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Project description:Analysis of gene expression changes in tumour epithelium (DCIS and invasive breast cancer) and stroma both immediately surrounding the lesions and more distantly. Total RNA obtained from Formalin Fixed Paraffin Embedded archival material and the individual compartments (stroma and epithelium) compared independently across the samples. Sample abbreviation key: BC = breast cancer DCIS = ductal carcinoma in situ IDC = invasive ductal carcinoma RM = remote metastasis S = stroma NS = near stroma.
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Project description:Vascular endothelial growth factor (VEGF) is expressed robustly in human colon neoplasia and is a major new "rational" target of therapy for cancers of the colon and other organs. Nonetheless, the mechanism(s) of action of VEGF-targeted therapies and the biologic roles of VEGF in tumorigenesis have not been well defined. We used a transgenic approach to directly test the hypothesis that augmented VEGF expression can drive progression of intestinal neoplasia.Transgenic mouse lines were generated with moderate (vilVEGF1) and high (vilVEGF2) VEGF expression from the intestinal epithelium. vilVEGF1 mice were bred to Min mice (adenomatous polyposis coli [APC] +/-). Colon epithelial cells from an APC patient were cocultured with endothelial cells and fibroblasts.vilVEGF mice were generally healthy but displayed red small intestines. Vessels were larger and more numerous in the submucosa but not the mucosa. The mucosa showed striking stromal and epithelial hypercellularity, with increased epithelial proliferation. Many crypts formed cysts composed of relatively undifferentiated epithelial cells surrounded by cells with endothelial and myofibroblast markers. Compared with Min controls, vilVEGF1-Min mice developed 6-fold more intestinal adenomas of all sizes, with more advanced histologic features. Polycystic masses were also observed. Coculture of human colonocytes with endothelial cells and fibroblasts directly stimulated colonocyte proliferation.Augmented VEGF expression from intestinal epithelium potently stimulated cross talk with mesenchymal cells and proliferation of normal and neoplastic epithelium. These effects of VEGF, largely occurring prior to the canonical angiogenic switch in tumors, may be in part independent of angiogenesis.
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Project description:PurposeTo investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells.MethodsDot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo-/- mice, which, like humans, cannot synthesize ascorbate de novo.ResultsTreatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome-3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo-/- mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor.ConclusionsAscorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD).
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Project description:To identify tumor compartment-specific microRNA expression in stage I non-small cell lung cancer in humans, surgically resected and formalin-fixed tumor tissues were used in laser capture microdissection to isolate tumor epithelia and stroma. Total RNA extracted from the microdissectates was analyzed for microRNA expression using the 7th generation miRCURY™ locked nucleic acid microarray platform (Exiqon®, Vedbaek, Denmark).
2014-12-31 | GSE42425 | GEO
Project description:Lgr5 has been identified as a marker of the stem/progenitor cells in the murine ovary and oviduct by lineage tracing. However, little is known regarding LGR5 expression or its functional significance in human ovary tissues. Here, using RNA in situ hybridization and/or immunohistochemistry, we thoroughly investigated LGR5 expression in normal human ovaries, fallopian tubes and various ovarian tumors. We discovered that LGR5 expression is negligible in the human ovary surface epithelium, whereas ovarian stromal cells normally express low levels of LGR5. Remarkably, fallopian tube epithelium, inclusion cysts and serous cystadenomas with a Müllerian phenotype expressed high levels of LGR5, and LGR5 expression was restricted to PAX8+/FOXJ1- secretory cells of the tubal epithelium. Strong stromal LGR5 expression without epithelial LGR5 expression was consistently observed in the path from serous cystadenoma to serous borderline tumor to low grade serous carcinoma (LGSC). Unlike LGSC, high grade serous carcinoma (HGSC), clear cell carcinoma, endometrioid carcinomas displayed various epithelial-stromal LGR5 expression. Notably, high levels of LGR5 expression were observed in serous tubal intraepithelial carcinoma, which slightly declined in invasive HGSC. LGR5 expression was significantly associated with improved progression-free survival in HGSC patients. Moreover, in vitro assays demonstrated that LGR5 expression suppressed tumor proliferation and migratory capabilities. Taken together, these findings indicate a tumor-suppressive role for LGR5 in the progression of HGSC.
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Project description:ObjectiveTo determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns.DesignIn vitro study.SettingUniversity research laboratory.Patient(s)Endometrial biopsies were obtained from premenopausal women.Intervention(s)Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls.Main outcome measure(s)Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures.Result(s)Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion.Conclusion(s)This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro.
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