Search

Human existence is intrinsically community-oriented. Persons find themselves as responsible in community. This is a classical and Christian insight that is supported by significant contemporary philosophers such as Gabriel Marcel and Emmanuel Levinas. This article makes the claim that to thrive as a person is to belong; indeed, that it is the experience of belonging that satisfies the human need for meaning, value, and purpose. The article proceeds by considering the term ‘community.’ In itself, ‘community’ is a common sense term. However, it is also a symbol with dimensions of meaning beyond common sense implicit within it. For the sake of a richer understanding of belonging, this article proposes to discuss some of those dimensions under three headings: Dynamism; Communion; and Consummation. ‘Dynamism’ relates to the members of a community whose mutual participation constitutes the characteristics of that community. ‘Communion’ considers the emergence of genuine community, as opposed to general ...

A lack of host-derived SPARC promotes disease progression in an intraperitoneal (IP) ID8 mouse model of epithelial ovarian cancer (EOC). Since orthotopic injection (OT) of ID8 cells better recapitulates high-grade serous cancer, we examined the impact of host-derived SPARC following OT injection. Sparc−/− and wild-type (WT) mice were injected with ID8 cells either OT or IP and tumors were analyzed at the moribund stage. Sparc−/− mice had reduced survival and fewer well-defined abdominal lesions compared with WT controls after IP injection, whereas no differences were observed in survival or abdominal lesions between Sparc−/− and WT mice after OT injection. No differences in mass or collagen content were observed in ovarian tumors between OT-injected Sparc−/− and WT mice. The abdominal wall of the IP-injected Sparc−/− mice exhibited immature and less abundant collagen fibrils compared with WT mice both in injected and non-injected controls. In contrast to human EOC, SPARC was expressed by the tumor cells but was absent in reactive stroma of WT mice. Exposure to the ovarian microenvironment through OT injections alters the metastatic behaviour of ID8 cells, which is not affected by the absence of host-derived SPARC.

The discovery of occult invasive and intra-epithelial tubal carcinomas in BRCA1 mutation carriers undergoing prophylactic surgery has implicated the fallopian tube epithelium as the source of serous cancer. However, little is known of the early molecular events of serous oncogenesis, or why cancers in BRCA1 mutation carriers are found preferentially in tissues which are responsive to reproductive hormones. We hypothesize that molecular alterations present in morphologically normal tubal epithelium from BRCA1 heterozygotes reflect the earliest events in serous carcinogenesis and may be markers of increased cancer risk as well as targets for risk reduction. Genetic profiling of microdissected tubal epithelium from histologically normal BRCA1 mutation carriers and controls was performed. We sought to define a signature which differentiated BRCA1 mutant tubal epithelium from women with low risk of developing ovarian cancer. Molecular differences between the follicular and luteal phases were prominent and, by using filtering techniques and a two-way ANOVA without a False Discovery Rate correction, we identified 440 probe sets with a more than two-fold change in gene expression related to BRCA1 mutation status. Using gene ontology and known associations to cancer pathways, we selected five genes for further analysis by qPCR and immunohistochemistry, and were able to demonstrate statistically significant differentiation of BRCA1 and control cases in an independent set of cases. The altered expression profiles in histologically normal tubal epithelium from BRCA1 heterozygotes suggest that these cells may respond differently to microenvironmental stresses. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

// James B. Greenaway 1 , Carl Virtanen 2 , Kata Osz 1 , Tamas Revay 1 , Daniel Hardy 3 , Trevor Shepherd 4 , Gabriel DiMattia 4 , Jim Petrik 5 1 Department of Biomedical Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada 2 Princess Margaret Genomics Centre, University Health Network, Toronto, ON, M5G 1L7, Canada 3 Department of Ob/Gyn and Physiology and Pharmacology, Children’s Health Research Institute, Western University, London, ON, N6A 5C1, Canada 4 Department of Ob/Gyn and Oncology, Anatomy and Cell Biology, London Regional Cancer Program, Western University, London, ON, N6A 4L6, Canada 5 Department of Biomedical Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada Correspondence to: Jim Petrik, email: jpetrik@uoguelph.ca Keywords: ovarian cancer, mevalonate pathway, simvastatin, p53 Received: January 12, 2016 Accepted: June 04, 2016 Published: June 17, 2016 ABSTRACT Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer and often is not detected until late stages when cancer cells transcoelomically metastasize to the abdomen and typically become resistant to therapy resulting in very low survival rates. We utilize an orthotopic, syngeneic mouse model to study late stage disease and have discovered that the tumor cells within the abdominal ascites are irreversibly re-programmed, with an increased tumorigenicity and resistance to apoptosis. The goal of this study was to characterize the reprogramming that occurred in the aggressive ascites-derived cells (28-2 cells) compared to the original cell line used for tumor induction (ID8 cells). Microarray experiments showed that the majority of genes upregulated in the 28-2 cells belonged to the mevalonate pathway, which is involved in cholesterol biosynthesis, protein prenylation, and activation of small GTPases. Upregulation of mevalonate appeared to be associated with the acquisition of a p53 mutation in the ascites-derived cells. Treatment with simvastatin to inhibit HMG CoA reductase, the rate limiting enzyme of this pathway, induced apoptosis in the 28-2 cell line. Rescue experiments revealed that mevalonate, but not cholesterol, could inhibit the simvastatin-mediated effects. In vivo , daily intraperitoneal simvastatin treatment significantly regressed advanced stage disease and induced death of metastatic tumor cells. These data suggest that ovarian cancer cells become reprogrammed, with genetic mutations, and upregulation of the mevalonate pathway, which facilitates the development of advanced stage disease. The use of statins to inhibit HMGCR may provide novel therapeutic opportunities for the treatment of advanced stage EOC.

Real time RT-PCR was used to measure the changes in the rates of synthesis of mRNA encoding for growth hormone-1 (GH1) and -2 (GH2) and insulin-like growth factor-1 (IGF-1) and -2 (IGF-2), and whole embryo GH content was measured in early stage rainbow trout ( Oncorhynchus mykiss ) embryos reared at two incubation temperatures (8.5 and 6.0 °C). Particular attention was paid to the phase of embryo development that preceded the appearance of the pituitary gland. GH was present in zygotes, and there were no significant changes in whole embryo GH content of the two temperature treatment groups from fertilization ( t 0 ) until the time at which GH was detectable in the pituitary gland by immunostaining. The expression of the two GH genes decreased during the first 24 h post-fertilization, and then increased significantly by 17 dpf in embryos reared at both temperatures. There was a subsequent steep increase in the number of copies of GH1 and GH2 mRNA associated with the formation of the pituitary gland evident at 23 and 34 dpf in the 8.5 and 6.0 °C groups, respectively. The number of copies of mRNA encoding for IGF-1 and IGF-2 did not change during the first 24 h post-fertilization; however, there was a significant increase in the numbers of transcripts for both genes evident by 13 dpf in embryos reared at the two incubation temperatures. The differences in the timing of the increases in GH and IGF mRNA may suggest that IGF gene expression is not GH-dependent at that stage. Moreover, the increased expression of the GH genes prior to the formation of the pituitary gland suggests that tissues other than the pituitary are expressing these genes in early embryos. The pattern of changes in GH content was similar to the pattern of GH gene expression in embryos reared at the two incubation temperatures when the age of embryos was plotted using degree-days. There were no apparent compensatory responses in GH1, GH2, IGF-1 or IGF-2 gene expression related to altered growth rates. The number of copies of IGF-2 mRNA was higher than that of IGF-1 mRNA during the early developmental period; this is consistent with the hypothesis that IGF-2 predominates during embryonic development. A differential expression of GH2 and GH1 was also observed with the overall copy numbers of GH2 mRNA being consistently higher than those of GH1.

Angiogenesis does not normally occur in most adult tissues. However, in the ovary, there are cyclical vascular changes including angiogenesis that involve the interaction of numerous cytokines and growth factors. Angiogenic processes are regulated by a balance between pro- and antiangiogenic factors. The purpose of this study was to determine the expression of the antiangiogenic thrombospondin family and proangiogenic vascular endothelial growth factor (VEGF) in various sizes of healthy bovine follicles. Ovaries were collected from slaughterhouse animals and healthy follicles were sorted based on size ( 1.0 cm, large). Thrombospondin (TSP) protein levels were significantly higher in small follicles. Immunohistochemistry confirmed the granulosa layer as the primary area within the follicle involved in TSP generation and that small follicles had the highest proportion of immunopositive cells. TSP-1 and -2 mRNA levels were significantly higher in small follicles than either medium or large follicles. TSP colocalized with CD36 on granulosa cells (GC) in the follicle and in cultured cells. In contrast with TSP, VEGF expression increased during growth and development of the follicle. FSH stimulated GC expression of TSP, while LH had no effect. In summary, TSP-1 and -2 were coordinately expressed in the extravascular compartment of the ovary during early follicle development. VEGF was inversely expressed, with expression increasing as follicles developed. Regulated expression and localization of these proteins suggests that they may be involved in regulating growth and development of the follicle in a novel fashion.

Vascular endothelial growth factor (VEGF) is a potent mitogen and cytoprotective factor for vascular endothelial cells. Although VEGF is ubiquitously expressed, its role in nonvascular tissues is poorly understood. VEGF interacts with various cell surface receptors to mediate its cellular effects. It previously has been thought that the VEGF receptor Flk-1/KDR, its main signaling receptor, was expressed exclusively by endothelial cells. However, in the present study using bovine and rodent models, we demonstrate that VEGF and Flk-1/KDR are coexpressed in ovarian granulosa cells. VEGF and Flk-1/KDR mRNA and protein were both detectable in follicle tissue sections and in vitro cultured granulosa cells. Expression of both ligand and receptor increased in healthy follicles throughout follicular development. VEGF treatment of serum-starved and cytokine-exposed granulosa cells resulted in enhanced survival, and this cytoprotection was ameliorated when Flk-1/KDR signaling was inhibited. Reduced expression of Flk-1/KDR was also associated with the onset and progression of follicle atresia, suggesting involvement in follicular health in vivo. The results of this study demonstrate for the first time expression of Flk-1/KDR in ovarian granulosa cells and identify a novel extravascular role for VEGF and its receptor in ovarian function.

This paper will consider both the pragmatic and intellectual context of the medieval problem of order, politically and philosophically. The struggle for order in the Middle Ages was about more than the contest for jurisdiction between the empire and the church. What emerged from beneath the clamor for legitimate authority was the growing assertion of a more subtle third center of authority, the individual. This paper aims at articulating and charting the emergence of what is called existential authority, drawing on the insights of Eric Voegelin among others.

One of the difficulties in studying ovarian cancer historically has been the lack of a suitable animal model that replicates the human disease. Mouse models that utilize intraperitoneal implantation of tumorigenic cells lack interaction between the transformed ovarian epithelial cells and the ovarian stroma, which we have shown to be an integral component in replicating the etiology seen in human epithelial ovarian cancer (Greenaway, Gynecol Oncol 108:385-394, 2008). Xenograft models generally require the use of immunocompromised hosts, which then eliminates the influence of the immune system in disease progression, which also has been shown to be an important part of the progression of epithelial ovarian cancer (EOC). In this chapter, we describe the generation and optimization of an orthotopic, syngeneic mouse model and illustrate the importance of facilitating epithelial-stromal cell interaction to more closely replicate human EOC.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is often not diagnosed until late stages due to its asymptomatic nature. Women diagnosed with EOC typically undergo surgical debulking followed by chemotherapy; however, disease recurrence often occurs. In this study, we evaluated the ability of the thrombospondin-1 mimetic peptide, ABT-898, to regress established, late-stage tumors in a mouse model of human EOC. Ovarian tumors were induced and ABT-898 treatment was initiated at time points that were representative of late stages of the disease to study tumor regression. ABT-898 induced tumor regression and reduced the morbidity of treated animals compared with controls. Analysis of tumors from ABT-898–treated animals showed reduced abnormal tumor vasculature, decreased expression of the proangiogenic compound VEGF, and reduced tumor tissue hypoxia. ABT-898 treatment initiated at late-stage disease also significantly prolonged disease-free survival compared with control animals. Results from this study show that ABT-898 is capable of regressing established ovarian tumors in an animal model of the disease. As most women are detected at advanced stage EOC, ABT-898 may improve our treatment of ovarian cancer. Mol Cancer Ther; 10(10); 1876–85. ©2011 AACR.

Tumor development is a complex process that relies on interaction and communication between a number of cellular compartments. Much of the mass of a solid tumor is comprised of the stroma which is richly invested with extracellular matrix. Within this matrix are a host of matricellular proteins that regulate the expression and function of a myriad of proteins that regulate tumorigenic processes. One of the processes that is vital to tumor growth and progression is angiogenesis, or the formation of new blood vessels from preexisting vasculature. Within the extracellular matrix are structural proteins, a host of proteases, and resident pro- and antiangiogenic factors that control tumor angiogenesis in a tightly regulated fashion. This paper discusses the role that the extracellular matrix and ECM proteins play in the regulation of tumor angiogenesis.

Although osteopontin (OPN) is up-regulated in inflammatory bowel diseases, its role in disease pathogenesis remains controversial. The objective of this study was to determine the role of OPN in host responses to a non-invasive bacterial pathogen, Citrobacter rodentium, which serves as a murine infectious model of colitis. OPN gene knockout and wild-type mice were infected orogastrically with either C. rodentium or Luria-Bertani (LB) broth. Mouse-derived OPN(+/+) and OPN(-/-) fibroblasts were incubated with C. rodentium and attaching-effacing lesions were demonstrated using transmission electron microscopy and immunofluorescence. Colonic expression of OPN was increased by C. rodentium infection of wild-type mice. Furthermore, colonic epithelial cell hyperplasia, the hallmark of C. rodentium infection, was reduced in OPN(-/-) mice, and spleen enlargement by infection was absent in OPN(-/-) mice. Rectal administration of OPN to OPN(-/-) mice restored these effects. There was an 8- to 17-fold reduction in bacterial colonization in OPN(-/-) mice, compared with wild-type mice, which was accompanied by reduced attaching-effacing lesions, both in infected OPN(-/-) mice and OPN(-/-) mouse fibroblasts. Moreover, adhesion pedestals were restored in OPN(-/-) cells complemented with human OPN. Therefore, lack of OPN results in decreased pedestal formation, colonization, and colonic epithelial cell hyperplasia responses to C. rodentium infection, indicating that OPN impacts disease pathogenesis through bacterial attachment and altered host immune responses.

Epithelial ovarian cancer (EOC) comprises approximately 90% of ovarian cancers and arises from the surface epithelium. Typical treatment of EOC involves cytoreductive surgery combined with chemotherapy. More recent therapies have targeted the tumor vasculature using antiangiogenic compounds such as thrombospondin-1 (TSP-1). TSP-1 mimetic peptides such as ABT-510 have been created and have been in various clinical trials. We have previously shown that ABT-510 reduces abnormal vasculature associated with tumor tissue and increases the presence of mature blood vessels. It has been hypothesized that treatment with antiangiogenic compounds would allow increased delivery of cytotoxic agents and enhance treatment. In this study, we evaluated the potential role of ABT-510 and various chemotherapeutics (cisplatin and paclitaxel) on tumor progression, angiogenesis, and the benefits of combinational treatments on tissue uptake and perfusion using an orthotopic syngeneic mouse model of EOC. Animals were treated with ABT-510 (100 mg/kg per day) alone or in combination with cisplatin (2 mg/kg per 3 days) or paclitaxel (10 mg/kg per 2 days) at 60 days after tumor induction. Radiolabeled and fluorescently labeled paclitaxel demonstrated a significant increase in tumor uptake after ABT-510 treatment. Combined treatment with ABT-510 and cisplatin or paclitaxel resulted in a significant increase in tumor cell and tumor endothelial cell apoptosis and a resultant decrease in ovarian tumor size. Combined treatment also regressed secondary lesions and eliminated the presence of abdominal ascites. The results from this study show that through vessel normalization, ABT-510 increases uptake of chemotherapy drugs and can induce regression of advanced ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and also one of the most poorly understood. Other health issues that are affecting women with increasing frequency are obesity and diabetes, which are associated with dysglycemia and increased blood glucose. The Warburg Effect describes the ability of fast-growing cancer cells to preferentially metabolize glucose via anaerobic glycolysis rather than oxidative phosphorylation. Recent epidemiological studies have suggested a role for hyperglycemia in the pathogenesis of a number of cancers. If hyperglycemia contributes to tumour growth and progression, then it is intuitive that antihyperglycemic drugs may also have an important antitumour role. Preliminary reports suggest that these drugs not only reduce available plasma glucose, but also have direct effects on cancer cell viability through modification of molecular energy-sensing pathways. This review investigates the effect that hyperglycemia may have on EOC and the potential of antihyperglycemic drugs as therapeutic adjuncts.

Objective. Ovarian cancer is the fourth leading cause of cancer-related deaths among women and is among the least understood of all cancers. The objective of this study was to determine the effect of ovarian epithelial and stromal cell interaction in a mouse model of epithelial ovarian cancer (EOC) that closely resembled the human disease. Methods. A mouse model of EOC was generated by orthotopic injection of an ID8 mouse ovarian surface epithelial cell line (MOSEC) under the ovarian bursa of syngeneic mice and tissue was collected to evaluate factors contributing to the formation and development of ovarian tumors. Results. By 90 days post-injection, mice were moribund and had developed large primary ovarian tumors, secondary tumors within the peritoneal cavity, and extensive ascites fluid production. Tumors were hypervascularized and were characterized as serous epithelial carcinomatosis, which replicates the most common form of human ovarian cancer. Cells isolated from ascites fluid were more proliferative with increased expression of survival factors compared to original MOSEC cells and cells obtained from the abdomen following intraperitoneal injection. Orthotopic injection of these cells under the ovarian bursa resulted in more aggressive tumorigenesis, with mice becoming moribund at 60 days post-injection compared with 90 days post-injection with the original ID8-MOSEC cell line. Discussion. This study describes the generation of an orthotopic, syngeneic model of ovarian cancer, which replicates the phenotype of the human disease. Expression of angiogenic and proliferative factors, and the interaction of epithelial cells with the ovarian stroma are important factors in ovarian tumorigenesis.

VEGF is a potent pro-angiogenic factor whose effects are opposed by a host of anti-angiogenic proteins, including thrombospondin-1 (TSP-1). We have previously shown that VEGF has important extravascular roles in the ovary and that VEGF and TSP-1 are inversely expressed throughout the ovarian cycle. To date, however, a causal interaction between TSP-1 and VEGF has not been identified. Here, we show that TSP-1 has a direct inhibitory effect on VEGF by binding the growth factor and internalizing it via LRP-1. Mice lacking TSP-1 are subfertile and exhibited ovarian hypervascularization and altered ovarian morphology. Treatment of ovarian cells with TSP-1 decreased VEGF levels and rendered the cells more susceptible to TNFalpha-induced apoptosis. Knockdown of TSP-1, through RNA interference, resulted in overexpression of VEGF and reduced cytokine-induced apoptosis. In conclusion, we demonstrate a direct inhibitory effect of TSP-1 on VEGF in the ovary. TSP-1's regulation of VEGF appears to be an important mediator of ovarian angiogenesis and follicle development.

It is established that inflammation can create a protumorigenic environment. Many sources of chronic inflammation, including viral and bacterial, have been associated with accelerated tumorigenesis. An epidemiological link has been discovered between chronic inflammatory diseases and ovarian cancer suggesting that inflammation can increase the risk of epithelial ovarian cancer (EOC) potentially by synergizing with the local ovarian inflammation associated with ovulation. The purpose of this study is to identify the impact of prolonged exposure to chronic low-grade inflammation on epithelial ovarian cancer cell viability in vitro and EOC tumor progression in vivo. We hypothesize that this level of systemic inflammation will enhance the growth and survival of epithelial ovarian tumors by increasing angiogenesis, cell survival and metastatic capability. We believe these effects will occur in part due to the interactions between the malignant ovarian surface epithelial cells and the various immune cells recruited to the stromal microenvironment of the tumor. The first objective to examine these relationships was to determine the effect of a proinflammatory environment in an in vitro model using normal ovarian surface epithelium (NOSE) and transformed human ovarian epithelial cell lines CAOV-3, ES-2, OVCAR-3 and SKOV-3. Cells were exposed to proinflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α and in response to this exposure, the ovarian cancer cells showed enhanced viability and proliferation. The next objective involves examining the influence of the epithelial-stromal interactions of tumor cells by utilizing a co-culture model to initiate communication between the human epithelial cell lines in objective one with a differentiated macrophage cell line. In additional trials with transformed murine epithelial and microvascular cell lines we have identified the expression of Toll-like receptor 4 (TLR4) using reverse transcription-PCR. TLR4 is the receptor through which the bacterial endotoxin lipopolysaccharide (LPS) acts as an inflammatory agent. Based on these preliminary results, we propose to evaluate the role of chronic inflammation in the progression of EOC in an established mouse model using LPS to induce a low-grade level of chronic inflammation. Analysis of tumor cell survival, angiogenesis, and local and systemic inflammation will be evaluated western blot, PCR, ELISA assay and immunofluorescence. Evaluation of the relationship between chronic, systemic inflammation and the progression of EOC may be useful in developing treatment approaches for EOC, specifically in terms of anti-inflammatory therapies, and could provide insight into the role of inflammation in the progression of other human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 409. doi:10.1158/1538-7445.AM2011-409

Background: High-grade serous ovarian cancer is rarely diagnosed at an early and potentially curable stage, effective early detection and preventative strategies are few. Recently the discovery of occult invasive and intraepithelial tubal carcinomas in BRCA1 mutation carriers, who are at high risk of serous cancer, undergoing prophylactic surgery has focused attention on the fallopian tube epithelium as the cell of origin and has led to the reporting of putative serous cancer precursor lesions. However, little is known of the early molecular events of serous oncogenesis, or why cancers in BRCA 1 mutation carriers are found preferentially in tissues which are responsive to reproductive hormones. We hypothesize that molecular alterations are present in morphologically normal tubal epithelium from BRCA1 heterozygotes, and that these changes may reflect the earliest alterations in serous carcinogenesis and may be markers of increased cancer risk as well as targets for risk reduction. To identify cancer predisposition candidate genes of high-risk fallopian tube epithelium, we compared gene expression profiles of microdissected tubal epithelium from BRCA1 mutation carriers, control women and patients with HGSC. Methodology: Snap-frozen tissues were selected from the UHN Biobank; control and BRCA cases controlled for age, ovarian cycle status at surgery, and hormone therapy. Cases included 12 BRCA1 mutation carriers and 12 control women, undergoing salpingo-oophorectomy for reasons other than adnexal malignancy or family history, 15 HGSC and 8 contralateral normal tubes. Epithelium was microdissected in all cases using laser capture, RNA extracted and cDNA amplified. The gene expression profiles were generated using Affymetrix Human Genome U133 Plus 2.0 Array. Candidate genes were validated by qPCR and IHC on 2 fallopian tube TMAs. IHC was scored using Spectrum. Statistical analysis was performed using ANOVA (p Results: We focused the microarray analyses on identifying differential expression between BRCA1 heterozygotes and controls. Comparisons between FTE-nonBRCA and FTE-BRCA, resulted in 440 probe sets with more than 2-FC in gene expression. We selected 5 genes to validate by qPCR and IHC, based on gene ontology and known associations to cancer pathways. Conclusions: These results show that histological normal fallopian tube epithelium from BRCA1 heterozygotes have altered gene expression and these differences are most pronounced in the post-ovulatory phase of the ovarian cycle in pre-menopausal women, suggesting that factors associated with the luteal phase are implicated in the increased risk of HGSC in BRCA1 mutation carriers. We also show that genes involved in inflammation pathways are up-regulated in mutation carriers and that these genetic signatures are maintained in HGSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2835. doi:10.1158/1538-7445.AM2011-2835

Ovarian follicles undergo pronounced morphological changes, alternating between periods of growth and regression. The equine follicle will grow to an average of 45mm in diameter at ovulation, and during the phase of growth, there is an increase in blood supply to the follicle. Vascular endothelial growth factor (VEGF) is a cytokine that interacts with tyrosine kinase receptors to stimulate angiogenesis, endothelial cell proliferation and vascular permeability. The aim of the study was to evaluate the expression and localization of VEGF and the VEGF-receptor 2 (VEGF-R2) in equine follicles. Ovaries were collected from a slaughterhouse. Granulosa cells from follicles were pooled regardless of the size of the follicles. Western blots were performed using protein extracted from granulosa cells and follicular fluid. Blots were probed with rabbit anti-human VEGF and rabbit anti-mouse VEGF-R2 antibodies and visualized with chemiluminescence. Total RNA was extracted from granulosa cells and integrity of the RNA samples was tested by the amplification of β-actin. Complementary DNA was synthesized by reverse transcription, followed by polymerase chain reaction amplification of cDNA encoding with bovine primer sequences for VEGF and VEGF-R2. The PCR product was resolved on 1% agarose gel and the resulting VEGF and VEGF-R2 bands were sequenced. Immunostaining for VEGF and VEGF-R2 was performed on fixed, paraffin-embedded sections of follicle wall from follicles larger than 30mm. Western blot analysis of granulosa cell lysates revealed 22kDa bands for VEGF, and 210kDa bands for VEGF-R2. VEGF protein was present in follicular fluid, whereas VEGF-R2 was not detectable. RT-PCR experiments revealed the presence of VEGF and VEGF-R2 mRNA in isolated granulosa cells. Sequencing demonstrated 93% and 99% homology to known sequences of equine VEGF and VEGF-R2, respectively. Immunofluorescence experiments performed on dissected equine follicles localized VEGF to the granulosa cell layer and sporadically to the theca cell layer. VEGF-R2 co-localized with VEGF in the granulosa cells, and was relatively absent in the theca layer. The present study detected novel expression patterns for VEGF and VEGF-R2 in equine ovarian follicles. The results of these experiments suggest an extra-vascular role for the VEGF family in follicle development. Future studies will be directed at studying the genomic and proteonomic profiles of follicles during the selection of the dominant follicle in mares.