Andrew K. Edwards

The microRNAome of pregnancy: deciphering miRNA networks at the maternal-fetal interface.

MicroRNAs (miRNAs) post-transcriptionally regulate a vast network of genes by inhibiting mRNA translation. Aberrant miRNA expression profiles have been implicated in pathologies and physiological processes including pregnancy and angiogenesis. Using our established model of implantation failure and spontaneous fetal loss in pigs (Sus scrofa), 236 miRNAs were profiled and compared between 1) non-pregnant and pregnant endometrium, 2) maternal and fetal tissues, and 3) viable and growth-arrested conceptus attachment sites by microarray and Real-Time PCR. Many significant differences in miRNA expression were observed between each of the aforementioned comparisons, and several were validated by PCR. Results indicated which miRNAs were important during pregnancy, which were elevated on the maternal or fetal side of the maternal-fetal interface, and they implicated the maternal expression of miR-10a, 27a, 29c, 323, 331-5p, 339-3p, 374b-5p, and 935 in the spontaneous loss observed in pigs. Several putative mRNA targets of the miRNAs (elevated in endometrium associated with arresting conceptuses) were assessed by quantitative Real-Time PCR and were depressed, supporting their regulation by miRNAs. Finally, targets were clustered by function to obtain ranked lists of gene networks that indicated which pathways/physiological processes might be important in non-pregnant (extracellular matrix factors) versus pregnant endometrium (nuclear transcription factor regulation), maternal (blood vessel development) versus fetal (neuronal differentiation) tissue, and healthy (extracellular matrix factors) versus arresting (GRAM domain) conceptus attachment sites. Overall, we demonstrate the presence of miRNAs on both sides of the maternal-fetal interface, implicate them in spontaneous fetal loss, and present a unique glimpse into the vast microRNAome of pregnancy.

IL-17A Contributes to the Pathogenesis of Endometriosis by Triggering Proinflammatory Cytokines and Angiogenic Growth Factors.

Endometriosis is a chronic, inflammatory disease characterized by the growth of endometrial tissue in aberrant locations outside the uterus. Neoangiogenesis or establishment of new blood supply is one of the fundamental requirements of endometriotic lesion survival in the peritoneal cavity. IL-17A is emerging as a potent angiogenic and proinflammatory cytokine involved in the pathophysiology of several chronic inflammatory diseases such as rheumatoid arthritis and psoriasis. However, sparse information is available in the context of endometriosis. In this study, we demonstrate the potential importance of IL-17A in the pathogenesis and pathophysiology of endometriosis. The data show a differential expression of IL-17A in human ectopic endometriotic lesions and matched eutopic endometrium from women with endometriosis. Importantly, surgical removal of lesions resulted in significantly reduced plasma IL-17A concentrations. Immunohistochemistry revealed localization of IL-17A primarily in the stroma of matched ectopic and eutopic tissue samples. In vitro stimulation of endometrial epithelial carcinoma cells, Ishikawa cells, and HUVECs with IL-17A revealed significant increase in angiogenic (vascular endothelial growth factor and IL-8), proinflammatory (IL-6 and IL-1β), and chemotactic cytokines (G-CSF, CXCL12, CXCL1, and CX3CL1). Furthermore, IL-17A promoted tubulogenesis of HUVECs plated on Matrigel in a dose-dependent manner. Thus, we provide the first evidence, to our knowledge, that endometriotic lesions produce IL-17A and that the removal of the lesion via laparoscopic surgery leads to the significant reduction in the systemic levels of IL-17A. Taken together, our data show a likely important role of IL-17A in promoting angiogenesis and proinflammatory environment in the peritoneal cavity for the establishment and maintenance of endometriosis lesions.

Animal models for anti-angiogenic therapy in endometriosis

Endometriosis is a gynecological disease characterized by the growth of endometrium outside of the uterine cavity. It is often associated with dysmenorrhea, dyspareunia, pelvic pain and infertility. One of the key requirements for endometriotic lesions to survive is development of a blood supply to support their growth. Indeed, dense vascularization is characteristic feature of endometriotic lesions. This has led to the idea that suppression of blood vessel growth (anti-angiogenic therapy) may be a successful therapeutic approach for endometriosis. Potential effectiveness of anti-angiogenic therapies has been assessed in some animal models but there are no reports of human clinical trials. Without understanding the specific mechanism by which endometriosis lesions establish a new blood supply, short-term animal experiments will have limited value for translation into human medicine. Further, it is crucial to use appropriate animal models to assess efficacy of anti-angiogenic compounds. Syngeneic and autologous rodent models, where endometrial fragments are auto-transplanted into the peritoneal cavity are commonly used in anti-angiogenic therapy studies. Another approach is xenograft models where human endometrium is engrafted into immunodeficient mice. Here we review the animal models and experimental techniques used to evaluate anti-angiogenic therapies for endometriosis. We also review our own work on the role of stromal cell derived factor-1 in the recruitment of endothelial progenitor cells in endometriotic lesion angiogenesis, and the effects of the anti-angiogenic peptide ABT-898, a thrombospondin-1 mimetic, on endometriotic lesion growth and vascular development.

Selection and Validation of Reference Genes for miRNA Expression Studies during Porcine Pregnancy

MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.

Blocking of stromal cell-derived factor-1 reduces neoangiogenesis in human endometriosis lesions in a mouse model.

Endometriosis affects 5–10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Establishing new blood supply is a fundamental requirement for endometriosis lesion growth. Endothelial progenitor cells (EPCs), recruited by stromal cell–derived factor‐1 (SDF‐1), contribute to neoangiogenesis in endometriotic lesions. We hypothesized that SDF‐1 is central to the neoangiogenesis and survival of endometriotic lesions, and blocking of SDF‐1 will reduce vascularization of lesions in a mouse model.

An Overview of Molecular and Cellular Mechanisms Associated with Porcine Pregnancy Success or Failure

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.

Distinct microRNA expression in endometrial lymphocytes, endometrium, and trophoblast during spontaneous porcine fetal loss

Endometrial lymphocytes are recruited to the porcine maternal-fetal interface by conceptus-derived signals. The transiently recruited lymphocytes adopt a specialized phenotype in the endometrium that regulates various placental physiological processes, including angiogenesis. Small non-coding RNAs, microRNAs (miRNAs) are emerging as principal bio-molecules regulating the development of lymphocytes and their angiogenic functions. However, no information is available in the context of endometrial lymphocytes in pregnancy. We hypothesize that miRNAs are involved in the development of endometrial lymphocytes and their angiogenic functions at the porcine maternal-fetal interface. Using a targeted Q-PCR approach for selected miRNAs involved in immune cell development, angiogenesis, and anti-angiogenesis, we conducted a study to screen endometrial lymphocytes associated with healthy and spontaneously arresting conceptus attachment sites (CAS) at two well-defined periods of fetal loss. Comparisons were made with endometrium and trophoblasts associated with healthy and arresting CAS. In addition, levels of putative mRNA targets and subsequent functional clustering of genes were studied in order to predict the biological mechanisms affected. We found several significant differences for miRNAs involved in immune cell development and angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between endometrial lymphocytes associated with healthy and arresting CAS. Significant differences were also found in endometrium and trophoblasts for some miRNAs (miR-20b, miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, selected mRNA targets showed differential expression in all groups. Our data, although associative, are the first to unravel the selected miRNAs involved in immune cell development and provide insights into their possible regulation in abortive pregnancy.

Thrombospondin-1 mimetic peptide ABT-898 affects neovascularization and survival of human endometriotic lesions in a mouse model.

Endometriosis is a common cause of pelvic pain and infertility in women, and a common indication for hysterectomy, yet the disease remains poorly diagnosed and ineffectively treated. Because endometriotic lesions require new blood supply for survival, inhibiting angiogenesis could provide a novel therapeutic strategy. ABT-898 mimics the antiangiogenic properties of thrombospondin-1, so we hypothesized that ABT-898 will prevent neovascularization of human endometriotic lesions and that ABT-898 treatment will not affect reproductive outcomes in a mouse model. Endometriosis was induced in BALB/c-Rag2(-/-)Il2rg(-/-) mice by surgical implantation of human endometrial fragments in the peritoneal cavity. Mice received daily injections of ABT-898 for 21 days. Flow cytometry was performed to measure circulating endothelial progenitor cells in peripheral blood. Cytokines were measured in plasma samples. Half of the ABT-898-treated and control mice were euthanized to assess neovascularization of endometriotic lesions, using CD31(+) immunofluorescence. The remaining mice were mated and euthanized at gestation day 12. Endometriotic lesions increased circulating endothelial progenitor cells 13 days after engraftment, relative to baseline. Endometriotic lesions from ABT-898-treated mice exhibited reduced neovascularization, compared with controls, and lesions had fewer CD31(+) microvessels. Chronic treatment with ABT-898 did not lead to any fetal anomalies or affect litter size at gestation day 12, compared with controls. Our results suggest that ABT-898 inhibits neovascularization of human endometriotic lesions without affecting mouse fecundity.

Laser Capture Microdissection for Gene Expression Analysis

Laser capture microdissection (LCM) is an excellent and perhaps the only platform to isolate homogeneous cell populations from specific microscopic regions of heterogeneous tissue section, under direct microscopic visualization. The basic operations of the LCM system are based on (a) microscopic visualization of phenotypically identified cells of interest, (b) selective adherence of cells to a melting thermolabile film/membrane using a low-energy infrared laser (IR system) or photovolatization of cells within a selected region (UV system), (c) capturing or catapulting of structurally intact cells from a stained tissue section. RNA/DNA or protein can be extracted from the cell or tissue fragments for downstream applications to quantitatively study gene expression. This method can be applied to many downstream analyses including but not limited to quantitative real-time polymerase chain reaction (PCR), microarray, DNA genotyping, RNA transcript profiling, generation of cDNA library, mass spectrometry analysis, and proteomic discovery.The application of LCM is described here to specifically and reliably obtain a homogeneous cell population in order to extract RNA to study microRNA expression by quantitative real-time PCR.

A peptide inhibitor of synuclein-γ reduces neovascularization of human endometriotic lesions

Endometriosis is a chronic painful gynecological condition characterized by adherence and growth of endometrium outside of the uterine cavity. Neovascularization is essential to the developing endometriosis lesion to support its growth. Synuclein-γ (SNCG), a protein implicated in cellular proliferation, is associated with a broad range of malignancies as well as endometriosis. We hypothesized that SNCG plays an important role in the neovascularization and growth of endometriosis and blocking of SNCG will interfere with survival of endometriotic lesions in a mouse model. We developed SP012, a novel 12 amino acid peptide inhibitor of SNCG. SP012 inhibited three-dimensional endothelial cell tube formation in a dose-dependent manner. Using intravital microscopy, SP012 was shown to be successfully delivered to human endometriotic lesions in a xenograft mouse model in vivo. Alymphoid (BALB/c-Rag2-/-Il2rγ-/- lacking T, B and NK cells) mice were surgically induced with human endometriotic lesions and treated with SP012 or phosphate-buffered saline control. SP012 treated endometriotic lesions had decreased growth, development and vascularization at the time of necroscopy. Endometriotic lesions treated with SP012 also had fewer isolectin (+) microvessels. These results, using a mouse model, indicate that SNCG plays a role in the neovascularization and subsequent growth of human endometriotic lesions. Targeting SNCG function using peptide inhibitor might provide a potential therapeutic option for the treatment of endometriosis in the future.