The role of TGF-ß in the pathophysiology of peritoneal endometriosis.

BACKGROUND: Endometriosis is estimated to affect 6-10% of women of reproductive age and it is associated with chronic pelvic pain, dysmenorrhoea and subfertility. It is currently managed surgically or medically but symptoms recur in up to 75% of cases and available medical treatments have undesirable side effects. Endometriosis is defined as the presence of endometrial tissue outside the uterus with lesions typically found on the peritoneum. The aetiology of endometriosis is uncertain but there is increasing evidence that transforming growth factor (TGF)-ß plays a major role. OBJECTIVE AND RATIONALE: A descriptive review was undertaken of the published literature on the expression pattern of TGF-ß ligands and signalling molecules in women with and without endometriosis, and on the potential roles of TGF-ß signalling in the development and progression of peritoneal endometriosis. The current understanding of the TGF-ß signalling pathway is summarized. SEARCH METHODS: We searched the Pubmed database using the terms 'transforming growth factor beta' and 'endometriosis' for studies published between 1995 and 2016. The initial search identified 99 studies and these were used as the basic material for this review. We also extended our remit for important older publications. In addition, we searched the reference lists of studies used in this review for additional studies we judged as relevant. Studies which were included in the review focused on peritoneal endometriosis only as increasing evidence suggests that ovarian and deep endometriosis may have a differing pathophysiology. Thus, a final 95 studies were included in the review. OUTCOMES: TGF-ß1 is reported to be increased in the peritoneal fluid, serum, ectopic endometrium and peritoneum of women with endometriosis compared to women without endometriosis, and TGF-ß1-null mice have reduced endometriosis lesion growth when compared to their wild-type controls. Studies in mice and women have indicated that increasing levels of TGF-ß ligands are associated with decreased immune cell activity within the peritoneum, together with an increase in ectopic endometrial cell survival, attachment, invasion and proliferation, during endometriosis lesion development. TGF-ß1 has been associated with changes in ectopic endometrial and peritoneal cell metabolism and the initiation of neoangiogenesis, further fuelling endometriosis lesion development. WIDER IMPLICATIONS: Together these studies suggest that TGF-ß1 plays a major role in the development of peritoneal endometriosis lesions and that targeting this pathway may be of therapeutic potential.

Reduced Transforming Growth Factor-ß Activity in the Endometrium of Women With Heavy Menstrual Bleeding.

Context: Heavy menstrual bleeding (HMB) is common and incapacitating. Aberrant menstrual endometrial repair may result in HMB. The transforming growth factor (TGF)-ß superfamily contributes to tissue repair, but its role in HMB is unknown. Objective: We hypothesized that TGF-ß1 is important for endometrial repair, and women with HMB have aberrant TGF-ß1 activity at menses. Participants/Setting: Endometrial biopsies were collected from women, and menstrual blood loss objectively measured [HMB >80 mL/cycle; normal menstrual bleeding (NMB) <80 mL]. Design: Immunohistochemistry and reverse transcription polymerase chain reaction examined endometrial TGF-ß1 ligand, receptors, and downstream SMADs in women with NMB and HMB. The function and regulation of TGF-ß1 were examined using cell culture. Results: TGFB1 mRNA was maximal immediately prior to menses, but no differences detected between women with NMB and HMB at any cycle stage. Histoscoring of TGFB1 revealed reduced staining in the stroma during menses in women with HMB (P < 0.05). There were no significant differences in TGFBR1/2 or TGFBR1/2 immunostaining. Cortisol increased activation of TGFB1 in the supernatant of human endometrial stromal cells (HES; P < 0.05) via thrombospondin-1. Endometrial SMAD2 and SMAD3 were lower in women with HMB during menstruation (P < 0.05), and decreased phosphorylated SMAD2/3 immunostaining was seen in glandular epithelial cells during the late secretory phase (P < 0.05). Wound scratch assays revealed increased repair in HES cells treated with TGF-ß1 versus control (P < 0.05). Conclusions: Women with HMB had decreased TGF-ß1 and SMADs perimenstrually. Cortisol activated latent TGF-ß1 to enhance endometrial stromal cell repair. Decreased TGF-ß1 activity may hinder repair of the denuded menstrual endometrium, resulting in HMB.

ID2 mediates the transforming growth factor-ß1-induced Warburg-like effect seen in the peritoneum of women with endometriosis.

STUDY QUESTION: Is inhibitor of DNA-binding protein 2 (ID2) a mediator of the transforming growth factor (TGF)-ß1-induced Warburg-like effect seen in the peritoneum of women with endometriosis? SUMMARY ANSWER: The TGF-ß1-induced changes in the metabolic phenotype of peritoneal mesothelial cells from women with endometriosis are mediated through the ID2 pathway. WHAT IS KNOWN ALREADY: TGF-ß1 induces the metabolic conversion of glucose to lactate via aerobic glycolysis (the 'Warburg effect') in the peritoneum of women with endometriosis, through increased expression of the transcription factor hypoxia inducible factor α (HIF-1α). ID proteins are transcriptional targets of TGF-ß1. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Expression of ID2 was investigated in luteal phase peritoneal biopsies from women with regular menstrual cycles, with and without endometriosis (n = 8-10 each group) by quantitative RT-PCR (qRT-PCR) and immunohistochemistry. ID2 mRNA expression in primary human peritoneal mesothelial cells (HPMC) and immortalized mesothelial cells (MeT-5A) was assessed by qRT-PCR (n = 6). The effects of TGF-ß1 and ID2 siRNA on HIF-1α mRNA expression and lactate secretion was assessed using qRT-PCR and a colorimetric lactate assay. MAIN RESULTS AND THE ROLE OF CHANCE: ID2 is localized to peritoneal mesothelial and stromal cells of women with and without endometriosis. ID2 mRNA expression is lower in peritoneum adjacent to the endometriosis lesions compared to distal sites (P < 0.01). Exposure of HPMC and MeT-5A cells to physiological concentrations of TGF-ß1 decreases ID2 mRNA expression (P < 0.01, P < 0.001, respectively, versus control). ID2 knockdown increases HIF-1α mRNA expression (P < 0.01) and lactate secretion (P < 0.05 versus scrambled control) to the same degree as with exposure to TGF-ß1. LIMITATIONS, REASONS FOR CAUTION: Primary human cell cultures and a cell line were used in this study, and thus the results may not fully represent the situation in vivo. The results should also be replicated using a larger number of samples. WIDER IMPLICATIONS OF THE FINDINGS: Novel therapeutics that target the TGFß/ID pathway offer a potential role in the treatment of endometriosis. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was funded by a Wellbeing of Women research grant (R42533) awarded to A.W.H., J.K.B. and W.C.D.; and an MRC Centre Grant G1002033. V.J.Y. received grant support from Federation of Women Graduates (134225) and a PhD studentship from the College of Medicine and Veterinary Medicine at the University of Edinburgh. There are no competing interests to declare.

Peritoneal VEGF-A expression is regulated by TGF-ß1 through an ID1 pathway in women with endometriosis.

VEGF-A, an angiogenic factor, is increased in the peritoneal fluid of women with endometriosis. The cytokine TGF-ß1 is thought to play a role in the establishment of endometriosis lesions. Inhibitor of DNA binding (ID) proteins are transcriptional targets of TGF-ß1 and ID1 has been implicated in VEGF-A regulation during tumor angiogenesis. Herein, we determined whether peritoneal expression of VEGF-A is regulated by TGF-ß1 through the ID1 pathway in women with endometriosis. VEGF-A was measured in peritoneal fluid by ELISA (n = 16). VEGF-A and ID1 expression was examined in peritoneal biopsies (n = 13), and primary peritoneal and immortalized mesothelial cells (MeT5A) by immunohistochemistry, qRT-PCR and ELISA. VEGF-A was increased in peritoneal fluid from women with endometriosis and levels correlated with TGF-ß1 concentrations (P < 0.05). VEGF-A was immunolocalized to peritoneal mesothelium and TGF-ß1 increased VEGFA mRNA (P < 0.05) and protein (P < 0.05) in mesothelial cells. ID1 was increased in peritoneum from women with endometriosis and TGF-ß1 increased concentrations of ID1 mRNA (P < 0.05) in mesothelial cells. VEGF-A regulation through ID1 was confirmed by siRNA in MeT5A cells (P < 0.05). Our data supports role for ID1 in the pathophysiology of endometriosis, as an effector of TGFß1 dependent upregulation of VEGF-A, and highlights a novel potential therapeutic target.

The peritoneum is both a source and target of TGF-ß in women with endometriosis.

Transforming growth factor-ß (TGF-ß) is believed to play a major role in the aetiology of peritoneal endometriosis. We aimed to determine if the peritoneum is a source of TGF-ß and if peritoneal TGF-ß expression, reception or target genes are altered in women with endometriosis. Peritoneal fluid, peritoneal bushings and peritoneal biopsies were collected from women with and without endometriosis. TGF-ß1, 2 and 3 protein concentrations were measured in the peritoneal fluid. TGF-ß1 was measured in mesothelial cell conditioned media. Control peritoneum and peritoneum prone to endometriosis (within Pouch of Douglas) from women without disease (n = 16) and peritoneum distal and adjacent to endometriosis lesions in women with endometriosis (n = 15) and were analysed for TGF-ß expression, reception and signalling by immunohistochemistry, qRT-PCR and a TGF-ß signalling PCR array. TGF-ß1 was increased in the peritoneal fluid of women with endometriosis compared to those without disease (P<0.05) and peritoneal mesothelial cells secrete TGF-ß1 in-vitro. In women with endometriosis, peritoneum from sites adjacent to endometriosis lesions expressed higher levels of TGFB1 mRNA when compared to distal sites (P<0.05). The TGF-ß-stimulated Smad 2/3 signalling pathway was active in the peritoneum and there were significant increases (P<0.05) in expression of genes associated with tumorigenesis (MAPK8, CDC6), epithelial-mesenchymal transition (NOTCH1), angiogenesis (ID1, ID3) and neurogenesis (CREB1) in the peritoneum of women with endometriosis. In conclusion, the peritoneum, and in particular, the peritoneal mesothelium, is a source of TGF-ß1 and this is enhanced around endometriosis lesions. The expression of TGF-ß-regulated genes is altered in the peritoneum of women with endometriosis and this may promote an environment favorable to lesion formation.

Transforming growth factor-ß induced Warburg-like metabolic reprogramming may underpin the development of peritoneal endometriosis.

CONTEXT: TGF-ß is believed to play a major role in the etiology of peritoneal endometriosis. In tumors, TGF-ß induces the metabolic conversion of glucose to lactate via glycolysis, a process referred to as the "Warburg effect." Lactate increases cell invasion, angiogenesis, and immune suppression, all crucial steps in the development of endometriosis. OBJECTIVE: The aim of this study was to determine whether TGF-ß induces a "Warburg-like" effect in peritoneal endometriosis. DESIGN: The study was informed by human tissue analysis and cel culture. SETTING: The study was conducted at the university research institute. PATIENTS OR OTHER PARTICIPANTS: We studied women undergoing surgical investigation for endometriosis. INTERVENTIONS: Concentrations of lactate and TGF-ß1 in peritoneal fluid (n = 16) were measured by commercial assay. Expression of genes implicated in glycolysis was measured in endometrial and peritoneal biopsies (n = 31) by quantitative RT-PCR and immunohistochemistry. The effect of TGF-ß1 on primary human peritoneal mesothelial cells (n = 6) and immortalized mesothelial (MeT-5A) cells (n = 3) was assessed by quantitative RT-PCR, Western blot, and commercial assays. MAIN OUTCOME MEASURES: Lactate, TGF-ß1, and markers of glycolysis were measured. RESULTS: Concentrations of lactate in peritoneal fluid paralleled those of TGF-ß1, being significantly higher in women with endometriosis compared to women without (P < .05). Endometriosis lesions expressed higher levels of glycolysis-associated genes HIF1A, PDK1, and LDHA than eutopic endometrium, and adjacent peritoneum had higher levels of HIF1A and SLC2A1 than peritoneum from women without disease (P < .05 to P < .001). Exposure of mesothelial cells to TGF-ß1 increased production of lactate (P < .05), increased HIF1A mRNA (P < .05), and protein, and increased concentrations of mRNAs encoded by glycolysis-associated genes (LDHA, PDK1, SLC2A1; P < .05). CONCLUSIONS: A change in the metabolic phenotype of endometriosis lesions and peritoneal mesothelium in women with endometriosis may favor development of endometriosis.

The role of the peritoneum in the pathogenesis of endometriosis.

BACKGROUND Endometriosis affects 6-10% of women of reproductive age and is associated with chronic pelvic pain, dysmenorrhoea, dyspareunia and infertility. Endometriosis is defined by the presence of endometrial tissue outside the uterus, most commonly attached to the pelvic peritoneum. The endometrium in women with endometriosis is reported to be altered and there is increasing evidence that the phenotype of the pelvic peritoneum may also play a role in the establishment and maintenance of the disease. The aim of this review is to discuss the putative role of the pelvic peritoneum in the pathophysiology of peritoneal endometriosis. METHODS A review was undertaken of the published literature on (i) the anatomy and physiology of the peritoneum and (ii) the potential roles played by peritoneal cells in the establishment and maintenance of peritoneal endometriosis. The current understanding of the biology of peritoneal endometriosis is summarized and the potential interaction of the peritoneum with ectopic endometrial cells in endometriosis is highlighted. RESULTS Several studies indicate that differential expression of peritoneal mesothelial adhesion factors occurs in women with endometriosis, providing potential ectopic endometrial cell attachment sites for the establishment of endometriosis lesions. Changes in the peritoneal mesothelial cell phenotype, including loss of tight junctions, may allow ectopic cells to bind to, or early lesions to invade into, the extracellular matrix. Epithelial-to-mesenchymal transition of peritoneal mesothelial cells may also lead to an increase in lesion invasion and formation of fibrotic tissue in and around the lesion. There is evidence that the peritoneal mesothelium may also play a role in the invasion potential of ectopic cells by production of MMPs increasing local tissue remodelling. Peritoneal immune scavenging function may be lowered in women with endometriosis; for example there is a notable increase in macrophage-derived secretion products in women with endometriosis associated with increases in cell proliferation, cell adhesion and neovascularization. CONCLUSIONS The pelvic peritoneum appears to play a key role in the development and maintenance of endometriosis.