Fibroblast-to-myofibroblast Transdifferentiation Research Articles

The role of fibrosis in endometriosis: a systematic review.

Fibrosis is an important pathological feature of endometriotic lesions of all subtypes. Fibrosis is present in and around endometriotic lesions, and a central role in its development is played by myofibroblasts, which are cells derived mainly after epithelial-to-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT). Transforming growth factor-β (TGF-β) has a key role in this myofibroblastic differentiation. Myofibroblasts deposit extracellular matrix (ECM) and have contracting abilities, leading to a stiff micro-environment. These aspects are hypothesized to be involved in the origin of endometriosis-associated pain. Additionally, similarities between endometriosis-related fibrosis and other fibrotic diseases, such as systemic sclerosis or lung fibrosis, indicate that targeting fibrosis could be a potential therapeutic strategy for non-hormonal therapy for endometriosis. This review aims to summarize the current knowledge and to highlight the knowledge gaps about the role of fibrosis in endometriosis. A comprehensive literature overview about the role of fibrosis in endometriosis can improve the efficiency of fibrosis-oriented research in endometriosis. A systematic literature search was performed in three biomedical databases using search terms for 'endometriosis', 'fibrosis', 'myofibroblasts', 'collagen', and 'α-smooth muscle actin'. Original studies were included if they reported about fibrosis and endometriosis. Both preclinical in vitro and animal studies, as well as research concerning human subjects were included. Our search yielded 3441 results, of which 142 studies were included in this review. Most studies scored a high to moderate risk of bias according to the bias assessment tools. The studies were divided in three categories: human observational studies, experimental studies with human-derived material, and animal studies. The observational studies showed details about the histologic appearance of fibrosis in endometriosis and the co-occurrence of nerves and immune cells in lesions. The in vitro studies identified several pro-fibrotic pathways in relation to endometriosis. The animal studies mainly assessed the effect of potential therapeutic strategies to halt or regress fibrosis, for example targeting platelets or mast cells. This review shows the central role of fibrosis and its main cellular driver, the myofibroblast, in endometriosis. Platelets and TGF-β have a pivotal role in pro-fibrotic signaling. The presence of nerves and neuropeptides is closely associated with fibrosis in endometriotic lesions, and is likely a cause of endometriosis-associated pain. The process of fibrotic development after EMT and FMT shares characteristics with other fibrotic diseases, so exploring similarities in endometriosis with known processes in diseases like systemic sclerosis, idiopathic pulmonary fibrosis or liver cirrhosis is relevant and a promising direction to explore new treatment strategies. The close relationship with nerves appears rather unique for endometriosis-related fibrosis and is not observed in other fibrotic diseases. N/A.

Augmentation of scleral glycolysis promotes myopia through histone lactylation

Myopia is characterized of maladaptive increases in scleral fibroblast-to-myofibroblast transdifferentiation (FMT). Scleral hypoxia is a significant factor contributing to myopia, but how hypoxia induces myopia is poorly understood. Here, we showed that myopia in mice and guinea pigs was associated with hypoxia-induced increases in key glycolytic enzymes expression and lactate levels in the sclera. Promotion of scleral glycolysis or lactate production induced FMT and myopia; conversely, suppression of glycolysis or lactate production eliminated or inhibited FMT and myopia. Mechanistically, increasing scleral glycolysis-lactate levels promoted FMT and myopia via H3K18la, and this promoted Notch1 expression. Genetic analyses identified a significant enrichment of two genes encoding glycolytic enzymes, ENO2 and TPI1. Moreover, increasing sugar intake in guinea pigs not only induced myopia but also enhanced the response to myopia induction via the scleral glycolysis-lactate-histone lactylation pathway. Collectively, we suggest that scleral glycolysis contributes to myopia by promoting FMT via lactate-induced histone lactylation.

The heterogeneity of fibrosis and angiogenesis in endometriosis revealed by single-cell RNA-sequencing

Fibrosis, angiogenesis and chronic inflammation are the inherent characteristics of endometriosis (EMS). The cellular heterogeneity of ectopic and non-ectopic endometrium by single-cell RNA-sequencing (scRNA-seq)at secretory phase without the disturbance of hormone drugs hasn't been explored so far. In this study, scRNA-seq was adopted to explore the properties of ectopic endometrium (ECE), eutopic endometrium (EUE) and normal endometrium (NOE) at secretory phase. We found that (i) The proportion of myofibroblasts, pericytes, endothelial cells and macrophages in ECE overwhelms that of non-ectopic tissues (EUE and NOE), and Myofibro.C2 was the predominant myofibroblast sub-cluster in ECE. (ii) Myofibroblasts were mainly fibroblast-to-myofibroblast transdifferentiation (FMT) and pericytes were endothelial cell-dependent differentiation in ECE. (iii) Both myofibroblasts and pericytes had a low differentiation potential. (iv) The increased inflammation score, deceased NK cells, T cell exhaustion score and antigen-presenting capacity in ECE confirmed the inflammatory properties and immunodeficiency of ECE. These findings suggested that myofibroblasts, pericytes and macrophages may be the potential targets for anti-fibrotic, anti-angiogenic and anti-inflammatory therapy of EMS.

Activation of α7 nicotinic acetylcholine receptor retards the development of endometriosis

BackgroundWomen with endometriosis have been shown to have a reduced vagal tone as compared with controls and vagotomy promoted while vagus nerve stimulation (VNS) decelerated the progression of endometriosis in mice. Extensive research also has shown that the activation of the cholinergic anti-inflammatory pathway by VNS activates α7 nicotinic acetylcholine receptor (α7nAChR), potently reducing inflammation. Yet whether α7nAChR plays any role in endometriosis is unknown. We evaluated its expression in normal endometrium, ovarian and deep endometriotic lesions, and evaluated its role in the development of endometriosis.MethodsImmunohistochemistry analyses of α7nAChR in endometriotic lesions as well as control endometrium, and quantification of tissue fibrosis by Masson trichrome staining were performed. Mouse experiments were conducted to evaluate the impact of α7nAChR activation or suppression on lesional progression and possible therapeutic effect. Finally, in vitro experiments were conducted to evaluate the effect of activation of α7nAChR on epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM) and fibrogenesis in an endometriotic epithelial cell line and primary endometriotic stromal cells derived from ovarian endometrioma tissue samples.ResultsImmunostaining of α7nAChR was significantly reduced in human endometriotic epithelial cells as compared with their counterpart in normal endometrium. Lesional α7nAChR staining levels correlated negatively with lesional fibrosis and the severity of dysmenorrhea. The α7nAChR agonist significantly impeded the development of endometriotic lesions in mouse models possibly through hindrance of EMT and FMT. It also demonstrated therapeutic effects in mice with induced deep endometriosis. Treatment of endometriotic epithelial and stromal cells with an α7nAChR agonist significantly abrogated platelet-induced EMT, FMT and SMM, and suppressed cellular contractility and collagen production.Conclusionsα7nAChR is suppressed in endometriotic lesions, and its activation by pharmacological means can impede EMT, FMT, SMM, and fibrogenesis of endometriotic lesions. As such, α7nAChR can be rightfully viewed as a potential target for therapeutic invention.Trial registrationNot applicable.

Tetramethylpyrazine Retards the Progression and Fibrogenesis of Endometriosis

The development of more efficacious, non-hormonal therapeutics for endometriosis is still an unmet medical need begging to be fulfilled. Growing evidence indicates that endometriotic lesions are wounds undergoing repeated tissue injury and repair, and, as such, platelets play an important role in lesional progression. Tetramethylpyrazine (TMP), a compound derived from a herb that has been used for thousands of years to combat “blood stasis” in traditional Chinese medicine, is a prescription drug in China for the treatment of cerebrovascular disorders. We tested the hypothesis that TMP can decelerate lesional progression through arresting epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), and fibrogenesis. We found in our in vitro experiments that TMP treatment suppresses platelet-induced EMT, FMT, cellular contractility, and collagen production in a concentration-dependent manner. We also showed that in a mouse model of endometriosis, treatment with TMP significantly reduced lesion weight and the extent of lesional fibrosis and improved hyperalgesia, mostly likely through the reduction of lesional aggregation of platelets and the lesional expression of markers of EMT, FMT, and fibrogenesis. In light of our results and in view of its excellent safety profiles, TMP appears to be a promising drug candidate for treating endometriosis.

Higher fibrotic content of endometriotic lesions is associated with diminished prostaglandin E2 signaling.

PurposeWhile the prevailing view holds that the prostaglandin E2 (PGE2) signaling plays a vital role in endometriosis, PGE2 also is known to be anti‐fibrotic. We investigated the immunostaining of COX‐2, EP2, and EP4, along with fibrotic content in ovarian endometrioma (OE) and deep endometriosis (DE) lesions, and in OE lesions from adolescent and adult patients. In addition, we evaluated the effect of substrate stiffness on the expression of COX‐2, EP2, and EP4 in endometrial stromal cells.MethodsImmunohistochemistry analysis of COX‐2, EP2, and EP4, along with the quantification of lesional fibrosis, was conducted for OE and DE lesion samples and also OE lesion samples from adolescent and adult patients. The effect of substrate rigidity on fibroblast‐to‐myofibroblast transdifferentiation (FMT) and the expression of COX‐2, EP2, and EP4, with or without TGF‐β1 stimulation, were investigated.ResultsThe immunostaining of COX‐2, EP2, and EP4 was substantially reduced in endometriotic lesions as lesions became more fibrotic. Both TGF‐β1 stimulation and stiff substrates induced FMT and reduced the expression of COX‐2, EP2, and EP4.ConclusionsSince fibrosis is a common feature of endometriosis, our results thus cast doubts on the use of therapeutics that suppresses the PGE2 signaling pathway, either by inhibiting COX‐2 or EP2/EP4.

PGE2 and Thrombin Induce Myofibroblast Transdifferentiation via Activin A and CTGF in Endometrial Stromal Cells.

Endometriosis is characterized by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal hemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, menstruation-related factors, PGE2 and thrombin (P/T), a PAR1 agonist induced epithelial-mesenchymal transition (EMT) of endometrial cells under hypoxia. However, the molecular mechanisms by which P/T induce development of endometriosis have not been fully characterized. To investigate the effects of P/T, RNA extracted from endometrial stromal cells (ESCs) treated with P/T were subjected to RNA sequence analysis, and identified activin A, FOS, and GATA2 as upregulated genes. Activin A increased the expression of connective tissue growth factor (CTGF) and mesenchymal marker genes in ESCs. CTGF induced the expression of fibrosis marker type I collagen, fibronectin, and α-smooth muscle actin (αSMA), indicating fibroblast to myofibroblast transdifferentiation (FMT) of ESCs. In addition, activin A, FOS, GATA2, CTGF, and αSMA were localized in endometriosis lesions. Taken together, our data show that P/T induces changes resembling EMT and FMT in ectopic ESCs derived from retrograde menstruation, and that these are associated with fibrotic changes in the lesions. Pharmacological means that block P/T-induced activin A and CTGF signaling may be strategies to inhibit fibrosis in endometriotic lesions.

Carbon nanotubes promote alveolar macrophages toward M2 polarization mediated epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation

With substantial progress of nanotechnology, carbon nanotubes (CNTs) are widely used in a variety of industrial and commercial applications. There is rising concern about potential adverse health effects, such as pulmonary fibrosis, related to inhalation of CNTs. The detailed cellular and molecular mechanisms of pulmonary fibrosis induced by CNTs are still not clear. Epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT) are considered as critical events in pathogenesis of pulmonary fibrosis. Alveolar macrophages (AMs) polarization plays a key role of regulating EMT and FMT in pulmonary fibrosis. In this study, we applied CNTs to stimulate primary mouse AMs under M1 or M2 polarization conditions, then analyzed the proportion of F4/80+CD11c+ or F4/80+CD206+ AMs, mRNA expression and activities of iNOS or Arg-1, as well as mRNA expression and content of TNF-α and IL-6 or TGF-β and IL-10 to evaluate dynamic phenotypic and functional changes of AMs. Single-walled CNT (SWCNT), short-type multi-walled CNT (MWCNT), and long-type MWCNT exposure at dose of 50 µg/ml promote AMs polarization toward M1 phenotype at early stage, while promote AMs polarization toward M2 phenotype at late stage. The roles of AMs polarization during development of EMT and FMT were further investigated by conditioned medium (CM) experiments. CNTs-activated M2 AMs promote progression of EMT and FMT via secreting TGF-β. Furthermore, up-regulating IRF4 may be involved in CNTs-induced M2 AMs polarization. In conclusion, this study demonstrates a new insight that CNTs exposure promotes AMs polarization toward M2 phenotype which facilitate EMT and FMT through secreting TGF-β.

Diagnosing Deep Endometriosis Using Transvaginal Elastosonography.

Transvaginal ultrasound (TVUS) and MRI are currently two mainstream imaging techniques used to diagnose deep endometriosis (DE) with comparable accuracy, but there is still ample room for improvement. As endometriotic lesions progress to fibrosis concomitant with the increase in tissue stiffness, transvaginal elastosonography (TVESG) is well-suited for diagnosing DE. To test the hypothesis that lesional stiffness as measured by TVESG correlates with the extent of lesional fibrosis, the markers of progression, hormonal receptor expression, and vascularity, we recruited 30 patients suspected to have DE who went through pelvic examination, TVUS and/or MRI, and TVESG and were ultimately diagnosed by histology. Their lesional tissue samples were subjected to immunohistochemistry analysis of markers for epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), estrogen and progesterone receptors (ERβ and PR), microvessel density (MVD), and vascularity, as well as quantification of lesional fibrosis. We found that pelvic examination, TVUS, and MRI detected 83.3%, 66.7%, and 83.3% of all DE cases, respectively, while TVESG detected them all. The lesions missed by pelvic exam, TVUS and MRI were significantly smaller than those detected but nonetheless had higher lesional stiffness. Lesional stiffness correlated closely and positively with the extent of lesional fibrosis, negatively with the markers of EMT, MVD, vascularity, and PR expression, but positively with the marker for FMT and ERβ. Thus, through the additional use of information on differential stiffness between DE lesions and their surrounding tissues, TVESG improves diagnostic accuracy, provides a ballpark estimate on the developmental stage of the lesions, and may help clinicians choose the best treatment modality.

Scutellarin Suppresses Platelet Aggregation and Stalls Lesional Progression in Mouse With Induced Endometriosis.

Platelets play an important role in the development of endometriosis. Scutellarin is a flavonoid isolated from a medicinal herb traditionally used as a potent antiplatelet agent. In this study, we sought to evaluate its potential therapeutic effect, if any, in mice with induced endometriosis. Endometriosis was induced in 27 female Balb/c mice by intraperitoneal injection of uterine fragments. Two weeks after the induction, the 27 mice were randomly divided in equal sizes into 3 groups: untreated, which received only vehicle, and low-dose and high-dose groups, which received low- and high dose of scutellarin treatment. Hotplate test was administrated to all mice before endometriosis induction, and before and after the scutellarin treatment. Two weeks after the treatment, a blood sample was drawn before sacrifice and all lesions were harvested. The peripheral platelet activation rate and total lesion weight were assessed, and immunohistochemistry and histochemistry analyses were performed to evaluate the extent of proliferation, angiogenesis, fibroblast-to-myofibroblast transdifferentiation (FMT), and fibrosis in lesions. Compared with untreated mice, mice in both low-dose and high-dose groups had significantly reduced lesion weight and improved hyperalgesia. Scutellarin also reduced the peripheral-activated platelets rate and resulted in significantly reduced platelet aggregation, cellular proliferation, angiogenesis, the extent of FMT, and the extent of fibrosis in lesions. Thus, we conclude that scutellarin is efficacious in treating endometriosis in vivo by suppressing platelet aggregation, inhibiting proliferation, angiogenesis, and fibrogenesis, resulting in reduced lesion size and improved pain behavior. As such, scutellarin may be a potentially promising therapeutics for the treatment of endometriosis.

Neuropeptides Substance P and Calcitonin Gene Related Peptide Accelerate the Development and Fibrogenesis of Endometriosis

Endometriotic lesions are known to be hyperinnervated, especially in lesions of deep endometriosis (DE), which are frequently in close proximity to various nerve plexuses. DE lesions typically have higher fibromuscular content than that of ovarian endometriomas (OE) lesions, but the underlying reason remains elusive. Aside from their traditional role of pain transduction, however, whether or not sensory nerves play any role in the development of endometriosis is unclear. Here, we show that, thorough their respective receptors neurokinin receptor 1 (NK1R), calcitonin receptor like receptor (CRLR), and receptor activity modifying protein 1 (RAMP-1), neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) induce epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT) and further turn stromal cells into smooth muscle cells (SMCs) in endometriotic lesions, resulting ultimately in fibrosis. We show that SP and CGRP, or the rat dorsal root ganglia (DRG) supernatant, through the induction of NK1R and CGRP/CRLR/RAMP-1 signaling pathways, promoted EMT, FMT and SMM in endometriosis, resulting in increased migratory and invasive propensity, cell contractility, production of collagen, and eventually to fibrosis. Neutralization of NK1R and/or CGRP/CRLR/RAMP-1 abrogated these processes. Extended exposure of endometriotic stromal cells to SP and/or CGRP or the DRG supernatant induced increased expression of α-SMA, desmin, oxytocin receptor, and smooth muscle myosin heavy-chain. Finally, we show that DE lesions had significantly higher nerve fiber density, increased staining levels of α-SMA, NK1R, CRLR, and RAMP-1, concomitant with higher lesional fibrotic content than that of OE lesions. The extent of lesional fibrosis correlated positively with the staining levels of NK1R, CRLR, and RAMP-1, as well as the nerve fiber density in lesions. Thus, this study provides another piece of evidence that sensory nerves play an important role in promoting the development and fibrogenesis of endometriosis. It explains as why DE frequently have higher fibromuscular content than that of OE, highlights the importance of lesional microenvironment in shaping the lesional fate, gives more credence to the idea that ectopic endometrium is fundamentally wounds that go through repeated tissue injury and repair, and should shed much needed light into the pathophysiology of endometriosis.

Sensory nerve-derived neuropeptides accelerate the development and fibrogenesis of endometriosis.

Do sensory nerves play any role in the development of endometriosis? Sensory nerves participate in all major steps (epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT) and smooth muscle metaplasia (SMM)) in the development and fibrogenesis of endometriotic lesions. Endometriotic lesions are known to be hyperinnervated due to neurogenesis resulting from neutrophins secreted by endometriotic lesions and possibly platelets. These neutrophins seem to preferentially favour production of sensory neurons at the expense of sympathetic neurons. Three independent, yet complementary, prospective, randomized mouse experimentations were conducted. A total of 143 female Balb/C mice and 24 female immunodeficient nude Balb/C mice were used. The mice were sacrificed 2 or 4 weeks after the induction of endometriosis. In Experiment 1, 21 mice were randomly divided into three groups of equal size for sympathetic denervation, sensory denervation and controls. Denervation was carried out chemically. In Experiment 2, 24 nude mice were randomly divided into three equal-sized groups: the BEFORE and AFTER groups that respectively received surgical denervation 3 days before or after the induction of endometriosis by subcutaneous grafting of human endometriotic tissues, and the Control group that received a sham surgery without denervation 3 days before induction. For Experiments 1 and 2, all mice were sacrificed two weeks after induction of endometriosis. In Experiment 3, substance P (SP) and aprepitant, a potent and selective neurokinin 1 receptor (NK1R) antagonist, were used to activate and inhibit the NK1R signalling pathway, respectively. A total of 32 mice were randomly divided into four groups of equal sizes: control (CTL), SP, Before-Induction and After-Induction. One day before the induction of endometriosis, mice in CTL, SP and Before-Induction groups were infused with sterile saline, SP and aprepitant, respectively, via Alzet osmotic pumps. Two weeks after the induction, the After-induction group was infused with aprepitant in similar fashion. All mice were sacrificed four weeks after the induction of endometriosis. In all three experiments, the bodyweight and hotplate latency were evaluated before induction and sacrifice. In addition, all lesions were excised, weighed and processed for quantification and immunohistochemistry analysis of markers for EMT, FMT and SMM, and the extent of fibrosis was evaluated by Masson trichrome staining. In Experiment 1, chemical denervation of sympathetic and sensory nerves reduced the lesion weight by 43.2% (±23.1%) and 68.7% (±20.3%), respectively, as compared with controls. In particular, sensory denervation led to significantly greater reduction in lesion weight than sympathetic denervation. Sensory denervation also resulted in significantly improved hyperalgesia as compared with controls. In contrast, sympathetic denervation yielded only transient improvement in hyperalgesia. Both sympathetic and sensory denervation resulted in lower immunoreactivity against markers of proliferation and fibrosis, especially sensory denervation.In Experiment 2, surgical denervation before or after induction of endometriosis also decelerated the development of endometriosis, as manifested by significantly reduced lesion weight and extent of lesional fibrosis, along with improved hyperalgesia.In Experiment 3, NK1R activation by SP infusion accelerated lesional development, as evidenced by significantly increased lesional weight, more thorough progression of EMT, FMT, SMM, exaggerated lesional fibrosis and deteriorated hyperalgesia. In contrast, NK1R antagonism decelerated lesional development and improved hyperalgesia. N/A. This study is limited by the use of histologic and immunohistochemistry analyses only and the lack of molecular data. Since sensory nerves are known to be important in wound healing and fibrogenesis, our findings also give more credence to the notion that endometriotic lesions are wounds undergoing repeated tissue injury and repair. As such, sensory nerves or the NK1R signalling pathway in particular may be potential targets for intervention. This research was supported by Grants 81471434 (SWG), 81530040 (SWG), 81771553 (SWG), 81671436 (XSL) and 81871144 (XSL) from the National Natural Science Foundation of China and an Excellence in Centres of Clinical Medicine grant (2017ZZ01016) from the Science and Technology Commission of Shanghai Municipality. None of the authors have anything to disclose.

The establishment of a mouse model of deep endometriosis.

Is it possible to establish a mouse model of deep endometriosis (DE)? A mouse DE model that is macroscopically and microscopically similar to nodular lesions in humans can be constructed in as short as 3 weeks by intraperitoneal injection of uterine fragments along with the infusion of substance P (SP) and/or calcitonin gene-related peptide (CGRP). Although a baboon DE model was reported 5 years ago, its prohibitive cost and demand for facilities and expertise associated with the use of non-human primates put its use out of reach for most laboratories. A total of 48 female Balb/C mice were used for this study. Among them, 16 were randomly selected as donors that contributed uterine fragments, and the remaining 32 were recipient mice. The mice with induced endometriosis were followed up for 3-4 weeks. One day before the induction of endometriosis by intraperitoneal injection of uterine fragments, osmotic pumps were inserted into equal groups of recipient mice to infuse either sterile saline, SP, CGRP, or both SP and CGRP. The hotplate test was administrated to all mice at the baseline and before and after induction of endometriosis. Four (3 for the SP+CGRP group) weeks after induction, all mice were sacrificed. Their endometriotic lesions were excised, weighed and processed for histopathologic examination, and histochemistry, immunohistochemistry and immunofluorescence analyses of markers of proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM), mesothelial-mesenchymal transition (MMT) and endothelial-mesenchymal transition (EndoMT) were done. The extent of lesional fibrosis was evaluated by Masson trichrome staining. To further evaluate surrounding organ/tissue invasion, the peritoneal areas adhesive to the lesions were excised for immunohistochemical analysis. Endometriotic lesions in mice treated with SP and/or CGRP satisfied all requirements for DE, i.e. presence of endometrial epithelial and stromal cells, abundance of fibromuscular content, and encapsulation in surrounding tissues or organs. The lesion weight in the CGRP, SP and SP+CGRP groups was 1.62, 2.14 and 2.18-fold, respectively, heavier than that of control group. Concomitantly, the SP, CGRP and SP+CGRP groups had significantly shorter hotplate latency than that of control group. Lesions in mice treated with SP and/or CGRP, especially with SP+CGRP, exhibited characteristics consistent with EMT, FMT, SMM and extensive fibrosis, along with signs of MMT and EndoMT. Lesional invasion into surrounding tissues/organs was found to be 25.0, 75.0 and 87.5% in mice treated with CGRP, SP and SP+CGRP, but none in control mice. N/A. This study is limited by the use of histologic and immunohistochemistry analyses only and lacks molecular data. The establishment of a mouse DE model supports the idea that endometriotic lesions are wounds undergoing repeated tissue injury and repair and underscores the importance of microenvironments in shaping the lesions' destiny. In addition, signs consistent with MMT and EndoMT indicate that there may be more culpable factors that still remain unidentified and should be pursued in the future. Moreover, the close correlation between the extent of lesional fibrosis and markers of EMT, MMT, EndoMT, FMT and SMM as shown here should facilitate our understanding of the molecular mechanisms underlying the DE pathophysiology. Since this DE model is based on a biologically plausible and evidence-backed theory, it should shed much needed insight into the molecular mechanisms underlying the pathophysiology of DE. This research was supported by Grants 81471434 (S.W.G.), 81530040 (S.W.G.), 81771553 (S.W.G.), 81671436 (X.S.L.) and 81871144 (X.S.L.) from the National Natural Science Foundation of China. None of the authors has any conflict of interest to disclose.

Histological and Immunohistochemical Characterization of the Similarity and Difference Between Ovarian Endometriomas and Deep Infiltrating Endometriosis.

Ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE) have long been recognized to have different histology and, as such, postulated to be 2 separate disease entities. Few studies, however, have attempted to elucidate the causes for their differences. Making use of ectopic endometrial tissue samples from 25 and 20 women with OMA and DIE, respectively, and control endometrial tissue samples from 25 women without endometriosis, we conducted an immunohistochemical analysis to evaluate the expression of a group of carefully chosen markers for epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM), fibrosis, vascularity, hormonal receptors, and proteins involved in epigenetic modifications. We found that both OMA and DIE lesions exhibited the same cellular changes consistent with EMT, FMT, SMM, and fibrosis as already shown in animal models. Compared to OMA, DIE lesions underwent more thorough and extensive EMT, FMT, and SMM and, consequently, displayed significantly higher fibrotic content but less vascularity. The 2 conditions also showed different expression levels of hormonal receptors. Both OMA and DIE lesions, especially the latter, showed significantly higher staining of enhancer of zeste homolog 2, H3K9me3, and H3K27me3 than that of control endometrium, suggesting progressive epigenetic changes concomitant with cellular ones. Finally, proteins that are known to be involved in fibrogenesis, such as thymocyte differentiation antigen 1 and peroxisome proliferator-activated receptor γ , were also aberrantly expressed under both conditions. The many similarities shared by both OMA and DIE indicate that the 2 conditions may actually share the same pathogenesis/pathophysiology. Their differences, however, suggest that the source of these differences may result from the different lesional microenvironments.

Progressive development of endometriosis and its hindrance by anti-platelet treatment in mice with induced endometriosis

We have recently shown that platelets drive smooth muscle metaplasia (SMM) and fibrogenesis in endometriosis through epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT). To see whether this is true in vivo, this prospective, randomized, and serially evaluated mouse investigation was conducted. Endometriosis was induced in female Balb/C mice, which were then randomly divided into two groups: Tanshinone IIA (TAN) and control (CTL) groups. TAN mice were treated with TAN but CTL mice received none. Every week until the 6th week after induction, five mice from each group were killed. Lesion weight was measured and lesion samples were subjected to immunohistochemistry and histochemistry analysis of platelet aggregation (CD41), E-cadherin, TGF-β1, phosphorylated Smad3, α-SMA, collagen I, CCN2, LOX, desmin and SM-MHC, and the extent of fibrosis was evaluated by Masson trichrome staining. It was found that endometriotic lesions exhibited progressive cellular changes consistent with the progressive EMT, FMT, SMM, and fibrogenesis. TAN treatment resulted in significant hindrance of EMT, FMT, SMM and fibrogenesis, and reduced lesion weight (all P-values <0.05). These data corroborate the notion that endometriotic lesions undergo progressive EMT and FMT, giving rise to SMM and ultimately fibrosis. This understanding sheds new light onto the natural history of endometriosis.

Anti-platelet therapy holds promises in treating adenomyosis: experimental evidence

BackgroundRecently emerging evidence indicates that endometriotic lesions are wounds undergoing repeated tissue injury and repair (ReTIAR), and platelets induce epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), leading ultimately to fibrosis. Due to the commonality of cyclic bleeding as in endometriosis, adenomyotic lesions are also wounds that undergo ReTIAR, and we have recently provided evidence corroborating platelet-induced EMT, FMT and fibrogenesis in adenomyosis. This study sought to evaluate the effect of antiplatelet therapy in a mouse model of adenomyosis.MethodsAdenomyosis was induced in 57 female ICR mice with neonatal dosing of tamoxifen, while another 12 (group C) were dosed with solvent only, serving as a blank control. Starting from 4 weeks after birth, hotplate test was administrated to all mice every 4 weeks. At the 16th week, all mice with induced adenomyosis were randomly divided into 6 groups: untreated, low- and high-dose Ozagrel, low- and high-dose anti-mouse GPIbα polyclonal IgG antibody to deplete platelets, and isotype-matched inert IgG non-immune antibody. Group C received no treatment. After 3 weeks of treatment, they were hotplate tested again, their uterine horns and brains were harvested, and a blood sample was taken to measure the plasma corticosterone level by ELISA. The left uterine horn was used for immunohistochemistry analysis. The brainstem nucleus raphe magnus (NRM) sections were subjected to immunofluorescence staining for GAD65. The depth of myometrial infiltration and uterine contractility were evaluated.ResultsWe found that both Ozagrel treatment and platelet depletion dose-dependently suppressed myometrial infiltration, improved generalized hyperalgesia, reduced uterine contractility, and lowered plasma corticosterone levels, improved the expression of some proteins known to be involved in adenomyosis and slowed down the process of fibrogenesis. It also elevated the number of GAD65-expressing neurons in the brainstem NRM, possibly boosting the GABAergic inhibition of pain due to adenomyosis.ConclusionThis study further provides evidence that platelets play important roles in the development of adenomyosis. Anti-platelet treatment is efficacious in suppression of myometrial infiltration, improving generalized hyperalgesia, reducing uterine hyperactivity and systemic corticosterone levels. Collectively, these results demonstrate that anti-platelet therapy seems to be promising for treating adenomyosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0198-1) contains supplementary material, which is available to authorized users.

Cellular Changes Consistent With Epithelial-Mesenchymal Transition and Fibroblast-to-Myofibroblast Transdifferentiation in the Progression of Experimental Endometriosis in Baboons.

We have recently shown that platelets play important roles in development of endometriosis and proposed that endometriotic lesions are essentially wounds that undergo repeated tissue injury and repair (ReTIAR). Further investigation indicated that endometriotic lesions, stimulated by platelet-derived transforming growth factor β1 (TGF-β1), activate the TGF-β1/Smad3 signaling pathway and undergo epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT), resulting in increased cellular contractility and collagen production and increased smooth muscle metaplasia (SMM), leading to fibrosis. Using serially dissected endometriotic tissue samples from baboons with induced endometriosis, we tested the hypothesis of progressive EMT, FMT, SMM, and fibrosis through TGF-β1/Smad activation using immunohistochemistry and immunoflurescence staining analyses. We found that platelets are aggregated in endometriotic lesions, and vimentin expression was increased in the epithelial compartment of the lesions as they progressively developed. We also found that the number of smooth muscle cells (SMCs) appeared to increase with time as lesions progressed and was concomitant with the increased vimentin-positive glandular epithelial cells in the lesions. As lesion development progressed, TGF-β1 and phosphorylated-Smad3 staining was elevated and the number of α-smooth muscle actin-positive myofibroblasts and highly differentiated SMCs increased in the stromal compartment, which correlated with the increasing extent of fibrosis. These results, taken together, provide support for the notion that ReTIAR occurs in the endometriotic lesions, resulting in EMT and FMT, leading to SMM and ultimately fibrosis as lesions progress. Consequently, our data also provide corroborative evidence that platelets drive the EMT and FMT in endometriotic lesions over time, promoting SMM and resulting ultimately in fibrosis in the endometriotic lesions. These findings cast a new light on the natural history of endometriosis which so far has been elusive.

Platelets drive smooth muscle metaplasia and fibrogenesis in endometriosis through epithelial–mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation

Smooth muscle metaplasia (SMM) and fibrotic tissues are frequently seen in endometriotic lesions, yet the mechanisms underlying their formation are poorly understood. In this study, we investigated the roles of activated platelets in driving epithelial–mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT) in endometriosis. Through in vitro experimentations, we found that activated platelets, through the release of TGF-β1 and the induction of TGF-β/Smad signaling pathway, promoted EMT and FMT in endometriosis, resulting in increased cell contractility, collagen production, and ultimately to fibrosis. TGF-β blockade reversed these processes. Prolonged exposure of endometriotic stromal cells to activated platelets induced increased expression of α-SMA as well as markers of differentiated smooth muscle cells. Consequently, endometriotic lesions and their microenvironment contain all the necessary molecular machinery to promote SMM and fibrogenesis. Our results suggest that endometriotic lesions are wounds that undergo repeated injury and healing, highlighting the importance of platelets in the development of endometriosis.

Corroborating evidence for platelet-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis.

Do platelets play any role in the development of adenomyosis? As in endometriosis, adenomyotic lesions show significantly increased platelet aggregation, increased expression of transforming growth factor (TGF)-β1, phosphorylated Smad3, markers of epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT), and smooth muscle metaplasia (SMM), in conjunction with increased fibrosis as compared with normal endometrium. Both EMT and FMT are known to play vital roles in fibrogenesis in general and in endometriosis in particular. EMT has been implicated in the development of adenomyosis. SMM is universally seen in endometriosis and also in adenomyosis, and is correlated positively with the extent of fibrosis. However, there has been no published study on the role of platelets in fibrogenesis in adenomyosis, even though adenomyotic lesions undergo repeated cycles of tissue injury and repair, which suggests the involvement of platelets and their possible roles in fibrogenesis. Cross-sectional studies of ectopic endometrial and control endometrial tissue samples from three sets of women with and without adenomyosis (n= 34 and 20, 12 and 10, and 8 and 8, respectively) were carried out from 2014 to 2015. Immunohistochemistry analysis of ectopic endometrial tissues from women with (n= 34) and without (n= 20) adenomyosis with respect to biomarkers of EMT, FMT and highly differentiated smooth muscle cells as well as TGF-β1, phosphorylated Smad3, markers of proliferation, angiogenesis and extracellular matrix (ECM) deposits. Masson trichrome staining, Van Gieson staining and Pico-Sirius staining were performed to evaluate and quantify the extent of fibrosis in lesions. Progesterone receptor isoform B (PR-B) staining also was performed. In addition, CD42b-positive platelets in ectopic (n= 12) and control (n= 10) endometrium were counted by confocal microscopy and compared. The protein expression levels of TGF-β1 and phosphorylated Smad3 in both ectopic (n= 8) and control (n= 8) endometrium were measured by western blot analysis. Immunofluorescent staining of both platelets and hepatocyte growth factor (HGF) was also performed for adenomyotic tissue samples (n= 10). Adenomyotic lesions had a significantly higher extent of platelet aggregation and increased staining for TGF-β1 and phosphorylated Smad3 (both P-values <0.001 versus control). In addition, E-cadherin staining was decreased while vimentin staining in adenomyotic epithelial cells was increased, along with increased staining of proliferating cell nuclear antigen, vascular endothelial growth factor and CD31 (all P-values <0.001), markers of proliferation and angiogenesis. Staining for α-SMA, a marker for myofibroblast, desmin, smooth muscle myosin heavy chain and oxytocin receptor was significantly increased in adenomyotic lesions versus control, concomitant with increased staining of collagen I and lysyl oxidase (all P-values <0.001). Histochemistry analysis indicates that the extent of fibrosis is high in adenomyotic lesions (P < 0.001), and the extent appeared to correlate negatively with the microvessel density (P < 0.05). PR-B staining was significantly decreased in adenomyotic lesion as compared with control endometrium (P < 0.001). Platelets and HGF were co-localized mostly in the stromal component of adenomyotic lesions, near the glandular epithelium. The results are limited by the cross-sectional nature of the study and the use of histochemistry and immunohistochemistry analyses only, but nonetheless is a validation of our previous finding in mouse experiments. The data presented are consistent with the notion that platelet-induced activation of the TGF-β/Smad signaling pathway may be a driving force in EMT, FMT and SMM in the development of adenomyosis, leading to fibrosis. This study provides the first piece of evidence that adenomyotic lesions are wounds that undergo repeated injury and healing, and, as such, platelets play critical roles in the development of adenomyosis by promoting proliferation, angiogenesis, increasing ECM deposits, and SMM, resulting in fibrosis. Platelets may also be involved in uterine hyperactivity and myometrial hyperinnervation. Our results provide one explanation as to why adenomyosis is a challenge for medical treatment, and shed new light onto the pathophysiology of adenomyosis. Support for data collection and analysis was provided by grants from the National Science Foundation of China. None of the authors has anything to disclose.

Transforming growth factor β1 signaling coincides with epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis in mice.

Do platelets have any role in the development of adenomyosis? Activated platelets coincide with the release of transforming growth factor (TGF)-β1 and induction of the TGF-β/Smad signaling pathway as well as evidence of epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT) in a mouse model of adenomyosis, resulting ultimately in fibrosis, as in adenomyosis. Both EMT and FMT are known to play vital roles in fibrogenesis in general and in endometriosis in particular. EMT has been implicated in the development of adenomyosis, but this was based primarily on cross-sectional observation. It is unclear as to whether adenomyotic lesions and their microenvironment have the machinery to promote EMT and FMT, resulting ultimately in fibrosis. There has not been any published study on the role of platelets in the development of adenomyosis, even though adenomyotic lesions undergo repeated cycles of tissue injury and repair, which implicates the involvement of platelets and constitutes an environment conducive for fibrogenesis. Adenomyosis was induced in 28 female ICR mice by neonatal dosing of tamoxifen. Another 32 were neonatally dosed without tamoxifen. These mice were sacrificed serially and their tissue samples were subsequently evaluated. Female ICR mice with and without induced adenomyosis were sacrificed in batch at 5, 10, 15, 42 and 60 days of age. The depth of myometrial infiltration of endometrial tissues was assessed and immunohistochemistry analysis of biomarkers of EMT and FMT, as well as TGF-β1, phosphorylated Smad3 (p-Smad3) and markers of proliferation, angiogenesis and extracellular matrix (ECM) deposits was performed in ectopic (for adenomyotic mice) and eutopic (controls) endometrial tissue samples. Masson trichrome and Van Gieson stainings were performed to quantify the extent of fibrosis in lesions. Progesterone receptor isoform B (PR-B) staining also was performed. While TGF-β1 immunoreactivity was consistently low in control endometrium, its level was increased dramatically starting from Day 10, along with the extent of platelet aggregation. Staining for TGF-β1 and p-Smad3 increased progressively as adenomyosis progressed, along with markers for proliferation, angiogenesis and ECM deposits. Consistently, staining of vimentin (a marker for stromal or mesenchymal cells) was also increased while that of E-cadherin (a marker for epithelial cells) was reduced. PR-B staining also decreased progressively. Starting from Day 42, α-SMA staining, a marker for myofibroblasts, was elevated in lesions, while in control endometrium, it was negative. Concomitantly, the extent of fibrosis also was increased. This study is limited by the use of histochemistry and immunohistochemistry analyses only and the lack of intervention. Like their endometriotic counterpart, adenomyotic lesions and their microenvironment may contain all the necessary molecular machinery to promote fibrogenesis. Platelet-induced activation of the TGF-β/Smad signaling pathway may be a driving force in EMT and FMT in the development of adenomyosis, leading to fibrosis. This study provides the first piece of evidence that adenomyotic lesions are wounds that undergo repeated injury and healing, and as such, platelets play critical roles in the development of adenomyosis. It suggests the potential for the use of anti-platelet therapy in the treatment of adenomyosis, and also opens a new venue for developing novel biomarkers for diagnostic or prognostic purposes. Support for data collection and analysis was provided by grants from the National Science Foundation of China. None of the authors has anything to disclose.