MSCs promote the efferocytosis of large peritoneal macrophages to eliminate ferroptotic monocytes/macrophages in the injured endometria.

BACKGROUND: Endometria are one of the important components of the uterus, which is located in the peritoneal cavity. Endometrial injury usually leads to intrauterine adhesions (IUA), accompanied by inflammation and cell death. We previously reported that both the endometrial ferroptosis was increased and monocytes/macrophages were involved in endometrial injury of IUA. Large peritoneal macrophages (LPMs) are recently reported to migrate into the injured tissues and phagocytose dead cells to repair the tissues. We previously demonstrated that mesenchymal stromal cells (MSCs) had made excellent progress in the repair of endometrial injury. However, it is unclear whether MSCs regulate the LPM efferocytosis against ferroptotic monocytes/macrophages in the injured endometria. METHODS: Here, endometrial injury in IUA mouse model was conducted by uterine curettage and LPS injection surgery and the samples were collected at different times to detect the changes of LPMs and ferroptotic monocytes/macrophages. We conducted LPMs depletion assay in vivo and LPMs and Erastin-induced ferroptotic THP-1 cells coculture systems in vitro to detect the LPM efferocytosis against ferroptotic monocytes/macrophages. The IUA model was treated with MSCs, and their effects on LPMs and endometrial repair were analyzed. Flow cytometry, western blotting, quantitative real-time PCR, immunohistochemical analysis, ELISA, and RNA-sequencing were performed. RESULTS: We found that LPMs migrated to the injured uteri in response to the damage in early phase (3 h), and sustained to a later stage (7 days). Astonishingly, we found that ferroptotic monocytes/macrophages were significantly increased in the injured uteri since 12 h after injury. Moreover, LPMs cocultured with Erastin-induced ferroptotic THP-1 cells in vitro, efferocytosis of LPMs against ferroptotic monocytes/macrophages was emerged. The mRNA expression profiles revealed that LPM efferocytosis against ferroptotic monocytes/macrophages was an induction of glycolysis program and depended on the PPARγ-HK2 pathway. Importantly, we validated that MSCs promoted the efferocytic capability and migration of LPMs to the injured uteri via secreting stanniocalcin-1 (STC-1). CONCLUSION: The data collectively demonstrated first the roles of LPMs via removal of ferroptotic monocytes/macrophages and provided a novel mechanism of MSCs in repairing the endometrial injury.

Vaginal reconstruction by collagen scaffolds loaded with vaginal epithelial and smooth muscle cells in pigs.

In women, a healthy and functional vagina is important for the maintenance of a good quality of life. Various factors, including congenital anomalies, cancer, trauma, infections, inflammation, or iatrogenic injuries, can lead to damage or loss of the vaginal structure, necessitating repair or replacement. Often, such reconstruction procedures involve the use of nonvaginal tissue substitutes, like segments of the large intestine or skin, which are less than ideal both anatomically and functionally. Therefore, there is an urgent need to develop new methods of vaginal reconstruction. In this study, we established a new method for isolation and expansion of vaginal epithelial and smooth muscle cells. Subsequently, collagen scaffolds designed for vaginal reconstruction were loaded with vaginal epithelial and smooth muscle cells in vitro and tested in vivo using the vaginal excision pig model. The results showed that the collagen scaffold loaded with vaginal epithelial and smooth muscle cells significantly promotes the reconstruction of the vagina compared with small intestinal submucosa (SIS) membrane or bare collagen scaffold. Notably, the reconstructed vaginal tissues exhibit remarkable similarity to their normal counterparts, encompassing not only the vaginal epithelium and smooth muscle but also the intricate networks of blood vessels and nerves. These compelling results underscore the feasibility of a tissue engineering approach in vaginal reconstruction, offering promising prospects for improving the quality of life in affected individuals.

Targeting CD301+ macrophages inhibits endometrial fibrosis and improves pregnancy outcome.

Macrophages are a key and heterogeneous cell population involved in endometrial repair and regeneration during the menstrual cycle, but their role in the development of intrauterine adhesion (IUA) and sequential endometrial fibrosis remains unclear. Here, we reported that CD301+ macrophages were significantly increased and showed their most active interaction with profibrotic cells in the endometria of IUA patients compared with the normal endometria by single-cell RNA sequencing, bulk RNA sequencing, and experimental verification. Increasing CD301+ macrophages promoted the differentiation of endometrial stromal cells into myofibroblasts and resulted in extracellular matrix accumulation, which destroyed the physiological architecture of endometrial tissue, drove endometrial fibrosis, and ultimately led to female infertility or adverse pregnancy outcomes. Mechanistically, CD301+ macrophages secreted GAS6 to activate the AXL/NF-κB pathway, upregulating the profibrotic protein synthesis. Targeted deletion of CD301+ macrophages or inhibition of AXL by Bemcentinib blunted the pathology and improved the outcomes of pregnancy in mice, supporting the therapeutic potential of targeting CD301+ macrophages for treating endometrial fibrosis.

Ferroptosis contributes to endometrial fibrosis in intrauterine adhesions.

Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a challenging clinical issue in reproductive medicine. We previously demonstrated that epithelial-mesenchymal transition (EMT) and fibrosis of endometrial stromal cells (HESCs) played a vital role in the development of IUA, but the precise pathogenesis remains elucidated. Ferroptosis has now been recognized as a unique form of oxidative cell death, but whether it is involved in endometrial fibrosis remains unknown. In the present study, we performed an RNA-seq of the endometria from 4 severe IUA patients and 4 normal controls. Enrichment analysis and protein-protein interactions (PPIs) network analysis of differentially expressed genes (DEGs) were conducted. Immunohistochemistry was used to assess ferroptosis levels and cellular localization. The potential role of ferroptosis for IUA was investigated by in vitro and in vivo experiments. Here, we demonstrated that ferroptosis load is increased in IUA endometria. In vitro experiments showed that erastin-induced ferroptosis promoted EMT and fibrosis in endometrial epithelial cells (P < 0.05), but did not lead to pro-fibrotic differentiation in endometrial stromal cells (HESCs). Cell co-culture experiments showed that erastin-stimulated epithelial cell supernatants promoted fibrosis in HESCs (P < 0.05). In vivo experiments suggested that elevation of ferroptosis level in mice by erastin led to mild endometrial EMT and fibrosis. Meanwhile, the ferroptosis inhibitor Fer-1 significantly ameliorated endometrial fibrosis in a dual-injury IUA murine model. Overall, our findings revealed that ferroptosis may serve as a potential therapeutic target for endometrial fibrosis in IUA.

MFG-E8 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells through GSK3ß/ß-catenin signaling pathway.

Fracture nonunion and bone defects are challenging for orthopedic surgeons. Milk fat globule-epidermal growth factor 8 (MFG-E8), a glycoprotein possibly secreted by macrophages in a fracture hematoma, participates in bone development. However, the role of MFG-E8 in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. We investigated the osteogenic effect of MFG-E8 in vitro and in vivo. The CCK-8 assay was used to assess the effect of recombinant human MFG-E8 (rhMFG-E8) on the viability of hBMSCs. Osteogenesis was investigated using RT-PCR, Western blotting, and immunofluorescence. Alkaline phosphatase (ALP) and Alizarin red staining were used to evaluate ALP activity and mineralization, respectively. An enzyme-linked immunosorbent assay was conducted to evaluate the secretory MFG-E8 concentration. Knockdown and overexpression of MFG-E8 in hBMSCs were established via siRNA and lentivirus vector transfection, respectively. Exogenous rhMFG-E8 was used to verify the in vivo therapeutic effect in a tibia bone defect model based on radiographic analysis and histological evaluation. Endogenous and secretory MFG-E8 levels increased significantly during the early osteogenic differentiation of hBMSCs. Knockdown of MFG-E8 inhibited the osteogenic differentiation of hBMSCs. Overexpression of MFG-E8 and rhMFG-E8 protein increased the expression of osteogenesis-related genes and proteins and enhanced calcium deposition. The active ß-catenin to total ß-catenin ratio and the p-GSK3ß protein level were increased by MFG-E8. The MFG-E8-induced enhanced osteogenic differentiation of hBMSCs was partially attenuated by a GSK3ß/ß-catenin signaling inhibitor. Recombinant MFG-E8 accelerated bone healing in a rat tibial-defect model. In conclusion, MFG-E8 promotes the osteogenic differentiation of hBMSCs by regulating the GSK3ß/ß-catenin signaling pathway and so, is a potential therapeutic target.

The UCMSC-bFGF/Scaffold System Accelerates the Healing of the Uterine Full-Thickness Injury.

Severe uterine injury is a major cause of endometrial scar formation and female infertility. At present, the methods for accelerating injured uterine healing are still lacking. Genetic engineering modification of mesenchymal stem cells (MSCs) has been shown great promise in preclinical studies on regeneration. Here, we constructed a type of umbilical cord MSCs (UC-MSCs) with overexpressed basic fibroblast growth factor (UCMSC-bFGF) and investigated the effects of the UCMSC-bFGF/scaffold on functional regeneration of the full-thickness defect uterus of the rat model. At days 7, 14, and 30 after treatments, the rats were killed and the injured uterus was observed. The structural and functional change of uterine was assessed by hematoxylin and eosin staining, immunohistochemical staining, and fertility experiment. The UCMSC-bFGF/scaffold group exhibited anti-inflammatory effect, and the number of CD45+ cell in the UCMSC-bFGF/scaffold group was significantly less than that in UC-MSCs/scaffold group and scaffold group, but higher than sham-operated group at day 7 postmending. At day 14, the UCMSC-bFGF/scaffold group exhibited dramatically proangiogenesis efficacy compared with UC-MSCs/scaffold group and scaffold group. At day 30, the endometrial thickness, structure of myometrium, and blood vessels in the UCMSC-bFGF/scaffold were better than those of the UC-MSCs/scaffold group and scaffold group, even close to sham-operated group. Implantation rate at injury region postoperation 30 days in the UCMSC-bFGF/scaffold group (8/16) was significantly higher than that in UC-MSCs/scaffold group (1/16) and scaffold group (0/16). Taken together, the UCMSC-bFGF/scaffold system suppressed local inflammation, promoted angiogenesis, and accelerated regeneration of the defected uterine wall, and thereby greatly shortened the healing time of the injured uterus. Impact statement In this study, we used umbilical cord mesenchymal stem cells (UC-MSCs) with stably overexpressed basic fibroblast growth factor (UCMSC-bFGF) to repair the full-thickness defect uterine wall of the rat model and found that the UCMSC-bFGF/scaffold system suppressed early acute inflammation after uterus injury, promoted angiogenesis, and accelerated regeneration of the injured uterine wall.

Clinical effectiveness of a pneumatic compression device combined with low-molecular-weight heparin for the prevention of deep vein thrombosis in trauma patients: A single-center retrospective cohort study.

BACKGROUND: To investigate the clinical effectiveness of a pneumatic compression device (PCD) combined with low-molecular-weight heparin (LMWH) for the prevention and treatment of deep vein thrombosis (DVT) in trauma patients. METHODS: This study retrospectively analyzed 286 patients with mild craniocerebral injury and clavicular fractures admitted to our department from January 2016 to February 2020. Patients treated with only LMWH served as the control group, and patients treated with a PCD combined with LMWH as the observation group. The incidence of DVT, postoperative changes in the visual analogue scale (VAS) score, and coagulation function were observed and compared between the two groups. Excluding the influence of other single factors, binary logistic regression analysis was used to evaluate the use of a PCD in the patient's postoperative coagulation function. RESULTS: After excluding 34 patients who did not meet the inclusion criteria, 252 patients were were included. The incidence of DVT in the observation group was significantly lower than that in the control group (5.6% vs. 15.1%, χ2=4.605, P<0.05). The postoperative VAS scores of the two groups were lower than those before surgery (P<0.05). The coagulation function of the observation group was significantly higher than that of the control group, with a better combined anticoagulant effect (P<0.05). There were no significant differences between the two groups in preoperative or postoperative Glasgow Coma Scale scores, intraoperative blood loss, postoperative infection rate, or length of hospital stay (P>0.05). According to logistic regression analysis, the postoperative risk of DVT in patients who received LMWH alone was 1.764 times that of patients who received LMWH+PCD (P<0.05). The area under the receiver operating characteristic (AUROC) curve of partial thromboplastin time (APTT) and platelet (PLT) were greater than 0.5, indicating that they were the influence indicators of adding PCD to prevent DVT. Excluding the influence of other variables, LMWH+PCD effectively improved the coagulation function of patients. CONCLUSIONS: Compared with LMWH alone, LMWH+PCD could improve blood rheology and coagulation function in patients with traumatic brain injury and clavicular fracture, reduce the incidence of DVT, shorten the length of hospital stay, and improve the clinical effectiveness of treatment.

Down-regulation of PBK inhibits proliferation of human endometrial stromal cells in thin endometrium.

BACKGROUND: Thin endometrium (TE) is a challenging clinical issue in the reproductive medicine characterized by inadequate endometrial thickness, poor response to estrogen and no effective treatments currently. At present, the precise pathogenesis of thin endometria remains to be elucidated. We aimed to explore the related molecular mechanism of TE by comparing the transcriptome profiles of late-proliferative phase endometria between TE and matched controls. METHODS: We performed a bulk RNA-Seq (RNA-sequencing) of endometrial tissues in the late-proliferative phase in 7 TE and 7 matched controls for the first time. Differential gene expression analysis, gene ontology enrichment analysis and protein-protein interactions (PPIs) network analysis were performed. Immunohistochemistry was used for molecular expression and localization in endometria. Human endometrial stromal cells (HESCs) were isolated and cultured for verifying the functions of hub gene. RESULTS: Integrative data mining of our RNA-seq data in endometria revealed that most genes related to cell division and cell cycle were significantly inhibited, while inflammation activation, immune response and reactive oxygen species associated genes were upregulated in TE. PBK was identified as a hub of PPIs network, and its expression level was decreased by 2.43-fold in endometria of TE patients, particularly reduced in the stromal cells, which was paralleled by the decreased expression of Ki67. In vitro experiments showed that the depletion of PBK reduced the proliferation of HESCs by 50% and increased the apoptosis of HESCs by 1 time, meanwhile PBK expression was inhibited by oxidative stress (reduced by 76.2%), hypoxia (reduced by 51.9%) and inflammatory factors (reduced by approximately 50%). These results suggested that the insufficient expression of PBK was involved in the poor endometrial thickness in TE. CONCLUSIONS: The endometrial transcriptome in late-proliferative phase showed suppressed cell proliferation in women with thin endometria and decreased expression of PBK in human endometrial stromal cells (HESCs), to which inflammation and reactive oxygen species contributed.

Defective autophagy contributes to endometrial epithelial-mesenchymal transition in intrauterine adhesions.

Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a common cause of uterine infertility. We previously demonstrated that partial epithelial-mesenchymal transition (EMT) and the loss of epithelial homeostasis play a vital role in the development of endometrial fibrosis. As a pro-survival strategy in maintaining cell and tissue homeostasis, macroautophagy/autophagy, conversely, may participate in this process. However, the role of autophagy in endometrial fibrosis remains unknown. Here, we demonstrated that autophagy is defective in endometria of IUA patients, which aggravates EMT and endometrial fibrosis, and defective autophagy is related to DIO2 (iodothyronine deiodinase 2) downregulation. In endometrial epithelial cells (EECs), pharmacological inhibition of autophagy by chloroquine (CQ) promoted EEC-EMT, whereas enhanced autophagy by rapamycin extenuated this process. Mechanistically, silencing DIO2 in EECs blocked autophagic flux and promoted EMT via the MAPK/ERK-MTOR pathway. Inversely, overexpression of DIO2 or triiodothyronine (T3) treatment could restore autophagy and partly reverse EEC-EMT. Furthermore, in an IUA-like mouse model, the autophagy in endometrium was defective accompanied by EEC-EMT, and CQ could inhibit autophagy and aggravate endometrial fibrosis, whereas rapamycin or T3 treatment could improve the autophagic levels and blunt endometrial fibrosis. Together, we demonstrated that defective autophagy played an important role in EEC-EMT in IUA via the DIO2-MAPK/ERK-MTOR pathway, which provided a potential target for therapeutic implications.Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; AMPK: adenosine 5'-monophosphate-activated protein kinase; AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; CDH1/E-cadherin: cadherin 1; CDH2/N-cadherin: cadherin 2; CQ: chloroquine; CTSD: cathepsin D; DIO2: iodothyronine deiodinase 2; DEGs: differentially expressed genes; EECs: endometrial epithelial cells; EMT: epithelial-mesenchymal transition; FN1: fibronectin 1; IUA: intrauterine adhesions; LAMP1: lysosomal associated membrane protein 1; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; T3: triiodothyronine; T4: tetraiodothyronine; TFEB: transcription factor EB; PBS: phosphate-buffered saline; TEM: transmission electron microscopy; TGFB/TGFß: transforming growth factor beta.

Deciphering the endometrial niche of human thin endometrium at single-cell resolution.

Thin endometrium has been widely recognized as a critical cause of infertility, recurrent pregnancy loss, and placental abnormalities; however, access to effective treatment is a formidable challenge due to the rudimentary understanding of the pathogenesis of thin endometrium. Here, we profiled the transcriptomes of human endometrial cells at single-cell resolution to characterize cell types, their communications, and the underlying mechanism of endometrial growth in normal and thin endometrium during the proliferative phase. Stromal cells were the most abundant cell type in the endometrium, with a subpopulation of proliferating stromal cells whose cell cycle signaling pathways were compromised in thin endometrium. Both single-cell RNA sequencing and experimental verification revealed cellular senescence in the stroma and epithelium accompanied by collagen overdeposition around blood vessels. Moreover, decreased numbers of macrophages and natural killer cells further exacerbated endometrial thinness. In addition, our results uncovered aberrant SEMA3, EGF, PTN, and TWEAK signaling pathways as causes for the insufficient proliferation of the endometrium. Together, these data provide insight into therapeutic strategies for endometrial regeneration and growth to treat thin endometrium.

The role of junctional adhesion molecule-C in trophoblast differentiation and function during normal pregnancy and preeclampsia.

INTRODUCTION: Junctional adhesion molecule-C (JAM-C) is an important regulator of many physiological processes, ranging from maintenance of tight junction integrity of epithelia to regulation of cell migration, homing and proliferation. Preeclampsia (PE) is a trophoblast-related syndrome with abnormal placentation and insufficient trophoblast invasion. However, the role of JAM-C in normal pregnancy and PE pathogenesis is unknown. METHODS: The expression and location of JAM-C in placentas were determined by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. The expression of differentiation and invasion markers were detected by qRT-PCR or western blot. The effects of JAM-C on migration and invasion of trophoblasts were examined using wound-healing and invasion assays. Additionally, a mouse model was established by injection of JAM-C-positive adenovirus to explore the effects of JAM-C in vivo. RESULTS: In normal pregnancy, JAM-C was preferentially expressed on cytotrophoblast (CTB) progenitors and progressively decreased when acquiring invasion properties with gestation advance. However, in PE patients, the expression of JAM-C was upregulated in extravillous trophoblasts (EVTs) and syncytiotrophoblasts (SynTs) of placentas. It was also demonstrated that JAM-C suppressed the differentiation of CTBs into EVTs in vitro. Consistently, JAM-C inhibited the migration and invasion capacities of EVTs through GSK3ß/ß-catenin signaling pathway. Importantly, Ad-JAMC-infected mouse model mimicked the phenotype of human PE. DISCUSSION: JAM-C plays an important role in normal placentation and upregulated JAM-C in placentas contributes to PE development.

circPTPN12/miR-21-5 p/∆Np63α pathway contributes to human endometrial fibrosis.

Emerging evidence demonstrates the important role of circular RNAs (circRNAs) in regulating pathological processes in various diseases including organ fibrosis. Endometrium fibrosis is the leading cause of uterine infertility, but the role of circRNAs in its pathogenesis is largely unknown. Here, we provide the evidence that upregulation of circPTPN12 in endometrial epithelial cells (EECs) of fibrotic endometrium functions as endogenous sponge of miR-21-5 p to inhibit miR-21-5 p expression and activity, which in turn results in upregulation of ΔNp63α to induce the epithelial mesenchymal transition (EMT) of EECs (EEC-EMT). In a mouse model of endometrium fibrosis, circPTPN12 appears to be a cofactor of driving EEC-EMT and administration of miR-21-5 p could reverse this process and improve endometrial fibrosis. Our findings revealed that the dysfunction of circPTPN12/miR-21-5 p/∆Np63α pathway contributed to the pathogenesis of endometrial fibrosis.

HMGA2-induced epithelial-mesenchymal transition is reversed by let-7d in intrauterine adhesions.

Intrauterine adhesions (IUAs), the leading cause of uterine infertility, are characterized by endometrial fibrosis. The management of IUA is challenging because the pathogenesis of the disease largely unknown. In this study, we demonstrate that the mRNA and protein levels of high mobility group AT-hook 2 (HMGA2) were increased by nearly 3-fold (P < 0.0001) and 5-fold (P = 0.0095) in the endometrial epithelial cells (EECs) of IUA patients (n = 18) compared to controls. In vivo and in vitro models of endometrial fibrosis also confirmed the overexpression of HMGA2 in EECs. In vitro cell experiments indicated that overexpression of HMGA2 promoted the epithelial-mesenchymal transition (EMT) while knockdown of HMGA2 reversed transforming growth factor-ß-induced EMT. A dual luciferase assay confirmed let-7d microRNA downregulated HMGA2 and repressed the pro-EMT effect of HMGA2 in vitro and in vivo. Therefore, our data reveal that HMGA2 promotes IUA formation and suggest that let-7d can depress HMGA2 and may be a clinical targeting strategy in IUA.

ΔNp63α-induced DUSP4/GSK3ß/SNAI1 pathway in epithelial cells drives endometrial fibrosis.

Epithelial homeostasis plays an essential role in maintaining endometrial function. But the epithelial role in endometrial fibrosis has been less studied. Previously, we showed that ectopic expression of ΔNp63α is associated with fibrosis process and epithelial dysfunction in endometria of patients with intrauterine adhesions (IUAs). Since ΔNp63α is profoundly involved in maintaining the epithelial homeostasis, we hereby focused on its roles in regulating the function and phenotype of endometrial epithelial cells (EECs) in context of endometrial fibrosis. We identified a typical type 2 epithelial-to-mesenchymal transition (EMT) in EECs from IUA patients and this process was induced by the forced expression of ΔNp63α in EECs. In transcriptomic analysis, we found that diverse signaling pathways regulated by ΔNp63α were involved in pro-EMT. We demonstrated that the DUSP4/GSK-3ß/SNAI1 pathway was critical in transducing the pro-EMT signals initiated by ΔNp63α, while bFGF reversed ΔNp63α-induced EMT and endometrial fibrosis both in vitro and in vivo by blocking DUSP4/GSK3ß/SNAI1 pathway. Taken together, our findings are important to understand the molecular mechanisms of endometrial fibrosis and to provide potential therapeutic targets.

Up-regulated cytotrophoblast DOCK4 contributes to over-invasion in placenta accreta spectrum.

In humans, a subset of placental cytotrophoblasts (CTBs) invades the uterus and its vasculature, anchoring the pregnancy and ensuring adequate blood flow to the fetus. Appropriate depth is critical. Shallow invasion increases the risk of pregnancy complications, e.g., severe preeclampsia. Overly deep invasion, the hallmark of placenta accreta spectrum (PAS), increases the risk of preterm delivery, hemorrhage, and death. Previously a rare condition, the incidence of PAS has increased to 1:731 pregnancies, likely due to the rise in uterine surgeries (e.g., Cesarean sections). CTBs track along scars deep into the myometrium and beyond. Here we compared the global gene expression patterns of CTBs from PAS cases to gestational age-matched control cells that invaded to the normal depth from preterm birth (PTB) deliveries. The messenger RNA (mRNA) encoding the guanine nucleotide exchange factor, DOCK4, mutations of which promote cancer cell invasion and angiogenesis, was the most highly up-regulated molecule in PAS samples. Overexpression of DOCK4 increased CTB invasiveness, consistent with the PAS phenotype. Also, this analysis identified other genes with significantly altered expression in this disorder, potential biomarkers. These data suggest that CTBs from PAS cases up-regulate a cancer-like proinvasion mechanism, suggesting molecular as well as phenotypic similarities in the two pathologies.

Down-regulation of B2R contributes to preeclampsia by inhibiting human trophoblast cell invasion and angiogenesis.

OBJECTIVE: Bradykinin B2 receptor (B2R) was decreased in early chorionic villi of pregnancies who progressed to severe preeclampsia (PE), suggesting downregulation of B2R may be involved in the pathogenesis of PE. The aim of this study was to investigate the possible roles of B2R in the pathophysiology of PE and its function in trophoblastic cells. STUDY DESIGN: The expression of B2R in placentas from patients with early-onset severe PE (sPE) and LPS induced PE-like rats were detected. The roles of B2R in HTR-8/SVneo cells migration and invasion were analyzed through transfecting B2R overexpressing plasmid vector or B2R-specific siRNA. The effect of HTR-8/SVneo cells culture supernatant with high and low expressing B2R on human umbilical vein endothelial cells (HUVEC) capillary formation ability was also investigated. RESULTS: We found that B2R expression was significantly decreased in placentas of patients with sPE and PE-like rats. In addition, siRNA-mediated down-regulation of B2R markedly inhibited the migration and invasion of HTR-8/SVneo cells. Conversely, over-expression of B2R significantly promoted the migration and invasion of HTR-8/SVneo cells. Furthermore, the culture supernatant from B2R-overexpressed-HTR-8/SVneo cells promoted the capillary formation of HUVEC through increasing placental growth factor (PlGF) levels, while the culture supernatant from si-B2R-HTR-8/SVneo cells had the opposite effects. CONCLUSIONS: The decrease of B2R in placentas leads to the dysfunction of invasion, migration and angiogenesis of trophoblasts, which may be involved in the pathogenesis of PE.

Metabolomic Analysis Reveals That the Mechanism of Astaxanthin Improves the Osteogenic Differentiation Potential in Bone Marrow Mesenchymal Stem Cells.

At present, little research has been done on the metabolic phenotype of the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. In this study, the effect of astaxanthin on improving osteogenic differentiation potential of mesenchymal stem cells was studied by metabolomics. Results showed that L-methionine, L-tyrosine, and 2-hydroxycinnamic acid were upregulated in MSCs treated with astaxanthin, while L-lysine, L-pipecolic acid, L-histidine, L-arginine, D-fructose, and L-aspartic acid were downregulated in samples treated with astaxanthin. In addition, astaxanthin exhibited a significant dose-dependent relationship with these markers. Metabolic pathway enrichment analysis revealed that AST mainly regulated phenylalanine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; and pantothenate and CoA biosynthesis during the process of osteogenic differentiation of MSCs. Furthermore, the staining results showed that astaxanthin could actively promote the osteogenic differentiation of mesenchymal stem cells. These findings clearly indicate that astaxanthin plays an important role in inducing osteogenic differentiation of mesenchymal stem cells. In addition, the changed metabolites can be used to monitor the differentiation process.

CSF1-associated decrease in endometrial macrophages may contribute to Asherman's syndrome.

PROBLEM: Asherman's syndrome (AS) is characterized by endometrial fibrosis leading to intrauterine adhesions and symptoms like hypomenorrhea, infertility, and recurrent pregnancy loss. Macrophages are key regulators of inflammation, tissue repair, regeneration, and fibrosis. However, the role of macrophages in AS remains unclear. METHOD OF STUDY: Endometrial biopsies of AS patients and controls were collected during the late proliferating phase of menstrual cycle. Fibrosis and proliferation markers were detected by Masson's trichrome staining and immunohistochemistry. Macrophages were examined by immunostaining and flow cytometry. The expression levels of CCL2, CSF1, CSF1R, and GM-CSF were detected by quantitative real-time polymerase chain reaction (q-PCR) and immunohistochemistry. A well-differentiated endometrial cell line Ishikawa (IK) was used for in vitro studies. Macrophages differentiating from THP-1 monocytic cells were polarized by IL-4/IL-13. Their culture supernatants (M(IL-4/13)-S) were applied to H2 O2 or bleomycin-damaged IK cells. RESULTS: In AS patients, endometrial stroma was replaced by fibrous tissue and cell proliferation was reduced. Macrophages in endometrial tissue were mainly alternative activated macrophages and their number was significantly decreased in AS patients. The CSF1 expression level was reduced in AS patients. M(IL-4/13)-S promoted the growth and migration of IK cells and inhibited H2 O2 -induced apoptosis. M(IL-4/13)-S protected IK cells from bleomycin-induced fibrosis. CONCLUSION: Macrophages are critical cells involved in the process of endometrial repair and fibrosis. The decreased amount of endometrial macrophages may be attributed to the reduced expression level of CSF1. Manipulation of macrophage activation/function may provide a novel therapeutic target for AS.

Vascular endothelial growth factor 165 inhibits pro-fibrotic differentiation of stromal cells via the DLL4/Notch4/smad7 pathway.

Endometrial fibrosis is the main pathological feature of Asherman's syndrome (AS), which is the leading cause of uterine infertility. Much is known about the expression of VEGF165 in luminal/glandular epithelial cells and stromal cells of the endometrium in normal menstrual cycles; however, less is known about the role and mechanism of VEGF165 in endometrial fibrosis. Herein, we report that VEGF165 is a key regulator in endometrial stromal cells to inhibit α-SMA and collagen 1 expression. Compared to human control subjects, patients with AS exhibited decreased VEGF165 expression in the endometrium along with increased fibrotic marker expression and collagen production. A fibrotic phenotype was shown in both mice with conditional VEGF reduction and VEGF165-deleted endometrial stromal cells. Exogenous VEGF165 could suppress TGFß1-induced α-SMA and collagen 1 expression in human primary endometrial stromal cells. However, this beneficial effect was hindered when the expression of smad7 or Notch4 was inhibited or when Notch signaling was blocked, suggesting that smad7 and Notch4 are essential downstream molecules for VEGFA functioning. Overall, our results uncover a clinical targeting strategy for VEGF165 to inhibit pro-fibrotic differentiation of stromal cells by inducing DLL4/Notch4/smad7, which paves the way for AS treatment.