Mesothelial-to-Mesenchymal Transition and Exosomes in Peritoneal Metastasis of Ovarian Cancer

Most patients with ovarian cancer (OvCA) present peritoneal disseminated disease at the time of diagnosis. During peritoneal metastasis, cancer cells detach from the primary tumor and disseminate through the intraperitoneal fluid. The peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by OvCA cells. Subsequent progression of tumors through the peritoneum leads to the accumulation into the peritoneal stroma of a sizeable population of carcinoma-associated fibroblasts (CAFs), which is mainly originated from a mesothelial-to-mesenchymal transition (MMT) process. A common characteristic of OvCA patients is the intraperitoneal accumulation of ascitic fluid, which is composed of cytokines, chemokines, growth factors, miRNAs, and proteins contained in exosomes, as well as tumor and mesothelial suspended cells, among other components that vary in proportion between patients. Exosomes are small extracellular vesicles that have been shown to mediate peritoneal metastasis by educating a pre-metastatic niche, promoting the accumulation of CAFs via MMT, and inducing tumor growth and chemoresistance. This review summarizes and discusses the pivotal role of exosomes and MMT as mediators of OvCA peritoneal colonization and as emerging diagnostic and therapeutic targets.

Clinical Factors Associated With the Culture of Primary Human Peritoneal Mesothelial Cell From Peritoneal Dialysate Effluent

Background: The culture of primary human peritoneal mesothelial cell (HPMC) provides us an in vitro tool to investigate peritoneal fibrosis and ultrafiltration failure. The aim of the present study was to establish the method of culturing HPMC and to explore clinical factors associated with the success rate of culture.Methods: HPMCs were aseptically harvested by centrifuge from peritoneal dialysate effluent (PDE) of patients with end-stage renal disease (ESRD) who underwent peritoneal dialysis (PD) catheterization for less than 2 weeks and were cultured in vitro. Cells were identified by simple morphological observation and immunofluorescent staining. Clinical data of PD patients was collected. Comparison between groups and binary logistic regression analysis were employed to explore the clinical factors associated with the success rate of culture.Results: The study included 36 patients (26 male (72.2%); mean age 53.9±15.6 years). HPMC from PDE successfully grew and survived in 22 patients. A typical cobblestone-like appearance was observed by inverted phase contrast microscope. Immunofluorescence staining showed positive expression of cytokeratin-18 (CK-18) and Vimentin. Comparison between groups demonstrated significant differences in diabetes (P=0.041), days from catheterization (P=0.002) and the use of erythrocyte lysate (P=0.019) between the two groups. Multivariate logistic regression analysis revealed that the success rate was correlated with days from catheterization (OR=0.318, 95%CI=0.107-0.946, P=0.039) and the level of C-reactive protein (CRP, OR=0.893, 95%CI=0.805-0.991, P=0.032). Conclusions: The method of culturing primary HPMC from PDE has been successfully established. The success rate of culture is correlated with CRP level and days from catheterization.

TMIGD1 Inhibited Abdominal Adhesion Formation by Alleviating Oxidative Stress in the Mitochondria of Peritoneal Mesothelial Cells

Background. Postoperative abdominal adhesion remains one of the frequent complications after abdominal surgery and lacks effective intervention. Peritoneal mesothelial cell injury and healing play crucial roles in the process of adhesion formation, and identifying this mechanism might provide new insight into possible new therapeutic strategies for this disease. Transmembrane and immunoglobulin domain-containing 1 (TMIGD1) has been proven to protect renal epithelial cells from injury induced by oxidative stress and has also been identified as a novel adhesion molecule. Here, we investigated the role of TMIGD1 and its possible mechanism in adhesion formation. Materials and Methods. Immunohistochemistry (IHC), qPCR, and immunofluorescence (IHF) were used to detect the expression of TMIGD1. The grade and tenacity score of adhesion were used to evaluate the adhesion formation conditions. A TMIGD1-overexpressing HMrSV5 cell line was established. MTT assay, Western blotting, Annexin V apoptosis analysis, and CK19 staining were used to measure mesothelial cell viability, apoptosis, and completeness. ROS and MDA detection were used to measure mesothelial cell oxidative stress levels. JC-1 staining, IHF, and transmission electron microscopy were performed to assess mitochondrial function. Scratch-wound and adhesion assays were used to evaluate the adhesion ability of mesothelial cells. Results. First, we showed that TMIGD1 was decreased in mouse abdominal adhesion tissue and peritoneal mesothelial cells. Second, TMIGD1 overexpression inhibited adhesion formation. Third, TMIGD1 overexpression protected mesothelial cells from hydrogen peroxide- (H2O2-) induced oxidative stress injury. Fourth, TMIGD1 overexpression alleviated oxidative stress by protecting the mitochondrial function of mesothelial cells. In addition, TMIGD1 overexpression enhanced mesothelial cell adhesion. Conclusion. Our findings suggest that TMIGD1 protects mesothelial cells from oxidative stress injury by protecting their mitochondrial function, which is decreased in regular abdominal adhesion tissue. In addition, TMIGD1 enhances peritoneal mesothelial cell adhesion to promote healing.

Involvement of STAT3 Signaling in High Glucose-Induced Epithelial Mesenchymal Transition in Human Peritoneal Mesothelial Cell Line HMrSV5

Background/Aims: Peritoneal fibrosis (PF) is a common complication in patients receiving long-term peritoneal dialysis, which results in damage to peritoneal functions. Epithelial-mesenchymal transition (EMT) is a key step in the early pathogenesis of PF. Increasing evidence has shown that signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in EMT and tissue fibrosis by interacting with distinct EMT-inducing molecules, including transforming growth factor (TGF)-β and advanced glycation end products (AGEs). This study investigated the involvement of STAT3 in the PF process. Methods: We used high glucose-treated human peritoneal mesothelial cell line HMrSV5 as an in vitro model to expose the peritoneal mesothelial cells to high-glucose dialysate. Expression of EMT markers was detected by qRT-PCR. Accumulation of methylglyoxal (MGO) and AGEs in the culture supernatant were measured by enzyme-linked immunosorbent assay. Phosphorylation of STAT3 was assessed by Western blot. Results: Results showed that high glucose upregulated TGF-β, increased the productions of MGO and AGEs, and induced EMT in HMrSV5 cells. High glucose also activated the STAT3 pathway. STAT3 inhibitor reduced the high glucose-induced EMT, via reducing TGF-β expression and repressing the accumulation of MGO and AGEs. Conclusion: Our results revealed a critical role for STAT3 signaling in high glucose-induced EMT in HMrSV5 cells, and suggested that inhibition of STAT3 might be a treatment for high glucose-induced fibrogenesis in PF.

Acetylation of HMGB1 by JNK1 Signaling Promotes LPS-Induced Peritoneal Mesothelial Cells Apoptosis

Increased high mobility group box 1 (HMGB1) in dialysis effluence is associated with the presence of peritoneal dialysis-related peritonitis in patients and peritoneal dysfunction in acute peritonitis mice model, but it remains unclear whether HMGB1 is involved in peritoneal mesothelial cell injury and functions via molecular posttranslational modifications by acetylation in this process. Here we first showed correlation between HMGB1 acetylation level in dialysis effluence of patients and occurrence of Gram-negative peritonitis. The increased level of acetylated HMGB1 was similarly observed under the lipopolysaccharides (LPS) treatment in both human peritoneal mesothelial cell line (HMrSV5) and mice visceral peritoneum tissue. Overexpression of wild-type, but not hypoacetylation mutant of HMGB1, enhanced LPS-induced apoptosis in HMrSV5 cells, which was accompanied by elevated protein levels of BAX and cleaved-caspase 3 compared to the control. Pretreatment of HMrSV5 cell with JNK inhibitor attenuated LPS-induced HMGB1 acetylation. Consistently, primary peritoneal mesothelial cells from Jnk1-/- mice showed a lower protein contents of acetylated HMGB1, fewer apoptosis, and decreased protein expression of BAX and cleaved-caspase3 after LPS exposure, as compared to those from wild-type mice. In conclusion, our data demonstrated HMGB1 promotes LPS-induced peritoneal mesothelial cells apoptosis, which is associated with JNK1-mediated upregulation of HMGB1 acetylation.

Sulfotanshinone IIA Sodium Ameliorates Glucose Peritoneal Dialysis Solution-Induced Human Peritoneal Mesothelial Cell Injury via Suppression of ASK1-P38-mediated Oxidative Stress

Background/Aims: Long-term use of high-glucose peritoneal dialysis solution (PDS) induces peritoneal mesothelial cell (PMC) injury, peritoneal dysfunction, and peritoneal dialysis (PD) failure in patients with end-stage renal disease. How to preserve PMCs in PD is a major challenge for nephrologists worldwide. In this study, we aimed to elucidate the efficacy and mechanisms of sulfotanshinone IIA sodium (Tan IIa) in ameliorating high-glucose PDS-induced human PMC injury. Methods: The human PMC line HMrSV5 was incubated with 4.25% PDS in vitro to mimic the high-glucose conditions in PD. Cellular viability was measured by Cell Counting Kit 8. Generation of superoxide and reactive oxygen species (ROS) was assessed using a Total ROS/Superoxide Detection Kit. Oxidative modification of protein was evaluated by OxyBlot Protein Oxidation Detection Kit. TUNEL (dT-mediated dUTP nick end labeling) assay and DAPI (4,6-diamidino-2-phenylindole) staining were used to evaluate apoptosis. Western blot analysis was performed to evaluate the efficacy and mechanisms of Tan IIa. Results: Tan IIa protected PMCs against PDS-induced injury as evidenced by alleviating changes in morphology and loss of cell viability. Consistent with their antioxidant properties, N-acetyl-L-cysteine (NAC) and Tan IIa suppressed superoxide and ROS production, protein oxidation, and apoptosis elicited by PDS. Apoptosis signal-regulating kinase 1 (ASK1)-p38 signaling was activated by PDS. Both Tan IIa and NAC suppressed ASK1 and p38 phosphorylation elicited by PDS. Moreover, genetic downregulation of ASK1 ameliorated cell injury and inhibited the phosphorylation of p38 and activation of caspase 3. Conclusion: Tan IIa protects PMCs against PDS-induced oxidative injury through suppression of ASK1-p38 signaling.