Peritoneal Mesothelial Cells Research Articles

Tamoxifen exerts anti-peritoneal fibrosis effects by inhibiting H19-activated VEGFA transcription

BackgroundPeritoneal dialysis (PD) remains limited due to dialysis failure caused by peritoneal fibrosis. Tamoxifen (TAM), an inhibitor of estrogen receptor 1 (ESR1), has been reported to treat fibrosis, but the underlying mechanism remains unknown. In this study, we sought to explore whether tamoxifen played an anti-fibrotic role by affecting transcription factor ESR1.MethodsESR1 expression was detected in the human peritoneum. Mice were daily intraperitoneally injected with 4.25% glucose PD dialysate containing 40 mM methylglyoxal for 2 weeks to establish PD-induced peritoneal fibrosis. Tamoxifen was administrated by daily gavage, at the dose of 10 mg/kg. Chromatin immunoprecipitation (ChIP) and dual‐luciferase reporter assay were performed to validate ESR1 bound H19 promoter. Gain-of-function and loss-of-function experiments were performed to investigate the biological roles of H19 on the mesothelial-mesenchymal transition (MMT) of human peritoneal mesothelial cells (HPMCs). Intraperitoneal injection of nanomaterial-wrapped 2′-O-Me-modified small interfering RNA was applied to suppress H19 in the mouse peritoneum. RNA immunoprecipitation and RNA pull-down assays demonstrated binding between H19 and p300. Exfoliated peritoneal cells were obtained from peritoneal dialysis effluent to analyze the correlations between ESR1 (or H19) and peritoneal solute transfer rate (PSTR).ResultsESR1 was increased significantly in the peritoneum after long-term exposure to PD dialysate. Tamoxifen treatment ameliorated high glucose-induced MMT of HPMCs, improved ultrafiltration rate, and decreased PSTR of mouse peritoneum. Tamoxifen reduced the H19 level by decreasing the ESR1 transcription of H19. Depletion of H19 reversed the pro-fibrotic effect of high glucose while ectopic expression of H19 exacerbated fibrotic pathological changes. Intraperitoneal injection of nanomaterial-wrapped 2′-O-Me-modified siRNAs targeting H19 mitigated PD-related fibrosis in mice. RNA immunoprecipitation (RIP) and RNA pull-down results delineated that H19 activated VEGFA expression by binding p300 to the VEGFA promoter and inducing histone acetylation of the VEGFA promoter. ESR1 and H19 were promising targets to predict peritoneal function.ConclusionsHigh glucose-induced MMT of peritoneal mesothelial cells in peritoneal dialysis via activating ESR1. In peritoneal mesothelial cells, ESR1 transcribed the H19 and H19 binds to transcription cofactor p300 to activate the VEGFA. Targeting ESR1/H19/VEGFA pathway provided new hope for patients undergoing peritoneal dialysis.Graphic

Phytohormone synthesis for PDH activation and phytochemical screening for the treatment of endometriosis

Introduction and Aim: Endometriosis is a challenging gynecological disorder that causes pelvic pain and infertility attributed to the prevalence of ectopic endometrial tissue outside the uterine tract. Endometrial tissue can travel to other areas of the body for inexplicable reasons. There are, nevertheless, certain patterns. Endometriosis could be treated in several ways, based on characteristics such as age, fertility, symptom severity, and disease degree. All endometriosis medications are available as contraceptives, restricting their use to pregnant mothers or intending to get children. By comparing human peritoneal mesothelial cells (HPMCs) derived from the pelvic peritoneum of women with endometriosis to HPMCs from women without endometriosis, the researchers found that HPMCs from women with endometriosis had reduced glycolysis, mitochondrial respiration, and pyruvate dehydrogenase (PDH) and improved lactate production. There is no treatment for endometriosis, even though various medications and management alternatives are available. The present study was carried out to find novel medications to treat endometriosis from medicinal plants which appear to be a promising endeavor. Methods and Materials: Phytohormone and phytochemical screening were performed to study the medication options that could possibly treat endometriosis. Phytochemicals were determined using GC-MS. Results: The phytohormone activity and phytochemical components of Mentha piperita, Curcuma longa, and Andrographis paniculata were identified using GC MS and FT-IR analysis. This initial data from the analytical methods will make it easier to perform follow-up research on finding bioactive ingredients, determining their efficacy through in vivo tests, and proving their safety and efficacy through clinical trials to treat endometriosis. Conclusion: Gibberellin, a phytohormone obtained from natural sources, is supposed to improve PDH activity and hence inhibit PDK. The demand for more modern medicines to increase survival in such cases remains unmet. As a result, there is an increasing interest in using herbal therapy to treat endometriosis.

Matrix metalloproteinase-10 deficiency has protective effects against peritoneal inflammation and fibrosis via transcription factor NFκΒ pathway inhibition

One of the most common causes of discontinued peritoneal dialysis is impaired peritoneal function. However, its molecular mechanisms remain unclear. Previously, by microarray analysis of mouse peritoneum, we showed that MMP (matrix metalloproteinase)-10 expression is significantly increased in mice with peritoneal fibrosis, but its function remains unknown. Chlorhexidine gluconate (CG) was intraperitoneally injected to wild-type and MMP-10 knockout mice to induce fibrosis to elucidate the role of MMP-10 on peritoneal injury. We also examined function of peritoneal macrophages and mesothelial cells obtained from wild-type and MMP-10 knockout mice, MMP-10-overexpressing macrophage-like RAW 264.7 cells and MeT-5A mesothelial cells, investigated MMP-10 expression on peritoneal biopsy specimens, and the association between serum proMMP-10 and peritoneal solute transfer rates determined by peritoneal equilibration test on patients. MMP-10 was expressed in cells positive for WT1, a mesothelial marker, and for MAC-2, a macrophage marker, in the thickened peritoneum of both mice and patients. Serum proMMP-10 levels were well correlated with peritoneal solute transfer rates. Peritoneal fibrosis, inflammation, and high peritoneal solute transfer rates induced by CG were all ameliorated by MMP-10 deletion, with reduction of CD31-positive vessels and VEGF-A-positive cells. Expression of inflammatory mediators and phosphorylation of NFκΒ subunit p65 at S536 were suppressed in both MMP-10 knockout macrophages and mesothelial cells in response to lipopolysaccharide stimulation. Overexpression of MMP-10 in RAW 264.7 and MeT-5A cells upregulated pro-inflammatory cytokines with phosphorylation of NFκΒ subunit p65. Thus, our results suggest that inflammatory responses induced by MMP-10 are mediated through the NFκΒ pathway, and that systemic deletion of MMP-10 ameliorates peritoneal inflammation and fibrosis caused by NFκΒ activation of peritoneal macrophages and mesothelial cells.

Iron increases the profibrotic properties of the senescent peritoneal mesothelial cells.

Treatment of anemia in peritoneal dialysis patients often requires intravenous iron supplementation. Iron diffuses into the peritoneal cavity and is injurious to the peritoneum. We studied how intermittent exposure to iron changes the properties of the senescent peritoneal mesothelial cells (MC). Replicative senescence was induced in MC in control medium (Con) or in control medium with intermittent exposure to iron isomaltoside 15 µg/dL (Con-IIS). After 10 passages properties of MC from both groups were compared to MC not exposed to replicative senescence. In senescent MC population doubling time was elongated, intracellular generation of free radicals and staining for β-galactosidase was stronger than in MC not exposed to replicative senescence. All these effects were stronger in MC intermittently exposed to IIS. In these cells intracellular iron content was also higher. Also expression of genes p21 and p53 was stronger in MC intermittently treated with IIS. In senescent cells higher release and expression of IL6 and TGFβ1 was observed and that effect was stronger in MC treated with iron. Senescent MC had reduced fibrinolytic activity, what may predispose to the peritoneal fibrosis. Synthesis of collagen was higher in senescent cells, more in MC treated with iron. MC aging results in change of their genotype and phenotype which lead to their profibrotic effect. Exposure to iron enhances these changes.

Ovarian cancer: Novel mechanisms and therapeutic targets regarding the microenvironment in the abdominal cavity.

Ovarian cancer is an intractable disease that is mostly diagnosed at an advanced stage and has a high recurrence rate. The early development of characteristic peritoneal dissemination via ascites contributes to a poor prognosis. Based on the "seed and soil" theory, ovarian cancer is considered to form a disseminated tumor that interacts with the peritoneum; superficial mesothelial cells are structurally important. Thus far, we have reported that peritoneal mesothelial cells, which originally are ecological defenses, transform into ovarian cancer-associated mesothelial cells, which are allies of cancer. They are found to be actively involved in the formation of a friendly "soil" that promotes the survival of "seeds" of ovarian cancer cells. We also demonstrated that the progression of ovarian cancer and the induction of its refractory nature are partially mediated through competition and cooperation between ovarian cancer and mesothelial cells. We believe that it is necessary to shift the aim of treatment strategies from solely targeting cancer cells to focusing on the crosstalk between the surrounding environment and ovarian cancer, an approach that ultimately aims to achieve "coexistence" with cancer through disease control.

Partial replacement of d-glucose with d-allose ameliorates peritoneal injury and hyperglycaemia induced by peritoneal dialysis fluid in rats.

Peritoneal dialysis (PD) is a crucial dialysis method for treating end-stage kidney disease. However, its use is restricted due to high glucose-induced peritoneal injury and hyperglycaemia, particularly in patients with diabetes mellitus. In this study, we investigated whether partially replacing d-glucose with the rare sugar d-allose could ameliorate peritoneal injury and hyperglycaemia induced by peritoneal dialysis fluid (PDF). Rat peritoneal mesothelial cells (RPMCs) were exposed to a medium containing d-glucose or d-glucose partially replaced with different concentrations of d-allose. Cell viability, oxidative stress and cytokine production were evaluated. Sprague-Dawley (SD) rats were administrated saline, a PDF containing 4% d-glucose (PDF-G4.0%) or a PDF containing 3.6% d-glucose and 0.4% d-allose (PDF-G3.6%/A0.4%) once a day for 4 weeks. Peritoneal injury and PD efficiency were assessed using immuno-histological staining and peritoneal equilibration test, respectively. Blood glucose levels were measured over 120 min following a single injection of saline or PDFs to 24-h fasted SD rats. In RPMCs, the partial replacement of d-glucose with d-allose increased cell viability and decreased oxidative stress and cytokine production compared to d-glucose alone. Despite the PDF-G3.6%/A0.4% having a lower d-glucose concentration compared to PDF-G4.0%, there were no significant changes in osmolality. When administered to SD rats, the PDF-G3.6%/A0.4% suppressed the elevation of peritoneal thickness and blood d-glucose levels induced by PDF-G4.0%, without impacting PD efficiency. Partial replacement of d-glucose with d-allose ameliorated peritoneal injury and hyperglycaemia induced by high concentration of d-glucose in PDF, indicating that d-allose could be a potential treatment option in PD.

Astragaloside IV ameliorates peritoneal fibrosis by promoting PGC-1α to reduce apoptosis in vitro and in vivo.

Prolonged exposure of the peritoneum to high glucose dialysate leads to the development of peritoneal fibrosis (PF), and apoptosis of peritoneal mesothelial cells (PMCs) is a major cause of PF. The aim of this study is to investigate whether Astragaloside IV could protect PMCs from apoptosis and alleviate PF. PMCs and rats PF models were induced by high glucose peritoneal fluid. We examined the pathology of rat peritoneal tissue by HE staining, the thickness of rat peritoneal tissue by Masson's staining, the number of mitochondria and oxidative stress levels in peritoneal tissue by JC-1 and DHE fluorescence staining, and mitochondria-related proteins and apoptosis-related proteins such as PGC-1α, NRF1, TFAM, Caspase3, Bcl2 smad2 were measured. We used hoechst staining and flow cytometry to assess the apoptotic rate of PMCs in the PF model, and further validated the observed changes in the expressions of PGC-1α, NRF1, TFAM, Caspase3, Bcl2 smad2 in PMCs. We further incubated PMCs with MG-132 (proteasome inhibitor) and Cyclohexylamine (protein synthesis inhibitor). The results demonstrated that Astragaloside IV increased the expression of PGC-1α by reducing the ubiquitination of PGC-1α. It was further found that the protective effects of Astragaloside IV on PMCs were blocked when PGC-1α was inhibited. In conclusion, Astragaloside IV effectively alleviated PF both in vitro and in vivo, possibly by promoting PGC-1α to enhance mitochondrial synthesis to reduce apoptotic effects.

Exosomal PPARγ derived from macrophages suppresses LPS-induced peritonitis by negative regulation of CD14/TLR4 axis.

Intercellular communication between macrophages and peritoneal mesothelial cells (PMCs) has been suggested as a key factor regulating peritonitis development. Here, we explored whether PPARγ (peroxisome proliferator-activated receptor gamma) can be packaged into macrophage exosomes to mediate intercellular communication and regulate peritonitis. Macrophage exosomes were isolated by ultracentrifugation and identified by nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of macrophage-derived exosomes was performed using mass spectrometry. Co-culture models of supernatants or exosomes with PMCs, as well as a mouse peritonitis model induced by lipopolysaccharide (LPS), were employed. In this study, using stable Raw264.7 cells overexpressing GFP-FLAG-PPARγ (OE-PPARγ), we found that PPARγ inhibited LPS-induced inflammatory responses in Raw264.7 cells and that PPARγ was incorporated into macrophage exosomes during this process. Overexpression of PPARγ mainly regulated thesecretion ofdifferentiallyexpressed exosomal proteins involved in the biological processes of protein transport, lipid metabolic process, cell cycle, apoptotic process, DNA damage stimulus, as well as the KEGG pathway of salmonella infection. Using co-culture models and mouse peritonitis model, we showed that exosomes from Raw264.7 cells overexpressing PPARγ inhibited LPS-induced inflammation in co-cultured human PMCs and in mice through downregulating CD14 and TLR4, two key regulators of the salmonella infection pathway. Pretreatment of the PPARγ inhibitor GW9662 abolished the anti-inflammatory effect of exosomes from Raw264.7 OE-PPARγ cells on human PMCs. These results suggested that overexpression of PPARγ largely altered the proteomic profile of macrophage exosomes and that exosomal PPARγ from macrophages acted as a regulator of intercellular communication to suppress LPS-induced inflammatory responses in vitro and in vivo via negatively regulating the CD14/TLR4 axis.

Effects of 101BHG-D01, a novel M receptor antagonism, on allergic rhinitis in animal models and its mechanism

Allergic rhinitis (AR) is a nasal mucosal disease with sneezing and nasal itching as the main symptoms. Although AR treatment continues to improve, there remains a lack of effective drugs. There are still controversies regarding whether anticholinergic drugs can effectively and safely relieve the symptoms of AR and reduce inflammation in the nasal mucosa. Here, we synthesized 101BHG-D01, which is a novel anticholinergic drug that mainly targets the M3 receptor and may reduce the adverse effects of other anticholinergic drugs on the heart. We evaluated the effects of 101BHG-D01 on AR and investigated the potential molecular mechanism of anticholinergic therapy for AR. We found that 101BHG-D01 effectively alleviated AR symptoms, reduced the infiltration of inflammatory cells and attenuated the expression of inflammatory factors (IL-4, IL-5, IL-13, etc.) in various AR animal models. In addition, 101BHG-D01 reduced the activation of mast cells and the release of histamine from rat peritoneal mesothelial cells (RPMCs) challenged by IgE. Moreover, 101BHG-D01 reduced the expression of MUC5AC in IL-13-challenged rat nasal epithelial cells (RNECs) and human nasal epithelial cells (HNEpCs). Furthermore, IL-13 stimulation significantly increased JAK1 and STAT6 phosphorylation, which was suppressed by 101BHG-D01. We demonstrated that 101BHG-D01 reduced mucus secretion and inflammatory cell infiltration in the nasal mucosa, which may occur through a reduction in activation of the JAK1-STAT6 signaling pathway, indicating that 101BHG-D01 is a potent and safe anticholinergic therapy for AR.

Galectin-1 promotes gastric cancer peritoneal metastasis through peritoneal fibrosis

BackgroundPeritoneal metastasis is one of the main causes of death in patients with gastric cancer (GC). Galectin-1 regulates various undesirable biological behaviors in GC and may be key in GC peritoneal metastasis.MethodsIn this study, we elucidated the regulatory role of galectin-1 in GC cell peritoneal metastasis. GC and peritoneal tissues underwent hematoxylin–eosin (HE), immunohistochemical (IHC), and Masson trichrome staining to analyze the difference in galectin-1 expression and peritoneal collagen deposition in different GC clinical stages. The regulatory role of galectin-1 in GC cell adhesion to mesenchymal cells and in collagen expression was determined using HMrSV5 human peritoneal mesothelial cells (HPMCs). Collagen and corresponding mRNA expression were detected with western blotting and reverse transcription PCR, respectively. The promoting effect of galectin-1 on GC peritoneal metastasis was verified in vivo. Collagen deposition and collagen I, collagen III, and fibronectin 1 (FN1) expression in the peritoneum of the animal models were detected by Masson trichrome and IHC staining.ResultsGalectin-1 and collagen deposition in the peritoneal tissues was correlated with GC clinical staging and were positively correlated. Galectin-1 enhanced the ability of GC cells to adhere to the HMrSV5 cells by promoting collagen I, collagen III, and FN1 expression. The in vivo experiments confirmed that galectin-1 promoted GC peritoneal metastasis by promoting peritoneal collagen deposition.ConclusionGalectin-1-induced peritoneal fibrosis may create a favorable environment for GC cell peritoneal metastasis.

#4645 HISTONE LACTYLATION ENHANCES PERITONEAL FIBROSIS BY INDUCING PERITONEAL MESOTHELIAL CELL SENESCENCE

Abstract Background and Aims Peritoneal fibrosis (PF) is a serious complication limiting the application of peritoneal dialysis (PD). Histone lactylation has recently been found to increase with glycolysis and intracellular lactate levels, thereby mediating chronic organ damage. This study investigated the role and mechanism of histone lactylation in high glucose peritoneal dialysate-induced peritoneal fibrosis in mice. Method The PF mouse model induced by high glucose peritoneal dialysate was constructed by daily intraperitoneal injection of 4.25% glucose peritoneal dialysate combined with 40 mM Methylglyoxal for 2 weeks. We also used a lactylation enhancer (rotenone) and a lactylation inhibitor (oxamate) to validate the effects of histone lactylation in vivo and in vitro. The parietal peritoneum samples and cultured peritoneal mesothelial cells in various groups were analyzed for evidence of senescence, fibrosis, glycolysis, and histone lactylation levels. In addition, we generated DcR2 knockout mice to further verify the effects of histone lactylation on peritoneal mesothelial cell (PMCs) senescence by clearing senescent cells. Results In the PF group, the results of Masson staining and immunohistochemistry revealed increased peritoneal thickness and expression of fibrosis markers (α-SMA, Collagen I, and Fibronectin) and senescence markers (P16, P21, and DcR2) compared to the control group. We found that lactate and glycolysis levels were significantly higher in the PF group compared to the control group, and further immunoprecipitation results also showed enhanced levels of histone lactylation modifications in the PF group. We further found that the level of lactylation modification of H3K18la site increased significantly, suggesting that H3K18la site may play an important role in promoting PF. Similarly, in vitro, we verified that peritoneal mesothelial cells (PMCs) exhibited enhanced levels of lactate and lactylation in response to stimulation by high glucose, while we found an increasing expression of cell senescence phenotype (P16, P21, and DcR2) and senescence-associated cytokines secretion (IL-1β, IL-6, and TNF-α). By enhancing or inhibiting lactylation, we found that PMCs senescence was significantly enhanced in the lactylation-enhanced group, while the lactylation-inhibited group alleviated PMCs senescence induced by high glucose peritoneal dialysate. In the PF model of DcR2 knockout mice, compared with the wild group, there was no significant difference in the level of histone lactylation, while peritoneal thickness and the expression of markers of fibrosis markers (α-SMA, Collagen I, and Fibronectin) were significantly reduced. Conclusion Histone lactylation leads to peritoneal fibrosis induced by high glucose peritoneal dialysate by promoting PMSCs senescence. Histone lactylation may be a vital mechanism of peritoneal dialysis-related peritoneal fibrosis.

#5837 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE PERITONEAL MESOTHELIAL AND ENDOTHELIAL CELL BARRIER

Abstract Background and Aims Solute transport across cellular levels is mediated by paracellular and transcellular pores, channels and carriers. Knowledge on their cell specific expression, regulation and function in peritoneal dialysis (PD) is limited. Method Polarized primary (HPMC) and immortalized human peritoneal mesothelial (MeT-5A), microvascular (HCMEC) and umbilical vein endothelial (HUVEC) cells underwent RNA sequencing and gene enrichment analysis. Key findings were confirmed by western blotting (WB), confocal and single molecule localization microscopy (SMLM). Arteriolar transcriptome and proteome datasets of non-CKD, CKD5 and PD children (n = 6/group) underwent targeted transport pathways analysis. Key transporter proteins were quantified in parietal peritoneum (n = 20–30/group) and related to peritoneal transport rates available in 23 children. Barrier function was studied in vitro (transepithelial electrical resistance, TER, and molecular weight dependent flux), ex vivo and in vivo. Results Junction, transmembrane and transcytotic transporter expression was highly cell type specific on RNA and protein level. Sealing Claudin (CLDN)1 was only expressed in mesothelial cells, sealing CLDN5 in endothelial cells. TER which reflects functional junction status, was 50% lower in HCMEC compared to HUVEC and the two mesothelial cell lines; 4- and 10-kDa dextran permeability was higher in HCMEC. At nanoscale, SMLM yielded highest distance of junction molecules in HCMEC and different spatial organisation, reflecting the low TER. In sheep peritoneum, removal of the mesothelium abolished tissue TER. In mice, short-term LPS exposure to modify mesothelial permeability resulted in faster transperitoneal 4- and 70-kDa dextran transport, suggesting a specific barrier function of the mesothelium. In human parietal peritoneum, total endothelial surface area per section was age dependently 1.5- to 2-fold higher than the respective mesothelial surface area, and further increased with double-chamber PD fluid, due to major hypervascularisation. Tight junction proteins CLDN-1 to -5, and -15, ZO-1, occludin and tricellulin, and transcellular transporter ENaC, PIT1, and SGLT1 were detected in mesothelial and arteriolar endothelial cells. In CKD mesothelial CLDN-1 and arteriolar CLDN-2 and -3 were more abundant than in non-CKD controls, and PD patients had highest mesothelial and arteriolar CLDN-1 and mesothelial CLDN-2, lower mesothelial and arteriolar claudin-3 and lower arteriolar ENaC. D/P creatinine and D/D0 glucose correlated with arteriolar CLDN-2 and with mesothelial CLDN-4 and -15, which are pore forming junctions, and for creatinine with mesothelial PIT-1, a sodium/phosphate co-transporter. Conclusion We provide the first in-depth analysis of peritoneal determinants of solute transport. The molecular expression pattern of the mesothelial and endothelial cell barrier and transporter proteins differs substantially. Disruption of the mesothelial layer increases peritoneal solute absorption rate. In the human peritoneum, peritoneal transporter status is modified by CKD and PD, and pore forming junction proteins are associated with dialytic solute transport function. These represent a promising target for therapeutic intervention.

#2629 ROS-SCAVENGING BLOCKS GLUCOSE INDUCED OXIDATIVE PERITONEAL CELL DAMAGE IN VITRO

Abstract Background and Aims Peritoneal dialysis (PD) is an effective method of renal replacement therapy (RRT). The long-term use of PD as RRT is limited due to adverse effects of high glucose-based PD fluids (PDFs) to the structure and function of the peritoneal membrane. Our group recently elucidated one pathological mechanism showing that glucose-based PDFs enhance oxidative DNA-damage in PD patients due to reduced thioredoxin (TRX) activity trigged by thioredoxin-interacting-protein (TXNIP) upregulation (Oberacker et al., 2022). Therefore, the aim of this in vitro study was to investigate drug treatments to mitigate oxidative stress and oxidative DNA-damage due to glucose-dependent induction of TXNIP expression. Method Using a mesotheliomal cell line (NCl-H28, ATCC) we performed in vitro studies mitigating reactive oxygen species (ROS) production and oxidative DNA damage as well. Cells were treated for three and eight hours with high-glucose media (1.5%, 2.3% and 4.25% glucose; glucose concentrations were adapted to PDFs from Fresenius Medical Care) compared to control media (normal glucose concentration). mRNA expression of TXNIP, TRX and further redox-related genes like superoxide dismutases (SODs) or catalase were analyzed by qPCR. Since TRX is negatively regulated by TXNIP, we investigated TRX activity in cell lysates. ROS production was monitored using a bioluminescent assay and immunofluorescence using the redox dye CellRoxGreen as well. The content of oxidative DNA-damage was investigated by immunofluorescence. Furthermore, we have preliminary analysis using primary human peritoneal mesothelial cells derived from PD effluent (PDF). Results We demonstrated a dose-dependent upregulation of round 1.5 fold TXNIP expression on mRNA level. However, no change in the expression of TRX, SODs and catalase could be observed. Interestingly, we observed a 20% reduction in TRX activity upon glucose stimulation compared to cells treated with control media. These findings are consistent with the previously published data on human peritoneal biopsies. Beyond, we demonstrated a dose-dependent increase in ROS production and increase in oxidative DNA-damage. However, ROS production and oxidative DNA damage could be prevented upon pre-incubation with the ROS-scavenger N-acetylcysteine (NAC). Conclusion Here, we show that enhanced levels of ROS and oxidative DNA damage due to glucose induced TXNIP expression could be diminished using ROS scavengers. These findings show that further antioxidative substances may be potential drugs, which could also be potential drug candidates for use in clinical practice to treat PD patients.

Spheroids as a model for endometriotic lesions.

The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a nonadherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells (12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared with spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in the presence of estradiol or pro-inflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.

TMT quantitative proteomics and network pharmacology reveal the mechanism by which asiaticoside regulates the JAK2/STAT3 signaling pathway to inhibit peritoneal fibrosis.

Traditional Chinese medicine, Centella asiatica (L.) Urb., has been extensively utilized in clinics to treat a variety of fibrotic disorders. Asiaticoside (ASI), as an important active ingredient, has attracted much attention in this field. However, the effect of ASI on peritoneal fibrosis (PF) is still unclear. Therefore, we evaluated the benefits of ASI for PF and mesothelial-mesenchymal transition (MMT) and revealed the underlying mechanisms. The objective of this investigation was to anticipate the potential molecular mechanism of ASI against peritoneal mesothelial cells (PMCs) MMT employing proteomics and network pharmacology, and to confirm it using in vivo and in vitro studies. The mesentery of peritoneal fibrosis mice and normal mice were analyzed quantitatively for proteins that were differentially expressed using a technique tandem mass tag (TMT). Next, the core target genes of ASI against PF were screened through network pharmacology analysis, and PPI and C-P‒T networks were constructed by Cytoscape Version 3.7.2. According to the findings of a GO and KEGG enrichment analysis of differential proteins and core target genes, the signaling pathway with a high correlation degree was selected as the key signaling pathway of ASI inhibiting the PMCs MMT for further molecular docking analysis and experimental verification. TMT-based quantitative proteome analysis revealed the identification of 5727 proteins, of which 70 were downregulated and 178 were upregulated. Among them, the levels of STAT1, STAT2, and STAT3 in the mesentery of mice with peritoneal fibrosis were considerably lower than in the control group, indicating a role for the STAT family in the pathogenesis of peritoneal fibrosis. Then, a total of 98 ASI-PF-related targets were identified by network pharmacology analysis. JAK2 is one of the top 10 core target genes representing a potential therapeutic target. JAK/STAT signaling may represent a core pathway mediating PF effects by ASI. Molecular docking studies showed that ASI had the potential to interact favorably with target genes involved in the JAK/STAT signaling pathway, such as JAK2 and STAT3.The experimental results showed that ASI could significantly alleviate Chlorhexidine Gluconate (CG)-induced peritoneal histopathological changes and increase JAK2 and STAT3 phosphorylation levels. In TGF-β1-stimulated HMrSV5 cells, E-cadherin expression levels were dramatically reduced whereas Vimentin, p-JAK2, α-SMA, and p-STAT3 expression levels were considerably increased. ASI inhibited the TGF-β1-induced HMrSV5 cell MMT, decreased the activation of JAK2/STAT3 signaling, and increased the nuclear translocation of p-STAT3, which was consistent with the effect of the JAK2/STAT3 pathway inhibitor AG490. ASI can inhibit PMCs MMT and alleviate PF by regulating the JAK2/STAT3 signaling pathway.

Mechanisms of human umbilical cord mesenchymal stem cells-derived exosomal lncRNA GAS5 in alleviating EMT of HPMCs via Wnt/β-catenin signaling pathway.

Prolonged peritoneal dialysis (PD) can result in epithelial-to-mesenchymal transition (EMT) and peritoneal fibrosis (PF), which can cause patients to discontinue PD. It is imperative to urgently investigate effective measures to mitigate PF. This study aims to reveal mechanisms of exosomal lncRNA GAS5 derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) on EMT of human peritoneal mesothelial cells (HPMCs) under high glucose (HG) conditions. HPMCs were stimulated with 2.5% glucose. The effects on EMT of HPMCs were observed by using an hUC-MSC conditioned medium (hUC-MSC-CM) and extracted exosomes. After hUC-MSCs were transfected with GAS5 siRNA, exosomes were extracted to act on HPMCs for detecting EMT markers, PTEN, and Wnt/β-catenin pathway, lncRNA GAS5 and miR-21 expressions in HPMCs. We found that HG could induce the EMT of HPMCs. Compared with the HG group, the hUC-MSC-CM could alleviate the EMT of HPMCs induced by HG through exosomes. Exosomes in the hUC-MSC-CM entered HPMCs, by transferring lncRNA GAS5 to HPMCs, which down-regulates miR-21 and up-regulates PTEN, thus finally alleviating EMT of HPMCs. The Wnt/β-catenin pathway plays an essential role in alleviating EMT of HPMCs by exosomes in the hUC-MSC-CM. By transferring lncRNA GAS5 to HPMCs, exosomes derived from hUC-MSCs may competitively bind to miR-21 to regulate suppression on target PTEN genes and alleviate EMT of HPMCs through the Wnt/β-catenin pathway. Exosomes from the hUC-MSCs-CM could alleviate the EMT of HPMCs induced by HG via regulating lncRNA GAS5/miR-21/PTEN through the Wnt/β-catenin signaling pathway.

Autophagy in peritoneal fibrosis.

Peritoneal dialysis (PD) is a widely accepted renal replacement therapy for patients with end-stage renal disease (ESRD). Morphological and functional changes occur in the peritoneal membranes (PMs) of patients undergoing long-term PD. Peritoneal fibrosis (PF) is a common PD-related complication that ultimately leads to PM injury and peritoneal ultrafiltration failure. Autophagy is a cellular process of "self-eating" wherein damaged organelles, protein aggregates, and pathogenic microbes are degraded to maintain intracellular environment homeostasis and cell survival. Growing evidence shows that autophagy is involved in fibrosis progression, including renal fibrosis and hepatic fibrosis, in various organs. Multiple risk factors, including high-glucose peritoneal dialysis solution (HGPDS), stimulate the activation of autophagy, which participates in PF progression, in human peritoneal mesothelial cells (HPMCs). Nevertheless, the underlying roles and mechanisms of autophagy in PF progression remain unclear. In this review, we discuss the key roles and potential mechanisms of autophagy in PF to offer novel perspectives on future therapy strategies for PF and their limitations.

Canagliflozin alleviates high glucose-induced peritoneal fibrosis via HIF-1α inhibition.

The cardioprotective effects of sodium-glucose cotransporter type 2 (SGLT2) inhibitors have been demonstrated in many studies. However, their benefits for end-stage kidney disease patients, particularly those on peritoneal dialysis, remain unclear. SGLT2 inhibition has shown peritoneal protective effects in some studies, but the mechanisms are still unknown. Herein, we investigated the peritoneal protective mechanisms of Canagliflozin in vitro by simulating hypoxia with CoCl2 in human peritoneal mesothelial cells (HPMCs) and rats by intraperitoneal injection of 4.25% peritoneal dialysate simulating chronic high glucose exposure. CoCl2 hypoxic intervention significantly increased HIF-1α abundance in HPMCs, activated TGF-β/p-Smad3 signaling, and promoted the production of fibrotic proteins (Fibronectin, COL1A2, and α-SMA). Meanwhile, Canagliflozin significantly improved the hypoxia of HPMCs, decreased HIF-1α abundance, inhibited TGF-β/p-Smad3 signaling, and decreased the expression of fibrotic proteins. Five-week intraperitoneal injection of 4.25% peritoneal dialysate remarkably increased peritoneal HIF-1α/TGF-β/p-Smad3 signaling and promoted peritoneal fibrosis and peritoneal thickening. At the same time, Canagliflozin significantly inhibited the HIF-1α/TGF-β/p-Smad3 signaling, prevented peritoneal fibrosis and peritoneal thickening, and improved peritoneal transportation and ultrafiltration. High glucose peritoneal dialysate increased the expression of peritoneal GLUT1, GLUT3 and SGLT2, all of which were inhibited by Canagliflozin. In conclusion, we showed that Canagliflozin could improve peritoneal fibrosis and function by ameliorating peritoneal hypoxia and inhibiting the HIF-1α/TGF-β/p-Smad3 signaling pathway, providing theoretical support for the clinical use of SGLT2 inhibitors in patients on peritoneal dialysis.

Modification of polypropylene mesh by titanium compound: An in Vivo and in Vitro study

Objective: Previous basic studies on the use of titanized polypropylene meshes in abdominal external hernia repair are not only limited, but also highly controversial. This study aims to investigate the modification effect of titanium compounds on polypropylene materials and compare the performance of two kinds of meshes both in vivo and in vitro.Methods: Human peritoneal mesothelial cells (HMrSV5), human epidermal fibroblasts (HSF), and human monocytic cells (THP-1) were cultured in vitro to simulate the abdominal external hernia environment. Titanized polypropylene meshes (Ti) and polypropylene mesh (Non-Ti) were co-cultured with the cells respectively. The effects of titanium compounds on cell growth were determined by cell activity and apoptosis, and the growth of cells on the mesh surface was assessed using a scanning electron microscope and a confocal microscope. In vivo experiments, different sizes titanized polypropylene meshes and polypropylene meshes were placed between the external oblique abdominal muscle and the internal oblique abdominal muscle, the parietal peritoneum, the serous layer of the descending colon, and the underside of the femoral nerve in rabbits. The effects of titanium compounds were evaluated by observing the anti-adhesion, anti-contraction, anti-fibrotic properties, and effects on nerves of the mesh.Results: Titanium compounds effectively reduced the effects of polypropylene material on cell growth, and improved the fixation and adhesion of HMrSV5, HSF and THP-1 (M0) on the surface of titanized polypropylene meshes. Furthermore, titanium compounds improved the adhesion, contraction and fibrosis of polypropylene material, as well as reduced nerve damage. This improvement demonstrated a regular trend with the type of titanized polypropylene meshes.Conclusion: The titanium compounds improved the biocompatibility of the polypropylene material, which was conducive to the fixation and adhesion of cells on the surface of the meshes, and alleviated the adhesion and contraction of the meshes, and the degree of tissue fibrosis, as well as the influence on nerves.

Abstract 2739: Genetic modification of mesothelial cells with full length membrane-bound TRAIL for ovarian cancer treatment

Abstract Mesothelial cells lining the peritoneum play an important role in epithelial ovarian cancer (EOC) locoregional spreading by promoting cancer cell proliferation, adhesion, and invasion into the peritoneal wall and organs. Peritoneal dissemination represents a critical first step in EOC progression, and we posit that genetic modification of mesothelial cells with full length membrane-bound TRAIL is a practical approach to intervention and will provide local control of EOC progression. Here, we stably overexpressed full length tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with a lentiviral vector in the immortalized mesothelial cell line, MeT5A. Overexpression of TRAIL on the cell surface of MeT5A was confirmed by flow cytometry, and no obvious changes in proliferation or morphology were observed when MeT5A/TRAIL cells were grown in culture. In co-culture experiments, however, MeT5A/TRAIL cells induced apoptosis of surrounding cancer cells and continued proliferating until the culture surface was confluent with mesothelial cells alone. Interestingly, MeT5A/TRAIL not only induced cell death of TRAIL-sensitive cancer cells, such as OVCAR3 and H460, but it also induced significant apoptosis of cancer cells previously deemed resistant to soluble TRAIL, such as A2780, and OVCAR8. This finding suggests that full length TRAIL (amino acid 1-281) expressed on the cell surface is capable of activating death receptor-mediated apoptotic pathways that are not reactive to the soluble form of TRAIL (amino acid 114-281), which was previously developed as a cancer therapeutic and thoroughly evaluated in animal models and clinical trials. When co-cultured with MeT5A/TRAIL, primary ovarian cancer cells from patients also underwent apoptosis as detected by flow cytometry and the upregulation of cleaved caspase-3, caspase-8, caspase-9, PARP, and Bid on Western blots. Next, we assessed the anticancer potential of mesothelial cells expressing membrane-bound TRAIL in a NSG xenograft mouse model of ovarian cancer. Intraperitoneal inoculation of MeT5A/TRAIL cells significantly reduced overall cancer burden in mice where OVCAR3, A2780, and OVCAR8 cancer cells were pre-established. Intraperitoneal gene delivery of TRAIL to resident peritoneal mesothelial cells utilizing a conditionally replicative adenovirus vector also prolonged survival of NSG mice bearing A2780 xenografts. Our data suggest that genetic modification of mesothelial cells with full length membrane-bound TRAIL is feasible and has therapeutic potential for disrupting EOC progression. Citation Format: Er Yue, Tonya Walser, Tamara Mirzapoiazova, Ravi Salgia, Edward W. Wang. Genetic modification of mesothelial cells with full length membrane-bound TRAIL for ovarian cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2739.