Human Peritoneal Mesothelial Cells Research Articles

Autophagy caused by oxidative stress promotes TGF-β1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells

Epithelial-to-mesenchymal transition (EMT) is one of the main causes of peritoneal fibrosis. However, the pathophysiological mechanisms of EMT, specifically its relationship with autophagy, are still unknown. This study aimed to evaluate the role of autophagy in transforming growth factor-beta 1 (TGF-β1)-induced EMT in human peritoneal mesothelial cells (HPMCs). Primary cultured HPMCs were treated with TGF-β1 (2 and 5 ng/mL) and changes in autophagy markers and the relationship between autophagy and EMT were evaluated. We also identified changes in EMT- and autophagy-related signaling pathways after autophagy and NADPH oxidase 4 (NOX4) inhibition. TGF-β1 increased the generation of NOX4 and reactive oxygen species (ROS) in HPMCs, resulting in mitochondrial damage. Treatment with GKT137831 (20 μM), a NOX1/4 inhibitor, reduced ROS in the mitochondria of HPMC cells and reduced TGF-β1-induced mitochondrial damage. Additionally, the indirect inhibition of autophagy by GKT137831 (20 μM) downregulated TGF-β1-induced EMT, whereas direct inhibition of autophagy using 3-methyladenine (3-MA) (2 mM) or autophagy-related gene 5 (ATG5) gene silencing decreased the TGF-β1-induced EMT in HPMCs. The suppressor of mothers against decapentaplegic 2/3 (Smad2/3), autophagy-related phosphoinositide 3-kinase (PI3K) class III, and protein kinase B (Akt) pathways, and mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular signal-regulated kinase (ERK) and P38, were involved in TGF-β1-induced EMT. Autophagy and NOX4 inhibition suppressed the activation of these signaling pathways. Direct inhibition of autophagy and its indirect inhibition through the reduction of mitochondrial damage by upstream NOX4 inhibition reduced EMT in HPMCs. These results suggest that autophagy could serve as a therapeutic target for the prevention of peritoneal fibrosis in patients undergoing peritoneal dialysis.

#2144 Expression profile induced by TGF-β1 in senescent human peritoneal mesothelial cells is less pronounced than in young cells

Abstract Background and Aims Transforming growth factor β1 (TGF-β1) is a recognized driver of both mesothelial-to-mesenchymal transition (MMT) and cellular senescence observed in vitro in human peritoneal mesothelial cells (HPMCs), and have a significant role in adverse peritoneal remodelling of patients treated with peritoneal dialysis (PD). We have previously shown that senescent HPMCs spontaneously acquire some phenotypic features of MMT which in young HPMCs are induced by TGF-β1. At the same time, senescent HPMCs appeared to be less responsive to TGF-β. It is not clear what determines which pathway is activated by TGF-β1. Here, we aimed to extend previous observations and used electron microscopy, transcriptomics, and proteomics to identify differences between young and senescent cells in their responsiveness to TGF-β. Method Replicative senescence of omentum-derived HPMCs isolated from 4 separate donors was induced by serial passages until cells ceased to grow and expressed senescence-associated β-galactosidase. Serum-starved young and senescent cells were treated in parallel with 1 ng/mL of TGF-β1 for 72 hours. Fixed cells were analyzed with a Jeol transmission electron microscope at 80 kV (Jeol, Tokyo, Japan). Cell lysates were analysed for global gene and protein expression using an integrated transcriptomic (Array-based) and proteomic (LC/MS-based) approach. Results Exposure to TGF-β1 led to phenotypic changes consistent with MMT in young, but not in senescent HPMCs (Fig. 1A). The omics analyses revealed that the response to TGF-β1 was associated with 89 genes being differentially expressed (55 down- and 34 up-upregulated) in young HPMCs and with 13 genes (5 down- and 8 up-regulated) in senescent HPMCs (Fig. 1B). In addition, TGF-β1 changed the expression of 131 proteins in young HPMCs (72 down- and 59 up-regulated) and 232 proteins in senescent HPMCs (127 down- and 105 up-regulated) (Fig. 1C). The responses to TGF-β identified at both the transcriptome and protein levels (Fig. 1D) and present in both young and senescent HPMCs included TGFBI (TGF-β-induced) and ITGB3 (β3 integrin), which were upregulated, and UPK1B (uroplakin 1B) and SEMA3B (semaphorin 3B), which were downregulated. Changes in response to TGF-β seen at the RNA and protein level included also VCAN (versican), ITGA11 (integrin α11), FN1 (fibronectin 1), CCNYL1 (Cyclin Y-like 1) and MYOZ2 (myozenin 2), however, these were observed only in young cells. A decrease in KLK7 (Kallikrein 7) gene expression after exposure to TGF-β was seen both in young and senescent HPMCs, with a decrease in KLK7 protein detected only in senescent cells. Finally, exposure to TGF-β led to an increase in THBS1 (thrombospondin 1) expression in young cells and at the protein level in senescent cells. Conclusion In conclusion, the profound morphological changes seen in senescent HPMCs are not significantly exacerbated by TGFβ. Likewise, changes in global gene and protein profiles in response to TGF-β appear to be less pronounced in senescent cells. Among the genes and proteins affected by TGF-β, those involved in angiogenesis and fibrosis deserve further attention as they may contribute to adverse peritoneal remodelling in PD patients.

Differential expression and clinical significance of IGF2BP3 in peritoneal dialysate of patients with varying duration of peritoneal dialysis.

This study aims to investigate the differential expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the peritoneal dialysate among patients with different durations of peritoneal dialysis and its association with the angiogenic marker vascular* endothelial growth factor (VEGF), the fibronectin (FN), and various clinical indicators. A cohort of 122 peritoneal dialysis patients was categorized into short-term (≤1 year, n = 33), mid-term (>1 and ≤5 years, n = 55), and long-term (>5 years, n = 34) groups based on dialysis duration. We utilized enzyme-linked immunosorbent assay (ELISA) and western blot assays to quantify the levels of IGF2BP3, VEGF, and FN in the dialysate. Our findings showed a progressive increase in IGF2BP3 levels with the duration of PD, with the long-term group exhibiting significantly higher levels than both the short-term and mid-term groups (p < 0.001). A positive correlation between IGF2BP3 and VEGF (r = 0.386, p = 0.013), as well as between IGF2BP3 and FN (r = 0.340, p = 0.030), was observed. IGF2BP3 levels also correlated positively with serum creatinine, calcium, and phosphorus levels. Invitro analysis further confirmed that IGF2BP3 expression is enhanced in human peritoneal mesothelial cells under high-glucose conditions (p < 0.05). The study highlights the potential of IGF2BP3 in PD effluent as a biomarker for monitoring PF progression, with its expression significantly correlated with the duration of PD (Pearson r = 0.897, p < 0.001). In conclusion, our results underscore a correlation between elevated IGF2BP3 levels and PD duration, suggesting the clinical significance of IGF2BP3 as a biomarker for PF progression.

Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

Background. The activation of complement is involved in monocyte recruitment in tuberculous pleural effusion (TPE), while the role of the cleavage product of complement C3 in this process needs further research. Methods. The expression of complement components in pleural biopsy specimens of TPE patients was measured. The concentration of cleavage products of complement was tested in TPE by ELISA. Moreover, the colocalizations of C3b and CR1, C3d and CR3, and CXCL12 and CXCR4 in monocytes and pleural mesothelial cells (PMCs) isolated from TPE were determined by an immunofluorescent assay. Monocyte chemotaxis assay was analyzed via transwell chambers. Results. Three pathways of the complement system were activated in tuberculous pleurisy. In patients with TPE, C3 lysis was more active than peripheral blood in pleural cavity. Tuberculous protein Mpt64 and anaphylatoxin C3a could significantly promote CXCL12 production in human PMCs isolated from TPE. C3b-CR1, C3d-CR3, and CXCL12-CXCR4 were colocalized in PMCs and monocytes from TPE. The recruitment of monocytes into TPE mediated by PMCs could be inhibited by anti-CR1, anti-CR3, and anti-CXCL12 monoclonal antibodies (mAbs). Conclusions. Complement activates strongly in TPE, and PMCs induced monocytes to the pleural cavity through C3a, C3b, and C3d.

Expression and significance of SIRT6 in human peritoneal dialysis effluents and peritoneal mesothelial cells.

Sirtuin 6 (SIRT6) can inhibit the fibrosis of many organs. However, the relationship between SIRT6 and peritoneal fibrosis (PF) in peritoneal dialysis (PD) remains unclear. We collected 110 PD patients with a duration of PD for more than 3months and studied the influence of PD duration and history of peritonitis on SIRT6 levels in PD effluents (PDEs). We also analyzed the relationship between SIRT6 levels in PDEs and transforming growth factor beta 1 (TGF-β1), IL-6, PD duration, peritoneal function, PD ultrafiltration (UF), and glucose exposure. We extracted human peritoneal mesothelial cells (HPMCs) from PDEs and measured the protein and gene expression levels of SIRT6, E-cadherin, vimentin, and TGF-β1 in these cells. Based on the clinical results, we used human peritoneal mesothelial cells lines (HMrSV5) to observe the changes in SIRT6 levels and mesothelial-to-mesenchymal transition (MMT) after intervention with PD fluid. By overexpressing and knocking down SIRT6 expression, we investigated the effect of SIRT6 expression on E-cadherin, vimentin, and TGF-β1 expression to elucidate the role of SIRT6 in mesothelial-to-epithelial transition in PMCs. Results: (1) With the extension of PD duration, the influence of infection on SIRT6 levels in PDEs increased. Patients with the PD duration of more than 5years and a history of peritonitis had the lowest SIRT6 levels. (2) SIRT6 levels in PDEs were negatively correlated with PD duration, total glucose exposure, TGF-β1, IL-6 levels, and the dialysate-to-plasma ratio of creatinine (Cr4hD/P), but positively correlated with UF. This indicates that SIRT6 has a protective effect on the peritoneum. (3) The short-term group (PD ≤ 1year) had higher SIRT6 and E-cadherin gene and protein levels than the mid-term group (1year < PD ≤ 5years) and long-term group (PD > 5years) in PMCs, while vimentin and TGF-β1 levels were lower in the mid-term group and long-term group. Patients with a history of peritonitis had lower SIRT6 and E-cadherin levels than those without such a history. (4) After 4.25% PD fluid intervention for HPMCs, longer intervention time resulted in lower SIRT6 levels. (5) Overexpressing SIRT6 can lead to increased E-cadherin expression and decreased vimentin and TGF-β1 expression in HPMCs. Knocking down SIRT6 expression resulted in decreased E-cadherin expression and increased vimentin and TGF-β1 expression in HPMCs. This indicates that SIRT6 expression can inhibit MMT in HPMCs, alleviate PF associated with PD, and have a protective effect on the peritoneum.

Integrative analysis of chromatin accessibility and transcriptome landscapes in the induction of peritoneal fibrosis by high glucose

BackgroundPeritoneal fibrosis is the prevailing complication induced by prolonged exposure to high glucose in patients undergoing peritoneal dialysis.MethodsTo elucidate the molecular mechanisms underlying this process, we conducted an integrated analysis of the transcriptome and chromatin accessibility profiles of human peritoneal mesothelial cells (HMrSV5) during high-glucose treatment.ResultsOur study identified 2775 differentially expressed genes (DEGs) related to high glucose-triggered pathological changes, including 1164 upregulated and 1611 downregulated genes. Genome-wide DEGs and network analysis revealed enrichment in the epithelial–mesenchymal transition (EMT), inflammatory response, hypoxia, and TGF-beta pathways. The enriched genes included VEGFA, HIF-1α, TGF-β1, EGF, TWIST2, and SNAI2. Using ATAC-seq, we identified 942 hyper (higher ATAC-seq signal in high glucose-treated HMrSV5 cells than in control cells) and 714 hypo (lower ATAC-seq signal in high glucose-treated HMrSV5 cells versus control cells) peaks with differential accessibility in high glucose-treated HMrSV5 cells versus controls. These differentially accessible regions were positively correlated (R = 0.934) with the nearest DEGs. These genes were associated with 566 up- and 398 downregulated genes, including SNAI2, TGF-β1, HIF-1α, FGF2, VEGFA, and VEGFC, which are involved in critical pathways identified by transcriptome analysis. Integrated ATAC-seq and RNA-seq analysis also revealed key transcription factors (TFs), such as HIF-1α, ARNTL, ELF1, SMAD3 and XBP1. Importantly, we demonstrated that HIF-1α is involved in the regulation of several key genes associated with EMT and the TGF-beta pathway. Notably, we predicted and experimentally validated that HIF-1α can exacerbate the expression of TGF-β1 in a high glucose-dependent manner, revealing a novel role of HIF-1α in high glucose-induced pathological changes in human peritoneal mesothelial cells (HPMCs).ConclusionsIn summary, our study provides a comprehensive view of the role of transcriptome deregulation and chromosome accessibility alterations in high glucose-induced pathological fibrotic changes in HPMCs. This analysis identified hub genes, signaling pathways, and key transcription factors involved in peritoneal fibrosis and highlighted the novel glucose-dependent regulation of TGF-β1 by HIF-1α. This integrated approach has offered a deeper understanding of the pathogenesis of peritoneal fibrosis and has indicated potential therapeutic targets for intervention.

Myocardin regulates fibronectin expression and secretion from human pleural mesothelial cells.

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-β induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-β stimulation at nucleus, suggesting that TGF-β facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.

SPHK1/S1P/S1PR pathway promotes the progression of peritoneal fibrosis by mesothelial-mesenchymal transition.

Long-term exposure to non-physiologically compatible dialysate inevitably leads to peritoneal fibrosis (PF) in patients undergoing peritoneal dialysis (PD), and there is no effective prevention or treatment for PF. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced after catalysis by sphingosine kinase (SPHK) 1/2 and activates signals through the S1P receptor (S1PR) via autocrine or paracrine. However, the role of SPHK1/S1P/S1PR signaling has never been elucidated in PF. In our research, we investigated S1P levels in peritoneal effluents and demonstrated the role of SPHK1/S1P/S1PR pathway in peritoneal fibrosis. It was found that S1P levels in peritoneal effluents were positively correlated with D/P Cr (r = 0.724, p < .001) and negatively correlated with 4 h ultrafiltration volume (r = -0.457, p < .001). S1PR1 and S1PR3 on peritoneal cells were increased after high glucose exposure invivo and invitro. Fingolimod was applied to suppress S1P/S1PR pathway. Fingolimod restored mouse peritoneal function by reducing interstitial hyperplasia, maintaining ultrafiltration volume, reducing peritoneal transport solute rate, and mitigating the protein expression changes of fibronectin, vimentin, α-SMA, and E-cadherin induced by PD and S1P. Fingolimod preserved the morphology of the human peritoneal mesothelial cells, MeT-5A, and moderated the mesothelial-mesenchymal transition (MMT) process. We further delineated that SPHK1 was elevated in peritoneal cells after high glucose exposure and suppression of SPHK1 in MeT-5A cells reduced S1P release. Overexpression of SPHK1 in MeT-5A cells increased S1P levels in the supernatant and fostered the MMT process. PF-543 treatment, targeting SPHK1, alleviated deterioration of mouse peritoneal function. In conclusion, S1P levels in peritoneal effluent were correlated with the deterioration of peritoneal function. SPHK1/S1P/S1PR pathway played an important role in PF.

Different calcium ion concentrations affect epithelial mesenchymal transformation of human peritoneal mesothelial cells via endoplasmic reticulum stress

To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation (EMT) of human peritoneal mesothelial cell (HPMC) via endoplasmic reticulum stress (ERS). HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting. Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2-ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2-ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2-ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2-ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/β-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/β-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/β-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/β-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2-ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2-ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2-ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2-ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/β-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/β-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/β-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/β-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05]. High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.

A protective role of nintedanib in peritoneal fibrosis through H19-EZH2-KLF2 axis via impeding mesothelial-to-mesenchymal transition.

Peritoneal fibrosis (PF), a common complication of long-term peritoneal dialysis, accounts for peritoneal ultrafiltration failure to develop into increased mortality. Nintedanib has previously been shown to protect against multi-organ fibrosis, including PF. Unfortunately, the precise molecular mechanism underlying nintedanib in the pathogenesis of PF remains elusive. The mouse model of PF was generated by chlorhexidine gluconate (CG) injection with or without nintedanib administration, either with the simulation for the cell model of PF by constructing high-glucose (HG)-treated human peritoneal mesothelial cells (HPMCs). HE and Masson staining were applied to assess the histopathological changes of peritoneum and collagen deposition. FISH, RT-qPCR, western blot and immunofluorescence were employed to examine distribution or expression of targeted genes. Cell viability was detected using CCK-8 assay. Cell morphology was observed under a microscope. RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays were applied to validate the H19-EZH2-KLF2 regulatory axis. Aberrantly overexpressed H19 was observed in both the mouse and cell model of PF, of which knockdown significantly blocked HG-induced mesothelial-to-mesenchymal transition (MMT) of HPMCs. Moreover, loss of H19 further strengthened nintedanib-mediated suppressive effects against MMT process in a mouse model of PF. Mechanistically, H19 could epigenetically repressed KLF2 via recruiting EZH2. Furthermore, TGF-β/Smad pathway was inactivated by nintedanib through mediating H19/KLF2 axis. In summary, nintedanib disrupts MMT process through regulating H19/EZH2/KLF2 axis and TGF-β/Smad pathway, which laid the experimental foundation for nintedanib in the treatment of PF.

Role of tenascin C in lesion formation in early peritoneal endometriosis

To identify cytokines or extracellular matrix components that contribute to adhesion to, and invasion of, the peritoneum, proximal to lesions in the early phase of endometriosis. Laboratory-based study. University Hospital and Laboratory of Animal Science. Five women with ovarian endometrioma, 138 wild-type (WT) C57BL/6N mice, and 48 Tenascin C (Tnc) knockout (TncKO) mice. To establish a murine endometriosis model, 20 pieces of minced uterine tissue fragments from each horn were administered intraperitoneally to syngeneic mice. Three days later, endometriotic lesions and peritoneal tissues were collected. Separately, we transfected human peritoneal mesothelial cells (HMrSV5) or human endometrial stromal cells (hESCs) with Tnc small interfering ribonucleic acid. We employed a polymerase chain reaction array to profile gene expression in the murine peritoneum, in both peritoneum distal to lesions and peritoneum surrounding lesions (PSL). The expression of upregulated genes in the PSL was verified in the peritoneal samples by real-time reverse transcription-polymerase chain reaction. TncKO mice were used to investigate the role of Tnc in the development of endometriosis. We evaluated the proliferative activity or inflammatory state of lesions by Ki67 or CD3 immunostaining. Intraperitoneal distribution of macrophages was assessed by fluorescence-activated cell sorting. Using Tnc small interfering ribonucleic acid, we examined the invasive capacity of hESCs in a coculture system with HMrSV5. Tnc gene expression was significantly higher in PSL than in peritoneum distal to lesions. The weight and number of TncKO lesions in TncKO hosts were lower than those of WT lesions in WT hosts. In contrast, the weight and number of nonattached TncKO lesions in TncKO hosts were higher than those of nonattached WT lesions in WT hosts. We observed decreased Ki67-positive cells or H-scores for CD3, a lower proportion of M1 macrophages, and a higher proportion of M2 macrophages in TncKO lesions in TncKO recipients. Silencing of Tnc expression in hESCs and HMrSV5 diminished the invasivity of hESCs. Tnc may be a crucial factor in the development of early peritoneal endometriosis.

O-GlcNAcylation regulates HIF-1α and induces mesothelial-mesenchymal transition and fibrosis of human peritoneal mesothelial cells

O-GlcNAcylation is a post-translational modification of proteins that regulates various biological processes. However, its involvement in peritoneal dialysis fibrosis remains unclear. This study aimed to investigate the impact of O-GlcNAcylation on human peritoneal mesothelial cells (HPMCs) cultured in control and high-glucose medium. To manipulate cellular conditions, we employed knockdown techniques targeting HIF-1α and OGT, along with the administration of pharmacological agents (PUGNAc, OSMI-1, MG-132, FG-4592, and HIF-1α inhibitor). Our findings revealed that elevated glucose levels increased global O-GlcNAcylation and the abundance of HIF-1α, α-SMA, fibronectin, and COL1A2. Conversely, the expression of E-Cadherin was decreased. Significantly, a positive correlation was observed between O-GlcNAcylation, HIF-1α, mesothelial-to-mesenchymal transition (MMT), and fibrosis in HPMCs. Notably, O-GlcNAcylation was found to regulate HIF-1α, thereby promoting MMT and fibrosis under high glucose conditions. Furthermore, we discovered that high glucose levels induced O-GlcNAcylation of HIF-1α, preventing its ubiquitination and proteasomal degradation. In summary, our study demonstrates the critical role of O-GlcNAcylation-mediated regulation of HIF-1α in MMT and fibrosis during peritoneal dialysis.

Preventive effect of culture supernatant of epithelial-like peritoneal mesothelial cells on peritoneal fibrosis.

Peritoneal fibrosis (PF) is a primary reason for discontinuing peritoneal dialysis, which involves characteristic changes of peritoneal mesothelial cells (PMCs). We previously reported preventive effects of implanting human epithelial-like PMCs (P-Epi) for mouse PF caused by mechanical peritoneum scrapings. In the present study, we analysed the preventive effects of culture supernatant of P-Epi in PF. Concentrated culture supernatant of P-Epi or human fibroblast-like PMCs (P-Fibro) or vehicles was injected into nude mice that had undergone mechanical scraping of the parietal and visceral peritoneum, and thickness and amount of adhesions were analysed. Although increased peritoneal adhesions and peritoneum thickening were observed in the vehicle-injected positive control group compared to the sham operation group, fewer number of adhesions and less thickness were observed in the mice treated with culture supernatant of P-Epi, but not P-Fibro, compared to the vehicle-injected positive controls. Immunofluorescent analysis revealed that the expression of extracellular matrix, type I collagen and fibronectin, was lower in the mice treated with culture supernatant of P-Epi than in the vehicle-injected positive controls. In addition, exosomes from P-Epi significantly reduced transforming growth factor-β (TGF-β)-induced expressions of type I collagen and fibronectin in 3T3 fibroblast cells. Collectively, culture supernatant of P-Epi has preventive effects on PF, thus cell therapy is not necessarily required. Further exploration of substances secreted by P-Epi and their protective mechanisms could lead to the development of therapeutic strategies to limit PF.

Role of Plasminogen Activator Inhibitor-1 (PAI-1) on Phenotype Transition and an Induction of Oxidative Stress in Human Peritoneal Mesothelial Cells (MCs)

Role of Plasminogen Activator Inhibitor-1 (PAI-1) on Phenotype Transition and an Induction of Oxidative Stress in Human Peritoneal Mesothelial Cells (MCs)

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.

The mTORC2/SGK1/NDRG1 Signaling Axis Is Critical for the Mesomesenchymal Transition of Pleural Mesothelial Cells and the Progression of Pleural Fibrosis.

Progressive lung scarring because of persistent pleural organization often results in pleural fibrosis (PF). This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to loculation. In PF, pleural mesothelial cells undergo mesomesenchymal transition (MesoMT) to become profibrotic, characterized by increased expression of α-smooth muscle actin and matrix proteins, including collagen-1. In our previous study, we showed that blocking PI3K/Akt signaling inhibits MesoMT induction in human pleural mesothelial cells (HPMCs) (1). However, the downstream signaling pathways leading to MesoMT induction remain obscure. Here, we investigated the role of mTOR complexes (mTORC1/2) in MesoMT induction. Our studies show that activation of the downstream mediator mTORC1/2 complex is, likewise, a critical component of MesoMT. Specific targeting of mTORC1/2 complex using pharmacological inhibitors such as INK128 and AZD8055 significantly inhibited transforming growth factor β (TGF-β)-induced MesoMT markers in HPMCs. We further identified the mTORC2/Rictor complex as the principal contributor to MesoMT progression induced by TGF-β. Knockdown of Rictor, but not Raptor, attenuated TGF-β-induced MesoMT in these cells. In these studies, we further show that concomitant activation of the SGK1/NDRG1 signaling cascade is essential for inducing MesoMT. Targeting SGK1 and NDRG1 with siRNA and small molecular inhibitors attenuated TGF-β-induced MesoMT in HPMCs. Additionally, preclinical studies in our Streptococcus pneumoniae-mediated mouse model of PF showed that inhibition of mTORC1/2 with INK128 significantly attenuated the progression of PF in subacute and chronic injury. In conclusion, our studies demonstrate that mTORC2/Rictor-mediated activation of SGK1/NDRG1 is critical for MesoMT induction and that targeting this pathway could inhibit or even reverse the progression of MesoMT and PF.

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.

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.

#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.