Primary Human Peritoneal Mesothelial Cells Research Articles

#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.

FC088: Molecular and Functional Characterization of the Mesothelial and Endothelial Cell Barrier in Health, Ckd and Peritoneal Dialysis

Abstract BACKGROUND AND AIMS Tight junctions (TJ) and transcellular ion channels and transporters define solute transport characteristics across cellular barriers, which is of particular interest in patients on peritoneal dialysis (PD). Little is known about their cell specific expression, and regulation in PD. We studied their expression in human endothelial and mesothelial cell lines and in paediatric peritoneal tissues. METHOD In vitro, polarized primary human peritoneal mesothelial cells (HPMC), immortalized mesothelial cells (MeT-5A), human umbilical vein endothelial cells (HUVEC) and human capillary microvascular endothelial cells (HCMEC) underwent RNA sequencing, and gene enrichment analysis (GSEA, ClueGO/Cluepedia) for functional annotation. Key findings were reconfirmed by western blotting and confocal laser scanning immunofluorescence microscopy. Transepithelial electrical resistance (TER) and permeability fluxes of fluorescent 4-, 10- and 70-kDa dextran were measured in Transwells. Ex vivo, whole transcriptome and proteome data from microdissected omental arterioles were used for targeted pathway analysis in non-CKD children, chronic kidney disease (CKD5) and on PD with low and high glucose degradation product (GDP) content (n = 6/group). Mesothelial and endothelial peritoneal solute transporting proteins were quantified in parietal peritoneum of independent paediatric non-CKD, CKD5 and PD cohorts by digital immunohistochemistry. RESULTS A total of 9853 of 12 760 transcripts were common between all four cell lines. A total of 631 transcripts were MeT-5A, 366 HPMC, 99 HUVEC and 87 HCMEC specific. Next to the tissue origin and transformation status, the transcripts reflected major differences in extracellular matrix, glycocalyx and adhesion organization between HCMEC and HUVEC, and extracellular matrix, migration, growth factor and immune response between HPMC and MeT-5A cells. While total counts of cell junction, transmembrane and endocytosis related transcripts were similar among cell lines, the specific TJ, transmembrane and endocytosis related transcript patterns, differed substantially between endothelial and mesothelial cells. Of the functionally well described sealing TJs, claudin (CLDN)1 was expressed in mesothelial cells, and CLDN5 in endothelial cells. Findings were reconfirmed by western blotting and immunofluorescence staining. Functionally, transepithelial resistance (TER) was 50% lower for HCMEC compared to HPMC, MeT-5A and HUVEC; 4-, 10- and 70-kDa dextran permeability was increased in HCMEC. Ex vivo, human arteriolar pathway analysis demonstrated upregulation of paracellular transport-related pathways in CKD5 versus non-CKD patients on protein level. Compared to CKD5, low GDP PD upregulated and high GDP PD downregulated these pathways on the transcriptome and protein levels. Transcellular transporter pathway regulation was variable. In the parietal peritoneum the endothelial surface area for transport was age dependently 1.5–2-fold higher than the mesothelial surface area and the ratio markedly increased with low GDP PD. Validation in parietal peritoneal tissues, reconfirmed arteriolar sealing TJ regulation. Arteriolar CLDN2, a paracellular pore forming cation and water transporter, correlated with D/PCreatinine (r = 0.58) and D/D0Glucose (r = –0.62), mesothelial pore forming cation transporter CLDN15 with D/PCreatinine (r = 0.57) and D/D0Glucose (–0.66). Transcellular transporters for sodium, glucose and phosphate were hardly affected by PD; phosphate transporter PIT1 abundance correlated with serum phosphate (r = –0.48). CONCLUSION We provide the first comprehensive analysis of the peritoneal paracellular and transcellular determinants of solute transporters and its regulation by CKD and PD. Mesothelial and endothelial cell barrier sealing and transporter abundance differed substantially, and associated with PD membrane function, with functional data suggesting a key role of both the mesothelial and endothelial cell barrier.

Patient-derived and artificial ascites have minor effects on MeT-5A mesothelial cells and do not facilitate ovarian cancer cell adhesion.

The presence of ascites in the peritoneal cavity leads to morphological and functional changes of the peritoneal mesothelial cell layer. Cells loose cell-cell interactions, rearrange their cytoskeleton, activate the production of fibronectin, and change their cell surface morphology in a proinflammatory environment. Moreover, ovarian cancer cell adhesion has been shown to be facilitated by these changes due to increased integrin- and CD44-mediated binding sites. In this study, the biological responsiveness of the human pleural mesothelial cell line MeT-5A to patient-derived and artificial ascites was studied in vitro and adhesion of ovarian cancer cells, i.e. SKOV-3 cells, investigated. Changes were mainly observed in cells exposed to artificial ascites containing higher cytokine concentrations than patient-derived ascites. Interestingly, reduced cell-cell interactions were already observed in untreated MeT-5A cells and effects on tight junction protein expression and permeability upon exposure to ascites were minor. Ascites induced upregulation of CDC42 effector protein 2 expression, which affects stress fiber formation, however significant F-actin reorganization was not observed. Moreover, fibronectin production remained unchanged. Analysis of mesothelial cell surface characteristics showed upregulated expression of intercellular adhesion molecule 1, slightly increased hyaluronic acid secretion and decreased microvillus expression upon exposure to ascites. Nevertheless, the observed changes were not sufficient to facilitate adhesion of SKOV-3 cells on MeT-5A cell layer. This study revealed that MeT-5A cells show a reduced biological responsiveness to the presence of ascites, in contrast to published studies on primary human peritoneal mesothelial cells.

AGEs induce epithelial to mesenchymal transformation of human peritoneal mesothelial cells via upregulation of STAT3.

Epithelial-mesenchymal transition (EMT) in peritoneum was induced during peritoneal dialysis (PD), which finally caused progressive fibrosis. However, the underlying mechanisms were not well elucidated. We established advanced glycation end-products (AGEs)-induced EMT model using primary human peritoneal mesothelial cells (HPMCs). The working concentration and time of AGEs were optimized. Then the expression and activation signal transducer and activator of transcription 3 (STAT3), a key factor of EMT in cancer, were detected. The regulation of STAT3 by miRNA was also explored. 50μg/ml AGEs treatment for 24h can successfully induce EMT in HPMCs. AGEs treatment upregulated and activated STAT3. miRNA-454, potentially targeting STAT3, was down-regulated in AGEs-treated HPMCs. Overexpression of miRNA-454 prevented AGEs- induced EMT in HPMCs. AGEs induce epithelial to mesenchymal transformation of Human peritoneal mesothelial cells via upregulation of STAT3.

Telomere length profiles in primary human peritoneal mesothelial cells are consistent with senescence

Mesothelial cell (MC) senescence contributes to malignancy and tissue fibrosis. The role of telomere erosion in MC senescence remains controversial, with evidence for both telomere-dependent and telomere-independent mechanisms reported. Single telomere length analysis revealed considerable telomere length heterogeneity in freshly isolated human peritoneal MCs, reflecting a heterogeneous proliferative history and providing high-resolution evidence for telomere-dependent senescence. By contrast the attenuated replicative lifespan, lack of telomere erosion and induction of p16 expression in in vitro-aged cells was consistent with stress-induced senescence. Given the potential pathophysiological impact of senescence in mesothelial tissues, high-resolution MC telomere length analysis may provide clinically useful information.

ID2 mediates the transforming growth factor-β1-induced Warburg-like effect seen in the peritoneum of women with endometriosis.

Is inhibitor of DNA-binding protein 2 (ID2) a mediator of the transforming growth factor (TGF)-β1-induced Warburg-like effect seen in the peritoneum of women with endometriosis? The TGF-β1-induced changes in the metabolic phenotype of peritoneal mesothelial cells from women with endometriosis are mediated through the ID2 pathway. TGF-β1 induces the metabolic conversion of glucose to lactate via aerobic glycolysis (the 'Warburg effect') in the peritoneum of women with endometriosis, through increased expression of the transcription factor hypoxia inducible factor α (HIF-1α). ID proteins are transcriptional targets of TGF-β1. Expression of ID2 was investigated in luteal phase peritoneal biopsies from women with regular menstrual cycles, with and without endometriosis (n=8-10 each group) by quantitative RT-PCR (qRT-PCR) and immunohistochemistry. ID2 mRNA expression in primary human peritoneal mesothelial cells (HPMC) and immortalized mesothelial cells (MeT-5A) was assessed by qRT-PCR (n=6). The effects of TGF-β1 and ID2 siRNA on HIF-1α mRNA expression and lactate secretion was assessed using qRT-PCR and a colorimetric lactate assay. ID2 is localized to peritoneal mesothelial and stromal cells of women with and without endometriosis. ID2 mRNA expression is lower in peritoneum adjacent to the endometriosis lesions compared to distal sites (P <0.01). Exposure of HPMC and MeT-5A cells to physiological concentrations of TGF-β1 decreases ID2 mRNA expression (P<0.01, P<0.001, respectively, versus control). ID2 knockdown increases HIF-1α mRNA expression (P < 0.01) and lactate secretion (P <0.05 versus scrambled control) to the same degree as with exposure to TGF-β1. Primary human cell cultures and a cell line were used in this study, and thus the results may not fully represent the situation in vivo. The results should also be replicated using a larger number of samples. Novel therapeutics that target the TGFβ/ID pathway offer a potential role in the treatment of endometriosis. None. This work was funded by a Wellbeing of Women research grant (R42533) awarded to A.W.H., J.K.B. and W.C.D.; and an MRC Centre Grant G1002033. V.J.Y. received grant support from Federation of Women Graduates (134225) and a PhD studentship from the College of Medicine and Veterinary Medicine at the University of Edinburgh. There are no competing interests to declare.

The Therapeutic Potential of Human Umbilical Mesenchymal Stem Cells From Wharton's Jelly in the Treatment of Rat Peritoneal Dialysis-Induced Fibrosis.

A major complication in continuous, ambulatory peritoneal dialysis in patients with end-stage renal disease who are undergoing long-term peritoneal dialysis (PD) is peritoneal fibrosis, which can result in peritoneal structural changes and functional ultrafiltration failure. Human umbilical mesenchymal stem cells (HUMSCs) in Wharton's jelly possess stem cell properties and are easily obtained and processed. This study focuses on the effects of HUMSCs on peritoneal fibrosis in in vitro and in vivo experiments. After 24-hour treatment with mixture of Dulbecco's modified Eagle's medium and PD solution at a 1:3 ratio, primary human peritoneal mesothelial cells became susceptible to PD-induced cell death. Such cytotoxic effects were prevented by coculturing with primary HUMSCs. In a rat model, intraperitoneal injections of 20 mM methylglyoxal (MGO) in PD solution for 3 weeks (the PD/MGO 3W group) markedly induced abdominal cocoon formation, peritoneal thickening, and collagen accumulation. Immunohistochemical analyses indicated neoangiogenesis and significant increase in the numbers of ED-1- and α-smooth muscle actin (α-SMA)-positive cells in the thickened peritoneum in the PD/MGO 3W group, suggesting that PD/MGO induced an inflammatory response. Furthermore, PD/MGO treatment for 3 weeks caused functional impairments in the peritoneal membrane. However, in comparison with the PD/MGO group, intraperitoneal administration of HUMSCs into the rats significantly ameliorated the PD/MGO-induced abdominal cocoon formation, peritoneal fibrosis, inflammation, neoangiogenesis, and ultrafiltration failure. After 3 weeks of transplantation, surviving HUMSCs were found in the peritoneum in the HUMSC-grafted rats. Thus, xenografts of HUMSCs might provide a potential therapeutic strategy in the prevention of peritoneal fibrosis. Significance: This study demonstrated that direct intraperitoneal transplantation of human umbilical mesenchymal stem cells into the rat effectively prevented peritoneal dialysis/methylglyoxal-induced abdominal cocoon formation, ultrafiltration failure, and peritoneal membrane alterations such as peritoneal thickening, fibrosis, and inflammation. These findings provide a basis for a novel approach for therapeutic benefits in the treatment of encapsulating peritoneal sclerosis.

Interference of peritoneal dialysis fluids with cell cycle mechanisms.

Peritoneal dialysis fluids (PDF) differ with respect to osmotic and buffer compound, and pH and glucose degradation products (GDP) content. The impact on peritoneal membrane integrity is still insufficiently described. We assessed global genomic effects of PDF in primary human peritoneal mesothelial cells (PMC) by whole genome analyses, quantitative real-time polymerase chain reaction (RT-PCR) and functional measurements. PMC isolated from omentum of non-uremic patients were incubated with conventional single chamber PDF (CPDF), lactate- (LPDF), bicarbonate- (BPDF) and bicarbonate/lactate-buffered double-chamber PDF (BLPDF), icodextrin (IPDF) and amino acid PDF (APDF), diluted 1:1 with medium. Affymetrix GeneChip U133Plus2.0 (Affymetrix, CA, USA) and quantitative RT-PCR were applied; cell viability was assessed by proliferation assays. The number of differentially expressed genes compared to medium was 464 with APDF, 208 with CPDF, 169 with IPDF, 71 with LPDF, 45 with BPDF and 42 with BLPDF. Out of these genes 74%, 73%, 79%, 72%, 47% and 57% were downregulated. Gene Ontology (GO) term annotations mainly revealed associations with cell cycle (p = 10(-35)), cell division, mitosis, and DNA replication. One hundred and eighteen out of 249 probe sets detecting genes involved in cell cycle/division were suppressed, with APDF-treated PMC being affected the most regarding absolute number and degree, followed by CPDF and IPDF. Bicarbonate-containing PDF and BLPDF-treated PMC were affected the least. Quantitative RT-PCR measurements confirmed microarray findings for key cell cycle genes (CDK1/CCNB1/CCNE2/AURKA/KIF11/KIF14). Suppression was lowest for BPDF and BLPDF, they upregulated CCNE2 and SMC4. All PDF upregulated 3 out of 4 assessed cell cycle repressors (p53/BAX/p21). Cell viability scores confirmed gene expression results, being 79% of medium for LPDF, 101% for BLPDF, 51% for CPDF and 23% for IPDF. Amino acid-containing PDF (84%) incubated cells were as viable as BPDF (86%). In conclusion, PD solutions substantially differ with regard to their gene regulating profile and impact on vital functions of PMC, i.e. on cells known to be essential for peritoneal membrane homeostasis.

Transforming growth factor-β induced Warburg-like metabolic reprogramming may underpin the development of peritoneal endometriosis.

TGF-β is believed to play a major role in the etiology of peritoneal endometriosis. In tumors, TGF-β induces the metabolic conversion of glucose to lactate via glycolysis, a process referred to as the "Warburg effect." Lactate increases cell invasion, angiogenesis, and immune suppression, all crucial steps in the development of endometriosis. The aim of this study was to determine whether TGF-β induces a "Warburg-like" effect in peritoneal endometriosis. The study was informed by human tissue analysis and cel culture. The study was conducted at the university research institute. We studied women undergoing surgical investigation for endometriosis. Concentrations of lactate and TGF-β1 in peritoneal fluid (n = 16) were measured by commercial assay. Expression of genes implicated in glycolysis was measured in endometrial and peritoneal biopsies (n = 31) by quantitative RT-PCR and immunohistochemistry. The effect of TGF-β1 on primary human peritoneal mesothelial cells (n = 6) and immortalized mesothelial (MeT-5A) cells (n = 3) was assessed by quantitative RT-PCR, Western blot, and commercial assays. Lactate, TGF-β1, and markers of glycolysis were measured. Concentrations of lactate in peritoneal fluid paralleled those of TGF-β1, being significantly higher in women with endometriosis compared to women without (P < .05). Endometriosis lesions expressed higher levels of glycolysis-associated genes HIF1A, PDK1, and LDHA than eutopic endometrium, and adjacent peritoneum had higher levels of HIF1A and SLC2A1 than peritoneum from women without disease (P < .05 to P < .001). Exposure of mesothelial cells to TGF-β1 increased production of lactate (P < .05), increased HIF1A mRNA (P < .05), and protein, and increased concentrations of mRNAs encoded by glycolysis-associated genes (LDHA, PDK1, SLC2A1; P < .05). A change in the metabolic phenotype of endometriosis lesions and peritoneal mesothelium in women with endometriosis may favor development of endometriosis.

PAR-1 Mediates the Thrombin-induced Mesothelial Cell Overproduction of VEGF and PAI-1

Thrombin mediates an excess in the production of neoangiogenetic (VEGF) and profibrotic (PAI-1) factors in human peritoneal mesothelial cells (HMC). The mechanisms leading to this overproduction have not been elucidated so far; in the context of peritoneal dialysis it can result in impaired peritoneal membrane function. This study was performed to evaluate the presence of the thrombin receptor protease-activated receptor-1 (PAR-1) in HMC and to characterize its function in the thrombin-dependent effects mentioned above. All experiments were performed using cultured primary HMC. Real-Time PCR and Western Blot were used to evaluate PAR-1; ELISA and Real-Time PCR were employed to examine PAR-1 effects on target mediators. We found that cultivated primary HMC show a basal presence of PAR-1. Stimulation with IL-1β induced an increase of the mesothelial PAR-1 expression whereas stimulation with glycosilated human serum albumin or the ligand thrombin itself resulted in decreased PAR-1 expression. Stimulation with the specific PAR-1 ligand TFLLR-NH(2) caused increased VEGF and PAI-1 levels similar to stimulation with thrombin, whereas preincubation with PAR-1 blocking antibodies ATAP2 and WEDE15 attenuated the thrombin-induced overproduction of VEGF and PAI-1. HMC express PAR-1 and the receptor is involved in thrombin effects on these cells. These findings may be a basis for pharmacological prevention of neoangiogenesis and adhesions in the context of peritoneal dialysis and peritonitis.

Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors

Background. Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC).Methods. Primary HPMC were isolated from omental biopsies and TLR expression detected by real-time polymerase chain reaction (PCR), reverse transcription (RT)–PCR and flow cytometry. The responsiveness of HPMC to specific bacterial TLR agonists was determined using chemokine production as a biological readout. The requirement for CD14 in HPMC responses to a clinically relevant Staphylococcus epidermidis cell-free supernatant (SES) was investigated using soluble CD14 or anti-CD14-blocking antibodies.Results. Real-time PCR detected TLR1-6 messenger RNA expression in HPMC and responses to TLR2/1 and TLR2/6 ligands and SES. No cell surface TLR4 expression or responses to lipopolysaccharide were detectable in HPMC, but they did respond to flagellin, a TLR5 ligand. SES-mediated responses were dependent on TLR2 but did not require CD14 in HPMC for optimal efficiency, unlike peripheral blood mononuclear cells. HPMC expression of TLR2 was also modulated by TLR2 ligands and inflammatory cytokines.Conclusions. These data suggest that mesothelial cell activation by TLR2/1, TLR2/6 and TLR5 contributes to bacterial recognition influencing the course of the infective process and has implications for improving treatment of infection in PD patients.

Benfotiamine Protects against Peritoneal and Kidney Damage in Peritoneal Dialysis

Residual renal function and the integrity of the peritoneal membrane contribute to morbidity and mortality among patients treated with peritoneal dialysis. Glucose and its degradation products likely contribute to the deterioration of the remnant kidney and damage to the peritoneum. Benfotiamine decreases glucose-induced tissue damage, suggesting the potential for benefit in peritoneal dialysis. Here, in a model of peritoneal dialysis in uremic rats, treatment with benfotiamine decreased peritoneal fibrosis, markers of inflammation, and neovascularization, resulting in improved characteristics of peritoneal transport. Furthermore, rats treated with benfotiamine exhibited lower expression of advanced glycation endproducts and their receptor in the peritoneum and the kidney, reduced glomerular and tubulointerstitial damage, and less albuminuria. Increased activity of transketolase in tissue and blood contributed to the protective effects of benfotiamine. In primary human peritoneal mesothelial cells, the addition of benfotiamine led to enhanced transketolase activity and decreased expression of advanced glycation endproducts and their receptor. Taken together, these data suggest that benfotiamine protects the peritoneal membrane and remnant kidney in a rat model of peritoneal dialysis and uremia.

Abstract 1527: Hepatocyte growth factor modulates adhesion of ovarian cancer cells to the peritoneum mesothelium through p70 S6 kinase

Abstract Ovarian cancer has the highest mortality of all gynecological cancers. These deaths are mainly due to the highly metastatic characteristic with already widespread peritoneal dissemination and malignant ascites at the time of diagnosis. However the molecular mechanisms that regulate ovarian cancer progression are poorly understood. A key step in this process is the adhesion of ovarian cancer cells to the peritoneal mesothelium. Hepatocyte growth factor (HGF) is present at high concentrations in ovarian cancer ascitic fluid. We show here a role for HGF signaling in the regulation of the peritoneal adhesion of ovarian cancer cells. HGF increased binding of ovarian cancer cells to peritoneal extracellular matrix (ECM) proteins fibronectin and laminin and their receptors α5, α6, and β1 integrin expression. Blockade of HGF resulted in reduced attachment of ovarian cancer cells to these ECM proteins, with a concomitant reduction of α5, α6, and β1 integrin expression, confirming that the effect was HGF specific. Similarly, the peritoneal ECM adhesion could be reversed by neutralizing antibodies to α5, α6, and β1 integrin. The adhesive phenotype and integrin expression activated by HGF could be blocked by specific inhibition of p70 S6 kinase (p70S6K) using short hairpin RNA or small molecule inhibitor, suggesting that HGF transmits the signal through p70S6K. Importantly, it also inhibited adhesion to primary human peritoneal mesothelial cells. These results identify a potential novel mechanism for HGF-induced peritoneal adhesion, which is mediated through p70S6K through regulation of α5, α6, and β1 integrin (This work is supported by the Hong Kong Research Grants Council Grant HKU7599/05M to A.S.T.W.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1527. doi:10.1158/1538-7445.AM2011-1527

Phosphorylation of glycogen synthase kinase-3beta induces epithelial mesenchymal transition in human peritoneal mesothelial cells

To investigate the role of phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) inducing epithelial mesenchymal transition in human peritoneal mesothelial cells (HPMC). Primary HPMC was harvested from human omental tissue and maintained under defined in vitro conditions. The expression of p-GSK-3beta and total GSK-3beta in HMPC was detected by Western blot after incubation with different concentrations (0, 5, 10, 20, and 40 mmol/L)of LiCl at different time points (0, 1, 3, 6, and 12 h). The protein expression of E-cadherin and alpha-SMA was also examined after treatment with 20 mmol/L LiCl according to different time courses. The intracellular distribution and expression of alpha-SMA were determined by indirect immunofluorescence. LiCl stimulated phosphorylation of GSK-3beta and the effect was time-dependent and concentration-dependent to limited extent (P<0.05). The expression of alpha-SMA increased (P<0.05) and the expression of E-cadherin decreased significantly (P<0.05) after 24 h stimulation by 20 mmol/L LiCl. The indirect immunoflurescence showed that the expression of alpha-SMA in HPMC increased significantly after 24 h incubation with 20 mmol/L LiCl. The phosphorylation of GSK-3beta leads HMPC to epithelial mesenchymal transition and provides new clue for the treatment of peritoneal fibrosis.

Normal and SV40 transfected human peritoneal mesothelial cells produce IL-6 and IL-8: implication for gynaecological disease.

High levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) have been demonstrated in the peritoneal fluid of benign and malignant gynaecological disease. Peritoneal monocytes and macrophages, endometrial cells, endometrial and peritoneal stromal cells and tumour cells produce these cytokines in vitro. To investigate whether normal human peritoneal mesothelial cells (HPMC) produce IL-6 and IL-8, HPMC were isolated from omental biopsies. Primary HPMC (P-HPMC) were transfected with pSV3-neo encoding SV40 large T antigen (T-HPMC) to generate sufficient cells. T-HPMC preserved the characteristics of P-HPMC as assessed by phase contrast microscopy, electron microscopy, immunocytochemistry and flow cytometry (FACS) analysis. T-HPMC retained a stable phenotype up to passage 14-19, whereas P-HPMC proliferated poorly and became senescent by passage 4-6. T-HPMC and P-HPMC constitutively expressed IL-6 and IL-8 at both protein and mRNA level. IL-6 and IL-8 production was stimulated by recombinant human interleukin-1beta (hIL-1beta) or human tumour necrosis factor-alpha (hTNF-alpha) alone in a dose-dependent manner. Moreover, hIL-1beta or hTNF-alpha up-regulated IL-6 and IL-8 gene expression as determined by competitive PCR. In contrast, human interferon-gamma (hIFN-gamma) or lipopolysaccharide (LPS) showed no effect. These data indicate that (1) T-HPMC lines mimic the morphological and functional features of P-HPMC, (2) P-HPMC and T-HPMC constituitively produce IL-6 and IL-8, which is enhanced by hIL-1beta and hTNF-alpha and (3) HPMC in vivo may participate in the pathogenesis of benign and malignant gynaecological disease.

Vascular endothelial growth factor production and regulation in human peritoneal mesothelial cells

Vascular endothelial growth factor (VEGF) was recently found in peritoneal effluents of peritoneal dialysis (PD) patients. It was suggested that human peritoneal mesothelial cells (HMC) contribute to the intraperitoneal production of VEGF, which may augment vascular permeability, vasodilation and neoangiogenesis in the peritoneal membrane. The present study was designed to assess the influence of proinflammatory cytokines, thrombin, d-glucose and glycated albumin in the regulation of VEGF synthesis in primary HMC cultures. VEGF antigen concentrations were measured in the cell supernatant by ELISA and VEGF mRNA expression was evaluated by real time RT-PCR. Incubation of HMC with interleukin-1alpha (IL-1alpha; 10 to 100 U/mL), tumor necrosis factor-alpha (TNF-alpha; 500 to 1000 U/mL) or thrombin (1 to 10 U/mL) resulted in a time (24 to 72 hours) and concentration dependent increase in VEGF synthesis. In contrast, d-glucose (30 to 90 mmol/L), which is commonly used as an osmotic agent in peritoneal dialysis, was not able to up-regulate VEGF expression. High glucose levels even decreased VEGF production. However, exposure of HMC to Amadori-modified glycated albumin, which is generated in the peritoneal cavity in the presence of glucose-based dialysis solutions, resulted in a dose and time dependent increase in VEGF mRNA expression and antigen secretion. These results demonstrate, to our knowledge for the first time, that glycated serum albumin, not glucose, increases VEGF production in HMC. HMC play an important role as a source of intraperitoneal VEGF synthesis, and VEGF expression also is up-regulated in the presence of proinflammatory cytokines and thrombin. Additionally, these results confirm clinical data that the continuous exposure of the peritoneal membrane to glucose-based dialysis solutions is an important stimulus for VEGF expression. However, it is not glucose per se, but nonenzymatic glycation products like glycated albumin that up-regulate VEGF expression.