Proliferation Of Human Peritoneal Mesothelial Cells Research Articles

MiR-29b may suppresses peritoneal metastases through inhibition of the mesothelial\u2013mesenchymal transition (MMT) of human peritoneal mesothelial cells

Peritoneal dissemination is a major metastatic pathway for gastrointestinal and ovarian malignancies. The miR-29b family is downregulated in peritoneal fluids in patients with peritoneal metastases (PM). We examined the effect of miR-29b on mesothelial cells (MC) which play critical a role in the development of PM through mesothelial-mesenchymal transition (MMT). Human peritoneal mesothelial cells (HPMCs) were isolated from surgically resected omental tissue and MMT induced by stimulation with 10 ng/ml TGF-β1. MiR-29b mimics and negative control miR were transfected by lipofection using RNAiMAX and the effects on the MMT evaluated in vitro. HPMC produced substantial amounts of miR-29b which was markedly inhibited by TGF-β1. TGF-β1 stimulation of HPMC induced morphological changes with decreased expression of E-cadherin and calretinin, and increased expression of vimentin and fibronectin. TGF-β1 also enhanced proliferation and migration of HPMC as well as adhesion of tumor cells in a fibronectin dependent manner. However, all events were strongly abrogated by simultaneous transfection of miR-29b. MiR-29b inhibits TGF-β1 induced MMT and replacement of miR-29b in the peritoneal cavity might be effective to prevent development of PM partly through the effects on MC.

Requirement of Histone Deacetylase 6 for Interleukin-6 Induced Epithelial-Mesenchymal Transition, Proliferation, and Migration of Peritoneal Mesothelial Cells.

Aims: Influenced by microenvironment, human peritoneal mesothelial cells (HPMCs) acquired fibrotic phenotype, which was identified as the protagonist for peritoneal fibrosis. In this study, we examined the role of histone deacetylase 6 (HDAC6) for interleukin-6 (IL-6) induced epithelial-mesenchymal transition (EMT), proliferation, and migration of HPMCs.Methods: The role of HDAC6 in IL-6-elicited EMT of HPMCs was tested by morphological observation of light microscope, immunoblotting, and immune-fluorescence assay; and the function of HDAC6 in proliferation and migration of HPMCs was examined by CCK-8 assay, wound healing experiment, and immunoblotting.Results: IL-6 stimulation significantly increased the expression of HDAC6. Treatment with tubastatin A (TA), a highly selective HDAC6 inhibitor, or silencing of HDAC6 with siRNA decreased the expression of HDAC6. Moreover, TA or HDAC6 siRNA suppressed IL-6-induced EMT, as evidenced by decreased expressions of α-SMA, Fibronectin, and collagen I and the preserved expression of E-cadherin in cultured HPMCs. Mechanistically, HDAC6 inhibition suppressed the expression of transforming growth factor β (TGFβ) receptor I (TGFβRI), phosphorylation of Smad3, secretion of connective tissue growth factor (CTGF), and transcription factor Snail. On the other hand, the pharmacological inhibition or genetic target of HDAC6 suppressed HPMCs proliferation, as evidenced by the decreased optical density of CCK-8 and the expressions of PCNA and Cyclin E. The migratory rate of HPMCs also decreased. Mechanistically, HDAC6 inhibition blocked the activation of JAK2 and STAT3.Conclusion: Our study illustrated that IL-6-induced HDAC6 not only regulated IL-6 itself downstream JAK2/STAT3 signaling but also co-activated the TGF-β/Smad3 signaling, leading to the change of the phenotype and mobility of HPMCs. HDAC6 could be a potential therapeutic target for the prevention and treatment of peritoneal fibrosis.

Abstract 2380: MiR29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells

Abstract Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). MiR-29 family is well known as tumor suppressor. Methods: MiR-29b mimics and negative control miRNA were transfected by lipofection method. Proliferation and migration of human gastric cancer cell lines, MKN45 and NUGC-4, were examined with MTT assay and transwell assay, respectively. MMT of human peritoneal mesothelial cells (HPMCs) was induced 10 ng/ml of TGF-β. Proliferation and migration of HPMCs were examined with the same methods, and MMT was evaluated with immunofluorescence staining for epithelial, mesothelial and mesenchymal markers. To evaluate the effect of miR-29b in vivo, we developed a highly metastatic subclone YTN16P2 from a murine gastric cancer cell line, YTN16, with in vivo selection, and inoculated the YTN16P2 in peritoneal cavity of syngenic C57BL/6 mice. MiR-29b mimics or negative control miRNA with atelocollagen were intraperitoneally (ip) injected every 3 days from tumor implantation. The mice were sacrificed at 14 days after tumor implantation, and formation of peritoneal metastasis were evaluated. Results: Transfection of miR-29b mimics significantly decreased the proliferation of MKN45 and NUGC-4 by 20-60% (p<0.01) as compared with negative control miRNA. Migration was inhibited more strongly by 90% (p<0.001). After 48 hr-culture with TGF-β, HPMCs apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMCs was decreased while vimentin tended to be upregulated by TGF-β. However, transfection of miR-29b mimics significantly suppressed the morphological changes and reduced the expression levels of vimentin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMCs. MiR-29b also decreased the proliferation and migration of HPMCs by 20% (p<0.05) and 90% (p<0.001) as compared with negative control miRNA. Finally, mice treated with miR-29b developed less PM nodules than those treaded with negative control miRNA and only atelocollagen. Conclusion: MiR-29b efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Yurie Futoh, Kohei Tamura, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Yuko Kumagai, Hideyo Miyato, Naohiro Sata, Joji Kitayama. MiR29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2380.

The exosomal integrin \u03b15\u03b21/AEP complex derived from epithelial ovarian cancer cells promotes peritoneal metastasis through regulating mesothelial cell proliferation and migration

PurposeEpithelial ovarian cancer (EOC) is one of the most malignant cancers in the gynecologic system. Many patients are diagnosed at an advanced stage with disseminated intra-peritoneal metastases. EOC spreads via both direct extension and trans-coelomic spread. However, the interplay between human peritoneal mesothelial cells (HPMCs) and EOC cells is still ambiguous. We hypothesize that integrins (ITG) in HPMCs may play important roles in EOC metastasis.MethodsThe expression of different integrin subtypes from HPMCs was assessed using Western blotting. The expression of integrin α5β1 (ITGA5B1) and its co-localization with asparaginyl endopeptidase (AEP) in HPMCs derived from EOC patients (EOC-HPMCs) were assessed using immunofluorescence. The role and mechanism of the exosomal ITGA5B1/AEP complex in HPMCs was assessed using both in vitro and in vivo assays. A retrospective study involving 234 cases was carried out to assess ITGA5B1 and AEP levels in circulating sera and ascites of EOC patients, as well as associations between ITGA5B1/AEP expression and overall survival.ResultsWe found that ITGA5B1was highly expressed and co-localized with AEP in EOC cells, and that the exosomal ITGA5B1/AEP complex secreted by EOC cells played an important role in the proliferation and migration of HPMCs. High levels of exosomal ITGA5B1/AEP were also found in circulating sera and ascites of EOC patients, and the expression of ITGA5B1/AEP in EOC tissues was found to be negatively associated with overall survival.ConclusionsOur data indicate that EOCs may regulate the function of HPMCs through exosomal ITGA5B1/AEP, which may be crucial for peritoneal metastasis.

Exosomal Integrin α5β1/AEP Complex Derived from EOC Promotes Peritoneal Metastasis Through Regulating Mesothelial Cells Proliferation and Migration

Purpose: Epithelial ovarian cancer (EOC) is one of the most malignant cancer in gynecologic system. Many patients are diagnosed at advanced stage with widely disseminated intra-peritoneal metastasis. EOC spreads via direct extension and transcoelomic spread. However, the interplay between human peritoneal mesothelial cells (HPMCs) and EOC cells is ambiguous. we hypothesize that integrin in HPMCs plays an important role in tumor initial metastasis. Experimental. Design: The expression of integrin different subtypes from HPMCs was validated using WB. The integrin α5β1 high expression and colocalization with asparaginyl endopeptidase (AEP) in EOC cells and HPMCs derived from EOC patients (EOC-HPMCs) was concluded by IF. The role and mechanism of exosomal integrin α5β1/AEP complex in HPMCs was elucidated using in vitro and in vivo. A retrospective study involving 234 patients showed levels of integrin α5β1/AEP in circulating serum and ascites of EOCs patients, and the association between integrin α5β1/AEP expression and overall survival. Results: The integrin α5β1 was highly expressed and co-localized with AEP in EOC cells, and exosomal integrin α5β1/AEP complex secreted by EOC cells played an important role in the proliferation and migration of HPMCs. High levels of exosomal integrin α5β1/AEP were also found in circulating serum and ascites of EOCs patients, and the expression of integrin α5β1/AEP in EOC tissues was negatively associated with overall survival. Conclusions: EOC regulated the function of HPMCs through exosomal integrin α5β1/AEP, which might be crucial for EOC peritoneal initial metastasis. Funding Statement: This study was supported by grants from the National Science Foundation of China (81372787, 81402042, 81602280), Shanghai Municipal Health Bureau (20154Y0109), Shanghai Science and Technology Development Foundation (17411968100), and the Training plan for scientific research of Renji Hospital (RJZZ13-021). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: The study protocol was reviewed and approved by the Institutional Review Board of Shanghai First Maternity and Infant Hospital, in accordance with the Declaration of Helsinki.

Endothelin-1 triggers human peritoneal mesothelial cells' proliferation via ERK1/2-Ets-1 signaling pathway and contributes to endothelial cell angiogenesis.

Dysfunction of the peritoneum as a dialysis organ may result from progressive membrane injury on peritoneal dialysis (PD). It has been increasingly recognized that the human peritoneal mesothelial cells (HPMCs) play a key role in peritoneal membrane early injury. Recently, it has been reported that bioincompatible PD fluid with high concentrations of glucose and glucose degradation products, low pH, high osmolality, and peritonitis stimulates HPMCs to release endothelin-1 (ET-1). ET-1 causes increased the release of vascular endothelial growth factor, which is important for tumor cell angiogenesis. We hypothesized that activating ET-1 might predict injury of peritoneal membrane. HPMCs were isolated from normal omentum. ERK1/2 and Ets-1 phosphorylation were measured by Western blot analysis. HPMC proliferation was detected by the bromodeoxyuridine (BrdU) assay. Capillary networks of tubes formed were photographed under a microscope, and five randomly selected fields from each well were analyzed for total capillary length by using Image J software. MEK-1 blocker significantly abolished the ERK1/2 activation by ET-1, which also triggered phosphorylation, thus activating the transcription factor Ets-1 downstream from ERK1/2. ET-1 was capable to induce HPMC proliferation, which could be attenuated by ET-1 antagonists. Antibody and small interfering RNA mediated blockade of Ets-1 had similar antiproliferative effects. Thus, ET-1 specifically triggered HPMC proliferation via ERK1/2-Ets-1 signaling pathway. VEGF production and endothelial cell angiogenesis were significantly in response to conditioned medium from HPMCs treated with ET-1. ET-1 triggered HPMC proliferation through the ERK1/2-Ets-1 signaling pathway and contributed to VEGF production and endothelial cell angiogenesis.

Methylglyoxal and Advanced Glycation End-Products Promote Cytokines Expression in Peritoneal Mesothelial Cells Via MAPK Signaling

BackgroundPeritoneal dialysis fluid degrades glucose into glucose degradation products that impair peritoneal mesothelial cell functions. These compounds are known to interfere with many cellular functions and to promote the formation of advanced glycation end products (AGEs). This study aimed to investigate the biological effects and the underlying mechanism of glucose degradation products and AGEs on mesothelial cells. MethodsCell proliferation was determined using [3H]-thymidine incorporation assay. Real-time polymerase chain reaction and enzyme linked immunosorbent assay (ELisA) were used to determine the mRNA and protein expression of cytokines. Reactive oxygen species production in mesothelial cells was determined by flow cytometry. Western blot was used to measure the protein expression of p38 MAPK. ResultsMethylglyoxal (MGO) and AGE-human serum albumin (AGE-HsA) inhibited human peritoneal mesothelial cells proliferation in a dose- and time-dependent manner. The mRNA and protein expression of cytokines including vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) was significantly increased after treatment with MGO and AGE-HSA. Also, the antioxidant N-acetylcysteine (NAC) inhibited MGO- or AGE-HSA-induced reactive oxygen species generation. Western blot showed that MGO and AGE increased the phosphorylation levels of p38 MAPK, which was significantly attenuated after treatment of NAC or p38 MAPK inhibitor SB203580. Furthermore, AGE- or MGO-induced increased expression of VEGF and MCP-1 was significantly reduced in the presence of NAC or SB203580. ConclusionsTogether, this study suggested that AGE or MGO promoted VEGF and MCP-1 expression through activation of p38 MAPK signaling.

Role of malignant ascites on human mesothelial cells and their gene expression profiles.

BackgroundMalignant ascites is often present at diagnostic in women with advanced ovarian cancer (OC) and its presence is associated with a worse outcome. Human peritoneal mesothelial cells (HPMCs) are key components of malignant ascites. Although the interplay between HPMCs and OC cells is believed to be critical for tumor progression, it has not been well characterized. The purpose of this study was to assess the effect of ascites on HPMCs and clarify the role of HPMCs in OC progression.MethodsHuman OC ascites and benign peritoneal fluids were assessed for their ability to stimulate HPMC proliferation. Conditioned medium from ascites- and benign fluid-stimulated HPMCs were compared for their ability to attenuate apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). We conducted a comparative analysis of global expression changes in ascites-stimulated HPMCs using Agilent oligonucleotide microarrays.ResultsAs compared to benign peritoneal fluids, malignant ascites stimulated the proliferation of HPMCs. TRAIL-induced apoptosis was attenuated in OC cells exposed to conditioned medium from ascites-stimulated HPMCs as compared to OC cells exposed to conditioned medium from benign fluid-stimulated HPMCs. A total of 649 genes were differentially expressed in ascites-stimulated HPMCs. Based on a ratio of more than 1.5-fold and a P < 0.05, 484 genes were up-regulated and 165 genes were down-regulated in ascites-exposed HPMCs. Stimulation of HPMCs with OC ascites resulted in differential expression of genes mainly associated with the regulation of cell growth and proliferation, cell death, cell cycle and cell assembly and organization, compared to benign peritoneal fluids. Top networks up-regulated by OC ascites included Akt and NF-κB survival pathways whereas vascular endothelial growth factor (VEGF) pathway was down-regulated.ConclusionsThe results of this study not only provide evidence supporting the importance of the interplay between cancer cells and HPMCs but also define the role that the tumor environment plays in these interactions.

Effect of various concentration calcium on human peritoneal mesothelial cells and protein expression of TNF-α

Objective To study the effect of calcium on human peritoneal mesothelial cells (HPMCs). Methods Proliferation abilities of HPMCs were assessed by tetrazolium salt colorimetry assay (MTT assay) and the levels of LDH in the supernatant were detected in all groups to evaluate the damage of HPMCs. The expression of TNF-α in cytoplasm was detected by immunohistochemistry. Results Calcium enhanced proliferation of HPMCs in a time-dependent manor(P＜0.01), especially calcium with 1.25 mmol/L(0.5098±0.016,0.6763±0.048) and 1.0 mmol/L(0.4853±0.016,0.6678±0.076). Calcium with different concentrations significantly increased the levels of LDH in HPMCs in a time-dependent manor, while the effect of calcium with 1.25 mmol/L was lowest(17.78±1.18,23.60±1.39,P＜0.01). Calcium with 2.0 mmol/L[(42.61±3.29)%] and 1.75 mmol/L[(33.32±1.88)%] significantly up-regulated the expression of TNF-α(P＜0.01) . Conclusions High calcium damaged HPMCs and up-regulated the expression of TNF-αby HPMCs, while physical calcium (1.25 mmol/L)can protect peritoneum and prevent peritoneal fibrosis. Key words: Calcium/AD; Peritoneum/CY/ME; Stromal cells/DE; Tumor necrosis factor-alpha/ME

Comparison of Icodextrin- and Glucose-Based Peritoneal Dialysis Fluids in Their Acute and Chronic Effects on Human Peritoneal Mesothelial Cells

Icodextrin-based peritoneal dialysis fluids (PDFs) display several features that may potentially improve their biocompatibility compared to conventional glucose-containing solutions. So far, however, the studies assessing the biocompatibility profile of icodextrin toward human peritoneal mesothelial cells (HPMC) has produced mixed results. The present study was performed to examine the acute and chronic impact of icodextrin on HPMC in vitro in comparison with standard glucose-based PDF. Omentum-derived HPMC were either acutely pre-exposed to or incubated chronically (for up to 10 days) in the presence of icodextrin-PDF. Parallel cultures were treated with conventional PDFs containing either 1.5% or 4.25% glucose. All fluids were tested at neutral pH. HPMC were assessed for viability, proliferation, IL-6 secretion and generation of reactive oxygen species (ROS). Incubation in the presence of icodextrin-PDF significantly reduced HPMC proliferation in a manner similar to that of 1.5% glucose-PDF. In addition, exposure to icodextrin-PDF impaired viability and IL-6 release from HPMC. This effect occurred both after the short pre-treatment with neat icodextrin-PDF for 1-4 hours and after prolonged incubation (up to 10 days) in media supplemented with icodextrin-PDF (1:1). The dysfunction of icodextrin-treated HPMC was of the magnitude that was between the effects exerted by 1.5%- and 4.25%-glucose PDF. Furthermore, exposure of HPMC to icodextrin-PDF induced a dose-dependent increase in ROS generation which was comparable to that produced by 1.5%-glucose PDF. Exposure to icodextrin-PDF may impair viability and function of HPMC. The detrimental effects of icodextrin-PDF are at least as serious as those produced by conventional heat-sterilized low glucose-based PDF.

TGF-β1 Induced by High Glucose is Controlled by Angiotensin-Converting Enzyme Inhibitor and Angiotensin II Receptor Blocker on Cultured Human Peritoneal Mesothelial Cells

Loss of peritoneal function is a major complication associated with long-term peritoneal dialysis. Observed changes include loss and degeneration of the mesothelium, submesothelial thickening, alterations in the structure and number of blood vessels, and reduplication of the vascular basement membrane. Exposure to high glucose concentrations in peritoneal dialysis solutions is known to cause injury to cultured human peritoneal mesothelial cells (HPMC) as a result of overexpression of transforming growth factor beta 1 (TGF-beta1). Previous studies have demonstrated that angiotensin II (AII) increases expression of TGF-beta1 in a number of different cell types; although this has not been demonstrated in HPMC. To clarify possible mechanisms involved in peritoneal fibrosis, we investigated whether HPMC expressed AII-forming pathway mRNA and whether increases in AII induced by high glucose contribute to the production of TGF-beta1. We also examined the effects of the angiotensin-converting enzyme inhibitor (ACEI) perindoprilat and the AII receptor blocker (ARB) candesartan on expression of TGF-beta1 and proliferation of HPMC. Expression of mRNA for the AII-forming pathway and TGF-beta1 in HPMC was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Levels of AII and TGF-beta1 following 48 hours of incubation of the cells in a range of glucose concentrations were measured by enzyme immunoassay and enzyme linked immunosorbent assay respectively. The effect of glucose on cell proliferation was examined using the water-soluble tetrazolium salt WST-1 and [3H]-thymidine uptake. We also investigated the effect of ACEI and ARB on the expression of TGF-beta1 and the proliferation of HPMC incubated at high glucose for 48 hours. AII-forming pathway mRNA was detected in HPMC, with expression of angiotensinogen, angiotensin-converting enzyme (ACE), AII type 1 receptor, and TGF-beta1 mRNA increasing following exposure to glucose according to glucose concentration. High glucose was also shown to increase the production of All and TGF-beta1 and decrease the proliferation of HPMC. In contrast, we found that both the ACEI and the ARB attenuated the increase in TGF-beta1 production and reduced cell proliferation caused by exposure to high glucose. These effects were greater with a combination of the two drugs. The present study provides evidence that (1) HPMC express mRNA for the AII-forming pathway; (2) ACEI and ARB inhibit the TGF-beta1 production induced by high glucose; (3) the AII-forming pathwayis one mechanism by which high glucose causes production of TGF-beta1. In addition to having antihypertensive and renal-protective effects, combination therapy with an ACEI and an ARB may also be effective in preventing loss of peritoneal function and decreasing peritoneal fibrosis.

Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells

Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells. Prolonged exposure of human peritoneal mesothelial cells (HPMC) to high glucose concentrations in peritoneal dialysate is the principal factor leading to matrix accumulation and thickening of the peritoneal membrane, accompanied by progressive deterioration of transport functions. These changes are mediated in part through protein kinase C (PKC) activation and the induction of transforming growth factor-beta 1 (TGF-beta 1). Emodin (3-methyl-1,6,8 trihydroxyanthraquinone) has previously been demonstrated to reduce cell proliferation and fibronectin synthesis in cultured mesangial cells. How emodin modulates glucose-induced abnormalities in HPMC has not been elucidated and thus constitutes the theme of this study. We investigated the effects of emodin on the expression of PKC alpha, TGF-beta 1, fibronectin, and collagen type I in HPMC, and its effects on HPMC proliferation under physiologic (5 mmol) or high (30 mmol) glucose concentrations. Exposure of HPMC cultured with 5 mmol or 30 mmol D-glucose to emodin (20 microg/mL) resulted in an initial lag of proliferation by 2.3 to 2.7 days, but did not affect cell viability or morphology at confluence. D-glucose (30 mmol) induced TGF-beta 1 secretion in a time-dependent manner (3.72 +/- 0.29 and 4.30 +/- 0.50 pg/microg cellular protein at 24 hours and 48 hours respectively, compared to 2.13 +/- 0.23 and 2.65 +/- 0.32 pg/microg cellular protein at 24 hours and 48 hours, respectively for 5 mmol glucose; P < 0.001 at both time points). Such induction was ameliorated by emodin (20 microg/mL) (TGF-beta 1 concentration 2.25 +/- 0.15 and 2.96 +/- 0.33 pg/microg cellular protein at 24 hours and 48 hours, respectively, in the presence of emodin and 30 mmol D-glucose; P < 0.001 compared to 30 mmol D-glucose alone at both time points). Induction of TGF-beta 1 synthesis by 30 mmol D-glucose was associated with induction of PKC alpha, phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor-1 (ATF-1), and increased fibronectin and type I collagen translation. Emodin abrogated all these effects of concentrated glucose. Immunohistochemical staining showed that 30 mmol D-glucose induced cytoplasmic, perinuclear, and extracellular fibronectin and type I collagen expression by HPMC. Emodin reduced 30 mmol D-glucose-induced cytoplasmic and extracellular matrix synthesis to near basal levels. Our findings demonstrate that emodin ameliorates the undesirable effects of concentrated glucose on HPMC via suppression of PKC activation and CREB phosphorylation, and suggest that emodin may have a therapeutic potential in the prevention or treatment of glucose-induced structural and functional abnormalities in the peritoneal membrane.

Dipyridamole inhibits PDGF-stimulated human peritoneal mesothelial cell proliferation

It has been proposed that proliferation of human peritoneal mesothelial cells (HPMCs) accompanied by collagen synthesis may contribute to the development of peritoneal fibrosis (PF) in patients of long-term continuous ambulatory peritoneal dialysis (CAPD). However, the precise molecular mechanism regulating HPMC proliferation has never been reported. Dipyridamole has been reported to have potential as an antiproliferative and antifibrotic agent. We investigated the mechanism and effect of dipyridamole in regulation of HPMC proliferation. HPMCs were cultured from human omentum by an enzyme digestion Cell proliferation was measured by the methyltetrazolium assay and intracellular cAMP was measured using an enzyme immunoassay kit. Cell-cycle distribution of HPMC was analyzed by flow cytometry. Extracellular signal-regulated protein kinase (p44/p42 ERK) activity and expressions of cell-cycle proteins (cyclin D(1), CDK4, pRB and p27(Kip1)) were determined by Western blotting. The addition of DP suppressed PDGF-stimulated HPMC proliferation by cell-cycle arrest at the G1 phase. The antimitogenic effect of dipyridamole was mediated through the cAMP pathway. PDGF (25 ng/mL) increased the ERK1/2 activity of HPMC within 15 minutes, which maximized at 30 minutes, and the pretreatment with dipyridamole (17 microg/mL) substantially reduced the ERK response to PDGF by approximately 78.5%. PDGF induced elevated protein levels of cyclin D(1), but the CDK4 protein level did not change. Dipyridamole and DBcAMP had no effect on the levels of cyclin D(1) and CDK4 in PDGF-stimulated HPMC. PDGF decreased p27(Kip1) and induced pRB phosphorylation of HPMC. In contrast, dipyridamole prevented PDGF-induced p27(Kip1) degradation and attenuated PDGF-stimulated pRB phosphorylation. Dipyridamole appears to inhibit PDGF-stimulated HPMC proliferation through attenuated ERK activity, preservation of p27(Kip1), and decreased pRB phosphorylation. Thus, dipyridamole may have therapeutic efficacy to prevent or alleviate PF.

Dipyridamole inhibits human peritoneal mesothelial cell proliferation in vitro and attenuates rat peritoneal fibrosis in vivo

Peritoneal fibrosis (PF) is one of the most serious complications after long-term continuous ambulatory peritoneal dialysis (CAPD). Proliferation of human peritoneal mesothelial cells (HPMC) and matrix over-production are regarded as the main processes predisposing to PF. Dipyridamole (DP) has been reported to have potential as an antiproliferative and antifibrotic agent. We thus investigated the effect of DP in inhibiting proliferation and collagen synthesis of HPMC. A rat model of peritonitis-induced PF was also established to demonstrate the in vivo preventive effect of DP. HPMC was cultured from human omentum by an enzyme digestion Cell proliferation was measured by the methyltetrazolium assay. Intracellular cAMP was measured using an enzyme immunoassay (EIA) kit. Total collagen synthesis was measured by (3)H-proline incorporation assay. Expression of collagen alpha1 (I) and collagen alpha 1 (III) mRNAs was determined by Northern blotting. The rat model of peritonitis-induced PF was developed by adding dextran microbeads (Cytodex, 8 mg/1 mL volume) to a standardized suspension (3 x 10(9)) of Staphylococcus aureus. DP was administrated via intravenous infusion (4 mg in 1 h) daily for seven days. Macroscopic grading of intraperitoneal adhesions and histological analyses of peritoneal thickness and collagen expression were performed. Addition of DP to HPMC cultures suppressed serum-stimulated cell proliferation and collagen synthesis. The antimitogenic and antifibrotic effects of DP appear to be predominantly mediated through the cAMP pathway, as DP increased intracellular cAMP in a dose-dependent manner. The macroscopic grade of intraperitoneal adhesion and peritoneal thickness were both significantly increased in animals treated with Cytodex plus S. aureus; on the other hand, DP attenuated these fibrotic changes with statistical significance (P < 0.01). Analysis of gene expression of collagen alpha 1 (I) and alpha1 (III) in the peritoneal tissue of experimental animals yielded similar results. This study suggests that dipyridamole may have therapeutic potential in treating peritoneal fibrosis.

Effects of Conventional and New Peritoneal Dialysis Solutions on Human Peritoneal Mesothelial Cell Viability and Proliferation

To investigate the biocompatibility of "new" peritoneal dialysis (PD) solutions with bicarbonate/lactate buffer, non glucose osmotic agents (icodextrin or amino acids), neutral pH, and low levels of glucose degradation products (GDPs). Using M199 culture medium as a control, we compared conventional and new PD solutions with respect to their effects on the viability of human peritoneal mesothelial cells (HPMCs) [using lactate dehydrogenase (LDH) release], on DNA damage in HPMCs [using single-cell gel electrophoresis (Comet assay)], and on HPMC proliferation (using [3H]-thymidine incorporation). The experiments were performed after cell growth was synchronized by incubation with serum-free media for 24 hours. The PD solutions tested included commercial 1.5% glucose and 4.25% glucose solutions with 40 mmol/L lactate (D 1.5 and D 4.25, respectively), 7.5% icodextrin (E), 1.1% amino acid (N), 1.5% glucose solution in a triple-chambered bag (Bio 1.5), 1.5% glucose solution in a dual-chambered bag with neutral pH (Bal 1.5), and 1.5% glucose and 4.25% glucose solution containing 25 mmol/L bicarbonate and 15 mmol/L lactate (P 1.5 and P 4.25, respectively). When HPMCs were continuously exposed to undiluted PD solutions, D 1.5, D 4.25, P 4.25, and E increased LDH release by more than 60% at 24 hours. All PD solutions tested increased LDH release by more than 75% at 96 hours. With 2-fold diluted PD solutions, only D 4.25 significantly increased LDH release at 96 hours, though not at 24 hours. When cells were exposed to undiluted PD solutions for 60 min and allowed to recover in M199 for up to 96 hours, LDH release was significantly higher at 24-96 hours in E (55%-69%) and D 1.5 (48%-72%) as compared with control [M199 (18%)]. Release of LDH was significantly lower with PD solutions containing lower levels of GDPs than those in D 1.5, suggesting that GDPs may have a role in cell viability. The D solutions (D 1.5 and D 4.25) and E solution also induced significant DNA damage. Both LDH release and DNA damage by D and E were significantly attenuated by adjusting the solution pH to 7.4, suggesting that low pH may be implicated in PD solution-induced DNA damage and cell death. When diluted 2-fold, D 1.5, D 4.25, and P 4.25 decreased [3H]-thymidine incorporation to 43%, 34%, and 41% of control, respectively, at 24 hours and to 45%, 26%, and 35% of control, respectively, at 96 hours. When cells were exposed to undiluted PD solutions for 5 minutes and allowed to recover in M199 for up to 96 hours, D1.5 and P 4.25--but not D 4.25--significantly inhibited cell proliferation at 24 hours. This effect was sustained up to 96 hours. The present in vitro data demonstrate that PD solutions with low pH, or high levels of GDPs, or both, promote HPMC death and DNA damage, and that PD solutions with high osmolality inhibit cell proliferation. Solutions with neutral pH, amino acids, and "low GDPs" appear to be more biocompatible than conventional PD solutions. These results require confirmation in in vivo animal and clinical studies.

Effect of osmotic solutes on human mesothelial cell proliferation in vitro.

We evaluated the acute effects of various solutions with high osmotic concentrations on the proliferation of human peritoneal mesothelial cells (HPMC) in vitro culture. HPMC proliferation was assessed by the 3Hthymidine incorporation. Damage to the HPMC membrane was measured by augmented release of the cytosolic enzyme lactic dehydrogenase (LDH). The various osmotic solutions included the crystalloid agents (glucose, raffinose, sorbitol, sucrose, mannitol, glycerol, a mixture of essential amino acids) at 50mM or 100mM and oncotic agents (dextran 70, maltodextrin, hydroxyethylstarch 20) at 3.5% or 7%.All of the solutions with high osmotic concentrations at neutral pH inhibited the growth of HPMC in a concentration-dependent way. The inhibition by the crystalloid agents was stronger than that by the oncotic agents. The amino acids caused very strong inhibition of HPMC growth. At the higher concentration (100mM), most of crystalloid agents also damaged the HPMC membrane, but no significant injury to HPMC was observed at the higher concentration (7%) of the oncotic agents.The results suggest that the toxic effect of the osmotic solutes on the proliferation of HPMC depends on the hyperosmolality. However, we cannot exclude a toxic metabolic effect on the cells. The oncotic solutes seem to be less toxic for mesothelial cell proliferation.

Glycerol Toxicity for Human Peritoneal Mesothelial Cells in Culture: Comparison with Glucose

Glycerol has been proposed as a substitute osmotic agent for glucose in peritoneal dialysis fluids. We have compared the effect of glycerol and glucose on the function of human peritoneal mesothelial cells (HPMC) in vitro. The viability of HPMC was not affected by glycerol (up to 250 mM), whereas it was reduced by glucose in a time- and dose-dependent manner, as assessed by the LDH release. Although the incubation of HPMC with glycerol induced a dose-dependent decrease in HPMC proliferation, the effect was significantly less inhibitory than that produced by glucose. In HPMC treated with 90 mM of glycerol or glucose the incorporation of [3H]-thymidine had reached 79.0 +/- 19.3% and 55.3 +/- 4.0% of the control (p < 0.05 and p < 0.01), respectively. As measured by the [methyl-14C]-choline incorporation, the intracellular amount of newly synthesized phospholipids was reduced from (cpm/microgram cellular protein) 147 +/- 58 in control HPMC to 59 +/- 15 in cells exposed to 90 mM of glucose (p < 0.01), but not affected by glycerol (163 +/- 65). On the other hand, both glycerol and glucose (90 mM) decreased the synthesis of proteins (as assessed by the [3H]-proline incorporation) and interfered with potassium (86Rb) transport mechanisms in HPMC. Our data suggest that there exist some possibly advantageous aspects of glycerol as far as mesothelial cell biocompatibility profile is concerned.