Mesothelial Monolayer Research Articles

Changes in peritoneal mesothelial cells phenotype after chronic exposure to glucose or N-acetylglucosamine

Glucose is commonly used as an osmotic solute in peritoneal dialysis fluids despite vast knowledge about deleterious peritoneal and systemic effects of that solute. N-acetylglucosamine (NAG) is a solute of the comparable size to glucose, with strong anti-inflammatory properties. We compared the chronic in vitro effect of both solutes on phenotype of peritoneal mesothelial cells. Experiments were performed of primary cultures of human peritoneal mesothelial cells, which were cultured over 4 weeks in medium supplemented either with glucose 45 mmol/L (GLU) or with NAG 45 mmol/L (NAG). Generation of reactive oxygen species (ROS) in cells was studied, as well as their ability to proliferate, synthesis of cytokines, fibronectin, and factors regulating peritoneal fibrinolysis. Cells cultured in the presence of glucose 45 mmol/L generated more ROS (+73% vs control, P < 0.01), whereas NAG did not stimulate generation of ROS. GLU caused hypertrophy of mesothelial cells (+53% vs control, P < 0.001) and prolonged their population doubling time (+16% vs control, P < 0.01); NAG did not cause significant changes in these parameters. Healing of mesothelial monolayer after mechanical injury was impaired in GLU treated cells: (-48% vs control, P < 0.001 and -40% vs NAG, P < 0.05). Synthesis of Il-6, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and fibronectin was higher in GLU group as compared with control: + 86%, P < 0.001, +38%, P < 0.05, +51%, P < 0.001, +38%, P < 0.05, respectively. In the presence of NAG, these parameters were comparable with the control group, but at the same time NAG stimulated synthesis of hyaluronan: +116% versus control, P < 0.001 and + 96% versus GLU, P < 0.01. Treatment with GLU resulted in decline of tissue plasminogen activator/plasminogen activator inhibitor-1 (t-PA/PAI-1) ratio by 23% versus control, P < 0.001, whereas NAG increased that parameter by 43%, P < 0.01 versus control. Glucose, contrary to NAG, induces oxidative stress and proinflammatory and profibrotic changes in mesothelial cells. NAG seems to be more biocompatible osmotic solute than glucose.

The use of Peritoneal Mesothelium as a Potential Source of Adult Stem Cells

At the dawn of the 21st century, classical curative medicine is being challenged by the fact that efforts to fight and prevent not a few diseases, are in many circumstances, beyond the power of the pharmacological armamentarium of the medical profession. On the other hand, replacement of lost function by mechanical or biophysical devices, or even by organ transplantation, prolongs life but generally derives in new and, at times, unsolvable problems. Regenerative therapy using stem cells began a revolutionary trend that may well change both the therapeutic approach to not a few of the diseases resulting from failing organs, as well as the fate and quality of life of millions of patients. The presence of pluripotent mesenchymal cells in the mesothelial monolayer as well as in the submesothelial connective tissue raises the possibility of using the peritoneal mesothelium in regenerative therapies. This perception of the problem is also based on observations made in humans as well as in laboratory animals showing bone, bone marrow, cartilaginous tissue, glomerular-like structures and creation of blood conducts, pathological situations (mesothelioma, sclerosing peritonitis), or after in vivo or ex vivo experimental interventions. The main concept emerging from this information is that peritoneal mesothelial cells are endowed with such a degree of plasticity that, if placed in the appropriate micro-environment, they have a remarkable potential to generate other mesenchymal-derived cell lines. Intensive research is required to define the best environmental conditions to take advantage of this plasticity and make the peritoneal mesothelium an actual option to be applied in regenerative medicine.

Possible involvement of TWIST in enhanced peritoneal metastasis of epithelial ovarian carcinoma

Loss of E-cadherin triggers peritoneal dissemination, leading to an adverse prognosis for most patients with epithelial ovarian carcinoma (EOC). Because TWIST mainly regulates the epithelial-to-mesenchymal transition and is one of the E-cadherin repressors, we investigated the possibility that TWIST expression affects peritoneal metastasis of EOC using siRNA technique. In the present study, we showed a correlation between TWIST expression and EOC cellular morphology. Furthermore, we demonstrated that the suppression of TWIST expression in EOC cells (HEY) alters the cellular morphology from a fibroblastic and motile phenotype to an epithelial phenotype, and inhibits the adhesion of these cells to mesothelial monolayers. To investigate the mechanism by which down-regulation of TWIST leads to inhibition of adhesion to mesothelial cells (MCs), expression of adhesion molecules (CD29, CD44 and CD54) were observed. Moreover, matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase, important markers associated with invasive and metastatic potential, were remarkably reduced. This findings suggests that reduced expression of TWIST suppresses the multistep process of peritoneal dissemination (detachment from the primary lesion, adhesion to MCs and invasion of MCs) and may be a potential therapeutic target for the treatment of this carcinoma.

The Influence of Reactive Oxygen Species on the Adhesion of Pancreatic Carcinoma Cells to the Peritoneum

Postoperative peritoneal carcinomatosis is a significant clinical problem after “curative” resection of pancreatic carcinoma. Peroperative surgical trauma activates a cascade of peritoneal defense mechanisms responsible for postoperative intra-abdominal tumor recurrence. Reactive oxygen species (ROS) play a pivotal role in this postoperative inflammatory reaction. This study explores the influence of ROS on adhesion of human pancreatic carcinoma cells to human mesothelial cells. Furthermore this study explores the influence of ROS on the presentation of adhesion molecules on Panc-1 and mesothelial cells. ROS were produced using the enzymatic reaction of xanthine with xanthine oxidase (X/XO). A reproducible in-vitro assay to study adhesion of human Panc-1 carcinoma tumor cells to a mesothelial cell monolayer of primary human mesothelial cells was used. Mesothelial monolayers were incubated with ROS produced prior to adhesion of the tumor cells. Incubation of the mesothelial cells with X/XO resulted in a significant increase (69.5%) in adhesion of Panc-1 in all patients. SOD/catalase, anti-oxidants, could reduce this increase by 56.7%. ROS significantly influenced the expression of the adhesion molecules ICAM-1, VCAM-1 and CD44h on mesothelial cells, but did not influence adhesion molecule expression on Panc-1. The ROS released during the post-operative inflammatory reaction may play an important role in the adhesion of pancreatic tumor cells to the mesothelium. Possibly by influencing adhesion molecule expression on mesothelial cells. Therefore ROS can partly be responsible for the enhanced post-operative intra-abdominal tumor recurrence.

Inflammatory Cytokines Stimulate the Adhesion of Colon Carcinoma Cells to Mesothelial Monolayers

Surgical handling of the peritoneum causes an inflammatory reaction, during which a potentially lethal cocktail of active mediators is produced, including cytokines and growth factors. The aim of this study was to investigate the effects of inflammatory cytokines on the interaction between tumor and mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the mesothelium with interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. Preincubation of the mesothelial monolayer with IL-1beta or TNF-alpha resulted in enhanced tumor cell adhesion of Caco2 and HT29 colon carcinoma cells. The amount of stimulation for the Caco2 cells was between 20% and 40% and for HT29 cells between 30% and 70%. Blocking experiments with anti-IL-1beta and anti-TNF-alpha resulted in significant inhibition of the cytokine-stimulated tumor cell adhesion. The presented results prove that IL-1beta and TNF-alpha are significant stimulating factors in tumor cell adhesion in vitro and may therefore account for tumor recurrence to the peritoneum in vivo.

Influence of Per-Operative Serum on Tumour Cell Adhesion in Vitro

In case of massive tissue damage, the ensuing inflammatory reaction produces a potentially lethal cocktail of active mediators. Beside the influence of these local factors on local tumour recurrence, we hypothesize that during surgery systemically produced mediators are potential stimulators of tumour cell adhesion as well. The aim of this study was to investigate the influence of serum samples, obtained at eight different time periods before, during, and after surgery on the interaction between human colon carcinoma cells and mesothelial cells. In the control situation, the average adhesion of the Caco-2 cells to the mesothelial monolayers was 29%. No enhancement in adhesion was observed after incubation of the mesothelial cells with the pre- or postoperative serum of the patients. This study suggests that systemic factors produced directly after surgery are not influencing tumour adhesion to mesothelial monolayers. Therefore, local tumour recurrence is probably only influenced by locally produced factors.

Surgery-Derived Reactive Oxygen Species Produced by Polymorphonuclear Leukocytes Promote Tumor Recurrence: Studies in an In Vitro Model

Tissue injury induces the acute phase response, aimed at minimizing damage and starting the healing process. Polymorphonuclear leukocytes (PMNs) respond to the presence of specific chemoattractants and begin to appear in large numbers. The aim of this study was to investigate the influence of reactive oxygen species (ROS) produced by PMNs on the interaction between colon carcinoma cells and mesothelial cells. An experimental human in vitro model was designed using Caco-2 colon carcinoma cells and primary cultures of mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the mesothelium with stimulated PMNs and unstimulated PMNs. Mesothelial cells were also incubated with xanthine/xanthine oxidase (X/XO) complex producing ROS after which adhesion of Caco-2 cells was investigated and the expression of adhesion molecules (ICAM-1, VCAM-1, and CD44) by means of enzyme immunoassay. In the control situation the average adhesion of Caco-2 cells to the mesothelial monolayers was 23%. Mesothelial monolayers incubated with unstimulated PMNs showed a 25% increase of tumor cell adhesion (P < 0.05). The adhesion of tumor to the monolayers incubated with the N-formyl-methionyl-leucyl-phenylalanine-stimulated PMNs increased with 40% (P < 0.01). Incubation of the mesothelium with X/XO resulted in an enhancement of adhesion of Caco-2 cells of 70% and an up-regulation of expression of ICAM-1, VCAM-1, and CD44. This study reveals an increase of tumor cell adhesion to the mesothelium induced by incubating the mesothelial monolayers with PMNs. PMNs are producing a number of products, like proteolytic enzymes, cytokines, and ROS. These factors up-regulate the expression of adhesion molecules and in that way stimulate the adhesion of tumor to the mesothelium.

Intercellular localization of occludins and ZO-1 as a solute transport barrier of the mesothelial monolayer

A hyperpermeable state has been observed in patients on long-term peritoneal dialysis. To understand the causes of the structural or functional changes and the progression of the fibrotic process, it is important to determine which region of the peritoneum exhibits these changes. The objectives of this study were to determine the solute permeability associated with cell-cell adhesion of human peritoneal mesothelial cells (HPMCs), to study the relationship between solute permeability and localizations of tight junction-associated proteins (TJPs: occludins and ZO-1), and to assess the effect of exogenous H2O2 supplementation. HPMCs were cultured on a Transwell until the transmesothelial electrical resistance (TER) reached a plateau. Solute permeation tests were conducted using fluorescein isothiocyanate - labeled dextran (molecular weight: 4, 10, 70, and 150 kDa) to calculate the solute permeability coefficient (SPC). Localization of TJPs was observed by a confocal laser scanning microscope after immunofluorescent staining. TER levels increased steadily, beginning at 97.5 +/- 0.7 ohms.cm2 and leveling off at 128 +/- 3.6 ohms.cm2 (n = 4). This was accompanied by the confluence of cells and the appearance of localized TJPs. SPC levels of the HPMC monolayer on the Transwell were reduced compared to those of the Transwell itself, indicating that the HPMC monolayer provided resistance against solute permeation. Exogenous H2O2 supplementation revealed an increased permeability accompanied with delocalization of TJPs, particularly occludins. The delocalization of occludins and ZO-1 at the intercellular space led to a decrease in intercellular binding capacity and thus triggered an increase in the solute permeability.

Time to reconsider saline as the ideal rinsing solution during abdominal surgery

Background Peritoneal mesothelial cells lining the peritoneal cavity play a primary role in prevention of formation of peritoneal adhesions, which depends mainly on their fibrinolytic activity. During surgical procedures, the abdominal cavity is in most cases rinsed with normal saline solution, which may modify the fibrinolytic activity of the peritoneal mesothelium and predispose to formation of adhesions. The goal of our experiments was to evaluate how normal saline and other solutions affect the fibrinolytic properties of the peritoneal mesothelial cells. Material and methods Experiments were performed on in vitro cultures of human peritoneal mesothelial cells. Mesothelial monolayers were exposed during 6 hours to the following solutions: culture medium (control), .9% NaCl, Hanks solution, Earles solution, new peritoneal dialysis fluid with low glucose degradation products (GDP) concentration (PDF), and peritoneal dialysis fluid with high concentration of GDP (PDF-GDP). Afterwards, morphology of the cells as well as leakage of lactate dehydrogenase (LDH) from their cytosol were evaluated. During the next 24 hours when the cells were cultured in standard medium synthesis of interleukin-6 (IL-6), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were studied. Results Mesothelial monolayers exposed to .9% NaCl or to PDF-GDP showed destruction of their morphology after 6 hours incubation and in case of PDF-GDP release of LDH from the cytosol was increased by 275% versus the control ( P < .05). During subsequent culture of all cells in standard medium, the release of IL-6 was decreased in cases of cells pretreated with .9% NaCl (−58%, P < .05) or PDF-GDP (−93%, P < .001). The release of t-PA was also reduced from cells pretreated with .9% NaCl (−71%, P < .01) or with PDF-GDP (−74%, P < .01) but increased after exposure of these cells to PDF (+35%, P < .05). Statistically significant decrease of PAI-1 synthesis was observed in cells preexposed to .9% NaCl (−69%, P < .01) or to PDF-GDP (−82%, P < .05). When changes in the PAI-1/t-PA ratio were calculated, a strong tendency for increase of that value was seen in cells pretreated with .9% NaCl or Earles salts solution but not with PDF. However, in cases of Hanks solution, a significant increase in the PAI-1/t-PA ratio was observed (+104%, P < .01). Conclusion Exposure of the peritoneal mesothelial cells to .9% NaCl, Hanks, Earles salts solution, or PDF-GDP results either in reduction of their viability or in loss of their fibrinolytic activity. Peritoneal dialysis fluid with a low content of glucose degradation products appears to be the optimal solution causing the least damage to mesothelial cells and therefore may be the ideal solution for rinsing the abdominal cavity with low risk of inducing deterioration of the mesothelial cells fibrinolytic activity and formation of adhesions. We postulate therefore that such hypertonic peritoneal dialysis fluids should be used not only during peritoneal dialysis but also for rinsing the abdominal cavity during any surgical procedures.

The Expression of Adhesion Molecules and the Influence of Inflammatory Cytokines on the Adhesion of Human Pancreatic Carcinoma Cells to Mesothelial Monolayers

Pancreatic cancer has a tremendously deplorable prognosis. Peritoneal dissemination frequently occurs after surgical resection of the tumor. Specific adhesion molecules may be of great importance in local tumor recurrence. These adhesion molecules may be influenced by inflammatory cytokines produced during surgery. The aim of this study was to investigate the effects of inflammatory cytokines, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), on the interaction between pancreatic tumor cells and mesothelial cells. An experimental in vitro model was designed using Panc-1, MiaPaCa-2, and BxPC-3 pancreatic carcinoma cell lines. Primary cultures of mesothelial cells were incubated with the inflammatory cytokines, and after the incubation, the adherence of the different pancreatic cell lines was measured. By means of immunocytochemical staining and enzyme immunoassay, the expression of adhesion molecules (ICAM-1, VCAM-1, and CD44) and counterparts (LFA-1 and VLA-4) was investigated. Preincubation of the mesothelial monolayer with IL-1beta or TNF-alpha resulted in enhanced tumor cell adhesion of the MiaPaCa-2 and BxPC-3 cells. The amount of stimulation for the MiaPaCa-2 cells was more than 100% versus the control situation and for BxPC-3 cells between 20% to 35%. IL-6 did not affect the tumor cell adhesion of the MiaPaCa-2 and BxPC-3 cells. The adherence of Panc-1 was not enhanced after preincubation of the mesothelial monolayers with the inflammatory cytokines. Mesothelial cells show a significant enhancement of expression of ICAM-1, VCAM-1, and CD44 after stimulation with IL-1beta and TNF-alpha. The presented results prove that IL-1beta and TNF-alpha are significant stimulating factors in pancreatic tumor cell adhesion in vitro and may therefore account for tumor recurrence to the peritoneum in vivo. The immunocytochemical staining results demonstrate that ICAM-1 and CD44 important adhesion molecules and interference with their function may decrease the incidence of peritoneal tumor recurrence after curative resection of pancreatic cancer.

The inhibition of tumor cell adhesion on human mesothelial cells (HOMC) by phospholipids in vitro

Intraperitoneal tumor cell adhesion to extracellular matrix and to mesothelial cells mediated by integrins is an important step in developing peritoneal carcinosis. In former animal studies, we could demonstrate that intraperitoneal treatment with a new phospholipid (PL) emulsion significantly reduces the amount of peritoneal carcinosis by adhesion prevention. This in vitro study tries to elucidate the influence of phospholipids on cells of the human gastric cancer cell line (NUGC-4) and the human rectal cancer cell line (HRT-18) adhering to mesothelial cells (HOMC) in a monolayer culture in vitro. HOMC cells were derived from omentum majus from patients undergoing elective abdominal surgery. Three passages of both cancer cell lines (NUGC-4 and HRT-18) were used. 1x10(5)/100 microl (HRT-18) or 1.2x10(5)/100 microl (NUGC-4) cells, according to forgoing dilution series, were pretreated with different concentrations of phospholipid emulsion (0.05, 0.1, 0.5, 0.75, 1% PL) stained with cell tracker chloromethyl-benzamidodialkylcarbocyanine (CM-DIL) and seeded into each well on the mesothelial monolayer. After 90 min, the number of adherent cells was counted by fluorescence microscopy at 530 and 620 nm. Additionally, flow cytometric analysis of integrin alpha3 and beta1 expression on the tumor cell surface after treatment with phospholipids was completed. We found a dose dependent effect of phospholipids on both tumor cell lines causing a reduction of cell-cell adhesion. Already low concentrations of phospholipids (PL 0.5) had a significant influence. The mean cell count could be reduced from 234+/-12/mm2 in controls to 124+/-41/mm2 (PL 0.5; NUG-4) and from 295+/-49/mm2 to 169+/-29/mm2 (PL 0.5; HRT-18), respectively. Additionally, the integrin alpha3 and beta1 expression on both cell lines could be reduced. Our results within the scope of published data indicate that adhesion prevention is capable to reduce peritoneal carcinosis.

Disaggregation and invasion of ovarian carcinoma ascites spheroids

BackgroundMalignant ascites often develops in advanced stages of ovarian carcinoma, consisting of single and aggregated tumor cells, or spheroids. Spheroids have commonly been used as tumor models to study drug efficacy, and have shown resistance to some chemotherapies and radiation. However, little is known about the adhesive or invasive capabilities of spheroids, and whether this particular cellular component of the ascites can contribute to dissemination of ovarian cancer. Here, we examined the invasive ability of ascites spheroids recovered from seven ovarian carcinoma patients and one primary peritoneal carcinoma (PPC) patient.MethodsAscites spheroids were isolated from patients, purified, and immunohistochemical analyses were performed by a pathologist to confirm diagnosis. In vitro assays were designed to quantify spheroid disaggregation on a variety of extracellular matrices and dissemination on and invasion into normal human mesothelial cell monolayers. Cell proliferation and viability were determined in each assay, and statistical significance demonstrated by the student's t-test.ResultsSpheroids from all of the patients' ascites samples disaggregated on extracellular matrix components, with the PPC spheroids capable of complete disaggregation on type I collagen. Additionally, all of the ascites spheroid samples adhered to and disaggregated on live human mesothelial cell monolayers, typically without invading them. However, the PPC ascites spheroids and one ovarian carcinoma ascites spheroid sample occasionally formed invasive foci in the mesothelial cell monolayers, suggestive of a more invasive phenotype.ConclusionWe present here in vitro assays using ascites spheroids that imitate the spread of ovarian cancer in vivo. Our results suggest that systematic studies of the ascites cellular content are necessary to understand the biology of ovarian carcinoma.

ICAM-1 Mediated Peritoneal Carcinomatosis, A Target for Therapeutic Intervention

Development of peritoneal metastasis is a significant issue in the treatment of abdominal cancers. Primary interaction between tumour cells and the mesothelium is a vital step in initiating this process. Our aim was to determine the role of the intercellular adhesion molecule-1 (ICAM-1) in mesothelial-tumour adhesion and the effectiveness of therapeutic intervention. Mesothelial cells were derived from omental tissue. ICAM-1 expression in resting state, in the presence of TNF-alpha or after the application of heparin or hyaluronan was determined by flow cytometry. Functional effects on tumour adhesion to a mesothelial monolayer were determined via a Calcein-AM in vitro adhesion assay. In vivo studies were performed utilising 30 WAG/rij rats, which underwent mini-laparotomy with the injection of 1 x 10(5 )CC 513 tumour cells intraperitoneally. Tumour growth was assessed macroscopically and microscopically by two independent examiners. Mesothelial cells expressed high level of ICAM-1, which was up-regulated by the presence of TNF-alpha. The introduction of heparin caused a decrease in ICAM-1 expression, however hyaluronan did not affect the expression. A significant decrease in tumour-mesothelial cell adhesion in vitro and complete aberration of tumour growth in vivo was observed with heparin application. In vitro studies showed utilisation of high molecular weight hyaluronan, which was more limited in vivo. These data imply that heparin may be used as a potential therapeutic through a defined molecular mechanism both in vitro and in vivo. Hyaluronan appears to function as a barrier and hence may be unreliable in blocking peritoneal recurrence.

Homogeneity of Mesothelial Cells with Lymphatic Endothelium: Expression of Lymphatic Endothelial Markers by Mesothelial Cells

Mesothelial cell monolayers cover the serous cavities and internal organs, and provide a protective low-friction interface between apposed organs and tissues. The mesothelium also regulates inflammation, fluid and cell exchange, and tissue repair in these compartments and possibly tumor metastasis. In the present study, a stable pleural mesothelial cell line (MIM) was isolated and characterized, and the expression of several lymphatic specific markers by these cells examined. MIM were isolated from mice stably expressing a temperature-sensitive SV40 large T antigen ('Immortomouse', strain: H-2K(b)-tsA58). These cells were compared with lymphatic endothelial cells (LEC) derived from the mesenteric adventitia of the Immortomouse. MIM and LEC expression of lymphatic-specific markers (Flt-4, LYVE-1, and Prox-1) was examined, and the tight junction protein (ZO-1) was studied by immunofluorescence and immunoblotting in these cells. LYVE-1, Prox-1, and Flt-4 were detected in both MIM and LEC, with Prox-1 and LYVE-1 more strongly expressed on LEC than MIM. Conversely, Flt-4 was more densely expressed on MIM than on LEC. Spatially, ZO-1 was prominent at MIM junctions, but was less well organized in LEC. MIM and LEC share several characteristic markers usually associated with lymphatic endothelium. MIM might be useful for studying the biology and pathology of mesothelial cells in vitro and help in the development of therapies for mesothelial-related diseases, such as mesothelioma and pleural effusion.

Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers

Ovarian carcinoma patients frequently develop malignant ascites containing single and aggregated tumor cells, or spheroids. Spheroids have been shown to be resistant to many therapies, but their contribution to ovarian cancer dissemination remains undetermined. We have previously shown that ascites spheroids adhere to extracellular matrix (ECM) proteins and live human mesothelial cells via beta1 integrin subunits. Here, we assessed the ability of spheroids that were generated from the human ovarian carcinoma cell line NIH:OVCAR5 to disseminate and invade in vitro. Spheroids were seeded on ECM proteins for 24 h. While laminin and type IV collagen stimulated some cell migration, spheroids completely disaggregated on type I collagen substrates. A monoclonal antibody against the beta1 integrin subunit significantly inhibited disaggregation on all proteins tested. To test their invasive ability, spheroids were added to monolayers of live human LP9 mesothelial cells. Within 24 h, the spheroids adhered and disaggregated on top of the monolayers, and within a week had established foci of invasion encompassing a 200-fold larger surface area. Addition of a monoclonal antibody against the beta1 integrin subunit drastically reduced spheroid invasion into the mesothelial cell monolayers. GM 6001, a broad-scale matrix metalloproteinase inhibitor, also significantly blocked spheroid invasion into the mesothelial cell monolayers. Epsilon-amino-N-caproic acid, a serine protease inhibitor, partially inhibited spheroid invasion. Based on their ability to attach to, disaggregate on, and invade into live human mesothelial cell monolayers, spheroids should thus be regarded as potential contributors to the dissemination of ovarian cancer.

Effects of Neutral pH and Reduced Glucose Degradation Products in a New Peritoneal Dialysis Solution on Morphology of Peritoneal Membrane in Rats

Background: In vitro studies have shown that pH and glucose degradation products (GDPs) in the dialysate are determinant factors for the biocompatibility of peritoneal dialysis (PD) treatment. The present study was thus designed to evaluate whether a newly developed PD solution, which features neutral pH levels and a low GDP concentration, influences tissue damage of the peritoneal membrane in an in vivo setting, and which factor is more critical to the histological changes. Methods: Rats were injected 3 times per day during 1 or 4 weeks with 10 ml of various PD fluids (group G, acidic pH, high GDPs; group S, neutral pH, low GDPs; or group A, acidic pH, low GDPs). When the experimental period was over, the mesothelial cell monolayers of the animals were taken and studied with population analysis, and peritoneal membranes were obtained from the abdominal wall for immunohistochemical examination with proliferating cell nuclear antigen (PCNA) and for measurement of thickness of the peritoneal specimens. Results: The density of the mesothelial cell monolayer and the number of fibroblast-like cells in group S were significantly less than in group G at 1 and 4 weeks’ injection. PCNA-positive nuclei in group S were significantly less than in group G for only the 1-week injection set (group G, 2.03 ± 0.95; group S, 0.85 ± 1.18 nuclei/1 × 10⁴ µm²). At 4 weeks, the peritoneal thickness of group S (6.32 ± 0.53 µm) was significantly less than that of group G (7.94 ± 0.77 µm), There was no significant difference between groups S and A throughout the whole study period except for the result of the number of fibroblast-like cells. Conclusion: These results indicate that a PD solution with a neutral pH and low GDPs proved more biocompatible with the peritoneal membrane than a solution with an acidic pH and high GDPs. Furthermore, the level of the GDPs has more impact on tissue damage of the peritoneal membrane than the pH in the short term.

A Short Review of Experimental Peritoneal Sclerosis: From Mice to Men

Peritoneal sclerosis has been induced in rodents in vivo by exposing the membrane to a variety of experimental interventions: asbestos, 0.1% chlorexidine, iron dextran, glucose degradation products, AGE deposits derived from uremia per se, sodium hypochlorite, lypopolysaccharide, low pH, pure water, silica or zymosan. With a few exceptions (pure water, chlorhexidine and low pH), the other substances mentioned operate setting out different degrees of oxidative stress. This short review describes several experimental interventions in rodents, aimed at acute exfoliation or long-term, sustained injury of the mesothelial monolayer performed by means of intraperitoneal injections of different oxidant agents. Acute exfoliation induced by deoxycholate resulted in a depopulated monolayer coincident with immediate alteration of the peritoneal permeability, evidenced by increased urea D/P ratio, higher glucose absorption rate, elevated albumin losses in the effluent and significant reduction of the ultrafiltration rate. In the long term (30 days), these manifestations of membrane failure persisted and coincided with substantial peritoneal sclerosis. Peritoneal sclerosis was also induced by IP injections of 0.125% trypsin and 6.6 mM/L solution of formaldehyde. Using the doughnut rat model of mesothelial regeneration, exposure to 4.25% glucose or 7.5% icodextrin solutions severely hampered repopulation of the monolayer, which was replaced by a thick sheet of fibrous tissue. It is concluded that peritoneal sclerosis derives mostly from sustained oxidative injury to the peritoneal membrane. Loss of the mesothelial monolayer is the first step in the chain of events leading to this complication.

Mechanisms of hyaluronan synthesis during wound healing process of the mesothelial cell monolayer

Mechanisms of hyaluronan synthesis during wound healing process of the mesothelial cell monolayer

Intercellular localizations of Occludins and ZO-1 solute transport barrier of the cultured mesothelial monolayer

Intercellular localizations of Occludins and ZO-1 solute transport barrier of the cultured mesothelial monolayer

Novel roles of the autocrine motility factor/phosphoglucose isomerase in tumor malignancy

Autocrine motility factor (AMF) stimulates cell motility in an autocrine manner and is related to tumor malignancy. AMF is a multifunctional molecule, also known as phosphoglucose isomerase and neuroleukin. Signal cascades of the AMF-stimulated motility and novel functions of this protein contributing to tumor malignancy have been presented recently. AMF stimulation activated small Rho-like GTPases and subsequently induced actin fiber rearrangement, which was removed by the C3 exoenzyme, a specific inhibitor of Rho. The expression of Jun N-terminal kinase (JNK)1, JNK2 and the Rho GDP dissociation inhibitor-beta was upregulated by AMF. The addition of AMF to culture medium stimulated the motility of the endothelial cells and the formation of tube-like structures in collagen gels. Highly AMF-expressing HT1080 cells induced aggressive angiogenesis in vivo. The expression of fms-like tyrosine kinase (Flt)-1, a vascular endothelial growth factor (VEGF) receptor, was enhanced in AMF-expressing tumors dependent on protein kinase C and phosphatidylinositol 3 kinase (PI3K) activation; meanwhile kinase insert domain-containing receptor, another receptor of VEGF, was not. Permeability of mesothelial and endothelial cell monolayers was increased by AMF, and numerous gaps were observed in the monolayers after treatment with AMF. AMF gene transfection transformed NIH3T3 cells to proliferate quickly and acquire anti-apoptosis ability induced by serum deprivation in a PI3K-dependent manner. The anti-apoptotic effect of AMF has been described by other authors who have shown that the AMF over-expressing cells were resistant to mitomycin-C-induced apoptosis showing regression of Apaf-1 and caspase-9 dependent on PI3K and MAP kinase. These novel functions of AMF makes it a likely target for cancer therapy.