Dissimilar peptide growth factors can induce normal human mesothelial cell multiplication

Summary Quiescent normal human mesothelial (NHM) cells will undergo one round of DNA synthesis when they are incubated in a defined medium consisting of LHC basal medium supplemented with hydrocortisone, insulin, transferrin, and one of the following peptide mitogens: epidermal growth factor; transforming growth factor beta (1 or 2); platelet derived growth factor (a,b heterodimer or b,b homodimer); fibroblast growth factor (acid or basic forms); interleukin 1 (alpha or beta forms); interleukin 2; interferon gamma; interferon beta; or cholera toxin. However, sustained cell multiplication does not occur unless the medium contains hydrocortisone, insulin, transferrin, any one of the above-listed peptide growth factors and high density lipoproteins. Growth can be increased twofold if the medium contains certain combinations of these mitogens and high density lipoproteins. The finding that NHM cells can respond to a broad spectrum of growth factors supports the possibility that an autocrine mechanism may be part of the mechanism that leads to transformation of these cells by asbestos.     

Synthesis and Assembly of the Hyaluronan-Containing Coats around Normal Human Mesothelial Cells

In this study we examined the capacity of normal human mesothelial (NHM) cells and human malignant mesothelioma cells to form hyaluronan-containing pericellular matrices or "coats." The assembly of the pericellular coats was visualized by a particle exclusion assay. We found that large hyaluronan-containing coats were formed around NHM cells whereas their transformed counterparts had no or very limited coats. The coats were removed by treatment with Streptomyces hyaluronidase, which specifically degrades hyaluronan. NHM cells exhibited hyaluronan-containing pericellular matrix within 5 h after seeding. The formation of the coats was stimulated by platelet-derived growth factor and epidermal growth factor. Interestingly, the assembly of the hyaluronan-dependent pericellular matrices was inhibited by the addition of hyaluronan dodecasaccharides. The inhibitory effect on the formation of the coats was due to a destabilization of pericellular matrix and not due to an inhibitory effect of hyaluronan dodecasaccharides on hyaluronan synthesis. In contrast, hyaluronan hexasaccharides, an inhibitor of the interaction between polymeric hyaluronan and its cell surface receptors, had no effect on the size of the coat. Thus, our results are compatible with the possibility that the pericellular matrix surrounding NHM cells consists of newly synthesized hyaluronan which is extruded from the cell and independent of hyaluronan receptors on the cell surface. The coat seems to be stabilized by interactions (hyaluronan-hyaluronan or hyaluronan-protein bridges) which can be prevented by hyaluronan dodecasaccharides.     

Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan-containing coats around normal human mesothelial cells

The composition and permeability of the pericellular coat surrounding normal human mesothelial (NHM) cells have been studied in vitro. NHM cells were grown in the presence of ³H-glucosamine and the amount of label recovered in hyaluronan and chondroitin sulfate was determined after selective enzymatic digestion of the polysaccharides in medium, pericellular, and intracellular pools. For comparison a similar analysis was carried out on mesothelioma cells (Mero-14). Of the labeled polysaccharides in the medium and pericellular pools of NHM cells about 80–90% could be ascribed to hyaluronan and only 3–5% to chondrojtin sulfate. In contrast, Mero-14 synthesized only minute amounts of hyaluronan whereas chondroitin sulfate corresponded to 61% of the total glycosaminoglycans in the culture. The results exclude a structure of the pericellular layer of NHM cells similar to the hyaluronan-proteoglycan aggregates found in cartilage. The permeability of the pericellular layer was tested by the exclusion of polystyrene microspheres and bacteria of diameter 0.1–3.0 μm, as well as erythrocytes of diameter 7 μm. While the erythrocytes were excluded the smaller particles penetrated the coat. By adding 0.5 mg/ml of aggregating cartilage proteoglycan to the medium particles of 0.3 μm or larger were also excluded. Thus exogenous proteoglycans can reinforce the structure of the pericellular layer. © 1995 Wiley-Liss Inc.     

Regulation of the human melanocortin 1 receptor expression in epidermal melanocytes by paracrine and endocrine factors and by ultraviolet radiation

The aim of this study is to investigate the regulation of the human melanocortin 1 receptor (MC1R) expression in cultured normal human melanocytes (NHM) by specific paracrine and endocrine factors, and by ultraviolet radiation (UVR). Treatment of NHM with alpha-melanotropin [alpha-melanocyte stimulating hormone (alpha-MSH)] increased MC1R mRNA level; the response was often more pronounced in NHM with a low (NHM-c) than in NHM with a high melanin content (NHM-b). Endothelin-1 increased MC1R mRNA level in NHM regardless of their melanin content. Basic fibroblast growth factor consistently up regulated MC1R mRNA level in NHM-b but not in NHM-c. Activation of protein kinase C by 12-0-tetradecanoylphorbol-13-acetate slightly increased, while stimulation of adenylate cyclase by forskolin markedly up-regulated the MC1R mRNA level. beta-Estradiol increased, and combined treatment with beta-estradiol and alpha-MSH further elevated, MC1R mRNA level in NHM-c and NHM-b. Testosterone reduced, while progesterone had no effect on, MC1R mRNA level. Agouti signaling protein reduced, and UVR down regulated dose-dependently MC1R mRNA level in NHM-b and NHM-c. This effect was reversed 24 h after irradiation with the lower doses of 7 or 14 mJ/cm2, but not after exposure to a higher, more cytotoxic dose of UVR. We conclude that the MC1R is regulated by paracrine factors, including its own ligands, by specific endocrine sex hormones, and by UVR. Differences in the responses of NHM to some of these factors suggest differential regulation of MC1R gene expression, which may contribute to the variation in constitutive and UV-induced cutaneous pigmentation in humans.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

Growth of normal and neoplastic rat pleural mesothelial cells in the presence of conditioned medium from neoplastic mesothelial cells

Several transformed cells have been demonstrated to secrete growth factors. We studied the effect of conditioned medium from neoplastic rat pleural mesothelial cells on normal and neoplastic mesothelial cell growth. The results showed that the concentrated conditioned medium stimulated neoplastic mesothelial cell growth but inhibited reversibly normal mesothelial cell growth.     

Isolation and characterization of rabbit peritoneal mesothelial cells

Four enriched mesothelial cell populations distinguished by different size and density were obtained when a unit gravity sedimentation procedure was applied to pronase-dispersed rabbit peritoneal mesothelial cells. Cell integrity was confirmed by trypan blue, ultrastructural and biosynthetic analyses, as well as by explantation in tissue culture. The enriched mesothelial cells displayed the immunocytochemical, ultrastructural and growth characteristics of native mesothelial cells. This isolation and separation procedure should provide a valuable experimental tool to study the pathobiology of mesothelial cells.     

Mesothelial cells differentiate into fibroblast-like cells under the scirrhous gastric cancer microenvironment and promote peritoneal carcinomatosis in vitro and in vivo

Peritoneal metastases are one reason for the poor prognosis of scirrhous gastric cancer (SGC), and myofibroblast provides a favorable environment for the peritoneal dissemination of gastric cancer. The aim of this study was to determine whether myofibroblast originates from peritoneal mesothelial cells under the influence of the tumor microenvironment. Immunohistochemical studies of peritoneal biopsy specimens from patients with peritoneal lavage cytological (+) status demonstrate the expression of the epithelial markers cytokeratin in fibroblast-like cells entrapped in the stroma, suggesting that these cells stemmed from local conversion of mesothelial cells. To confirm this hypothesis in vitro, we co-incubated mesothelial cells with SGC or non-SGC to investigate morphology and function changes. As we expected, mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype with loss of epithelial morphology and decrease in the expression of cytokeratin and E-cadherin when exposed to conditioned medium from HSC-39, and the induction of mesothelial cells can be abolished using a neutralizing antibody to transforming growth factor-beta1 (TGF-β1) as well as by pre-treatment with SB431542. Moreover, we found that these mesothelial cells-derived cells exhibit functional properties of myofibroblasts, including the ability to increase adhesion and invasion of SGC. In summary, our current data demonstrated that mesothelial cells are a source of myofibroblasts under the SGC microenvironment which provide a favorable environment for the dissemination of gastric cancer; TGF-β1 produced by autocrine/paracrine in peritoneal cavity may play a central role in this pathogenesis.     

Transforming growth factor-beta1 produced by ovarian cancer cell line HRA stimulates attachment and invasion through an up-regulation of plasminogen activator inhibitor type-1 in human peritoneal mesothelial cells

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.     

High Adhesion of Tumor Cells to Mesothelial Monolayers Derived from Peritoneal Wash of Disseminated Gastrointestinal Cancers

The role of the mesothelial layer in the peritoneal spreading of cancer cells is only partially clarified. Here we attempted to better define the mesothelial contribution to the tumor cell adhesion using a direct adhesion test applied to human primary cultures of mesothelial cells (HPMCs) derived from the peritoneal washes of patients with gastric and colorectal cancers. Gastric and colon carcinoma cells were seeded on different mesothelial monolayers and quantitative fluorescence analysis was performed to analyze their growth and adhesive properties. The adhesion of the cancer cells was not affected by the origin of the HPMCs when derived from patients with different cancers or with benign disease. In contrast, the high levels of ICAM1 expression and ROS production, which characterize these senescent mesothelial cells, enhanced the tumor cell adhesion. These results suggest that the mesothelial adhesive properties are dependent on the cell senescence, while are not affected by the tumor environment. The use of peritoneal washes as a source to isolate HPMCs provides a practical and reliable tool for the in vitro analysis of the mesothelial conditions affecting the peritoneal carcinomatosis.     

Rat pleural mesothelial cells adapted to serum-free medium as a model for the study of growth factor effects

Mesothelial cells are the putative progenitors of mesotheliomas and cell lines have been used as tools to study the responses of these cells to various stimuli, including growth factors. The present study was undertaken to develop a rat mesothelial cell line capable of sustained growth under serum-free conditions with the object of avoiding the possible confounding effects of undefined serum components. Responses of mesothelial cells to epidermal growth factor were shown to differ under serum-free versus low-serum culture conditions. In contrast, a cell line, SFM1, adapted to growth in serum-free medium was characterized and found to exhibit responses to growth factors similar to the responses reported for human cell lines. This new line should prove to be a useful model for the study of these cells in vitro .     

Rat adipose tissue-derived stem cells attenuate peritoneal injuries in rat zymosan-induced peritonitis accompanied by complement activation

Background aimsIn patients receiving peritoneal dialysis, fungal or yeast peritonitis has a poor prognosis. In rat peritoneum with mechanical scraping, severe peritonitis can be induced by zymosan, a component of yeast (Zy/scraping peritonitis). Administration of rat adipose tissue-derived stromal cells (ASCs) potentially can improve several tissue injuries. The present study investigated whether rat ASCs could improve peritoneal inflammation in Zy/scraping peritonitis.MethodsRat ASCs were injected intraperitoneally on a daily basis in rats with Zy/scraping peritonitis.ResultsPeritoneal inflammation accompanied by accumulation of inflammatory cells and complement deposition was suppressed by day 5 after injection of rat ASCs. The peritoneal mesothelial layer in Zy/scraping peritonitis with rat ASC treatment was restored compared with the peritoneal mesothelial layer without rat ASC treatment. Injected rat ASCs co-existed with mesothelial cells in the sub-peritoneal layer. In vitro assays showed increased cellular proliferation of rat mesothelial cells combined with rat ASCs by co-culture assays, confirming that fluid factors from rat ASCs might play some role in facilitating the recovery of rat mesothelial cells. Hepatocyte growth factor was released from rat ASCs, and administration of recombinant hepatocyte growth factor increased rat mesothelial cell proliferation.ConclusionsBecause the peritoneal mesothelium shows strong expression of membrane complement regulators such as Crry, CD55 and CD59, restoration of the mesothelial cell layer by rat ASCs might prevent deposition of complement activation products and ameliorate peritoneal injuries. This study suggests the therapeutic possibilities of intraperitoneal rat ASC injection to suppress peritoneal inflammation by restoring the mesothelial layer and decreasing complement activation in fungal or yeast peritonitis.     

Spontaneously immortalized mouse mesothelial cells display characteristics of malignant transformation

Abstract. Objectives: Mesotheliomas occur in occult serous cavities after chronic exposure of mesothelial cells to asbestos fibres. Molecular events that contribute to the development of this cancer are therefore not readily accessible for study. We have used in vitro culture systems to study and compare induced and spontaneous transformation events in primary mouse mesothelial cells. Materials and methods: Mouse mesothelial cells were cultivated until small populations of proliferating cells emerged from senescing cultures. Spontaneously transformed cultures of cells were characterized and compared to malignantly transformed cells. Results: Human mesothelial cells had a finite lifespan of 10–15 population doublings when cultured in vitro; mouse mesothelial cells typically exhibit this same pattern. Here, we show that mouse mesothelial cells can be cultured for extended periods and that these cells can transform spontaneously. Lines of spontaneously transformed cells generated in this study are immortal and growth factor‐independent. They display the salient characteristic features of transformation, including increased proliferation rate, lack of contact inhibition, aneuploidy and ability to grow in anchorage‐independent conditions. A subset of these cell lines developed into tumours in syngeneic mice. Comparative gene expression analysis demonstrated that spontaneously transformed cell lines were more closely related to neoplastic cells than to primary cells. Conclusion: These findings have implications for interpretation of in vitro transformation studies, demonstrating broad similarity between spontaneous and induced genetic changes.     

Phenotypic and functional characteristics of porcine peritoneal mesothelial cells

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.     

Cytophotometric determination of DNA in mesotheliomas and reactive mesothelial cells

Cytophotometry was used to study the nuclear DNA content of cells in Feulgen-stained effusion specimens from 18 patients with mesothelioma and 14 patients with reactive mesothelial proliferations. The mean DNA content (MDNA) of mesothelioma cells was significantly higher than that of reactive mesothelial cells (P less than .001). Other parameters reflecting the DNA content also differed significantly between the two kinds of cells, including (1) the ratio of mean mesothelial DNA to mean lymphocyte DNA, (2) the percentage of mesothelial cells with DNA content exceeding three times the lymphocyte MDNA and (3) the coefficient of variation of the DNA content. Since these parameters were highly correlated, only one was accepted in a stepwise linear discriminant model for distinguishing reactive from mesotheliomatous effusions. The model correctly classified all of the reactive effusions studied and 89% of the mesotheliomatous effusions. These results indicate that DNA analysis, using the Feulgen stain and cytophotometry, yields criteria that may be useful in distinguishing benign reactive mesothelial cells from malignant mesothelioma in effusions when used in conjunction with other traditional parameters.     

ERK1/2 and p38 MAP kinase control MMP-2, MT1-MMP, and TIMP action and affect cell migration: a comparison between mesothelioma and mesothelial cells

Pleural malignant mesothelioma is a locally aggressive tumor of mesothelial cell origin. In other tumor types high expression of matrix metalloproteinase (MMP)-2, together with membrane-type1-MMP (MT1-MMP), and low levels of the tissue inhibitor of MMP (TIMP)-2 have been correlated with aggressive tumor progression and low survival rates. Therefore, we compared the expression and activation of these three factors and their regulation by two mesothelioma associated growth factors, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)-beta1 in six human mesothelioma and one mesothelial cell line. Polymerase chain reaction (PCR), immunoblotting, zymography, and small inhibitory RNAs (siRNA) were used to study gene expression, protein activation, and signal transduction. To proof the relevance of our in vitro data immunohistochemistry was performed in tissue sections. PDGF-BB induced, while TGF-beta1 inhibited cell proliferation. PDGF-BB was a chemoattractant for mesothelial cells, and its effect was increased in the presence of TGF-beta1. TGF-beta1 stimulated the de novo synthesis of pro-MMP-2 in both cell types. Pro-MMP-2 synthesis involved p38 MAP kinase. In cell culture and tissue sections only mesothelial cells expressed MT1-MMP. Migration of mesothelioma cells was dependent on the presence of MT1-MMP. Migration, but not proliferation of mesothelioma cells was inhibited by oleoyl-N-hydroxylamide, TIMP-2, and siRNA for MT1-MMP. Our data suggest that in mesothelioma cells the phosphorylation of p38 MAP kinase is deregulated and is involved in pro-MMP-2 expression. Mesothelioma progression depends on an interaction with mesothelial cells that provide MT1-MMP necessary to activate pro-MMP-2 to facilitate migration through an extracellular matrix (ECM) layer.     

Interindividual differences in leg muscle mass and pyruvate kinase activity correlate with interindividual differences in jumping performance of Hyla multilineata

Frog jumping is an excellent model system for examining the structural basis of interindividual variation in burst locomotor performance. Some possible factors that affect jump performance, such as total body size, hindlimb length, muscle mass, and muscle mechanical and biochemical properties, were analysed at the interindividual (intraspecies) level in the tree frog Hyla multilineata. The aim of this study was to determine which of these physiological and anatomical variables both vary between individuals and are correlated with interindividual variation in jump performance. The model produced via stepwise linear regression analysis of absolute data suggested that 62% of the interindividual variation in maximum jump distance could be explained by a combination of interindividual variation in absolute plantaris muscle mass, total hindlimb muscle mass (excluding plantaris muscle), and pyruvate kinase activity. When body length effects were removed, multiple regression indicated that the same independent variables explained 43% of the residual interindividual variation in jump distance. This suggests that individuals with relatively large jumping muscles and high pyruvate kinase activity for their body size achieved comparatively large maximal jump distances for their body size.