Peritoneal malignant mesothelioma: Slippery like an eel to diagnose on cytology-case series of 3 cases.

INTRODUCTION: Peritoneal malignant mesothelioma is an extremely rare tumor and is a difficult diagnosis to be made on cytology alone. We report 3 cases where the cytologic features were misdiagnosed as carcinoma/lymphoma but histopathology and immunohistochemistry (IHC) established the diagnosis of malignant mesothelioma. CLINICAL DETAILS: Case 1 was a 60-year-old man with multiloculated ascites and omental caking. Peritoneal fluid was reported as malignant on cytology but was misclassified as adenocarcinoma. Case 2, a 45-year-old man with ascites and peritoneal nodularity, radiologically mimicking peritoneal carcinomatosis, was also reported positive for malignancy on ascitic fluid cytology. Fine-needle aspiration (FNAC) from omental fat revealed signet ring cells, thus misleading to cytologic diagnosis of adenocarcinoma. Case 3 was a 63-year-old man with perisplenic mass with extensive omental caking and peritoneal nodularity that was also suspected to be peritoneal carcinomatosis on radiology. FNAC smears from perisplenic mass showed sheets of plasmacytoid cells. On cytology, the differential diagnoses offered were neuroendocrine tumor or non-Hodgkin lymphoma. The diagnosis of malignant mesothelioma was established only after IHC on histopathologic sections in all these cases. None of our patients had history of prior asbestos exposure. CONCLUSION: In such clinical scenarios, with radiology suggesting peritoneal carcinomatosis, the cytologic features need corroboration by IHC/fluorescence in situ hybridization on cell block or biopsy to correctly identify malignant mesothelioma and differentiate it from metastatic carcinomatous deposits and benign mesothelial proliferation.

Hematopoietic Stem Cell Requirement for Macrophage Regeneration Is Tissue Specific.

Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRMΦ have undergone intense scrutiny, in which now most TRMΦ are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity macrophages (MΦ) (large and small peritoneal MΦ) whose origins and relationship to both fetal and adult long-term (LT) HSCs have not been fully investigated. In this study, we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for large and small peritoneal MΦ, in which the initial wave of peritoneal MΦ is seeded from yolk sac-derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRMΦ, but their requirement is tissue specific in the peritoneum and brain.

A Possible Inhibitory Role of Sialic Acid on MUC1 in Peritoneal Dissemination of Clear Cell-Type Ovarian Cancer Cells.

The role of sialic acids on MUC1 in peritoneal dissemination of ovarian cancer cells was investigated. A human ovarian carcinoma cell line, ES-2, was transfected with full-length MUC1 containing 22 or 42 tandem repeats. These transfectants were less adherent to monolayers of patient-derived mesothelial cells than ES-2/mock transfectants. When these cells were inoculated into the abdominal cavity of female nude mice, mice that had received the transfectants showed better survival. When the transfectants were mixed with sialidase and injected, the survival was poorer, whereas when they were mixed with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid, a sialidase inhibitor, the survival was significantly prolonged. These behaviors, concerned with peritoneal implantation and dissemination observed in vitro and in vivo, were dependent on the expression of MUC1. Therefore, sialic acid linked to MUC1 in the form, at least in part, of sialyl-T, as shown to be recognized by monoclonal antibody MY.1E12, is responsible for the suppression of adhesion of these cells to mesothelial cells and the suppression of peritoneal implantation and dissemination.

Co-culture with peritoneum mesothelial stem cells supports the in vitro growth of mouse ovarian follicles.

The important roles played by the ovarian microenvironment and cell interactions in folliculogenesis suggest promising approaches for in vivo growth of ovarian follicles using appropriate scaffolds containing suitable cell sources. In this study, we have investigated the growth of early preantral follicles in the presence of decellularized mesenteric peritoneal membrane (MPM), peritoneum mesothelial stem cells (PMSCs), and conditioned medium (CM) of PMSCs. MPM of mouse was first decellularized; PMSCs were isolated from MPM and cultured and their conditioned medium (CM) was collected. Mouse follicles were separated into four groups: (1) culture in base medium (control), (2) culture in decellularized MPM (DMPM), (3) co-culture with PMSCs (Co-PMSCs), and (4) culture in CM of PMSCs (CM-PMSCs). Qualitative and quantitative assessments were performed to evaluate intact mesenteric peritoneal membrane (IMPM) as well as decellularized ones. After culturing the ovarian follicles, follicular and oocyte diameter, viability, eccentric oocyte percentage, and estradiol hormone amounts were evaluated. Quantitative and qualitative evaluations confirmed removal of cells and retention of the essential fibers in MPM after the decellularization process. Follicular parameters showed that Co-PMSCs better support in vitro growth and development of ovarian follicles than the other groups. The eccentric rate and estradiol production were statistically higher for the Co-PMSCs group than for the CM-PMSCs and control groups. Although the culture of early preantral follicles on DMPM and CM-PMSCs could improve in vitro follicular growth, co-culture of follicles with PMSCs showed even greater improvements in terms of follicular growth and diameter.

The clinicopathologic significance of Tks5 expression of peritoneal mesothelial cells in gastric cancer patients.

BACKGROUND: Gastric cancer (GC) patients frequently develop peritoneal metastasis. Recently, it has been reported that peritoneal mesothelial cells (PMCs) activated by GC cells acquire a migratory capacity and promote GC cell invasion. The invasiveness of PMCs reportedly depends on the activity of Tks5, an adaptor protein required for invadopodia formation. However, the relationship between clinicopathologic features and Tks5 expression in PMCs has been poorly documented. In this study, we evaluated the clinicopathologic significance of the Tks5 expression of PMCs in GC patients. MATERIALS AND METHODS: A total of 110 GC patients who underwent gastrectomy were enrolled in this study. Tks5 expressions in PMCs from the greater omentum, lesser omentum and retroperitoneum were evaluated by immunohistochemistry. We analyzed the correlation between Tks5 expressions in PMCs and the patients' clinicopathologic features. RESULTS: Tks5 expression was found in 71 (64.5%) of the 110 patients, while 39 (35.5%) were Tks5-negative. Tks5 positivity was significantly (p = 0.038) associated with a greater tumor depth (i.e., T3/4 compared with T1/T2). Peritoneal recurrence was found in 12 of 98 cases within 3 years of surgery. The 3-year peritoneal recurrence-free survival (PRFS) rate in Tks5-positive cases was significantly poorer than that in Tks5-negative cases (80.1% vs 97.4%, p = 0.024). Multivariate analysis revealed that Tks5 positivity and lymph node metastasis were independent factors for PRFS. CONCLUSION: Tks5 is frequently expressed in PMCs in advanced-stage gastric cancer. Tks5 might be a useful predictor for peritoneal recurrence in GC patients.

Effects of TGF-ß1 Receptor Inhibitor GW788388 on the Epithelial to Mesenchymal Transition of Peritoneal Mesothelial Cells.

We investigated the effectiveness of the transforming growth factor beta-1 (TGF-ß) receptor inhibitor GW788388 on the epithelial to mesenchymal transition (EMT) using human peritoneal mesothelial cells (HPMCs) and examined the effectiveness of GW788388 on the peritoneal membrane using a peritoneal fibrosis mouse model. HPMCs were treated with TGF-ß with or without GW788388. Animal experiments were conducted on male C57/BL6 mice. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate. GW788388 was administered by once-daily oral gavage. The morphological change, cell migration, and invasion resulted from TGF-ß treatment, but these changes were attenuated by cotreatment with GW788388. TGF-ß-treated HPMCs decreased the level of the epithelial cell marker and increased the levels of the mesenchymal cell markers. Cotreatment with GW788388 reversed these changes. Phosphorylated Smad2 and Smad3 protein levels were stimulated with TGF-ß and the change was attenuated by cotreatment with GW788388. For the peritoneal fibrosis mice, thickness and collagen deposition of parietal peritoneum was increased, but this change was attenuated by cotreatment with GW788388. GW788388, an orally available potent TGF-ß receptor type 1 inhibitor, effectively attenuated TGF-ß-induced EMT in HPMCs. Cotreatment with GW788388 improved peritoneal thickness and fibrosis, and recovered peritoneal membrane function in a peritoneal fibrosis mouse model.

C1 inhibitor mitigates peritoneal injury in zymosan-induced peritonitis.

Peritonitis, due to a fungal or bacterial infection, leads to injury of the peritoneal lining and thereby forms a hazard for the long-term success of peritoneal dialysis (PD) and remains a lethal complication in patients with PD. This study investigated whether C1 inhibitor (C1-INH) could protect against the progression of peritoneal injuries with five daily administrations of zymosan after mechanical scraping of the rat peritoneum to mimic fungal peritonitis. Severe peritoneal injuries were seen in this model, accompanied by fibrinogen/fibrin exudation and peritoneal deposition of complement activation products such as activated C3 and C5b-9. However, intraperitoneal injection of C1-INH decreased peritoneal depositions of activated C3 and C5b-9, ameliorated peritoneal thickening, reduced the influx of inflammatory cells, and prevented the production of peritoneal fibrous layers with both one and two doses of C1-INH each day. Our results suggest that C1-INH might be useful to protect against peritoneal injuries after causes of peritonitis such as fungal infection. This clinically available agent may thus help extend the duration of PD.NEW & NOTEWORTHY Peritoneal injuries associated with peritonitis comprise an important issue to prevent long-term peritoneal dialysis (PD) therapy. Here, we showed that C1 inhibitor (C1-INH), as an anticomplement agent, protected against peritoneal injuries in a peritonitis animal model related to fungal infection. Therefore, C1-INH might be useful to protect against peritoneal injuries after peritonitis due to fungal infection. This clinically available agent may thus help extend the duration of PD.

Native and IgE-primed rat peritoneal mast cells exert pro-inflammatory activity and migrate in response to yeast zymosan upon Dectin-1 engagement.

Mast cells (MCs) play an essential role in host defense, primarily because of their location, their ability to pathogen destruction via several mechanisms, and the pattern recognition receptors they express. Even though most data is available regarding MC activation by various bacteria- or virus-derived molecules, those cells' activity in response to constituents associated with fungi is not recognized enough. Our research aimed to address whether Saccharomyces cerevisiae-derived zymosan, i.e., ß-(1,3)-glucan containing mannan particles, impacts MC activity aspects. Overall, the obtained results indicate that zymosan has the potential to elicit a pro-inflammatory response of rat peritoneal MCs. For the first time ever, we provided evidence that zymosan induces fully mature MC migration, even in the absence of extracellular matrix (ECM) proteins. Moreover, the zymosan-induced migratory response of MCs is almost entirely a result of directional migration, i.e., chemotaxis. We found that zymosan stimulates MCs to degranulate and generate lipid mediators (cysLTs), cytokines (IFN-α, IFN-ß, IFN-γ, GM-CSF, TNF), and chemokine (CCL2). Zymosan also upregulated mRNA transcripts for several cytokines/chemokines with pro-inflammatory/immunoregulatory activity. Moreover, we documented that zymosan activates MCs to produce reactive oxygen species (ROS). Lastly, we established that the zymosan-induced MC response is mediated through activation of the Dectin-1 receptor. In general, our results strongly support the notion that MCs contribute to innate antifungal immunity and bring us closer to elucidate their role in host-pathogenic fungi interactions. Besides, provided findings on IgE-sensitized MCs appear to indicate that exposure to fungal zymosan could affect the severity of IgE-dependent disorders, including allergic ones.

Flow Cytometric Characterization of Murine B Cell Development.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

Higher expression of KCNK10 (TREK-2) K+ channels and their functional upregulation by lipopolysaccharide treatment in mouse peritoneal B1a cells.

Innate-like CD5+ B1a cells localized in serous cavities are activated by innate stimuli, such as lipopolysaccharide (LPS), leading to T cell-independent antibody responses. Although ion channels play crucial roles in the homeostasis and activation of immune cells, the electrophysiological properties of B1a cells have not been investigated to date. Previously, in the mouse B cell lymphoma cells, we found that the voltage-independent two-pore-domain potassium (K2P) channels generate a negative membrane potential and drive Ca2+ influx. Here, we newly compared the expression and activities of K2P channels in mouse splenic follicular B (FoB), marginal zone B (MZB), and peritoneal B1a cells. Next-generation sequencing analysis showed higher levels of transcripts for TREK-2 and TWIK-2 in B1a cells than those in FoB or MZB cells. Electrophysiological analysis, using patch clamp technique, revealed higher activity of TREK-2 with the characteristic large unitary conductance (~ 250 pS) in B1a than that in FoB or MZB cells. TREK-2 activity was further increased by LPS treatment (>2 h), which was more prominent in B1a than that in MZB or FoB cells. The cytosolic Ca2+ concentration of B cells was decreased by high-K+-induced depolarization (ΔRKCl (%)), suggesting the basal Ca2+ influx to be driven by negative membrane potential. The LPS treatment significantly increased the ΔRKCl (%) in B1a, though not in FoB and MZB cells. Our study was the first to compare the K2P channels in mouse primary B cell subsets, elucidating the functional upregulation of TREK-2 and augmentation of Ca2+ influx by the stimulation of Toll-like receptor 4 in B1a cells.

Cellular and humoral peritoneal immunity to Mesocestoides vogae metacestode infection in mice.

BACKGROUND: Here, Mesocestoides (M.) vogae infection in mice is proposed as a suitable experimental model for studying the immunity in the peritoneal cavity of mice. METHODS: To investigate the kinetics of immune parameters in M. vogae-infected mice, we detected, using flow cytometry, the expression of selected lymphoid and myeloid markers within the peritoneal cell population at day 0, 3, 6, 10, 14, 19, 25, 30 and 35 post-infection. Then, using ELISA, we analyzed the cytokine IFN-γ, TGF-ß, IL-4 and IL-10 responses and the levels of anti-M. vogae IgG and IgM antibodies in the peritoneal lavage fluid. Cells isolated from the peritoneal cavity were subjected to further molecular analysis. To assess cell activation, peritoneal cells were exposed to LPS, and culture supernatants were collected and assayed for the level of cytokines and production of nitrite. Ly6C+ and Ly6G+ cells were isolated using MACS from the peritoneal cells at day 35 post-infection. Both MACS-isolated subsets were co-cultured with preactivated T cells to measure their suppressive capacity. Next, the role of parasite excretory-secretory antigens in induction of CD11b+ myeloid cells with the suppressive phenotype and the production of IL-10 was examined. RESULTS: In the peritoneal cavity an initial increase of CD11b+Gr-1+F4/80highMHC IIhigh cells, NK, NKT cells and CD8+ cytotoxic T cells was observed in the first week of infection. At day 14 post-infection, an increase in the number of myeloid CD11b+Gr-1+ cells was detected, and most of this cell population expressed low levels of F4/80 and MHC II in later stages of infection, suggesting the impairment of antigen-presenting cell functions, probably through the excretory-secretory molecules. Moreover, we confirmed that peritoneal Gr1+ cells (Ly6C+ and Ly6G+ population) are phenotypically and functionally consistent with myeloid-derived suppressor cells. Metacestode infection elicited high levels of IL-10 and upregulated STAT-3 in peritoneal cells. A higher level of IgM suggests that this isotype may be predominant and is involved in the host protection. CONCLUSIONS: Mesocestoides vogae tetrathyridia induced the recruitment of immunosuppressive cell subsets, which may play a key role in the downregulation of immune response in long-term parasitic diseases, and excretory-secretory antigens seem to be the main regulatory factor.

Empagliflozin, a sodium glucose cotransporter-2 inhibitor, ameliorates peritoneal fibrosis via suppressing TGF-ß/Smad signaling.

Sodium glucose cotransporter-2 (SGLT-2) inhibitor has been reported to exert a glucose-lowering effect in the peritoneum exposed to peritoneal dialysis solution. However, whether SGLT-2 inhibitors can regulate peritoneal fibrosis by suppressing TGF-ß/Smad signaling is unclear. We aimed to (i) examine the effect of the SGLT-2 inhibitor empagliflozin in reducing inflammatory reaction and preventing peritoneal dialysis solution-induced peritoneal fibrosis and (ii) elucidate the underlying mechanisms. High-glucose peritoneal dialysis solution or transforming growth factor ß1 (TGF-ß1) was used to induce peritoneal fibrosis in vivo, in a mouse peritoneal dialysis model (C57BL/6 mice) and in human peritoneal mesothelial cells in vitro, to stimulate extracellular matrix accumulation. The effects of empagliflozin and adeno-associated virus-RNAi, which is used to suppress SGLT-2 activity, on peritoneal fibrosis and extracellular matrix were evaluated. The mice that received chronic peritoneal dialysis solution infusions showed typical features of peritoneal fibrosis, including markedly increased peritoneal thickness, excessive matrix deposition, increased peritoneal permeability, and upregulated α-smooth muscle actin and collagen I expression. Empagliflozin treatment or downregulation of SGLT-2 expression significantly ameliorated these pathological changes. Inflammatory cytokines (TNF-α, IL-1ß, IL-6) and TGF-ß/Smad signaling-associated proteins, such as TGF-ß1 and phosphorylated Smad (p-Smad3), decreased in the empagliflozin-treated and SGLT-2 downregulated groups. In addition, empagliflozin treatment and downregulation of SGLT-2 expression reduced the levels of inflammatory cytokines (TNF-α, IL-1ß, IL-6), TGF-ß1, α-smooth muscle actin, collagen I, and p-Smad3 accumulation in human peritoneal mesothelial cells. Collectively, these results indicated that empagliflozin exerted a clear protective effect on high-glucose peritoneal dialysis-induced peritoneal fibrosis via suppressing TGF-ß/Smad signaling.

PDCD4 regulates apoptosis in human peritoneal mesothelial cells and promotes gastric cancer peritoneal metastasis.

OBJECTIVE: Programmed cell death 4 (PDCD4) is a tumor suppressor gene, however, the function and regulatory mechanism remain to be discovered. The connection between tumorigenesis and apoptosis is one of the most important foci of cancer research. Our study aimed to explore the connections between PDCD4-mediated apoptosis of human peritoneal mesothelial cells (HPMC) and peritoneal metastasis in gastric cancer. METHODS: The PDCD4 expression in 31 pairs of HPMC and tumor tissues was assessed by immunohistochemistry and RT-PCR. In cell experiments, we monitored gastric cancer cell migration with a Transwell chamber assay when PDCD4 was silenced in HPMC. Subsequently, apoptosis of HPMC was detected by a flow cytometric assay and western blotting. After analyzing cytokines in culture supernatants from gastric cancer with enzyme-linked immunosorbent assays (ELISAs), transforming growth factor-beta 1 (TGF-ß1) was abundant in the culture supernatants of gastric cancer. Then, PDCD4 expression in HMrSV5 cells was analyzed by western blotting after retreatment with different concentrations of TGF-ß1. Moreover, apoptosis of peritoneal mesothelial cells treated with TGF-ß1 was detected according to the above methods. RESULTS: In human metastatic peritoneal tissues, the expression of PDCD4 was significantly lower than that in normal tissues. At the same time, decreased expression of PDCD4 in HPMC was associated with increased migration capacity of gastric cancer cells. Moreover, suppressing the expression of PDCD4 promoted apoptosis in mesothelial cells which may be regulated by TGF-ß secreted from gastric cancer cells. CONCLUSIONS: These data suggested that decreased expression of PDCD4 significantly promoted apoptosis in human peritoneal mesothelial cells, thus inducing peritoneal metastasis, and that TGF-ß1 secreted from gastric cancer cells may have played a crucial role.

Effect of aquaporin 1 on mouse peritoneal mesothelial cells after a long-term peritoneal dialysis.

Aquaporin 1 (AQP1) is one member of the aquaporin family, also the deeply studied one. It is widely located on the endothelial cells, but the effect of AQP1 on the peritoneal mesothelial cells (PMCs) after long-term peritoneal dialysis (PD) has not been reported before. We divided normal mice into two groups, control group and dialysis group, to confirm the fibrotic changes and expression of APQ1 on peritoneal mesothelial cells. Then we assigned normal mice and AQP1 knockout mice into four groups: Control group, normal dialysis group, AQP1 knockout control group and AQP1 knockout dialysis group. The two dialysis groups received 4.25% glucose dialysis for 28 days. We found that mice in both dialysis groups showed peritoneal fibrotic changes, which were most severe in the AQP1 knockout dialysis group; the peritoneal thickness in the AQP1 knockout dialysis group was also thicker than that in the dialysis group (P < .05). We used electron microscopy to detect ultrastructural changes and observed changes in microvilli and vacuolar degeneration in mesothelial cells from all groups except the control group. The basement membranes were damaged in the AQP1 knockout dialysis group, and peritoneal mesothelial cells were disrupted and detached in this group. Together our findings indicate that AQP1 plays an important role in maintaining the physiological functions of peritoneal mesothelial cells, and AQP1 can protect mesothelial cells during dialysis.

Neutrophil recruitment by chemokines Cxcl1/KC and Cxcl2/MIP2: Role of Cxcr2 activation and glycosaminoglycan interactions.

Chemokines play a crucial role in combating microbial infection by recruiting blood neutrophils to infected tissue. In mice, the chemokines Cxcl1/KC and Cxcl2/MIP2 fulfill this role. Cxcl1 and Cxcl2 exist as monomers and dimers, and exert their function by activating the Cxcr2 receptor and binding glycosaminoglycans (GAGs). Here, we characterized Cxcr2 G protein and ß-arrestin activities, and GAG heparan sulfate (HS) interactions of Cxcl1 and Cxcl2 and of the trapped dimeric variants. To understand how Cxcr2 and GAG interactions impact in vivo function, we characterized their neutrophil recruitment activity to the peritoneum, Cxcr2 and CD11b levels on peritoneal and blood neutrophils, and transport profiles out of the peritoneum. Cxcl2 variants compared with Cxcl1 variants were more potent for Cxcr2 activity. Native Cxcl1 compared with native Cxcl2 and dimers compared with native proteins bound HS with higher affinity. Interestingly, recruitment activity between native Cxcl1 and Cxcl2, between dimers, and between the native protein and the dimer could be similar or very different depending on the dose or the time point. These data indicate that peritoneal neutrophil recruitment cannot be solely attributed to Cxcr2 or GAG interactions, and that the relationship between recruited neutrophils, Cxcr2 activation, GAG interactions, and chemokine levels is complex and highly context dependent. We propose that the ability of Cxcl1 and Cxcl2 to reversibly exist as monomers and dimers and differences in their Cxcr2 activity and GAG interactions coordinate neutrophil recruitment and activation, which play a critical role for successful resolution of inflammation.

Mast Cell Degranulation Increases Mouse Mast Cell Protease 4-Dependent Vasopressor Responses to Big Endothelin-1 But Not Angiotensin I.

Mouse mast cell protease 4 (mMCP-4), the murine functional analog to the human chymase, is a serine protease synthesized and stored in mast cell secretory granules. Our previous studies reported physiologic and pathologic roles for mMCP-4 in the maturation and synthesis of the vasoactive peptide endothelin-1 (ET-1) from its precursor, big ET-1. The aim of this study was to investigate the impact of mast cell degranulation or stabilization on mMCP-4-dependent pressor responses after the administration of big ET-1 or angiotensin I (Ang I). In anesthetized mice, mast cell degranulation induced by compound 48/80 (C48/80) or stabilization by cromolyn enhanced or repressed, respectively, the dose-dependent vasopressor responses to big ET-1 in wild-type (WT) mice but not in mMCP-4 knockout mice in a chymase inhibitor (TY-51469)-sensitive fashion. In addition, mMCP-4-dependent hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was depleted or enhanced in peritoneal mast cells isolated from mice pretreated with C48/80 or cromolyn, respectively. Furthermore, C48/80 or cromolyn markedly increased or abolished, respectively, ET-1 (1-31) conversion from exogenous big ET-1 in WT mice peritoneal fluid-isolated mast cells, in vitro. Finally, the vasopressor responses to Ang I were unaffected by mast cell activation or stabilization, whereas those induced by the angiotensin-converting enzyme-resistant Ang I analog, [Pro11, D-Ala12] Ang I, were potentiated by C48/80. Altogether, the present study shows that mast cell activation enhances the mMCP-4-dependent vasoactive properties of big ET-1 but not Ang I in the mouse model. SIGNIFICANCE STATEMENT: The current work demonstrates a significant role for mast cell stability in the cardiovascular pharmacology of big endothelin-1 but not angiotensin I in the murine systemic circulation.

LGR4 maintains HGSOC cell epithelial phenotype and stem-like traits.

OBJECTIVE: High-grade serous ovarian cancer (HGSOC) is lethal mainly due to extensive metastasis. Cancer cell stem-like properties are responsible for HGSOC metastasis. LGR4, a G-protein-coupled receptor, is involved in the maintenance of stem cell self-renewal and activity in some human organs. METHODS: TCGA and CCLE databases were interrogated for gene mRNA in ovarian cancer tissues and cell lines. Gain and loss of functions of LGR4, ELF3, FZD5 and WNT7B were performed to identify their roles in ovarian cancer cell epithelial phenotype and stem-like properties. In vivo experiments were performed to observe the effect of LGR4 on ovarian cancer cell growth and peritoneal seeding. The binding of ELF3 to LGR4 gene promoter was investigated by dual-luciferase reporter assays and ChIP. RESULTS: LGR4 was shown to be overexpressed in HGSOCs and maintain the epithelial phenotype of HGSOC cells. LGR4 knockdown suppressed POU5F1, SOX2, PROM1 (CD133) and ALDH1A2 expression. Furthermore, LGR4 knockdown reduced CD133+ and ALDH+ subpopulations and impaired tumorisphere formation. To the contrary, LGR4 overexpression enhanced POU5F1 and SOX2 expression and tumorisphere formation capacity. LGR4 knockdown inhibited HGSOC cell growth and peritoneal seeding in xenograft models. Mechanistically, LGR4 and ELF3, an epithelium-specific transcription factor, formed a reciprocal regulatory loop, which was positively modulated by WNT7B/FZD5 ligand-receptor pair. Consistently, knockdown of ELF3, WNT7B, and FZD5, respectively, disrupted HGSOC cell epithelial phenotype and stem-like properties. CONCLUSION: Together, these data demonstrate that WNT7B/FZD5-LGR4/ELF3 axis maintains HGSOC cell epithelial phenotype and stem-like traits; targeting this axis may prevent HGSOC metastasis.

How Relevant Are Bone Marrow-Derived Mast Cells (BMMCs) as Models for Tissue Mast Cells? A Comparative Transcriptome Analysis of BMMCs and Peritoneal Mast Cells.

Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs.

Transforming growth factor-ß1 induces connective tissue growth factor expression and promotes peritoneal metastasis of gastric cancer.

Transforming growth factor-ß1 (TGF-ß1) is involved in human cancer development and progression. Nonetheless, the role of TGF-ß1 as regards peritoneal metastasis of gastric cancer has not been completely characterized. In the present study, we investigated the exact role of TGF-ß1 on peritoneal metastasis of gastric cancer. The results indicated that human peritoneal mesothelial cells (HPMCs) exposed to TGF-ß1 or serum-free conditional medium (SF-CM) of SGC7901 that produced a large amount of TGF-ß1 became exfoliated, apoptosis and exhibited signs of injury, and the tumor-mesothelial cell adhesion significantly increased. Connective tissue growth factor (CTGF) expression was also increased when HPMCs were exposed to TGF-ß1 or SF-CM of SGC7901. However, these effects were significantly decreased when HPMCs were exposed to SF-CM of SGC7901-TGFßS, a TGF-ß1 knockdown stable cell line. Animal studies revealed that nude mice injected with SGC7901-TGFßS cells featured a smaller number of peritoneal seeding nodules and lower expression of CTGF in ascites than the control cell lines. These findings suggest that TGF-ß1 promotes peritoneal metastasis of gastric cancer and induces CTGF expression. Therefore, blockage of TGF-ß1 or TGF-ß1 signaling pathway might prevent and treat peritoneal metastasis of gastric cancer.

Reprogramming of Mesothelial-Mesenchymal Transition in Chronic Peritoneal Diseases by Estrogen Receptor Modulation and TGF-ß1 Inhibition.

In chronic peritoneal diseases, mesothelial-mesenchymal transition is determined by cues from the extracellular environment rather than just the cellular genome. The transformation of peritoneal mesothelial cells and other host cells into myofibroblasts is mediated by cell membrane receptors, Transforming Growth Factor ß1 (TGF-ß1), Src and Hypoxia-inducible factor (HIF). This article provides a narrative review of the reprogramming of mesothelial mesenchymal transition in chronic peritoneal diseases, drawing on the similarities in pathophysiology between encapsulating peritoneal sclerosis and peritoneal metastasis, with a particular focus on TGF-ß1 signaling and estrogen receptor modulators. Estrogen receptors act at the cell membrane/cytosol as tyrosine kinases that can phosphorylate Src, in a similar way to other receptor tyrosine kinases; or can activate the estrogen response element via nuclear translocation. Tamoxifen can modulate estrogen membrane receptors, and has been shown to be a potent inhibitor of mesothelial-mesenchymal transition (MMT), peritoneal mesothelial cell migration, stromal fibrosis, and neoangiogenesis in the treatment of encapsulating peritoneal sclerosis, with a known side effect and safety profile. The ability of tamoxifen to inhibit the transduction pathways of TGF-ß1 and HIF and achieve a quiescent peritoneal stroma makes it a potential candidate for use in cancer treatments. This is relevant to tumors that spread to the peritoneum, particularly those with mesenchymal phenotypes, such as colorectal CMS4 and MSS/EMT gastric cancers, and pancreatic cancer with its desmoplastic stroma. Morphological changes observed during mesothelial mesenchymal transition can be treated with estrogen receptor modulation and TGF-ß1 inhibition, which may enable the regression of encapsulating peritoneal sclerosis and peritoneal metastasis.