Human Peritoneal Mesothelial Cells Research Articles

FC090: Stevia as an Alternative Osmotic Agent for Peritoneal Dialysis Fluid

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is a renal replacement technique that requires repeated contact of the peritoneum with hyperosmolar fluids. Unfortunately, it generates metabolic and biomechanical insults that promote structural and functional alterations of the peritoneal membrane (PM). PM is lined by a mesothelial cells (MCs) monolayer that is in direct contact with the PD fluids (PDFs). Repeated PDF exposure causes a mesothelial–mesenchymal transition (MMT) of the MCs, as well as an inflammatory response, angiogenesis and fibrosis of the peritoneal tissue. For decades, glucose (Gl) has been the most commonly used osmotic agent, and Gl itself is responsible for the aforementioned changes. Furthermore, Gl degradation products (GDPs) contribute to deleterious effects on the PM [1]. Therefore, there is an unmet need for more biocompatible osmotic agents able to better maintain the structure and function of the PM. Here we evaluated the biocompatibility of Stevia (STV), a compound of natural origin with a mixture of glycosides. STV has a molecular weight close to the theoretical ideal weight for an osmotic agent in PD [2] and it was used instead of Gl for PDF. METHODS The osmotic capacity of STV-containing fluids was analysed using an in vitro device with artificial membranes and compared to Gl-based fluids. In order to investigate the STV biocompatibility, human omental peritoneal mesothelial cells (HOMCs) were cultured in the presence of different types of Gl-based PDFs (Conventional 4.25% Gl solution with a high GDP level and biocompatible 2.3% Gl solution with low GDPs level), STV-based fluids or TGF-b for 48 h. Mice submitted to surgery of an intraperitoneal catheter insertion were treated for 40 days with STV- or Gl-based PDFs. Peritoneal tissues were collected to determine thickness, MMT, angiogenesis and inflammatory markers, as well as effluents to analyse infiltrating cells. RESULTS Experimentally, using artificial membranes we demonstrated that 1, 0.75 and 0.5% STV-based fluids have a maintained and prolonged osmotic capacity similar to commercial PDFs containing Gl concentrations between 1.5 and 2.3%. Moreover, 1.5% STV-based fluid showed an osmotic capacity higher than a PDF with Gl 2.3% and reached values similar to Gl 4.25% at longer time points. In addition, HOMCs exposed to STV-based fluids showed higher viability and did not induce MMT. Gl-based fluids induced upregulation of mesenchymal markers (VEGF, fibronectin, snail) and also downregulation of the epithelial marker E-cadherin in the case of the highest concentration of Gl (4.25%). None of these markers were significantly affected by STV treatment. In vivo, STV-based fluids preserved MCs monolayer and induced less PM thickness (Figure 1), angiogenesis, leukocyte infiltration and MMT than Gl-based fluids. Furthermore, STV solutions did not promote the release of pro-inflammatory cytokines by HOMCs nor by cells present in mice parietal peritoneal tissue in contrast to Gl-based PDFs. CONCLUSIONS STV-based fluids showed better biocompatibility than Gl-based PDFs in vivo and ex vivo since STV did not induce PM thickness, MMT, angiogenesis or the release of pro-inflammatory cytokines.

Myo5b Transports Fibronectin-Containing Vesicles and Facilitates FN1 Secretion from Human Pleural Mesothelial Cells.

Pleural mesothelial cells (PMCs) play a central role in the progression of pleural fibrosis. As pleural injury progresses to fibrosis, PMCs transition to mesenchymal myofibroblast via mesothelial mesenchymal transition (MesoMT), and produce extracellular matrix (ECM) proteins including collagen and fibronectin (FN1). FN1 plays an important role in ECM maturation and facilitates ECM-myofibroblast interaction, thus facilitating fibrosis. However, the mechanism of FN1 secretion is poorly understood. We report here that myosin 5b (Myo5b) plays a critical role in the transportation and secretion of FN1 from human pleural mesothelial cells (HPMCs). TGF-β significantly increased the expression and secretion of FN1 from HPMCs and facilitates the close association of Myo5B with FN1 and Rab11b. Moreover, Myo5b directly binds to GTP bound Rab11b (Rab11b-GTP) but not GDP bound Rab11b. Myo5b or Rab11b knockdown via siRNA significantly attenuated the secretion of FN1 without changing FN1 expression. TGF-β also induced Rab11b-GTP formation, and Rab11b-GTP but not Rab11b-GDP significantly activated the actin-activated ATPase activity of Myo5B. Live cell imaging revealed that Myo5b- and FN1-containing vesicles continuously moved together in a single direction. These results support that Myo5b and Rab11b play an important role in FN1 transportation and secretion from HPMCs, and consequently may contribute to the development of pleural fibrosis.

Cell Type-Specific Anti-Adhesion Properties of Peritoneal Cell Treatment with Plasma-Activated Media (PAM)

Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as a therapeutic option based on cell type-specific anti-adhesive responses. Primary human peritoneal fibroblasts and mesothelial cells were isolated, characterized and exposed to different PAM dosages. Cell type-specific PAM effects on different cell components were identified by contact- and marker-independent Raman imaging, followed by thorough validation by specific molecular biological methods. The investigation revealed cell type-specific molecular responses after PAM treatment, including significant cell growth retardation in peritoneal fibroblasts due to transient DNA damage, cell cycle arrest and apoptosis. We identified a therapeutic dose window wherein specifically pro-adhesive peritoneal fibroblasts were targeted, whereas peritoneal mesothelial cells retained their anti-adhesive potential of epithelial wound closure. Finally, we demonstrate that PAM treatment of peritoneal fibroblasts reduced the expression and secretion of pro-adhesive cytokines and extracellular matrix proteins. Altogether, we provide insights into biochemical PAM mechanisms which lead to cell type-specific pro-therapeutic cell responses. This may open the door for the prevention of pro-adhesive clinical disorders.

Gastric cancer cell-derived extracellular vesicles elevate E2F7 expression and activate the MAPK/ERK signaling to promote peritoneal metastasis through the delivery of SNHG12

Cancer cell-derived extracellular vesicles (EVs) have extensive application in the formation of their environment, including metastasis. This study explored the ability of gastric cancer (GC) cell-derived EVs-mediated microRNA-129-5p/E2F transcription factor 7/mitogen-activated protein kinase/extracellular regulated protein kinase (miR-129-5p/E2F7/MAPK/ERK) axis to affect the peritoneal metastasis of GC by delivering lncRNA small nucleolar RNA host gene 12 (SNHG12). EV-derived lncRNA and SNHG12/miR-129-5p/E2F7 network were determined by bioinformatics analysis. The regulatory relationship among SNHG12, miR-129-5p, and E2F7 was verified using a combination of dual-luciferase reporter gene, RNA immunoprecipitation, and RNA pull-down assays. The SNHG12, miR-129-5p, and E2F7 expression was measured by RT-qPCR. After GC cell-derived EVs were isolated and co-cultured with human peritoneal mesothelial cells (HPMCs), the uptake of EVs by HPMCs was observed under laser scanning confocal microscopy. Cell viability and apoptosis were examined using cell counting kit-8 and flow cytometry, respectively. Western blot analysis was performed to measure the mesothelial–mesenchymal transition (MMT)-related protein expression. The pathological and morphological characteristics of metastatic tumors in nude mice were observed by hematoxylin–eosin staining. A high SNHG12 expression was correlated with the poor prognosis of patients with GC. GC-derived EVs led to increased HPMC apoptosis and MMT by transferring SNHG12, whereas the knockdown of SNHG12 annulled the aforementioned results. SNHG12 sponged miR-129-5p to boost E2F7 expression and activate the MAPK/ERK signaling, thus inducing HPMC apoptosis and MMT. In vivo experiments further verified that EVs derived from GC cells promoted peritoneal metastasis in nude mice. GC cell-derived EVs elevated the E2F7 expression and activated the MAPK/ERK signaling to promote peritoneal metastasis through the delivery of SNHG12.

Role of IGF-1R in epithelial-mesenchymal transdifferentiation of human peritoneal mesothelial cells.

Peritoneal fibrosis (PF) is caused by epithelial-mesenchymal transdifferentiation (EMT) in the peritoneum under high glucose (HG) conditions. The study aimed to explored the role of Insulin-like growth factor 1 receptor (IGF-1R) in the regulation of EMT in human peritoneal mesothelial cells (HPMCs). We used HG peritoneal dialysis fluid (PDF) to induce in vivo PF in mice, and treated HPMCs with HG in vitro to stimulate EMT. In the mice, the higher the glucose concentration in the dialysate, the more obvious the peritoneal tissue thickening and the more that collagen was deposited. The in vitro study indicated that the expression of IGF-1R, α-SMA, vimentin was upregulated, while the expression of occludin, ZO-1, and E-cadherin was downregulated in HPMCs under HG and IGF-1R overexpression conditions. Conversely, the expression of IGF-1R, α-SMA, and vimentin was downregulated, while the expression of occludin, ZO-1, and E-cadherin was upregulated in IGF-1R-underexpressed HPMCs under HG conditions. The cell migration abilities were increased, while the cell adhesion abilities were reduced in HPMCs under HG and IGF-1R overexpression conditions. In contrast, cell migration abilities were reduced, while cell adhesion abilities were increased in IGF-1Runderexpressed HPMCs under HG conditions. Targeting at IGF-1R may provide novel insights into the prevention and treatment of PF.

Angiogenic Role of Mesothelium-Derived Chemokine CXCL1 During Unfavorable Peritoneal Tissue Remodeling in Patients Receiving Peritoneal Dialysis as Renal Replacement Therapy.

Peritoneal dialysis (PD) is a valuable ‘home treatment’ option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy.

Adverse effects of iron toward the peritoneal mesothelial cells are reversible

Evaluation of the effects of the drugs used for iron supplementation on the peritoneal mesothelial cells. Acute effects during18-h incubation of iron sucrose, ferric carboxymaltose, and iron isomaltoside in concentrations 1, 5, and 20 ug/dl on properties of the human peritoneal mesothelial cells in in vitro culture were evaluated, and their reversibility after the following culture for 14 days in the iron-free medium was studied. All studied compounds reduced viability of mesothelial cells and proliferation rate and increased intracellular oxidative stress and iron content in the cytosol. Secretion of monocyte chemoattractant protein-1 was increased and tissue plasminogen activator (t-PA) decreased. After 14 days of culture in iron-free medium, reversibility of all these effects was observed. Iron compounds impair the functional properties of mesothelial cells in a dose-dependent manner, but these effects are reversible after the following culture of the cells in an iron-free medium.

Calponin 1 contributes to myofibroblast differentiation of human pleural mesothelial cells.

Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, how these transformed mesothelial cells contribute to lung fibrosis remains unclear. Here, we investigated the mechanism of contractile myofibroblast differentiation of PMCs. Transforming growth factor-β (TGF-β) induced marked upregulation of calponin 1 expression, which was correlated with notable cytoskeletal rearrangement in human PMCs (HPMCs) to produce stress fibers. Downregulation of calponin 1 expression reduced stress fiber formation. Interestingly, induced stress fibers predominantly contain α-smooth muscle actin (αSMA) associated with calponin 1 but not β-actin. Calponin 1-associated stress fibers also contained myosin II and α-actinin. Furthermore, focal adhesions were aligned with the produced stress fibers. These results suggest that calponin 1 facilitates formation of stress fibers that resemble contractile myofibrils. Supporting this notion, TGF-β significantly increased the contractile activity of HPMCs, an effect that was abolished by downregulation of calponin 1 expression. We infer that differentiation of HPMCs to contractile myofibroblasts facilitates stiffness of scar tissue in pleura to promote pleural fibrosis (PF) and that upregulation of calponin 1 plays a central role in this process.

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

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

Role of reactive oxygen species in high concentration glucose-induced growth inhibition of human peritoneal mesothelial cells.

To investigate the effects and mechanism of high concentration glucose (HG), exogenous hydrogen peroxide (H2O2), and antioxidants on the cell growth (cell proliferation) of human peritoneal mesothelial cells (HPMCs). All tests were conducted on cultured HPMCs (HMrSV5) in vitro. Various concentrations of glucose (0.1%, 1.35%, and 3.86%), H2O2 (0.5 and 0.1 mmol/L), and antioxidants (pyruvate and catalase) were used in cell culture. Moreover, in order to study the interaction between these factors, HG and H2O2, HG and antioxidants, HG, H2O2, and antioxidants, were used respectively. After 12-24 h, phase-contrast microscopy was used to examine the morphological changes of HPMCs. DNA synthesis was detected by 3H-thymidine incorporation to measure cell proliferation, and flow cytometry was used to evaluate the proportion of cells in G1 phase. Furthermore, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) was utilized to determine the mRNA expression of p21Waf1 and p27Kip1 [cyclin-dependent kinase inhibitors (CKIs)], while immunocytochemistry (ICC) and Western blotting were employed to measure the protein expression of p21Waf1 and p27Kip1. HG or low-dose exogenous H2O2 resulted in hypertrophy and senescence of HPMCs, resulting in similar morphological changes. Both HG and exogenous H2O2 (0.5 mmol/L) inhibited the proliferation of HPMCs and led to G1 phase arrest of HPMCs. The proportion of cells in G1 phase increased. Moreover, HG enhanced the toxic effects of exogenous H2O2. Both HG and exogenous H2O2 increased the expression of p21Waf1 and p27Kip1 . The addition of an antioxidant in HG medium arrested cells in the G1 phase and improved the inhibited cell proliferation. Both HG and exogenous H2O2 treatments can induce growth inhibition of HPMCs by arresting cell cycle progression, which is partially due to the increased expression of p21Waf1 and p27Kip1 . Thus, the effects of HG might be associated with endogenous reactive oxygen species (ROS), and it might be beneficial to use antioxidants in peritoneal dialysis (PD).

Inhibition of LPS-mediated TLR4 activation abrogates gastric adenocarcinoma-associated peritoneal metastasis.

Surgical resection, the cornerstone of curative intent treatment for gastric adenocarcinoma, is associated with a high rate of infection-related post-operative complications, leading to an increased incidence of metastasis to the peritoneum. However, the mechanisms underlying this process are poorly understood. Lipopolysaccharide (LPS), an antigen from Gram-negative bacteria, represents a potential mechanismvia induction of local and systemic inflammation through activation of Toll-like receptor (TLR).Here, we use both a novel ex vivo model of peritoneal metastasis and in vivo animal models to assess gastric cancer celladhesion to peritoneum both before and after inhibition of the TLR4 pathway.We demonstrate that activation of TLR4 by either LPS or Gram-negative bacteria (E. coli) significantly increases the adherence of gastric cancer cells to human peritoneal mesothelial cells, and that this increased adherence is abrogated by inhibition of the TLR4 signal cascade and downstream TAK1 and MEK1/2 pathways. We alsodemonstrate that the influence of LPS on adherence extends to peritoneal tissue and metastatic spread. Furthermore, we show that loss of TLR4 at the site of metastasis reduces tumor cell adhesion, implicating the TLR4 signaling cascade in potentiating metastatic adhesion and peritoneal spread.These results identify potential therapeutic targets for the clinical management of patients undergoing resection for gastric cancer.

Mitoquinone treatment for the prevention of surgical adhesions via regulation of the NRF2/HO-1 signaling pathway in mice

BackgroundPostoperative adhesion is a common cause of long-term morbidity after abdominal or pelvic surgery. The development of postoperative adhesion involves oxidative stress, inflammatory response, and collagen deposition mechanisms. Here, we demonstrate that mitoquinone could be useful for the treatment of postoperative adhesion. MethodsA murine adhesion model was established by induction of peritoneal ischemic buttons. Mice received different doses of mitoquinone via the tail vein. All the ischemic buttons were dissected at 1 day and 7 days after surgery to investigate the effect of mitoquinone in the early and late stage of the adhesion process, respectively. Human peritoneal mesothelial cells were treated with H2O2 to examine the potential mechanisms of mitoquinone in oxidative insult. ResultsPostoperative adhesion scores were markedly decreased in mitoquinone-treated mice compared with the control mice. The degree of oxidative stress, inflammatory injury, and collagen deposition were also significantly reduced in the mitoquinone-treated mice. The expression of plasminogen-activating inhibitor, interleukin-1, interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, malondialdehyde, and nitric oxide was decreased, while the expression of tissue-type plasminogen activator, glutathione, superoxide dismutase, and Nrf2 was increased in the peritoneal ischemic buttons after mitoquinone treatment. Cellular reactive oxygen species and the canonical inflammatory pathway were inhibited in mitoquinone-treated human peritoneal mesothelial cells after H2O2 challenge. Mechanistically, mitoquinone was found to enhance the activity of Nrf2 and heme oxygenase-1 and to induce nuclear translocation of Nrf2 in human peritoneal mesothelial cells. ConclusionThe mitochondria-targeting antioxidant molecule mitoquinone attenuates postoperative adhesion formation by inhibiting oxidative stress, inflammation, and collagen accumulation, and therefore provides a therapeutic agent for the management of surgical adhesion.

Exosomal ANXA2 derived from ovarian cancer cells regulates epithelial-mesenchymal plasticity of human peritoneal mesothelial cells.

Ovarian cancer, one of the malignant gynaecological tumours with the highest mortality rate among female reproductive system, is prone to metastasis, recurrence and chemotherapy resistance, causing a poor prognosis. Exosomes can regulate the epithelial‐mesenchymal plasticity of tumour cells, remodel surrounding tumour microenvironment, and affect tumour cell proliferation, invasion and metastasis. However, the function and mechanism of exosomes in the intraperitoneal implantation of ovarian cancer remain unclear. In this study, exosomal annexin A2 (ANXA2) derived from ovarian cancer cells was co‐cultured with human peritoneal mesothelial (HMrSV5) cells; functional experiments were conducted to explore the effects of exosomal ANXA2 on the biological behaviour of HMrSV5 and the related mechanisms. This study showed that ANXA2 in ovarian cancer cells can be transferred to HMrSV5 cells through exosomes, exosomal ANXA2 can not only promote the migration, invasion and apoptosis of HMrSV5 cells, but also regulates morphological changes and fibrosis of HMrSV5 cells. Furthermore, ANXA2 promotes the mesothelial‐mesenchymal transition (MMT) and degradation of the extracellular matrix of HMrSV5 cells through PI3K/AKT/mTOR pathway, finally affects pre‐metastasis microenvironment of ovarian cancer, which provides a new theoretical basis for the mechanism of intraperitoneal implantation and metastasis of ovarian cancer.

DOCK2 Promotes Pleural Fibrosis by Modulating Mesothelial to Mesenchymal Transition.

Mesothelial to mesenchymal transition (MesoMT) is one of the crucial mechanisms underlying pleural fibrosis, which results in restrictive lung disease. DOCK2 (dedicator of cytokinesis 2) plays important roles in immune functions; however, its role in pleural fibrosis, particularly MesoMT, remains unknown. We found that amounts of DOCK2 and the MesoMT marker α-SMA (α-smooth muscle actin) were significantly elevated and colocalized in the thickened pleura of patients with nonspecific pleuritis, suggesting the involvement of DOCK2 in the pathogenesis of MesoMT and pleural fibrosis. Likewise, data from three different pleural fibrosis models (TGF-β [transforming growth factor-β], carbon black/bleomycin, and streptococcal empyema) consistently demonstrated DOCK2 upregulation and its colocalization with α-SMA in the pleura. In addition, induced DOCK2 colocalized with the mesothelial marker calretinin, implicating DOCK2 in the regulation of MesoMT. Our invivo data also showed that DOCK2-knockout mice were protected from Streptococcus pneumoniae-induced pleural fibrosis, impaired lung compliance, and collagen deposition. To determine the involvement of DOCK2 in MesoMT, we treated primary human pleural mesothelial cells with the potent MesoMT inducer TGF-β. TGF-β significantly induced DOCK2 expression in a time-dependent manner, together with α-SMA, collagen 1, and fibronectin. Furthermore, DOCK2 knockdown significantly attenuated TGF-β-induced α-SMA, collagen 1, and fibronectin expression, suggesting the importance of DOCK2 in TGF-β-induced MesoMT. DOCK2 knockdown also inhibited TGF-β-induced Snail upregulation, which may account for its role in regulating MesoMT. Taken together, the current study provides evidence that DOCK2 contributes to the pathogenesis of pleural fibrosis by mediating MesoMT and deposition of neomatrix and may represent a novel target for its prevention or treatment.

Three newly established immortalized mesothelial cell lines exhibit morphological phenotypes corresponding to malignant mesothelioma epithelioid, intermediate, and sarcomatoid types, respectively

BackgroundMalignant mesothelioma (MM) is a very aggressive tumor that develops from mesothelial cells, mainly due to asbestos exposure. MM is categorized into three major histological subtypes: epithelioid, sarcomatoid, and biphasic, with the biphasic subtype containing both epithelioid and sarcomatoid components. Patients with sarcomatoid mesothelioma usually show a poorer prognosis than those with epithelioid mesothelioma, but it is not clear how these morphological phenotypes are determined or changed during the oncogenic transformation of mesothelial cells.MethodsWe introduced the E6 and E7 genes of human papillomavirus type 16 and human telomerase reverse transcriptase gene in human peritoneal mesothelial cells and established three morphologically different types of immortalized mesothelial cell lines.ResultsHOMC-B1 cells exhibited epithelioid morphology, HOMC-A4 cells were fibroblast-like, spindle-shaped, and HOMC-D4 cells had an intermediate morphology, indicating that these three cell lines closely mimicked the histological subtypes of MM. Gene expression profiling revealed increased expression of NOD-like receptor signaling-related genes in HOMC-A4 cells. Notably, the combination treatment of HOMC-D4 cells with TGF-β and IL-1β induced a morphological change from intermediate to sarcomatoid morphology.ConclusionsOur established cell lines are useful for elucidating the fundamental mechanisms of mesothelial cell transformation and mesothelial-to-mesenchymal transition.

Thalidomide Suppresses Angiogenesis Through the Signal Transducer and Activator of Transcription 3/SP4 Signaling Pathway in the Peritoneal Membrane.

Peritoneal angiogenesis is the key pathophysiological factor that limits peritoneal ultrafiltration during peritoneal dialysis (PD) in uremic patients. Thalidomide has been confirmed to inhibit angiogenesis by inhibiting the secretion of vascular endothelial growth factor (VEGF), but the exact mechanism by which thalidomide inhibits vascular proliferation during PD is still unclear. Here, the objective of the present study was to investigate whether the reduction in VEGF production by human peritoneal mesothelial cells (HPMCs) was controlled by thalidomide. Stimulation of HPMCs with IL-6 in combination with soluble IL-6 receptor (sIL-6R) promoted VEGF expression and secretion, but these effects were attenuated by thalidomide treatment through a transcriptional mechanism that involved signal transducer and activator of transcription 3 (STAT3) and SP4. Conditioned medium from HPMCs cultured with thalidomide inhibited angiogenic endothelial tube formation, which could be further blocked by silencing SP4 and promoted by overexpressing SP4. In vivo, induction of peritoneal angiogenesis in sham rats, sham+PD rats, 5/6 nephrectomy (5/6Nx) rats, 5/6Nx+PD rats, and 5/6Nx+PD rats intraperitoneally treated with thalidomide showed that thalidomide was involved in the control of several key endothelial–specific targets, including VEGFR2, VEGFR3, SP4, and STAT3 expression and new vessel formation, confirming the role of thalidomide and STAT3/SP4 signaling in these processes. Taken together, these findings identify a novel mechanism that links thalidomide, STAT3/SP4 signaling, and angiogenesis in the peritoneal membrane.

LncRNA GAS5 Competitively Combined With miR-21 Regulates PTEN and Influences EMT of Peritoneal Mesothelial Cells via Wnt/β-Catenin Signaling Pathway.

ObjectiveEpithelial-mesenchymal transition (EMT) is an important factor leading to peritoneal fibrosis (PF) in end-stage renal disease (ESRD) patients. The current research aimed to evaluate the effect of long non-coding RNA growth arrest-specific 5 (lncRNA GAS5) in human peritoneal mesothelial cells (HPMCs) EMT and explore the potential molecular mechanisms.Materials and MethodsHPMCs were cultured under control conditions or with high glucose (HG). The cells were then treated with lncRNA GAS5, lncRNA GAS5 siRNA, with or without miR-21 inhibitor and PTEN transfection. Expression of lncRNA GAS5, miR-21, α-SMA, Vimentin, E-cadherin, phosphatase and tensin homolog deleted on chromosome ten (PTEN), Wnt3a, and β-catenin were measured by real time PCR and Western blotting. Bioinformatics analyses were used to test the specific binding sites between the 3′ UTR of the PTEN gene, miR-21, and lncRNA GAS5. Rescue experiments were performed to confirm the lncRNA GAS5/miR-21/PTEN axis in HPMC EMT.ResultsWe found that HG-induced EMT decreased lncRNA GAS5 and that overexpression of lncRNA GAS5 can attenuate EMT in HPMCs. In addition, lncRNA GAS5 regulated HG-induced EMT through miR-21/PTEN. Cotransfection of miR-21 inhibitors remarkably increased PTEN expression and attenuated EMT in lncRNA GAS5 knockdown HPMCs. Moreover, rescue experiments showed that overexpression of PTEN attenuated the EMT effects of lncRNA GAS5 siRNA in HPMCs. We also confirmed that the Wnt/β-catenin pathway was stimulated in lncRNA GAS5/miR-21/PTEN-mediated EMT.ConclusionOur research showed that lncRNA GAS5 competitively combined with miR-21 to regulate PTEN expression and influence EMT of HPMCs via the Wnt/β-catenin signaling pathway. This study provides novel evidence that lncRNA GAS5 may be a potential therapeutic target for HPMC EMT.

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

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

Blockade of Autophagy Prevents the Development and Progression of Peritoneal Fibrosis.

Peritoneal fibrosis (PF) is a major cause of ultrafiltration failure in long-term peritoneal dialysis (PD) patients. Nevertheless, limited measures have been shown to be effective for the prevention and treatment of PF. Some views reveal that activation of autophagy ameliorates PF but others demonstrate that autophagy promotes PF. It is obvious that the role of autophagy in PF is controversial and further studies are needed. Here, we investigated the role of autophagy in rat models of PF and damaged cultured human peritoneal mesothelial cells (HPMCs). Autophagy was highly activated in fibrotic peritoneum from two PF rat models induced by 4.25% peritoneal dialysate fluid (PDF) and 0.1% chlorhexidine gluconate (CG). Blockade of autophagy with 3-MA effectively prevented PF in both models and reversed epithelial to mesenchymal transition (EMT) by down-regulating TGF-β/Smad3 signaling pathway and downstream nuclear transcription factors Slug and Snail. Treatment with 3-MA also inhibited activation of EGFR/ERK1/2 signaling pathway during PF. Moreover, 3-MA prominently decreased STAT3/NF-κB-mediated inflammatory response and macrophage infiltration, and prevented peritoneal angiogenesis through downregulation of β-catenin signal. In addition, TGF-β1 stimulation up-regulated autophagic activity as evidenced by the increased autophagosome in vitro. Exposure of HPMCs to TGF-β1 resulted in the induction of EMT and activation of TGF-β/Smad3, EGFR/ERK1/2 signaling pathways. Treatment with 3-MA blocked all these responses. In addition, delayed administration of 3-MA was effective in reducing EMT induced by TGF-β1. Taken together, our study indicated that autophagy might promote PF and 3-MA had anti-fibrosis effect in vivo and in vitro. These results suggest that autophagy could be a potential target on PF therapy for clinical patients with long-term PD.

The prognosis and risk factors of baseline high peritoneal transporters on patients with peritoneal dialysis.

The relationship between baseline high peritoneal solute transport rate (PSTR) and the prognosis of peritoneal dialysis (PD) patients remains unclear. The present study combined clinical data and basic experiments to investigate the impact of baseline PSTR and the underlying molecular mechanisms. A total of 204 incident CAPD patients from four PD centres in Shanghai between 1 January 2014 and 30 September 2020 were grouped based on a peritoneal equilibration test after the first month of dialysis. Analysed with multivariate Cox and logistic regression models, baseline high PSTR was a significant risk factor for technique failure (AHR 5.70; 95% CI 1.581 to 20.548 p = 0.008). Baseline hyperuricemia was an independent predictor of mortality (AHR 1.006 95%CI 1.003 to 1.008, p < 0.001) and baseline high PSTR (AOR 1.007; 95%CI 1.003 to 1.012; p = 0.020). Since uric acid was closely related to high PSTR and adverse prognosis, the in vitro experiments were performed to explore the underlying mechanisms of which uric acid affected peritoneum. We found hyperuricemia induced epithelial‐to‐mesenchymal transition (EMT) of cultured human peritoneal mesothelial cells by activating TGF‐β1/Smad3 signalling pathway and nuclear transcription factors. Conclusively, high baseline PSTR induced by hyperuricaemia through EMT was an important reason of poor outcomes in CAPD patients.