Epithelial-to-mesenchymal Transition Of Mesothelial Cells Research Articles

Hypoxia - as a Possible Regulator of the Activity of Epicardial Mesothelial Cells After Myocardial Infarction.

Aim To study the effect of hypoxia on the activity of epithelial-mesenchymal transition (EMT) in epicardial cells, which provides formation of a specialized microenvironment.Material and methods This study used a model of experimental myocardial infarction created by ligation of the anterior descendent coronary artery. The activity of epicardial cells after a hypoxic exposure was studied with the hypoxia marker, pimonidazole, bromodeoxyuridine, immunofluorescent staining of heart cryosections, and in vitro mesothelial cell culture.Results The undamaged heart maintained the quiescent condition of mesothelial cells and low levels of their proliferation, extracellular matrix protein production, and of the EMT activity. Acute ischemic injury induced moderate hypoxia in the epicardial/subepicardial region. This caused a global rearrangement of this region due to the initiation of EMT in cells, changes in the cell composition, and accumulation of extracellular matrix proteins. We found that the initiation of EMT in mesothelial cells may result in the formation of smooth muscle cell precursors, fibroblasts, and a population of Sca-1+ cardiac progenitor cells, which may both participate in construction of new blood vessels and serve as a mesenchymal link for the paracrine support of microenvironmental cells. In in vitro experiments, we showed that 72‑h hypoxia facilitated activation of EMT regulatory genes, induced dissembling of intercellular contacts, cell uncoupling, and increased cell plasticity.Conclusion The epicardium of an adult heart serves as a "reparative reserve" that can be reactivated by a hypoxic exposure. This creates a basis for an approach to influence the epicardium to modulate its activity for regulating reparative processes.

Paricalcitol attenuates TGF‐β1–induced phenotype transition of human peritoneal mesothelial cells (HPMCs)viamodulation of oxidative stress and NLRP3 inflammasome

Phenotype transition of mesothelial cells, such as epithelial-to-mesenchymal transition (EMT), is one of the early mechanisms of peritoneal fibrosis, which is mediated by oxidative stress and inflammation. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein oligomer that promotes the maturation of IL-1β and IL-18. Paricalcitol is reported to exert an anti-inflammatory effect; however, there are no studies as to whether paricalcitol modulates the activation of NLRP3 inflammasome. We investigated the role of NLRP3 inflammasome in peritoneal EMT with an exploration of the effect of paricalcitol on oxidative stress, NLRP3 inflammasome, and EMT of mesothelial cells. TGF-β1-induced EMT in human peritoneal mesothelial cells (HPMCs) was associated with an up-regulation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and procaspase-1, with an increased production of IL-1β and IL-18, which was ameliorated by small interfering (si)NLRP3, siASC, caspase inhibitors, or neutralizing antibodies for IL-1β and IL-18. TGF-β1 enhanced reactive oxygen species generation with an increase in NADPH oxidase (NOX) activity and mitochondrial NOX4 production. Paricalcitol alleviated TGF-β1-induced EMT and the NLRP3 inflammasome, which was associated with a down-regulation of NOX activity by interfering with p47phox and p22phox interaction and mitochondrial NOX4 production in HPMCs. Taken together, paricalcitol ameliorated EMT of HPMCs via modulating an NOX-dependent increase in the activity of NLRP3 inflammasome. Paricalcitol could be a novel approach to protect the peritoneum from the development of EMT and peritoneal fibrosis.-Ko, J., Kang, H.-J., Kim, D.-A., Ryu, E.-S., Yu, M., Lee, H., Lee, H. K., Ryu, H.-M., Park, S.-H., Kim, Y.-L., Kang, D.-H. Paricalcitol attenuates TGF-β1-induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome.

Olive leaf extract counteracts epithelial to mesenchymal transition process induced by peritoneal dialysis, through the inhibition of TGFβ1 signaling.

The mesothelial cells (MCs) play an important role in the morpho-functional alterations of the peritoneal membrane (PM) undergoing peritoneal dialysis (PD). MCs, through the epithelial-mesenchymal transition process (EMT), progressively acquire a myofibroblast-like phenotype, promoting peritoneal fibrosis (PF) and failure of peritoneal membrane function. Transforming growth factor β1 (TGFβ1), through canonical and non-canonical pathways, promotes the epithelial-mesenchymal transition (EMT) process leading to PF. To investigate the therapeutic potential of an olive leaf extract (OLE) on preserving peritoneal membrane function, we evaluated the effect of OLE on the TGFβ1-induced EMT in mesothelial cells, Met5A, and elucidated the underlying molecular mechanisms. As assessed by changes in the expression of epithelial, mesenchymal, and fibrotic cell markers (such as E-cadherin, N-cadherin, α-SMA, fibronectin, vimentin), levels of matrix metalloproteinases (MMP2 and MMP9), and cell migration, OLE inhibited the TGFβ1-induced EMT. Importantly, the beneficial effect of OLE was mediated by reduction of the TGFβ1-induced activation of Smad2/3 signaling and the mitigation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Smad/non-Smad signaling pathways, activated by TGFβ1, both reduce expression of epithelial marker E-cadherin which has a crucial role in EMT initiation. Interestingly, we observed that in presence of OLE activity of the E-cadherin, promoter was increased and concomitantly OLE reduced the nuclear content of its co-repressor SNAIL. Our results suggest the potential therapeutic of OLE to counteract fibrotic process in peritoneal dialysis patients.

Hydrogen sulfide inhibits epithelial-mesenchymal transition in peritoneal mesothelial cells

Peritoneal fibrosis (PS) determines the long-term outcome of peritoneal dialysis (PD). We previous confirmed that hydrogen sulfide (H2S) inhibited PS, but its cellular mechanism was not fully elucidated. Epithelial-mesenchymal transition (EMT) of mesothelial cells (MCs) is an important cellular event of PS, we therefore investigated whether EMT can be affected by H2S in MCs. Rats were treated with 4.25% -glucose PD fluids plus lipopolysaccharide for 28 days to produce PS, and NaHS (56 μg/kg.d) was given simultaneously. NaHS (56 μg/kg.d) reduced the deposition of collagen in the submesothelial zone compared with the PS group. In primarily cultured rat MCs, 4.25% -glucose PD fluid induced EMT in MCs featured as loss of ZO-1 and Cytokeratin, and increase of α-SMA, plasminogen activator inhibitor 1, fibronectin and TGF-β1 proteins. PD fluid also increased IL-6 and monocyte chemotactic protein-1 mRNA expressions as well as the phosphorylation of Smad2/3 and Smad3. NaHS (50–300 μmol/L) reversed the above alterations with the optimal dose at 100 μmol/L. Thus, exogenous H2S improves PS by inhibiting EMT in MCs. The anti-EMT effect of H2S is associated with the inhibition of inflammation and TGF-β1-Smad signal pathway.

RhoA/Rho-kinase triggers epithelial-mesenchymal transition in mesothelial cells and contributes to the pathogenesis of dialysis-related peritoneal fibrosis

Peritoneal fibrosis (PF) with associated peritoneal dysfunction is almost invariably observed in long-term peritoneal dialysis (PD) patients. Advanced glycation end products (AGEs) are pro-oxidant compounds produced in excess during the metabolism of glucose and are present in high levels in standard PD solutions. The GTPase RhoA has been implicated in PF, but its specific role remains poorly understood. Here, we studied the effects of RhoA/Rho-kinase signaling in AGEs-induced epithelial-mesenchymal transition (EMT) in human peritoneal mesothelial cells (HPMCs), and evaluated morphological and molecular changes in a rat model of PD-related PF. Activation of RhoA/Rho-kinase and activating protein-1 (AP-1) was assessed in HPMCs using pull-down and electrophoretic mobility shift assays, respectively, while expression of transforming growth factor-β, fibronectin, α-smooth muscle actin, vimentin, N-cadherin, and E-cadherin expression was assessed using immunohistochemistry and western blot. AGEs exposure activated Rho/Rho-kinase in HPMCs and upregulated EMT-related genes via AP-1. These changes were prevented by the Rho-kinase inhibitors fasudil and Y-27632, and by the AP-1 inhibitor curcumin. Importantly, fasudil normalized histopathological and molecular alterations and preserved peritoneal function in rats. These data support the therapeutic potential of Rho-kinase inhibitors in PD-related PF.

HIF-1α mediates Hypoxia-induced epithelial–mesenchymal transition in peritoneal mesothelial cells

The epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells plays a pivotal role in the development of peritoneal fibrosis. The pathological effects of hypoxia on mesothelial cell EMT have not been fully elucidated. In this study, we, therefore, investigated the effects of hypoxia on EMT in mesothelial cells. Human mesothelial (MeT-5A) cells and primary-cultured rat mesothelial cells were cultured under hypoxic conditions (1% O2) for up to 72 h. Changes in cell type were then determined by investigating changes in morphology and in expression of epithelial (E-cadherin and occludin) and mesenchymal (fibronectin-1, vimentin and α-smooth muscle actin) cell markers. In some cases, MeT-5A cells were cultured under hypoxic conditions with a HIF-1α inhibitor and then assessed for changes in morphology and for altered expression of signaling molecules, such as HIF-1α, Snail-1, vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2). Levels of HIF-1α, Snail-1, VEGF, and MMP-2 in Met-5A cells were increased by hypoxia. Levels of epithelial cell markers were decreased and those of mesenchymal cell markers were increased. Cell morphology also changed from a cobblestone-like monolayer to spindle-shaped fibroblast-like cells in response to hypoxia. Inhibition of HIF-1α signaling by a HIF-1α inhibitor abrogated these changes. The cell marker and morphological changes induced by hypoxia were also observed in primary-cultured rat mesothelial cells. We can conclude that hypoxia induces EMT in mesothelial cells by activating HIF-1α. This finding indicates that hypoxia has pivotal roles in the development of peritoneal fibrosis in peritoneal dialysis patients.

Role of transforming growth factor-β1 in epithelial-mesenchymal transition of mesothelial cells and its effect on peritoneal metastasis of gastric cancer

To elucidate the role of transforming growth factor-beta1(TGF-β1) in epithelial-mesenchymal transition of mesothelial cells and peritoneal metastasis of gastric cancer. HMrSV5 cells, a human peritoneal mesothelial cell line, were incubated with TGF-β1, and their morphological changes were observed by phase contrast microscopy. Expressions of α-smooth muscle actin (α-SMA), vimentin, cytokeratin, E-cadherin, phosphorylated-Smad2 and Smad2 were examined by Western blotting. After fibroblastic-like mesothelial cells were co-incubate with HSC-39 cells(gastric cancer cell line), the adhesion and invasion potential of HSC-39 were evaluated by adhesion and invasion assay in vitro. Few mesothelial cells converted to spindle fibroblast-like morphology for 24 h, and remarkable phenotypic changes were observed at 72 h of TGF-β1 activation. TGF-β1 could induce α-SMA and vimentin expression, and down-regulate cytokeratin and E-cadherin expression in mesothelial cells (P<0.05). TGF-β1 induced phosphorylation of Smad2 within 15 min of stimulation, reached a maximum at 30 min after treatment and remained high level during the experiment without affecting total Smad2 expression(P>0.05). The percentage of HSC-39 gastric cancer cells adhered were significantly increased as compared to the control. When the mesothelial cells were treated by TGF-β1 for 72 h, the increased adhesion percentage was(146±17)%(P<0.05). After fibroblastic-like mesothelial cells co-incubated with HSC-39 cells for 48 h, more cancer cells [(61.1±11.4) cells/view field] invaded the coated membrane as compared to the control group [(31.9±8.1) cells/view field] (P<0.05). TGF-β1 can induce the transition of mesothelial cells into myofibroblasts and Smad2 signal pathway may play a role in this transition, which is associated with increased adhesion and invasiveness of gastric cancer cells, and provides favorable environment for the dissemination of gastric cancer.

Influence of Bicarbonate/Low-GDP Peritoneal Dialysis Fluid (Bicavera) on in Vitro and Ex Vivo Epithelial-to-Mesenchymal Transition of Mesothelial Cells

Peritoneal membrane damage induced by peritoneal dialysis (PD) is largely associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs), which is believed to be a result mainly of the glucose degradation products (GDPs) present in PD solutions. This study investigated the impact of bicarbonate-buffered, low-GDP PD solution (BicaVera: Fresenius Medical Care, Bad Homburg, Germany) on EMT of MCs in vitro and ex vivo. Omentum-derived MCs were incubated with lactate-buffered standard PD fluid or BicaVera fluid diluted 1:1 with culture medium. Ex vivo studies: From 31 patients randomly distributed to either standard or BicaVera solution and followed for 24 months, effluents were collected every 6 months for determination of EMT markers in effluent MCs. Culturing of MCs with standard fluid in vitro resulted in morphology change to a non-epithelioid shape, with downregulation of E-cadherin (indicative of EMT) and strong induction of vascular endothelial growth factor (VEGF) expression. By contrast, in vitro exposure of MCs to bicarbonate/low-GDP solution had less impact on both EMT parameters. Ex vivo studies partially confirmed the foregoing results. The BicaVera group, with a higher prevalence of the non-epithelioid MC phenotype at baseline (for unknown reasons), showed a clear and significant trend to gain and maintain an epithelioid phenotype at medium- and longer-term and to show fewer fibrogenic characteristics. By contrast, the standard solution group demonstrated a progressive and significantly higher presence of the non-epithelioid phenotype. Compared with effluent MCs having an epithelioid phenotype, MCs with non-epithelioid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin and VEGF. In comparing the BicaVera and standard solution groups, MCs from the standard solution group showed significantly higher secretion of interleukin 8 and lower secretion of collagen I, but no differences in the levels of other EMT-associated molecules, including fibronectin, VEGF, E-cadherin, and transforming growth factor β1. Peritonitis incidence was similar in both groups. Functionally, the use of BicaVera fluid was associated with higher transport of small molecules and lower ultrafiltration capacity. Effluent MCs grown ex vivo from patients treated with bicarbonate/low-GDP BicaVera fluid showed a trend to acquire an epithelial phenotype, with lower production of proinflammatory cytokines and chemokines (such as interleukin 8) than was seen with MCs from patients treated with a lactate-buffered standard PD solution.

Fluid Flow Stress Affects Peritoneal Cell Kinetics: Possible Pathogenesis of Peritoneal Fibrosis

Peritoneal fibrosis is an essential precursor condition to the development of encapsulating peritoneal sclerosis (EPS). This serious complication leads to a high mortality rate in peritoneal dialysis (PD) patients. Although several factors, including highly concentrated glucose in the dialysis solution, are believed to be potent agents for peritoneal fibrosis, the underlying mechanism remains unclear. During PD, the dialysis solution continuously generates fluid flow stress to the peritoneum under peristalsis and body motion. Fluid flow stress has been implicated as playing a critical role in the physiologic responses of many cell types. We therefore hypothesized that fluid flow stress may be involved in the pathogenesis of peritoneal fibrosis leading to EPS. To generate fluid flow stress, culture containers were placed on a rotatory shaker in a thermostatic chamber. In this system, the shaker rotated at a speed of 25 rpm with a radius of 1.5 cm. Mesothelial cells were cultured in low-glucose (1000 mg/L) or high-glucose (4500 mg/L) complete medium with and without flow stress. Fluid flow stress promoted hyperplasia and epithelial-mesenchymal transition (EMT) of mesothelial cells independent of glucose concentration. Fluid flow stress inhibited expression of ERK (extracellular signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) in mesothelial cells. Administration of ERK and p38 MAPK inhibitors replicated the stress-induced morphology of mesothelial cells. The present data indicate that fluid flow stress promotes hyperplasia and EMT of mesothelial cells via the MAPK axis, suggesting that fluid flow stress may be involved in the pathogenesis of peritoneal fibrosis.

Human peritoneal mesothelial cell transformation into myofibroblasts in response to TGF-ß1 in vitro

Peritoneal dissemination is one of the leading causes of death in gastric cancer patients. The interaction between carcinoma cells and the peritoneal lining may play a key role in tumor peritoneal dissemination. Human peritoneal mesothelial cells are a monolayer of squamous epithelial cells covering the peritoneal cavity and forming serosal membranes. The precise role of mesothelial cells in the peritoneal dissemination of gastric cancer remains to be identified. Expression of TGF-ß1, a cytokine known for its capacity to induce proliferative and transformative changes in cells, has been correlated with peritoneal metastasis and TNM stages of gastric cancer. High levels of TGF-ß1 in the subperitoneal milieu may play a key role in the transition of normal mesothelial cells to myofibroblasts. Here, we demonstrate that mesothelial cells activated by TGF-ß1 undergo epithelial-mesenchymal transition (EMT) and that the transition of mesothelial cells to myofibroblasts is dependent on Smad2 signaling. EMT of mesothelial cells was marked by up-regulation of α-smooth muscle actin and vimentin expression. Cytokeratin and E-cadherin expression decreased over time in transformed mesothelial cells. Knockdown of Smad2 gene by siRNA silencing significantly suppressed the transition of mesothelial cells to myofibroblasts. We conclude that when exposed to TGF-ß1 mesothelial cells undergo EMT which involves Smad2 signaling. Furthermore, mesothelial cells may be the possible source of myofibroblasts in peritoneal fibrosis and provide a favorable environment for the dissemination of gastric cancer.

Low-GDP peritoneal dialysis fluid ('balance') has less impact in vitro and ex vivo on epithelial-to-mesenchymal transition (EMT) of mesothelial cells than a standard fluid

Peritoneal membrane deterioration during peritoneal dialysis (PD) is associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MC), which is believed to be mainly due to glucose degradation products (GDPs) present in PD solutions. Here we investigate the impact of GDPs in PD solutions on the EMT of MC in vitro and ex vivo. For in vitro studies, omentum-derived MC were incubated with standard PD fluid or low-GDP solution diluted 1:1 with culture medium. For ex vivo studies, 33 patients, who were distributed at random to either the 'standard' or the 'low GDP' groups, were followed over 24 months. Effluents were collected every 6 months to determine EMT markers in effluent MC. Exposure of MC to standard fluid in vitro resulted in morphological change into a non-epitheloid shape, down-regulation of E-cadherin, indicative of EMT, and in a strong induction of vascular endothelial growth factor (VEGF) expression. In contrast, in vitro exposure of MC to low-GDP solution did not lead to these phenotype changes. This could be confirmed ex vivo, as the prevalence of non-epitheloid phenotype of MC in the standard group was significantly higher with increasing PD duration and MC isolated from this group showed significantly higher levels of EMT-associated molecules including fibronectin, collagen I, VEGF, IL-8 and TGF-β levels when compared with the low-GDP group. Over time, the expression of E-cadherin also decreased in the standard but increased in the low-GDP group. In addition, the levels of EMT-associated molecules (fibronectin, VEGF and IL-8) increased in the standard but decreased in the low-GDP group. A similar trend was also observed for collagen I and for TGF-β (for the first year), but did not reach global statistical significance. Accordingly, effluent MC with non-epitheloid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin, collagen I, VEGF and IL 8 when compared with MC with epitheloid phenotype. The incidence of peritonitis did not significantly influence these results. Drop-out due to technique failure was less in the 'balance' group. The functional, renal and peritoneal evaluation of patients being treated with either standard or 'balance' fluid did not show any significant difference over time. MC from PD effluent of patients treated with a PD fluid containing low GDP levels show fewer signs of EMT and the respective molecules than MC from patients treated with standard fluid, indicating a better preservation of the peritoneal membrane structure and a favourable outcome in patients using low-GDP fluid. It also confirms the hypothesis that the protection of EMT by GDP-reduced fluids is also present in vivo.

BMP-7 blocks mesenchymal conversion of mesothelial cells and prevents peritoneal damage induced by dialysis fluid exposure

During peritoneal dialysis (PD), mesothelial cells (MC) undergo an epithelial-to-mesenchymal transition (EMT), and this process is associated with peritoneal membrane (PM) damage. Bone morphogenic protein-7 (BMP-7) antagonizes transforming growth factor (TGF)-beta1, modulates EMT and protects against fibrosis. Herein, we analysed the modulating role of BMP-7 on EMT of MC in vitro and its protective effects in a rat PD model. Epitheliod or non-epitheliod MC were analysed for the expression of BMP-7, TGF-beta1, activated Smads, epithelial cadherin (E-cadherin), collagen I, alpha smooth muscle cell actin (alpha-SMA) and vascular endothelial growth factor (VEGF) using standard procedures. Rats were daily instilled with PD fluid with or without BMP-7 during 5 weeks. Histological analyses were carried out in parietal peritoneum. Fibrosis was quantified with van Gieson or Masson's trichrome staining. Vasculature, activated macrophages and invading MC were quantified by immunofluorescence analysis. Quantification of infiltrating leukocytes and MC density in liver imprints was performed by May-Grünwald-Giemsa staining. Hyaluronic acid levels were determined by ELISA. MC constitutively expressed BMP-7, and its expression was downregulated during EMT. Treatment with recombinant BMP-7 resulted in blockade of TGF-beta1-induced EMT of MC. We provide evidence of a Smad-dependent mechanism for the blockade of EMT. Exposure of rat peritoneum to PD fluid resulted in inflammatory and regenerative responses, invasion of the compact zone by MC, fibrosis and angiogenesis. Administration of BMP-7 decreased the number of invading MC and reduced fibrosis and angiogenesis. In contrast, BMP-7 had no effect on inflammatory and regenerative responses, suggesting that these are EMT-independent, and probably upstream, processes. Data point to a balance between BMP-7 and TGF-beta1 in the control of EMT and indicate that blockade of EMT may be a therapeutic approach to ameliorate peritoneal membrane damage during PD.

Cyclooxygenase-2 Mediates Dialysate-Induced Alterations of the Peritoneal Membrane

During peritoneal dialysis (PD), exposure of the peritoneal membrane to nonphysiologic solutions causes inflammation, ultimately leading to altered structure and function. Myofibroblasts, one of the cell types that contribute to dysfunction of the peritoneal membrane, can originate from mesothelial cells (MCs) by epithelial-to-mesenchymal transition (EMT), a process that has been associated with an increased rate of peritoneal transport. Because cyclooxygenase-2 (COX-2) is induced by inflammation, we studied the role of COX-2 in the deterioration of the peritoneal membrane. We observed that nonepithelioid MCs found in peritoneal effluent expressed higher levels of COX-2 than epithelioid MCs. The mass transfer coefficient for creatinine correlated with MC phenotype and with COX-2 levels. Although COX-2 was upregulated during EMT of MCs in vitro, COX-2 inhibition did not prevent EMT. In a mouse model of PD, however, COX-2 inhibition with Celecoxib resulted in reduced fibrosis and in partial recovery of ultrafiltration, outcomes that were associated with a reduction of inflammatory cells. Furthermore, PD fluid with a low content of glucose degradation products did not induce EMT or COX-2; the peritoneal membranes of mice treated with this fluid showed less worsening than mice exposed to standard fluid. In conclusion, upregulation of COX-2 during EMT may mediate peritoneal inflammation, suggesting COX-2 inhibition as a potential strategy to ameliorate peritoneal deterioration in PD patients.