Manuel López-Cabrera

Epithelial to Mesenchymal Transition and Peritoneal Membrane Failure in Peritoneal Dialysis Patients: Pathologic Significance and Potential Therapeutic Interventions

Peritoneal dialysis (PD) is a form of renal replacement and is based on the use of the peritoneum as a semipermeable membrane across which ultrafiltration and diffusion take place. Nevertheless, continuous exposure to bioincompatible PD solutions and episodes of peritonitis or hemoperitoneum cause acute and chronic inflammation and injury to the peritoneal membrane, which progressively undergoes fibrosis and angiogenesis and, ultimately, ultrafiltration failure. The pathophysiologic mechanisms that are involved in peritoneal functional impairment have remained elusive. Resident fibroblasts and infiltrating inflammatory cells have been considered the main entities that are responsible for structural and functional alterations of the peritoneum. Recent findings, however, demonstrated that new fibroblastic cells may arise from local conversion of mesothelial cells (MC) by epithelial-to-mesenchymal transition (EMT) during the inflammatory and repair responses that are induced by PD and pointed to MC as protagonists of peritoneal membrane deterioration. Submesothelial myofibroblasts, which participate in inflammatory responses, extracellular matrix accumulation, and angiogenesis, can originate from activated resident fibroblasts and from MC through EMT. This heterogeneous origin of myofibroblasts reveals new pathogenic mechanisms and offers novel therapeutic possibilities. This article provides a comprehensive review of recent advances on understanding the mechanisms that are implicated in peritoneal structural alterations, which have allowed the identification of the EMT of MC as a potential therapeutic target of membrane failure.

The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression

Hepatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell-dependent (NF-AT-dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.

Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation.

Increased nitric oxide (NO) production may contribute to the pathological changes featuring in some inflammatory diseases, but the role of NO in chronic viral hepatitis is still unknown. We compared the inducible NO synthase (NOS2) expression in the liver of patients with chronic viral hepatitis with that of both nonviral liver disease and histologically normal liver. NOS2 expression was assessed by immunohistochemical and in situ hybridization studies of liver biopsy sections. An intense hepatocellular NOS2 reactivity was detected in chronic viral hepatitis, whereas it was weakly or not observed in nonviral liver disease or normal liver, respectively. In addition, we determined whether the hepatitis B virus (HBV) might regulate the synthesis of this enzyme. NOS2 mRNA and protein levels as well as enzyme activity were assessed in cytokine-stimulated HBV-transfected and untransfected hepatoma cells. Transfection with either HBV genome or HBV X gene resulted in induction of NOS2 mRNA expression, and the maximal induction of this transcript and NO production was observed in cytokine-stimulated HBV-transfected cells. These results indicate that hepatotropic viral infections are able to upregulate the NOS2 gene expression in human hepatocytes, suggesting that NO may mediate important pathogenic events in the course of chronic viral hepatitis.

The Tight Junction-Associated Protein Occludin Is Required for a Postbinding Step in Hepatitis C Virus Entry and Infection

ABSTRACT The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.

Hepatitis C virus envelope components alter localization of hepatocyte tight junction–associated proteins and promote occludin retention in the endoplasmic reticulum

Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ‐associated proteins occludin, claudin‐1, and Zonula Occludens protein‐1 (ZO‐1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ‐associated function, measured as FITC‐dextran paracellular permeability, of genomic replicon‐containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon‐α (IFN‐α) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ‐associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ‐associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co‐localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co‐immunoprecipitation and pull‐down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot‐like accumulation of occludin in infected Huh7 cells. Conclusion: We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ‐associated proteins, which may provide new insights for HCV‐related pathogenesis. (HEPATOLOGY 2008.)

Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis

Peritoneal fibrosis is one of the most common morphological changes observed in continuous ambulatory peritoneal dialysis (CAPD) patients. Both resident fibroblasts and new fibroblast-like cells derived from the mesothelium by epithelial-to-mesenchymal transition are the main cells involved fibrogenesis. In order to establish markers of peritoneal impairment and pathogenic clues to explain the fibrogenic process, we conducted an immunohistochemical study focused on peritoneal fibroblasts. Parietal peritoneal biopsies were collected from four patient groups: normal controls (n=15), non-CAPD uremic patients (n=17), uremic patients on CAPD (n=27) and non-renal patients with inguinal hernia (n=12). To study myofibroblastic conversion of mesothelial cells, α-smooth muscle actin (SMA), desmin, cytokeratins and E-cadherin were analyzed. The expression of CD34 by fibroblasts was also analyzed. Fibroblasts from controls and non-CAPD uremic patients showed expression of CD34, but no myofibroblastic or mesothelial markers. The opposite pattern was present during CAPD-related fibrosis. Expression of cytokeratins and E-cadherin by fibroblast-like cells and α-SMA by mesothelial and stromal cells supports that mesothelial-to-myofibroblast transition occurs during CAPD. Loss of CD34 expression correlated with the degree of peritoneal fibrosis. The immunophenotype of fibroblasts varies during the progression of fibrosis. Myofibroblasts seem to derive from both activation of resident fibroblasts and local conversion of mesothelial cells.

Transcriptional regulation of the gene encoding the human C-type lectin leukocyte receptor AIM/CD69 and functional characterization of its tumor necrosis factor-alpha-responsive elements.

The human activation antigen CD69 is a member of the C-type animal lectin superfamily that functions as a signal-transmitting receptor. Although the expression of CD69 can be induced in vitro on cells of most hematopoietic lineages with a wide variety of stimuli, in vivo it is mainly expressed by T-lymphocytes located in the inflammatory infiltrates of several human diseases. To elucidate the mechanisms that regulate the constitutive and inducible expression of CD69 by leukocytes, we isolated the promoter region of the CD69 gene and carried out its functional characterization. Sequence analysis of the 5′-flanking region of the CD69 gene revealed the presence of a potential TATA element 30 base pairs upstream of the major transcription initiation site and several putative binding sequences for inducible transcription factors (NF-κB, Egr-1, AP-1), which might mediate the inducible expression of this gene. Transient expression of CD69 promoter-based reporter gene constructs in K562 cells indicated that the proximal promoter region spanning positions −78 to +16 contained the cis-acting sequences necessary for basal and phorbol 12-myristate 13-acetate-inducible transcription of the CD69 gene. Removal of the upstream sequences located between positions −78 and −38 resulted in decreased promoter strength and abolished the response to phorbol 12-myristate 13-acetate. We also found that tumor necrosis factor-α (TNF-α) is capable of inducing the surface expression of the CD69 molecule as well as the promoter activity of fusion plasmids that contain 5′-flanking sequences of the CD69 gene, suggesting that this cytokine may regulate in vivo the expression of CD69. In addition, cotransfection experiments demonstrated that the CD69 gene promoter can be activated by the NF-κB/Rel family members c-Rel and RelA. The deletion of the sequence spanning positions −255 to −170 abolished both the response to TNF-α and the transactivation by NF-κB. These results indicate that the NF-κB-binding site located at position −223 is necessary for the TNF-α-induced expression of the CD69 gene. Mobility shift assays showed that the two NF-κB motifs located in the proximal promoter region (positions −223 and −160) bind various NF-κB-related complexes, including the heterodimers p50/RelA and p50/c-Rel and homodimers of p50 (KBF-1) and RelA. Our findings help to explain the regulated synthesis of CD69 in vivo and suggest that TNF-α has a key role in the expression of this molecule at sites of chronic inflammation.

Blocking TGF-β1 Protects the Peritoneal Membrane from Dialysate-Induced Damage

During peritoneal dialysis (PD), mesothelial cells undergo mesothelial-to-mesenchymal transition (MMT), a process associated with peritoneal-membrane dysfunction. Because TGF-β1 can induce MMT, we evaluated the efficacy of TGF-β1-blocking peptides in modulating MMT and ameliorating peritoneal damage in a mouse model of PD. Exposure of the peritoneum to PD fluid induced fibrosis, angiogenesis, functional impairment, and the accumulation of fibroblasts. In addition to expressing fibroblast-specific protein-1 (FSP-1), some fibroblasts co-expressed cytokeratin, indicating their mesothelial origin. These intermediate-phenotype (Cyto(+)/FSP-1(+)) fibroblasts had features of myofibroblasts with fibrogenic capacity. PD fluid treatment triggered the appearance of CD31(+)/FSP-1(+) and CD45(+)/FSP-1(+) cells, suggesting that fibroblasts also originate from endothelial cells and from cells recruited from bone marrow. Administration of blocking peptides significantly ameliorated fibrosis and angiogenesis, improved peritoneal function, and reduced the number of FSP-1(+) cells, especially in the Cyto(+)/FSP-1(+) subpopulation. Conversely, overexpression of TGF-β1 in the peritoneum by adenovirus-mediated gene transfer led to a marked accumulation of fibroblasts, most of which derived from the mesothelium. Taken together, these results demonstrate that TGF-β1 drives the peritoneal deterioration induced by dialysis fluid and highlights a role of TGF-β1-mediated MMT in the pathophysiology of peritoneal-membrane dysfunction.

Epithelial-to-mesenchymal transition of mesothelial cells is an early event during peritoneal dialysis and is associated with high peritoneal transport.

Ultrafiltration (UF) failure is a consequence of long-term peritoneal dialysis (PD). Fibrosis, angiogenesis, and vasculopathy are causes of this functional disorder after 3-8 years on PD. Epithelial-to-mesenchymal transition (EMT) of mesothelial cell (MC) is a key process leading to peritoneal fibrosis with functional deterioration. Our purpose was to study the peritoneal anatomical changes during the first months on PD, and to correlate them with peritoneal functional parameters. We studied 35 stable PD patients for up to 2 years on PD, with a mean age of 45.3+/-14.5 years. Seventy-four percent of patients presented loss of the mesothelial layer, 46% fibrosis (>150 microm) and 17% in situ evidence of EMT (submesothelial cytokeratin staining), which increased over time. All patients with EMT showed myofibroblasts, while only 36% of patients without EMT had myofibroblasts. The number of peritoneal vessels did not vary when we compared different times on PD. Vasculopathy was present in 17% of the samples. Functional studies were used to define the peritoneal transport status. Patients in the highest quartile of mass transfer area coefficient of creatinine (Cr-MTAC) (>11.8 ml min(-1)) showed significantly higher EMT prevalence (P=0.016) but similar number of peritoneal vessels. In the multivariate analysis, the highest quartile of Cr-MTAC remained as an independent factor predicting the presence of EMT (odds ratio 12.4; confidence interval: 1.6-92; P=0.013) after adjusting for fibrosis (P=0.018). We concluded that, during the first 2 PD years, EMT of MCs is a frequent morphological change in the peritoneal membrane. High solute transport status is associated with its presence but not with increased number of peritoneal vessels.

The hepatitis B virus X protein activates nuclear factor of activated T cells (NF-AT) by a cyclosporin A-sensitive pathway.

The X gene product of the human hepatitis B virus (HBx) is a transcriptional activator of various viral and cellular genes. We recently have determined that the production of tumor necrosis factor‐α (TNF‐α) by HBV‐infected hepatocytes is transcriptionally up‐regulated by HBx, involving nuclear factor of activated T cells (NF‐AT)‐dependent activation of the TNF‐α gene promoter. Here we show that HBx activates NF‐AT by a cyclosporin A‐sensitive mechanism involving dephosphorylation and nuclear translocation of the transcription factor. Luciferase gene expression assays demonstrated that HBx transactivates transcription through NF‐AT‐binding sites and activates a Gal4–NF‐AT chimeric protein. DNA–protein interaction assays revealed that HBx induces the formation of NF‐AT‐containing DNA‐binding complexes. Immunofluorescence analysis demonstrated that HBx induces the nuclear translocation of NF‐AT, which can be blocked by the immunosuppressive drug cyclosporin A. Furthermore, immunoblot analysis showed that the HBx‐induced activation and translocation of NF‐AT are associated with its dephosphorylation. Thus, HBx may play a relevant role in the intrahepatic inflammatory processes by inducing locally the expression of cytokines that are regulated by NF‐AT.