WO2004065602A1 - Method of identifying the epithelial-mesenchymal transition of mesothelial cells (emtmc) and identifying emtmc-modulating compounds, pharmaceutical compositions and use thereof in the diagnosis and treatment of diseases associated with emtmc - Google Patents

Abstract

The invention relates to a method of identifying the epithelial-mesenchymal transition of mesothelial cells (EMTMC) and identifying EMTMC-modulating compounds, pharmaceutical compositions and use thereof in the diagnosis and treatment of diseases associated with EMTMC. The inventive method is based on the fact that mesothelial cells play an active role in the structural and functional alteration of the peritoneum during peritoneal dialysis which leads to the epithelial-mesenchymal transition which in turn causes ultrafiltration failure. The aforementioned findings demonstrate a series of targets and markers which are intended for the development of novel peritoneal dialysis solutions, for the monitoring of patients undergoing said treatment and for the treatment of diseases associated with functional alterations to the mesothelium.

Description

 PROCEDURE FOR IDENTIFYING THE EPITHELIUM TRANSITION

MESENQUIMAL OF MESOTELIAL CELLS (TEMCM) E

IDENTIFY TEMCM MODULATING COMPOUNDS,

PHARMACEUTICAL COMPOSITIONS AND ITS USE IN THE DIAGNOSIS AND TREATMENT OF DISEASES THAT COURSE WITH TEMCM.

D E S C R I P C I ÓN

OBJECT OF THE INVENTION

This specification refers to a request for an Invention Patent, corresponding to. a procedure to identify the epithelial-mesenchymal transition of mesothelial cells (TEMCM) and identify modulatory compounds of TMCM, pharmaceutical compositions and their use in the diagnosis and treatment of diseases that occur with TEMCM, whose purpose is to be configured as a procedure for determine the different gene expression of biological markers associated with the epithelial-mesenchymal transition that occurs in the mesothelial cells of vertebrate animals, preferably human, affected by diseases or pathological processes, hereinafter ^• method of the present invention.

Another additional object of the present invention is a device that allows the tuning of the gene expression of biological markers associated with the epithelio-mesenchymal transition, and consequently of the process of the present invention.

A further object of the present invention, It is the use of the procedure and the device mentioned above in procedures to predict, diagnose and assess the evolution of diseases or pathological processes that occur with epithelial-mesenchymal transition of mesothelial cells.

Likewise, a further object of the present invention is a method of identifying a potential compound modulating the epithelial-mesenchymal transition of mesothelial cells.

A further object of the present invention consists of a pharmaceutical composition comprising at least one modulator compound of the epithelial-mesenchymal transition of mesothelial cells for the treatment of diseases or pathological processes that occur with the epithelial-mesenchymal transition of mesothelial cells .

The use of a modulating compound of the present invention in the preparation of said pharmaceutical composition constitutes a further object of this invention.

Finally, this invention presents as an additional object thereof the use of said pharmaceutical composition in the preparation of a medicament for the treatment or prevention of diseases or pathological processes that will occur with an epithelial-mesenchymal transition of mesothelial cells and that belong, between others, to the following group: ultrafiltration failure in patients undergoing peritoneal dialysis, flanges and post-surgical adhesions, Meigs syndrome, mesotheliomas, pericarditis, ascites, pleurisy, adhesive pachyipleuritis, encapsulating peritonitis, idiopathic peritoneal fibrosis and radiation fibrosis.

FIELD OF THE INVENTION

This invention has its field of application within the pharmacological sector for its application in the health sector.

BACKGROUND OF THE INVENTION

Ambulatory peritoneal dialysis (CAPD) is an alternative to hemodialysis for the treatment of end-stage renal patients (Krediet, 1999). The peritoneal membrane is covered by a monolayer of mesothelial cells, which have certain epithelial cell characteristics and act as a regulated permeability bar and secrete various substances involved in the regulation of peritoneal permeability and local immune defense.

(Brulez and Verbrugh, ^' 1995; Krediet, 1999). Unfortunately, repeated exposure to acid, hyperosmotic and hyperglycemic dialysis solutions causes chronic low-grade inflammation and damage to the peritoneum, which progressively denies mesothelial cells and degenerates into tissue fibrosis (Krediet, 1999). These structural changes are the major cause of ultrafiltration failure, which affects 20% of patients in CAPD (Chaimovitz, 1994). This functional disorder can also be accelerated by severe or recurrent episodes of peritonitis or hemoperitoneum (Chaimovitz, 1994; Selgas et al., 1994).

The pathophysiology of peritoneal failure during CAPD is not understood in detail. Mesenchymal cells immersed in the fibrotic stroma have been classically considered responsible for the development of fibrosis (Dobbie, 1992). However, the possible direct role of the mesothelial cell in this process had never been examined in detail. In this context, mesothelial cells in culture have the ability to change their morphology and produce components of the extracellular matrix in response to different stimuli (Connell and Rheinwald, 1983; Fang et al., 2000; .. Faull et al., 2001; Leavesley et al., 1999; Medcalf et al., 2001; Rampino et al., 2001; Yang et al., 1999). In addition, in vitro mesothelial cell treatment with media with high glucose concentration or proinflammatory cytokines induces TGF-β D expression (Ha et al., 2001) and decreases cadherin-E expression (Ito et al., 2000). The relevance of the TGF-β profibrotic growth factor (Massagué, 1998) in the ultrafiltration failure produced by CAPD has recently been evidenced in an in vivo rat model, in which the TGF-β gene was transduced to the peritoneum and caused a decrease in peritoneal function (Margetts et al., 2001).

In this protocol we demonstrate in vivo and ex vivo that mesothelial cells undergo an epithelial-mesenchymal transition, also called transdifferentiation, when they undergo peritoneal dialysis. Transdifferentiation is a complex and generally reversible process that begins with a break in intercellular junctions and a loss of Apico-basal polarity typical of epithelial cells, which are transformed into fibroblast cells with migratory, invasive and fibrogenic characteristics (Hay, 1995). Although transdifferentiation can be induced in culture in most epithelial cells with a wide range of stimuli, in vivo this process occurs only during embryonic development and in some pathological conditions such as wound repair or oral progression (Birchmeier et al., 1996; Hay, 1995). The cadherin-E intercellular adhesion molecule plays a central role in controlling the epithelial-mesenchymal transition since the loss of its expression or function correlates with the ability of epithelial cells to adopt a typically mesenchymal invasive and migratory morphology (Perl et al., 1998; Takeichi, 1995). Snail transcription factor is a potent repressor of cadherin-E transcription and inducer of transdifferentiation (Batlle et al., 2000; Cano et al., 2000; Carver et al., 2001). Therefore, phenotypic changes of mesothelial cells during CAPD may be directly related to the failure of the peritoneum barrier function, suggesting a fundamental role of these cells in the development of ultrafiltration failure in patients on peritoneal dialysis.

DESCRIPTION OF THE INVENTION

Peritoneal dialysis is becoming an increasingly common alternative to hemodialysis. However, during the dialysis procedure the mesothelial cells are subjected to high osmotic pressure and bioincompatible substances. Previous studies conducted using techniques Standard immunohistological peritoneum of patients in CAPD, show a total loss of mesothelial cell monolayer and tissue fibrosis, which may be responsible for the failure of the functionality of the peritoneal membrane (Krediet, 1999). Our data show that mesothelial cells undergo a transition from an epithelial to mesenchyne phenotype to during peritoneal dialysis with induction of Snail expression and a dramatic loss of expression of cadherin-E and cytokeratins. Mesothelial cells also acquire a ^• migratory phenotype with increased expression of α2 integrin. In vitro analysis suggests that the repair of wounds and pro-inflammatory and pro-inflammatory cytokines would be initiating factors of mesothelial transdifferentiation. In addition, these findings suggest a direct and active role of mesothelial cells in tissue fibrosis and failure in ultrafiltration, by local generation of new fibroblast cells that cause peritoneal fibrosis.

Previous studies have characterized epithelioid cells derived from effluent as indistinguishable from those obtained from omentum samples (Leavesley et al., 1999). However, already in the early stages of CAPD, mesothelial cells show a significant loss of their cubic morphology, observed both in vivo and ex vivo, accompanied by the induction of Snail that suppresses the expression of cadherin-E, even when the cells still have an epithelioid appearance. If peritoneal dialysis continues, chronic exposure to mechanical denudation and profibrotic factors such as TGF-β and inflammatory cytokines can induce the complete transition of these cells, which can be responsible for tissue fibrosis and subsequent failure in ultrafiltration. In this sense, patients with recurrent episodes of peritonitis, in which they express high levels of TGF-βD (Laí et al., 200Q) suffer from ultrafiltration failure more prematurely (Selgas et al., 1994).

The fact that mesothelial cells undergo an epithelial-mesenchymal transition during CAPD changes our perspective of the pathophysiology of ultrafiltration failure. Our data reveals a series of new markers such as Snail, E-cadherin or α2 integrin, which appear already altered in the early stages of the transdifferentiation process. In addition, the ICAM-1 adhesion molecule appears as a potential marker to discriminate mesothelial cells from fibroblasts.

In summary, the present invention is based on the fact that the inventors, when analyzing the differential expression of proteins in samples obtained from patients undergoing CAPD, observed that a series of proteins have an altered expression representative of the process of epithelio-mesenchymal transition of cells mesothelial (TEMCM), which makes them molecular markers of this transition, with diagnostic and predictive value of the failure or not of ultrafiltration that patients may suffer; and that they are involved in the etiopathogenesis of said alteration which simultaneously makes them possible therapeutic targets of diseases and pathological processes that occur with said epithelial-mesenchymal transition of mesothelial cells, and allows the development of new diagnostic procedures and treatment thereof.

Thus, an object of the present invention is a method for determining the different gene expression of biological markers associated with the epithelial-mesenchymal transition that occurs in the mesothelial cells of vertebrate animals, preferably human, affected by diseases or pathological processes, hereinafter procedure of the present invention, and characterized in that it is constituted by the following steps:

a) Extraction of the biological material from the aforementioned mesothelial cells, in the form of mRNA or protein,

b) Determination of the genomic or proteic fingerprint associated with said epithelial-mesenchymal transition,

c) Comparison with the genomic or proteomic fingerprint associated with a normal or physiological mesothelial state, and

d) The identification of said epithelial-mesenchymal transition if said fingerprint has a pattern of gene expression associated with said transition.

As used herein the term "different gene expression" refers to quantitative differences in gene expression levels and whether or not such biomarkers. The term "associated biological markers to said epithelial-mesenchymal transition "as used in the present invention refers to both the genes, in their mRNA form or their corresponding cDNA transcribed by RT-PCR, and to the proteins encoded by said genes associated with said transition and which are defined in Table I

Table I.- Genomic or proteomic fingerprint associated with said epithelial-mesenchymal transition

In the case of the ICAM-1 marker this represents a differential marker between mesothelial cells in any of its stages of transdifferentiation and fibroblasts.

As defined in the present invention the term "genomic or proteomic fingerprint associated with said epithelial-mesenchymal transition" indicates the pattern of gene expression defined by the set of gene expression alterations, increase or decrease, of the biological markers associated with this epithelial-mesenchymal transition defined in the present invention (Table I): increase in integrin α2, Snail, and decrease in integrin α3, cadherin E, cytokeratins, tetraspanin CD9 and tetraspanin CD151, preferably by the increase in Snail and decrease in cadherin E, or more preferably by the increase in Snáil. In addition, the ICAM-1 marker will always serve as a differential marker of the esothelial origin of the sample.

The term "diseases or pathological processes" as used in the present invention refers to diseases or pathological processes that will occur with an epithelio-mesenchymal transition of mesothelial cells and that belong, among others, to the following group: ultrafiltration failure in patients undergoing peritoneal dialysis, flanges and post-surgical adhesions, Meigs syndrome, mesotheliomas, pericarditis, ascites, pleurisy, adhesive pachyipleuritis, encapsulating peritonitis, idiopathic peritoneal fibrosis and radiation fibrosis. Additionally, the term "pathological process" as used in the present invention further refers to the epithelio-mesenchymal transition process that can take place in mesothelial cells in contact with potential compounds or solutions used in peritonal dialysis.

A further object of the present invention is a device, kit, a solution or chemical or biological composition that allows the development of the gene expression of the biological markers associated with the epithelial-mesenchymal transition and as a consequence of the procedure. of the present invention, preferably a biochip or microarray, either of genomic material, mRNA or cDNA, or of proteins. The technique of biochips or microarrays has become an important Biotechnology industry tool in the field of gene expression determination, in the diagnosis and prognosis of diseases, acogenomic far and infectious agents detection among others (Microarrays and DNA biochips. Technology Watch Report. GENOMA SPAIN / CIBT- FGUAM, 2002.www.gen-es.org; Haab et al. Protein microarray for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biology Vol. 2 No. 2; Below, Larissa et al. Immunophenotyping of leukemias using cluster of differentiation antibody microarray Cancer Research 61 June 1, 2001; Fung et al. Protein biochips for differential profiling. Analytical. Biotechnology 2001, 12: 65-69). On the other hand, a particular object of the present invention is a DNA biochip-type device that includes in the same nucleotide sequences that allow the determination of the gene expression of the mRNAs or cDNAs corresponding to the genes associated with the epithelial transition. Mesenchymal described in Table I. Additionally, a particular object of the present invention is a type device, biochip or microarray of antibody-based capture proteins (also called detection microarrays, which allow the detection of target proteins and their quantification) including specific antibodies, for example and among others those used in the present invention: the LIA1 / 1 anti-CD151, VJ1 / 20 anti-CD9, VJ1 / 18 anti-integrin α3, TEA1 / 41 and anti-integrin α2 antibody which allow the determination of the levels of expression of the proteins encoded by the genes of Table I. The microarrays of proteins based on prot Aeins, initially described in the early 1980s, have been the subject of attention in recent years. multiple groups and are being developed for different applications from the developments previously made for DNA biochips (Huang, RP Detection of multiple proteins in an ^' antibody-based protein microarray system. Journal of Immunological Methods 225 (2001): 1-13). It should be noted that within the scope of the present invention all those antibody-based protein biochips are included as capture agents, among others, which comprise monoclonal (hybridoma) or polyclonal antibodies, recombinant antibody fragments, combibodies, Fab and scFv fragments of antibodies, engineered scaffolds (domains of ligand binding proteins) and protein aptamers; -, and those based on non-protein ligands as capture agents, among others, nucleic acid aptamers or small molecules obtained by chemical synthesis, and which are described in the state of the art (Brent et al. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2, Nature 380 (1996): 548-550; Hesselberth J et al. In vitro selection of nucleic acids for diagnostic applications. Rev Mol Biotechnol 74 (2000): 15-25; http: // www. functionalgenomics.org.uk/sections/resources / protein arrays.htm; Kodadek T. Protein microarrays: prospects and problems. Che istry and Biology 8/2

(2001): 105-115). Likewise, all those compositions or kits for the realization of any other molecular biology technique that allow the determination of the gene expression of the biological markers associated with the epithelial-mesenchymal transition of mesothelial cells, among others, form part of the present invention. Northern blot and quantitative RT-PCR for the determination of genomic material and western blot for the determination of proteins. A further object of the present invention is the use of the method and the devices, compositions or kits mentioned above in procedures to predict, diagnose and assess the evolution of diseases or pathological processes that occur with epithelial-mesenchymal transition of mesothelial cells. A particular object of the present invention is the use of the procedure and the devices, compositions or kits mentioned above to predict, diagnose and assess the evolution of diseases or pathological processes in which the pathological process is the epithelial-mesenchymal transition process that It can take place in mesothelial cells in contact with potential compounds or solutions used in peritonal dialysis, and which allows the identification and selection of new compounds and solutions for peritoneal dialysis based on their lower induction capacity in mesothelial cells of the epithelial transition -mesenchymal, that is, greater biocompatibility (see example 7 of the present invention).

In addition, another additional object of the present invention is a method of identifying a potential compound modulating the epithelial-mesenchymal transition of mesothelial cells constituted by the following steps:

a) adding said potential modulating compound to a biological environment in which the epithelial-mesenchymal transition of mesothelial cells can be assessed,

b) determination of the evolution or not of said epithelial-mesenchymal transition, and

c) identification of a modulating compound when it is capable of modifying said epithelial-mesenchymal transition, either by inhibiting

(antagonist) or inducing (agonist) the expression or cellular signaling of the biological markers associated with this epithelial-mesenchymal transition defined in the present invention (Table I).

Related to the previous section, a particular object of the present invention is a method of identifying a potential compound modulating the epithelial-mesenchymal transition where the biological environment a) is constituted, inter alia, by Met5A mesothelial cells (see example 7 of the present invention) and / or where the determination of the evolution or not of said epithelial-mesenchymal transition b) is performed, among other possibilities, by determining the expression of the biological markers associated with this epithelial-mesenchymal transition defined in the present invention (Table I): increase in integrin α2, Snail, and decrease in integrin α3, cadherin E, cytokeratins, tetraspanin CD9 and tetraspanin CD151, preferably by the increase in Snail and decrease in cadherin E, or more preferably by the increase in Snail; or by studying the changes in the morphology of mesothelial cells by immunohistochemistry. In addition, the ICAM-1 marker will always serve as a differential marker of the mesothelial origin of the sample.

A further object of the present invention It consists of a pharmaceutical composition comprising, at least, a compound that modulates the epithelial-mesenchymal transition of mesothelial cells for the treatment of diseases or pathological processes that occur with the epithelial-mesenchymal transition of mesothelial cells. A particular object of the present invention is constituted by a pharmaceutical composition in which the modulating compound consists of an inhibitor of the expression or activity of the Snail repressor transcription factor.

The pharmaceutical composition provided by this invention comprises a therapeutically effective amount of at least one modulating compound of the present invention, together with at least one pharmaceutically acceptable excipient. Said pharmaceutical composition is useful for administration and / or application in the body of a mammal, preferably the human being.

The use of a modulating compound of the present invention in the preparation of said pharmaceutical composition constitutes a further object of this invention.

The modulating compounds of the present invention can be administered for the treatment of diseases or pathological processes by any means that allows contact between said compound and the site of action thereof in the body of a mammal.

The amount of therapeutically effective modulating compound of the present invention to be administered as well as its dosage to treat a pathological condition with said modulating compounds and / or Pharmaceutical compositions of the invention will depend on numerous factors, including age, patient condition, disease severity, route and frequency of administration, the modulating compound to be used, etc.

Pharmaceutical compositions containing the modulating compounds provided by this invention may be presented in any form of administration deemed appropriate, for example, orally, parenterally, rectally or topically, for which the pharmaceutically acceptable excipients necessary for the formulation will be included. of the desired administration form. A review of the different pharmaceutical forms of drug administration and of the excipients necessary to obtain them can be found, for example, in the "Galenica Pharmacy Treaty", C Faulí í Trillo, 1993, Luzán 5, SA Ediciones, Madrid.

On the other hand, this invention presents as an additional object thereof the use of said pharmaceutical composition in the preparation of a medicament for the treatment or prevention of diseases or pathological processes that will occur with an epithelial-mesenchymal transition of mesothelial cells and belonging , among others, to the following group: failure of ultrafiltration in patients undergoing peritoneal dialysis, flanges and post adhesions. Surgical, Meigs syndrome, mesotheliomas, pericarditis, ascites, pleurisy, adhesive pachyipleuritis, encapsulating peritonitis, idiopathic peritoneal fibrosis and radiation fibrosis. DESCRIPTION OF THE DRAWINGS

Figure 1: Mesothelial cells undergo morphological changes during the course of peritoneal dialysis. to. Photomicrographs of confluent onocaps of the different cell preparations used in the course of this study. Below are the flow cytometry histograms of the different cell types stained with antibodies against pancytokeratin or ICAM-1. The dotted line shows the negative control. b. The morphological changes of mesothelial cultures are directly related to the time the patient has been subjected to CAPÍ). The graph shows the variation in the prevalence of epithelioid cultures against non-epithelioids versus time in CAPD (n = 29 0-6 months; n = 15 β-12 months; n = 15> 12 months). The average in CAPD patients presenting cultures with epithelioid appearance was 7 + 1.15 SE months, compared to 13 + 2 SE months of non-epithelioid cultures (P = 0.011 in a Student test). In addition, a linear trend study gave a value of χ ² = 6,193 with p = 0.012 to the association of morphology with time, c. Photomicrographs of mesothelial cells derived from the effluent of the same patient during the course of a hemoperitoneum (Cl) episode or two months after the remission of the pathological process (C2) __.

Figure 2: Mesothelial cells undergo an epithelial-mesenchymal transition in vivo during peritoneal dialysis. to. Total cell lysates of the different mesothelial cell preparations were revealed by western-blot successively with antibodies against cadherin-E, cytokeratin or vimentin The chemiluminescence signal was quantified and normalized with respect to tubulin expression. The graphs represent the mean value ± 1 SE of four different experiments for each condition. b. Projections of series of confocal images of confluent monolayers of mesothelial cells derived from omento or fibroblast effluent stained with antibodies against cadherin-E, pancytoratin or vimentin or with phalloidin. The preparations show the phenotypic changes characteristic of an epithelial-mesenchymal transition. c. Vertical optical sections of confluent monolayers of omentum-derived mesothelial cells or effluent cells,. with epithelioid or fibroblastic f dye stained with the monoclonal antibody VJ1 / 20 anti-CD9, which reveals the three-dimensional structure of the cells.

Figure 3: Mechanical damage and treatment with TGF-β and IL-1 induce transdifferentiation of mesothelial cells in vitro. to. Confluent monolayers of omentum mesothelial cells were mechanically injured and their migration was continued for 2-3 d. Representative photomicrographs of the video sequences of two independent experiments are shown. Mesothelial cells acquire a fibroblastic appearance at the edge of the wound and adjacent areas (arrows). This transition is only local so that cells distanced from the edge of the wound maintain their epithelial morphology (panel C). Transdifferentiation is reversed once the wound has been repaired (panel F). b. Photomicrographs of untreated omentum derived mesothelial cells or treated with 0.5 ng / ml TGF-βl in combination or not with 2ng / ml IL-lβD for 48 hc Cell lysates Totals of the different mesothelial preparations treated or not for 48 h with 0.5 ng / ml TGF-β1 and 2 ng / ml IL-lβO were revealed by western blot with antibodies against cadherin-E, pancytokeratin, vimentin or tubulin. The chemiluminescence signal was quantified and normalized with respect to the values obtained in the omentum cells without treatment. A representative experiment of the three performed is shown.

Figure 4: Mesothelial transdifferentiation is associated with early expression of the Snail transcription factor. to. Mesothelial cells derived from omentum or effluent, from peritoneal dialysis (two samples for each type of preparation) were analyzed by RT-PCR to determine their expression of Snail and cadherin-E mRNA. b. Omento cells were stimulated with 0.5 ng / ml TGF-β1 and 2 ng / ml IL-lβ at different times and the expression of Snail and cadherin-E mRNA was analyzed by RT-PC. c. Omento confluent cell monolayers were subjected to mechanical damage and the expression of Snail mRNA and E-cadherin was analyzed at different times during the repair process.

Figure 5: Mesothelial transdifferentiation is associated with a greater expression of α2 integrin and a decrease in the expression of tetraspanins which induces a migratory phenotype, a. Total cell lysates of the different preparations of mesothelial cells were subjected to western blotting with antibodies against the α2 and α3 integrins or against tubulin. The chemiluminescence signal was quantified, normalized with respect to the values obtained with anti-tubulin and related to Omento cell expression levels. The graph represents the mean + 1 SE of four different samples for each condition. b. Histograms of flow cytology of the different preparations of stained mesothelial cells for β1, α2 and α3 integrins or for CD9 and CD151 tetraspanins. c. Total cell lysates of the different preparations of mesothelial cells treated or not for 48 hours with 0.5 ng / ml TGF-β1 and 2ng / ml IL-lβ DD were revealed by western-blot with antibodies against the integrins α2 or α3 integrin or against tubulin . The chemiluminescence signal was quantified and normalized with respect to tubulin expression in a representative experiment of four performed. d. Chemotaxis experiments at 10 ng / ml EGF and 10 Dg / ml haptotaxis of Collagen-1 or Laminin-5 performed with mesothelial cells treated or not for 48 hours with 0.5 ng / ml TGF-D1 and 2ng / ml IL-IDDy that migrated for 16 h at 37 ° C. The tests were performed in duplicate and are represented as the mean + 1S.D. of a representative experiment. Statistical analysis by the Student test gave the following P values: EGF Omento vs EGF T + IP = 0.3; Col vs Col T + IP = 0.006; LN5 vs LN5 T + IP = 0.0022; Epithelioid EGF vs EGF T + IP = 0.5; Col vs Col T + IP = 0.0Q27; LN-5 vs LN-5 T + IP = 0.036; Transitional EGF vs EGF T + IP = 0.19; Col vs. Col T + IP = 0.7, LN-5 vs LN5 T + IP = 0.00085; Fibroblast EGF vs EGF T + IP = 0.01; Col vs Col T + IP = 0.64; LN5 vs LN5 T + IP = 0.002; Omento EGF vs Epithelioid EGF P = 0.5; Omento EGF vs Transitional EGF P = 0.001; Omento EGF vs Fibroblastic EGFP = 0.00002.

I

Figure 6: Evidence of the epithelial-mesenchymal transition of mesothelial cells in peritoneal tissue of patients in CAPD. Analysis immunohistochemistry of peritoneal tissue samples stained with anti-cytokeratin (ac panels, 150x) or with ICAM-1 (panel d, 180x), and developed respectively with peroxidase or Fast-red. Panel a represents a control peritoneum of a patient undergoing unrelated abdominal surgery (of 8 examined). In panel b, an early patient (6 months) of peritoneal dialysis who already shows a loss of polarity in the mesothelial cell monolayer (representative of 9 samples corresponding to patients undergoing CAPD for a period of 0 to 9 months). Panel c (8 months) and the insert (34 months) represent the final stages in the process of transdifferentiation in patients undergoing peritoneal dialysis for long periods of time (representative of 8 samples obtained from dialysis patients between 8 and 77 months) In these samples, fibroblast mesothelial cells invading the fibrotic tissue are observed. These cells show staining for ICAM-1 (panel d, which corresponds to the same biopsy of panel c).

Figure 7: Biocompatibility tests: analysis of Snail expression in the Met5A mesothelial cell line exposed to different dialysis solutions. Monolayers of Met5A mesothelial cells were grown in the presence of different peritoneal dialysis solutions containing different buffers and different amounts of glucose. After 24 h the RNA was extracted and the expression of β-Actin and Snail was analyzed by quantitative RT-PCR. The results are expressed as relative units ± SE that result from dividing the Snail values by those of β-Actin, taking the baseline value as 1. PREFERRED EMBODIMENT OF THE INVENTION

Example 1: Mesothelial cells undergo morphological changes during peritoneal dialysis. ICAM-1 as a mesothelial marker.

Mesothelial cell cultures obtained from CAPD effluents show clearly distinguishable morphologies, from epithelioids, similar to mesothelial cells obtained from omentum, fibroblast and even mixed populations (Fig. La). The prevalence of non-epithelioid cells seems to be related either to the time that the patient has been subjected to CAPD (Fig. 1b), or to the processes of hemoperitoneum or peritonitis. Fibroblastic cells appeared sporadically in samples that presented hemorrhage or infiltrating lymphoid cells in the effluent, and a reversion to an epithelioid or transitional phenotype was observed (in 8 cases of 8) in cultures analyzed from the same patient once it had been resolved. the pathological process (Fig. lc).

To determine the nature of effluent derived cells, the expression of cytokeratins, a typically epithelial marker, and of

ICAM-1, which is expressed constitutively in cells

'. esoteliales (Suassúna et al., 1994). High cytokeratin expression was observed both in omentum-derived cells and in effluent cells with epithelioid appearance (Fig. La). The effluent cells showed a progressive reduction in the expression of cytokeratins, although even in fibroblast samples a small population of positive cells was maintained. In mixed cultures there was a Bimodal expression, while keratin was totally absent in omento fibroblasts. However, all preparations obtained from effluent showed a high and homogeneous expression of ICAM-1 regardless of morphology, including mixed cultures. On the contrary, the expression of ICAM-1 was undetectable in fibroblasts obtained from both omentum and skin (Fig. La), which demonstrates that fibroblastic effluent cells have a mesothelial origin and are not a contamination with fibroblasts of The biological sample.

Example 2: Mesothelial cells undergo an epithelial-mesenchymal transition in vivo during CAPD.

Morphological changes and decreased keratin expression of effluent-derived mesothelial cells could be indicative of an epithelial-mesenchymal transition (Hay, 1995). We analyzed by western-blot the expression of cadherin-E and the intermediate filament proteins cytokeratins and vimentin as markers of transdifferentiation. Cadherin-E expression decreased dramatically from omentum cells to epithelioid and non-epithelioid effluent (Fig. 2a). The expression of cytokeratins followed the same pattern while the expression of vimentin increased (Fig. 2a).

Trials using confocal microscopy demonstrated the loss of intercellular E-cadherin as well as the reorganization of the actin cytoskeleton of the cortical band typical of epithelial cells to a pattern of fibroblastic tension fibers (Fig. 2b). Cytokeratin was replaced by vimentin, although some fibroblast cells were still cytokeratin positive. Preparations stained with anti-CD9

(Fig. 2c), which is expressed in both apical microvilli and intercellular contacts (Yáñez-Mó et al., 2001), showed a gradual loss in epithelial cubic morphology, which is already evident in epithelioid mesothelial cells that they were about half as tall as omento cells in vertical confocal sections. Fibroblast cells lose contact inhibition and often stack in several layers.

Example 3: The mechanical damage and the treatment with TGF-β and IL-1 induce the differentiation of mesothelial cells reproducing the transition observed in vivo.

After in vitro mechanical denudation of a confluent monolayer of omentum cells it was observed that the mechanical stimulus was sufficient to induce mesothelial cell migration, and that the migrating cells underwent transient transdifferentiation showing a mesenchymal morphology that reverted to one aspect. epithelial once the wound is closed (Fig. 3a). This effect was locally confined to the edge of the wound and adjacent areas, while cells that were distanced from the damaged area were not modified (Fig. 3a, panel C), reinforcing the idea that the mechanical stimulus was itself just enough to induce transdifferentiation.

To determine whether TGF-β and IL-lβ, two cytokines detected from CAPD effluents primarily during episodes of peritonitis (Lai et al., 2000), were able to reproduce the changes Phenotypic observed clinically in vitro, mesothelial cultures derived from omentum were treated with TGF-β alone or in combination with IL-lβ. An additive effect of both stimuli was observed morphologically (Fig. 3b). Cadherin-E expression disappeared almost completely (Fig. 3c), and its location in the intercellular junctions was virtually undetectable by immunofluorescence (not shown). Cytokeratin expression also decreased, and IL-lβ also showed an additive effect. On the contrary, these treatments induced an increase in the expression of vimentin.

Example 4: .. Snail transcription factor expression in mesothelial cells that undergo an epithelial-mesenchymal transition.

Recently, a transcription factor called Snail has been described, which acts as a potent repressor of the expression of cadherin-E and therefore as an inducer of the epithelial-mesenchymal transition (Batlle et al., 2000; Cano et al., 2000; Carver et al., 2001). To determine if Snail expression was associated with the phenotypic changes observed in cells of the peritoneal membrane in a CAPD patient, RT-PCR analyzes were performed to estimate the expression of said transcription factor, as well as of E-cadherin, in mesothelial cells of effluent and omentum (Fig. 4a). No signal of Snail mRNA was detected in omentum cells, while in effluent preparations a progressive increase in the expression of Snail mRNA was found throughout the transdifferentiation process. A dramatic decrease in mRNA of cadherin-E was already observable in effluent cells with epithelioid appearance, consistent with protein expression data (Fig. 2a).

Stimulation of esothelial cultures with TGF-β plus IL-lβ revealed a rapid and transient induction of Snail mRNA. Cadherin-E mRNA was already decreased in the first time tested and remained almost undetectable even when Snail's expression had already declined (Fig. 4b). Similarly, after mechanical denudation of the cells, a transient induction of Snail's mRNA was observed, probably corresponding to the cells that undergo the transition at the edge of the damaged area. Because the majority of the cells were not involved in the repair process, no decrease in the mRNA of cadherin-E was observed in the total population (Fig. 4c).

Example 5: The transdifferentiation of mesothelial cells is accompanied by an increase in the expression of integrin α2 and the acquisition of a migratory phenotype.

The failure of ultrafiltration in CAPD is accompanied by peritoneal fibrosis. Therefore, we analyze the characteristics with respect to the extracellular matrix receptors of mesothelial preparations. _. Already in epithelioid-like effluent cells, an increase in the expression of integrin α2 was observed (Fig. 5a and 5b). On the contrary, the integrin α3 increased its expression in epithelioid cells and decreased in the last stages of the epithelial-mesenchymal transition (transitional and fibrolástíca preparations). Similarly, the CD9 and CD151 tetraspanins, which are associated with integrins, saw their expression diminished as transdifferentiation progressed. Similarly, TGF-β plus IL-lβ induced the Increase in α2 integrity in all mesothelial preparations, while α3 integrin increased in omentum and decreased in cells already in transition (Fig. 5c). IL-1β potentiated the effects of TGF-β.

Tetraspanins are functionally associated with cell migration (Hemler, 1998). Changes in the repertoire of integrins and the transition from a keratin-based cytoskeleton to one formed by vimentin could also alter the migratory capacity of mesothelial cells. Thus, in chemotaxis and haptotaxis tests, we observe that the transdifferentiation process is accompanied by a greater migratory capacity of the mesothelial cells (Fig 5d). In addition, treatment with TGF-β plus IL-1 increased collagen haptotaxis, the main binding of α2βl integrin. Migration to laminin-5 followed changes in the expression of its receptor, integrin α3β1 (Fig. 5d).

Example 6: Evidence of the epithelial-mesenchymal transition of mesothelial cells in the peritoneal tissue of patients in CAPD.

Our data suggest that mesothelial cells undergo an epithelial-mesenchymal transition during the course of CAPD. To confirm this fact in vivo, immunohistochemical staining of peritoneal biopsies of patients in CAPD was performed, confirming the loss of epithelial morphology of the mesothelial monolayer in the early stages of CAPD.

(Fig. 6b) (n = 9, 0-9 months in CAPD). In 8 biopsies of patients who had been dialyzed for 8 to 77 months, the mesothelial monolayer had disappeared and the cells ICAM-1 mesothelial and cytokeratin positive appeared embedded in fibrotic tissue and elongated appearance (Fig. 6c and 6d), which corresponded to non-epithelial effluent cultures.

Example 7: Snail expression is a marker to test the biocompatibility of different peritoneal dialysis solutions.

Since Snail is a marker associated with the progressive loss of filtration capacity of mesothelial cells, it was also used as a marker to study the biocompatibility of different peritoneal dialysis solutions. The analysis of Snail mRNA expression showed how the Bic4.5 solution induced a very marked increase in Snail expression, while the Bal2.3 solution hardly produced alterations in its expression. The rest of the solutions presented different capacities of induction of Snail.

METHODS

Patients and cells. Human mesothelial cells derived from effluent (25,569 cells ± 2,971

SE per bag) were obtained by centrifugation of the dialysis solution of 54 clinically stable patients chosen at random who had performed overnight exchange with dialysis solutions with 2.27% glucose, 1.25 / 1.75 M calcium. After 10-15 days the preparations reached confluence and were amplified (1: 2) 2-3 times. The morphology of the cultures was compared in confluent cell monolayers and remained stable for 2-3 passes. 85% of the cultures were obtained before the first episode of patient peritonitis Of 116 analyzed effluent cultures 62 had an epithelioid morphology, 28 were transitional, 20 fibroblastic and 6 cultures with mixed populations. The omentum cells were obtained by digestion with 0.05% Trypsin / 0.02% EDTA of omentum samples from 30 individuals undergoing abdominal surgery but not undergoing CAPD. Omento fibroblasts were obtained from three different samples of omento tissue after removing the mesothelial cells by extensive digestion of the sample (three rounds of 20 minutes with trypsin). All cells were cultured with Earle's M199 medium with 20% FCS, 50 IU / ml penicillin, 50 μg / l streptomycin, 2% Biqgro-2 (Biological Industries, Israel). For the experiments the cells were seeded on 50 μg / ml Collagen I without Biogro. Met5A cells were obtained from the ATCC and grown according to their recommendations. TGF-β1 and interleukin-lβ (IL-ββ) were from R&D (Minneapolis, MN), and the doses used are in the range of those detected in dialysis solutions during peritonitis processes (Lai et al., 2000 ).

This study was approved by the Ethics Committee of the Princess University Hospital, and informed consent was obtained from all donors.

Antibodies Monoclonal antibodies LIA1 / 1 anti-CD151, VJ1 / 20 anti-CD9, VJ1 / 18 anti-integrin α3, TS2 / 16 anti-integrin βl and TEAl / 41 anti-integrin α2 have been previously described (Yáñez-Mó et al ., 1998). The onoclonal anti-ICAM-1 HU5 / 3 was donated by Dr. FW Luscinskas (Brigham and omen's Hospital, Harvard Medical School, Boston, MA). The Rabbit polyclonal versus integrins 2 and α3 were yielded by Dr. G. Tarone (Universitá di Torino, Italy). The anti-cadherin-E was from Calbioche (La Jolla, CA); anti-vimeritin, anti-α tubulin and anti-pancytokeratin from Sigma (St. Louis, MO); and anti-ICAM-1 of Santa Cruz (Santa Cruz, CA).

Flow cytometry, immunohistochemistry and confocal microscopy. Flow cytometry and immunofluorescence assays were described in (Yáñez-Mó et al., 1998). Immunohistochemistry studies were performed on peritoneal tissue samples from 17 patients in CAPD and 8 control patients embedded in paraffin and by the method of estraptavidin-biotin (DAKO LSAB-2-Kit; DAKO, Carpentry, CA). As chromogens dia ^' minobenzidine and fast red were used.

Western Blot Mesothelial cell monolayers were used in RIPA buffer and equivalent amounts of protein were resolved by SDS-PAGE and western-blot as in (Yáñez-Mó et al., 1998). The band's luminescence signal was acquired with a Fuji LAS-1000 CCD camera (Fuji Photo Film Co, LTD; Tokyo, Japan) and quantified with Image Gauge V3.46 software.

RT-PCR Mesothelial RNA was extracted with RNAwiz (Abion, Aus ^' tin, TX). To obtain RNA from cells treated with dialysis solutions, a monolayer of Met5A cells was grown in the presence of different peritoneal dialysis solutions in the presence of 2% Biogro and 20% FCS. After 24 h the RNA was extracted. The cDNA was obtained from 1 μg of total RNA using the Applied Biosystems Kit (Foster City, CA). Snail amplification was performed with 40 cycles (40 at 95 ° C, 30 s at 53 ° C and 1 at 72 ° C) using oligonucleotides 1 (5 '-CACATCCTTCTCACTGCCATG-3') (SEQ NOl) and 2 (5'-GCATCTAAACTCTAGTCTGC-3 ') (SEQ N02). For the Snail assisted reaction, 30 reaction cycles were performed as before and a 1:50 dilution of the reaction was amplified by another 20 cycles (40 s at 95 ° C, 30 s at 60 ° C and 1 m at 72 ° C) when oligonucleotides 1 and 3 (5'-CCTGAGTGGGGTGGGAGCTTCC-3 ') (SEQ N03) (Cano et al., 2000). Cadherin-E PCR was carried out in 32 cycles as described in (Cano et al., 2000). Quantitative Snail PCR was performed using oligonucleotides 1 and 2 in a Lightcycler thermocycler (Roche, Mannheim, Germany) with 45 cycles (O at 95 ° C, 10 s at 533 ° C and 20 s at 72 ° C), using a Fast-Start kit (Roche).

Migration trials Chemotaxis and haptotaxis tests were performed on polycarbonate inserts with a pore size of 5 p (Costar, Corning, NY) coated or not coated on their basal face with 10 μg / ml of Collagen-1 or Laminin-5 (Rousselle et al., 1995) as described in (Lara-Pezzi et al., 2001).

Delayed time video microscopy. The videomicroscopy analysis was performed on an inverted microscope equipped with a Sony camcorder

SSC-M350CE CCD coupled to a Sony SVT-5000P time delay video recorder. The omentum cells were subjected to mechanical damage with an adapted scraper about 1500 microns wide and were filmed for 2-3 days until the wound was repaired in an incubator that kept the sample in conditions of

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Claims

RE IVINDI CAC I ON EN 1.- Procedure to identify the epithelial-mesenchymal transition of mesothelial cells (TEMCM) and identify modulatory compounds of TMCM, pharmaceutical compositions and their use in the diagnosis and treatment of diseases that occur with TEMCM, characterized by being constituted from the following Steps: a) extraction of the biological material from the aforementioned mesothelial cells, in the form of mRNA or protein, b) determination of the genomic or proteomic fingerprint associated with said epithelial-mesenchymal transition, c) comparison with the genomic or proteomic fingerprint associated with a normal or physiological mesothelial state, and d) the identification of said epithelio-mesenchymal transition if said fingerprint exhibits a pattern of gene expression associated with said transition. 2. Procedure to identify the eplithelial-mesenchymal transition of mesothelial cells (TEMCM) and identify modulatory compounds of TMCM, pharmaceutical compositions and their use in the diagnosis and treatment of diseases that occur with TEMCM, according to claim 1 characterized in that the determination of the footprint genomic or proteomic fingerprint associated with said epithelial-mesenchymal transition (b)) refers to the set of gene expression alterations, increase or decrease, of the biological markers associated with this epithelial-mesenchymal transition belonging to the following group: increase in α2 integrin, Snail and decrease of integrin α3, cadherin E, cytokeratins, tetraspanin CD9 and tetraspanin CD151, or preferably increase of Snail and decrease of cadherin E, or more preferably by the increase of Snail, using the determination of ICAM-1 as a differential marker of origin Sample mesothelial. 3. Device, ¹ composition or kit that allows the tuning of the determination of the genomic or proteomic fingerprint associated with the epithelial-mesenchymal transition according to claims 1 and 2 (b)) belonging, among others, to the following group : biochip or microarray, either of genomic material, mRNA or cDNA, or of proteins. 4. Use of the procedures according to claims 1 and 2 and device, composition or kit according to claim 3 in the preparation of a procedure aimed at predicting, diagnosing and assessing the evolution of diseases or pathological processes that occur with epithelial transition. mesenchymal mesothelial cells. 5. Use according to claim 4 characterized in that the diseases or pathological processes that occur with epithelio-mesenchymal transition of mesothelial cells belong, among others, to the following group: failure of the ultrafiltration in patients undergoing peritoneal dialysis, flanges and post-surgical adhesions, Meigs syndrome, mesothelium, pericarditis, ascites, pleurisy, adhesive pachyipleuritis, encapsulating peritonitis, idiopathic peritoneal fibrosis and radiation fibrosis. 6. Use according to claim 4 characterized in that the pathological process consists in the process of epithelial-mesenchymal transition that can take place in mesothelial cells in contact with potential compounds or solutions used in peritonal dialysis. 7.- Procedure for the identification of a potential compound modulating the epithelial-mesenchymal transition of the isothelial cells characterized in that it is constituted by the following stages: a) adding said potential modulating compound to a biological environment in which the epithelial-mesenchymal transition of mesothelial cells can be assessed, b) determination of the evolution or not of said epithelial-mesenchymal transition, and c) identification of a modulating compound when it is capable of modifying said epithelial-mesenchymal transition, either by inhibiting (antagonist) or inducing (agonist) the expression or cellular signaling of the biological markers associated with this epithelial-mesenchymal transition defined in the present invention 8. Method according to claim 7 characterized in that the biological environment a) is constituted, among others, by Met5A mesothelial cells and because the determination of the evolution or not of said epithelial-mesenchymal transition b) is performed, among other possibilities, by the determination of the expression of the biological markers associated with this epithelial-mesenchymal transition defined in the present invention: increase in α2 integrin, Snail and decrease in α3 integrin, cadherin E, cytokeratins, CD9 tetraspanin and CD151 tetraspanin, preferably by e-1 increase of Snail and decrease of cadherin E, or more preferably by the increase of Snail, using the determination of ICAM-1 as a differential marker of the mesothelial origin of the sample; or by studying the changes in the morphology of mesothelial cells by immunohistochemistry. 9.- Pharmaceutical composition comprising, at least, a compound modulating the epithelial-mesenchymal transition of mesothelial cells for the treatment of diseases or pathological processes that occur with the epithelial-mesenchymal transition of mesothelial cells. 10. Pharmaceutical composition according to claim 9 characterized in that the modulating compound consists of an inhibitor of the expression or activity of the repressor transcription factor Snail 11.- Use of a modulator compound of the Epithelial-mesenchymal transition of mesothelial cells in the preparation of a pharmaceutical composition according to claims 9 and 10. 12. Use of a pharmaceutical composition according to claims 9 and 10 in the preparation of a medicament for the treatment or prevention of diseases or pathological processes that will occur with an epithelial-mesenchymal transition of mesothelial cells and that belong, among others, to next group: ultrafiltration failure in patients undergoing peritoneal dialysis, flanges and post adhesions. Surgical, Meigs syndrome, mesotheliomas, pericarditis, ascites, pleurisy, adhesive pachyipleuritis, encapsulating peritonitis, idiopathic peritoneal fibrosis and radiation fibrosis.