WO2004065602A1 - Procede permettant d'identifier la transition epithelio-mesenchymateuse de cellules mesotheliales (temcm) et d'identifier des composes modulateurs de la tmcm, compositions pharmaceutiques et leur utilisation dans le diagnostic et le traitement de maladies liees a la temcm - Google Patents

Procede permettant d'identifier la transition epithelio-mesenchymateuse de cellules mesotheliales (temcm) et d'identifier des composes modulateurs de la tmcm, compositions pharmaceutiques et leur utilisation dans le diagnostic et le traitement de maladies liees a la temcm Download PDF

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WO2004065602A1
WO2004065602A1 PCT/ES2004/000017 ES2004000017W WO2004065602A1 WO 2004065602 A1 WO2004065602 A1 WO 2004065602A1 ES 2004000017 W ES2004000017 W ES 2004000017W WO 2004065602 A1 WO2004065602 A1 WO 2004065602A1
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epithelial
mesenchymal transition
mesothelial cells
cells
mesothelial
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WO2004065602B1 (fr
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Maria YÁNEZ MO
Enrique Lara Pezzi
Rafael SELGAS GUTIÉRREZ
Vicente ÁLVAREZ CHIVA
Francisco SÁNCHEZ-MADRID
Manuel LÓPEZ CABRERA
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Universidad Autónoma de Madrid
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5026—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell morphology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • 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.
  • TEMCM epithelial-mesenchymal transition of mesothelial cells
  • TMCM mesothelial cells
  • 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.
  • 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 .
  • 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.
  • This invention has its field of application within the pharmacological sector for its application in the health sector.
  • Ambulatory peritoneal dialysis 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.
  • 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).
  • TGF- ⁇ D expression Ha et al., 2001
  • cadherin-E expression Ito et al., 2000
  • TGF- ⁇ 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- ⁇ gene was transduced to the peritoneum and caused a decrease in peritoneal function (Margetts et al., 2001).
  • 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).
  • 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.
  • Peritoneal dialysis is becoming an increasingly common alternative to hemodialysis.
  • 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 ⁇ 2 integrin.
  • In vitro analysis suggests that the repair of wounds and pro-inflammatory and pro-inflammatory cytokines would be initiating factors of mesothelial transdifferentiation.
  • 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.
  • 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 ⁇ 2 integrin, which appear already altered in the early stages of the transdifferentiation process.
  • the ICAM-1 adhesion molecule appears as a potential marker to discriminate mesothelial cells from fibroblasts.
  • 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.
  • TEMCM epithelio-mesenchymal transition of cells mesothelial
  • 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:
  • 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” 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
  • ICAM-1 marker this represents a differential marker between mesothelial cells in any of its stages of transdifferentiation and fibroblasts.
  • 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 ⁇ 2, Snail, and decrease in integrin ⁇ 3, 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.
  • the ICAM-1 marker will always serve as a differential marker of the esothelial origin of the sample.
  • diseases or pathological processes 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.
  • 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.
  • 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.
  • 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 ⁇ 3, TEA1 / 41 and anti-integrin ⁇ 2 antibody which allow the determination of the levels of expression of the proteins encoded by the genes of Table I.
  • 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).
  • 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 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 ⁇ 2, Snail, and decrease in integrin ⁇ 3, 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.
  • the I increase in integrin
  • 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.
  • 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.
  • 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 Faul ⁇ ⁇ Trillo, 1993, Luzán 5, SA Ediations, Madrid.
  • 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
  • FIG. 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.
  • FIG. 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.
  • FIG. 3 Mechanical damage and treatment with TGF- ⁇ 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.
  • FIG. 4 Mesothelial transdifferentiation is associated with early expression of the Snail transcription factor.
  • 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.
  • Omento cells were stimulated with 0.5 ng / ml TGF- ⁇ 1 and 2 ng / ml IL-l ⁇ at different times and the expression of Snail and cadherin-E mRNA was analyzed by RT-PC.
  • 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.
  • FIG. 5 Mesothelial transdifferentiation is associated with a greater expression of ⁇ 2 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 ⁇ 2 and ⁇ 3 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.
  • 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.
  • 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).
  • FIG. 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 ⁇ -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 ⁇ -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.
  • cytokeratins a typically epithelial marker
  • ICAM-1 which is expressed constitutively in cells
  • Example 2 Mesothelial cells undergo an epithelial-mesenchymal transition in vivo during CAPD.
  • Fig. 2c which is expressed in both apical microvilli and intercellular contacts (Yá ⁇ ez-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- ⁇ and IL-1 induce the differentiation of mesothelial cells reproducing the transition observed in vivo.
  • Example 4 Snail transcription factor expression in mesothelial cells that undergo an epithelial-mesenchymal transition.
  • Snail 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).
  • 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).
  • Example 5 The transdifferentiation of mesothelial cells is accompanied by an increase in the expression of integrin ⁇ 2 and the acquisition of a migratory phenotype.
  • TGF- ⁇ plus IL-l ⁇ induced the Increase in ⁇ 2 integrity in all mesothelial preparations, while ⁇ 3 integrin increased in omentum and decreased in cells already in transition (Fig. 5c).
  • IL-1 ⁇ potentiated the effects of TGF- ⁇ .
  • 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- ⁇ plus IL-1 increased collagen haptotaxis, the main binding of ⁇ 2 ⁇ l integrin. Migration to laminin-5 followed changes in the expression of its receptor, integrin ⁇ 3 ⁇ 1 (Fig. 5d).
  • Example 6 Evidence of the epithelial-mesenchymal transition of mesothelial cells in the peritoneal tissue of patients in CAPD.
  • 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.
  • 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.
  • 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 ⁇ g / l streptomycin, 2% Biqgro-2 (Biological Industries, Israel). For the experiments the cells were seeded on 50 ⁇ g / ml Collagen I without Biogro. Met5A cells were obtained from the ATCC and grown according to their recommendations.
  • TGF- ⁇ 1 and interleukin-l ⁇ 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 ).
  • the anti-cadherin-E was from Calbioche (La Jolla, CA); anti-vimeritin, anti- ⁇ 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 were described in (Yá ⁇ ez-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.
  • RNAwiz Abion, Aus ' tin, TX.
  • RNAwiz Abion, Aus ' tin, TX.
  • 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 ⁇ g 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).
  • 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).
  • 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
  • the transcription factor snail is a repressor of E-Cadherin gene expression in epithelial tumor cells. Nat. Cell Biol. 2: 84-89. Birchmeier, C, W. Birchmeier, and B.B.-S. B. 1996. Epithelial-mesenchymal transitions in cancer progression. Minutes Anat. 156: 217-226.
  • Pentoxifylline inhibits human peritoneal mesothelial cell growth and collagen synthesis: effects on TGF-b.
  • HB-EGF is produced in the - peritoneal cavity and enhances mesothelial cell adhesion and igration.
  • High glucose-induced PKC activation mediates TGF-bl and fibronectin synthesis by peritoneal mesothelial cells. Kidney Int. 59: 463-470.
  • Yamakido 2000 Effect of glucose on intercellular junctions of cultured human peritoneal mesothelial cells. J. Mi. Soc. Nephrol. 11: 1969-1979. Krediet, RT 1999. The peritoneal membrane in chronic peritoneal dialysis patients. Kidney Int. 55: 341-356. Lai, KN, KB Lai, CW LAm, TM Chan, FK Li, and JC Leung. 2000. Changes of cytokine profiles during peritonitis in patients on continuous anmbulatory peritonela dialysys. Am. J. Kidney Di s. 35: 644-652. Lara-Pezzi, E., J. Serrador, M. Montoya, D.
  • HBx hepatitis B virus X protein
  • the hepatitis B virus X protein (HBx) induces a migratory phenotype in a CD44-dependent anner: possible role of HBx in, invasion and metastasis.
  • Epider al growth factor odifies the expression and function of extracellular atrix adhesion receptors expressed by peritoneal mesothelial cells from patients on CAPD.
  • Interleukin Ib stimulates the production of extracellular matrix in cultured human peritoneal mesothelial cells. Peri t. Dial Int. 19: 211-220.

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Abstract

L'invention concerne un procédé permettant d'identifier la transition épithélio-mésenchymateuse de cellules mésothéliales (TEMCM) et d'identifier des composés modulateurs de la TMCM, des compositions pharmaceutiques et leur utilisation dans le diagnostic et le traitement de maladies liées à la TEMCM. Ce procédé repose sur le fait que les cellules mésothéliales jouent un rôle actif dans l'altération structurelle et fonctionnelle du péritoine durant la dialyse péritonéale qui mène à la transition épithélio-mésenchymateuse empêchant l'ultrafiltration. Ces constatations ont permis de mettre en évidence une série de cibles et de marqueurs destinés à la mise en oeuvre de nouvelles solutions de dialyse péritonéale, à la surveillance des patients soumis à ce traitement et au traitement de maladies liées à des altérations fonctionnelles du mésothélium.
PCT/ES2004/000017 2003-01-17 2004-01-16 Procede permettant d'identifier la transition epithelio-mesenchymateuse de cellules mesotheliales (temcm) et d'identifier des composes modulateurs de la tmcm, compositions pharmaceutiques et leur utilisation dans le diagnostic et le traitement de maladies liees a la temcm WO2004065602A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007082967A1 (fr) * 2006-01-19 2007-07-26 Consejo Superior De Investigaciones Científicas Procédé d'identification d'un processus de fibrose rénale, utilisation des composés inhibiteurs de l'activité de snail dans l'élaboration de compositions pharmaceutiques, procédé d'identification desdits composés inhibiteurs, composition
WO2008107508A1 (fr) * 2007-03-08 2008-09-12 Consejo Superior De Investigaciones Cientificas Utilisation des composés inhibant l'activité de snail1 dans l'élaboration de compositions pharmaceutiques utilisées dans le traitement de chondrodysplasies, procédé d'identification de composés inhibiteurs, compositions pharmaceutiques, méthode de diagnostic de chondrs
US7939272B2 (en) 2007-10-03 2011-05-10 Osi Pharmaceuticals, Inc. Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US8048621B2 (en) 2007-10-03 2011-11-01 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US8093011B2 (en) 2005-03-16 2012-01-10 Haley John D Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US8377636B2 (en) 2007-04-13 2013-02-19 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to kinase inhibitors
US8383357B2 (en) 2005-03-16 2013-02-26 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
WO2017042253A1 (fr) * 2015-09-09 2017-03-16 Fresenius Medical Care Deutschland Gmbh Procédé et kit de diagnostic de transition épithélio-mésenchymateuse (tem) du péritoine
US9896730B2 (en) 2011-04-25 2018-02-20 OSI Pharmaceuticals, LLC Use of EMT gene signatures in cancer drug discovery, diagnostics, and treatment

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2161612A1 (es) * 1999-07-01 2001-12-01 Consejo Superior Investigacion Procedimiento para identificar un compuesto que inhiba la funcion represora de snail.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2161612A1 (es) * 1999-07-01 2001-12-01 Consejo Superior Investigacion Procedimiento para identificar un compuesto que inhiba la funcion represora de snail.

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOYER B. ET AL.: "Induction and regulation epithelial-mesenchymal transitions", BIOCHEM. PHARMACOL., vol. 60, no. 8, October 2000 (2000-10-01), pages 1091 - 1099 *
CANO A. ET AL.: "The transcription factor snail controls epithelial-mesenchymal transition by repressing E-cadherin expression", NAT. CELL. BIOL., vol. 2, no. 2, February 2000 (2000-02-01), pages 76 - 83, XP002941264, DOI: doi:10.1038/35000025 *
ITO T. ET AL.: "Effect of glucose on intercelular junction of cultured human peritoneal mesothelial cells", J. AM. SOC. NEPHROL., vol. 11, 2000, pages 1969 - 1979 *
YANEZO-MO M- ET AL.: "Peritoneal dialysis and epithelial to mesenchymal transition of mesothelial cells", N. ENGL. J. MED., vol. 348, no. 5, 30 January 2003 (2003-01-30), pages 403 - 413 *

Cited By (14)

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Publication number Priority date Publication date Assignee Title
US9244058B2 (en) 2005-03-16 2016-01-26 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US8383357B2 (en) 2005-03-16 2013-02-26 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US8093011B2 (en) 2005-03-16 2012-01-10 Haley John D Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
ES2310434A1 (es) * 2006-01-19 2009-01-01 Consejo Superior Investig. Cientificas Un procedimiento de identificacion de un proceso de fibrosis renal, uso de los compuestos inhibidores de la actividad de snail en la elaboracion de composiciones farmaceuticas, procedimiento de identificacion de dichos compuestos inhibidores, dichas composiciones farmaceuticas y.
WO2007082967A1 (fr) * 2006-01-19 2007-07-26 Consejo Superior De Investigaciones Científicas Procédé d'identification d'un processus de fibrose rénale, utilisation des composés inhibiteurs de l'activité de snail dans l'élaboration de compositions pharmaceutiques, procédé d'identification desdits composés inhibiteurs, composition
WO2008107508A1 (fr) * 2007-03-08 2008-09-12 Consejo Superior De Investigaciones Cientificas Utilisation des composés inhibant l'activité de snail1 dans l'élaboration de compositions pharmaceutiques utilisées dans le traitement de chondrodysplasies, procédé d'identification de composés inhibiteurs, compositions pharmaceutiques, méthode de diagnostic de chondrs
ES2310469A1 (es) * 2007-03-08 2009-01-01 Consejo Superior Investig. Cientificas Uso compuestos inhibidores actividad snail1 en elaboracion composiciones farmaceuticas utiles para tratamiento de condrodisplasias, procedimiento identificacion compuestos inhibidores, dichas composiciones farmaceuticas, procedimiento diagnostico condrodisplasias y aplicaciones.
US8377636B2 (en) 2007-04-13 2013-02-19 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to kinase inhibitors
US7939272B2 (en) 2007-10-03 2011-05-10 Osi Pharmaceuticals, Inc. Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US8048621B2 (en) 2007-10-03 2011-11-01 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US9896730B2 (en) 2011-04-25 2018-02-20 OSI Pharmaceuticals, LLC Use of EMT gene signatures in cancer drug discovery, diagnostics, and treatment
WO2017042253A1 (fr) * 2015-09-09 2017-03-16 Fresenius Medical Care Deutschland Gmbh Procédé et kit de diagnostic de transition épithélio-mésenchymateuse (tem) du péritoine
CN108449998A (zh) * 2015-09-09 2018-08-24 费森尤斯医疗护理德国有限责任公司 用于诊断腹膜的上皮向间充质转化(emt)的方法和试剂盒
CN108449998B (zh) * 2015-09-09 2021-07-06 费森尤斯医疗护理德国有限责任公司 用于诊断腹膜的上皮向间充质转化(emt)的方法和试剂盒

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