Manuel López Cabrera. Modelos experimentales para la investigación en DP

Transcription

1 Manuel López Cabrera Modelos experimentales para la investigación en DP

2 Manuel López Cabrera Transición Mesotelio Mesénquima (MMT) como Marcador de Enfermedad y como Diana Terapéutica en Fibrosis Peritoneal y Fallo de Membrana.

3 Structural alterations of the peritoneum associated with membrane failure 1.- Increase of ECM and fibrosis 2.-Increase of the number of peritoneal capillaries and vessel permeability and vasculopathy 3.- Loss of mesothelial cells monolayer (MMT)

4 * Acute: Peritonitis (IL-6) * Chronic: Composition of PD fluids (IL-17)

5 Myofibroblasts and infiltrating inflammatory cells are considered the main responsibles of structural and functional alterations of the peritoneum during PD

6 Inflammation and MMT establish a positive feedback loop during injury and repair responses Th17

7 Epithelial to Mesenchymal Transition (EMT) of MCs (MMT) in PDinduced Peritoneal Fibrosis? The EMT of mesothelial cells (Meothelial to Mesenchymal Transition, MMT) (in vivo and in vitro) was first described in a landmark paper published in 2003: CK CK CK ICAM-1

8 Resident quiescent fibroblasts in normal peritoneum are α-sma neg PARIETAL PERITONEUM α-sma

9 α-sma is expressed in activated fibroblasts located in the compact zone and in modified mesothelial cells α-sma (PD) -Expression in fibroblastic cells in 70.4 % (0% in controls) A B -Expression in mesothelial and modified mesothelial cells (57.9%) (0% in controls) C D E F

10 Activated fibroblasts in PD patients are α-sma + and co-express mesothelial markers α-sma CK Myofibroblasts (α-sma) SNAPSHOTS Partial co-expression of cytokeratins Their most probable origin is double: 1.- form resident fibroblasts activation 2.- from mesothelial cells by MMT

11 Mesothelial to Mesenchymal transition of MCs IN VITRO Omentum A TGFβ1 + IL-1β (48hr to 7 days) Omentum B Omentum C PD Fluids/Effluents " Omentum D

12 Mesothelial cells undergoing a MMT express both mesothelial and mesenchymal markers

13 Low GDPs dialysis fluids have less impact on MMT than standard bioincompatible fluids in vitro.

14 Mesothelial to Mesenchymal Transition (MMT) is a tightly regulated process

15 BMP-7 (Bone Morphogenic Protein-7), a natural antagonist of TGFβ, blocks ΜΜΤ in vitro induced with TGFβ BMP-7 blocks TGF -induced EMT of MC in vitro CONTROL TGF 1ng/ml TGF 1ng/ml + BMP-7 0.5µg/ml

16 TGFβ blocking peptide P17 blocks MMT in vitro

17 TGFβ blocking peptide P17 blocks MMT in vitro

18 Tamoxifen ameliorates MMT in vitro CONTROL TGF 1ng/ml TGF 1ng/ml +Tamo 3µM TGF 1ng/ml +Tamo 6µM

19 Pharmacological agents modulating MMT in vitro *Tamoxifen ( Modulator of estrogen Rec) *Paricalcitol (Vit. D Agonist) *Rapamycin (Inh. mtor) *Bosentan (Inh. ET-1 Receptors) *Macitentan (Inh. ET-1 Receptors)

20 The mesothelial cells undergo a MMT during PD fluid exposure. Ex vivo analysis Epitheliod effluent-derived MCs (Early MMT) Non-epitheliod effluent-derived MCs (Late MMT) Isolation of mesothelial cells from PD effluents (800 effluents from 150 PD patients)

21 Different Phenotypes of Effluent-derived MC Effluent MC Omentum Epitheliod Transitional Fibroblast-like Mixed Fibroblasts Non-epitheliod ICAM-1 Mesothelial cells undergoing a MMT ex vivo express both mesothelial and mesenchymal markers Loureiro et al. J Am Soc Nephrol 22: , 2011

22 TAK-1 Inhibitor reverts the mesenchymal phenotype of effluent non-epitheliod MCs (rmmt) Strippoli, el al. PLoS One 7(2): e

23 Smad-dependent signaling pathways of TGF-!1 and BMP-7 Loureiro J, et al., Nephrol Dial Transplant Apr;25(4): Loureiro J, et al., J Am Soc Nephrol Sep;22(9):

24 Smad-independent signaling in response to TGF-!1 and BMP-7 Strippoli, el al. PLoS One 7(2): e Strippoli, et al. J Cell Science: 123: Strippoli, et al. Dis Model Mech: 1:

25 Is MMT associated with peritoneal membrane failure? High peritoneal transport is associated with nonepitheliod phenotype of the effluent MC 0 % 76 %

26 Microarrays of MMT Ex vivo 9 epitheliod effluent-derived MCs (Early MMT) 8 non-epitheliod effluent-derived MCs (Late MMT) CONTROL POOL Microarrays analysis of EMT is performed by dye swap

27 Variations of gene expression in Epitheliod and Nonepitheliod MC (Ex vivo) Epith Non-epith Babelomics, Limma One-Class INDUCED Epith Non-epith REPRESSED Ruiz-Carpio et al. Sci Rep Mar 22;7:44941.

28 Variations of gene expression in Non-epitheliod vs Epitheliod (BIOMARKERS) Microarrays ex vivo MMT-Chip PATIENTS Diagnostic Prognostic Ruiz-Carpio et al. Sci Rep Mar 22;7:44941.

29 An ideal effluent biomarker for PD should encompass the following properties: 1. Detectable in peritoneal effluent 2. Local production within the peritoneal cavity 3. Involved in pathology of the peritoneal membrane 4. High sensitivity and specificity for the clinical outcome. TSP1, VEGFA, COL13, GREM1

30 Association of effluent levels and peritoneal transport (Cr-MTC) Ruiz-Carpio et al. Sci Rep Mar 22;7:44941.

31 Potential therapeutic interventions on MMT to prevent/reverse membrane failure

32 Peritoneal Dialysis Model in Mice Genetically Modified Mice (KO or Transgenic) CD69 KO Cav1 KO

33 TGFβ is considered the master molecule in PD-induced fibrosis and membrane failure and is also a key inducer of MMT

34 TGFβ blocking peptides ameliorate peritoneal fibrosis induced by dialysis fluid exposure and partially revert UFF. Control PDF PDF+P17 PDF+P144 Loureiro et al. J Am Soc Nephrol 22: , 2011

35 TGFβ blocking peptides ameliorate peritoneal angiogenesis induced by dialysis fluid exposure. Loureiro et al. J Am Soc Nephrol 22: , 2011

36 TGFβ blocking peptides decrease the accumulation of mesothelialderived fibroblasts induced by dialysis fluid exposure.

37 Pharmacological agents modulating MMT in vivo *Tamoxifen (Modulator estrogen receptor) Loureiro et al. PLoS One Apr23;8(4):e *Paricalcitol (Vit. D agonist) González-Mateo et al. PLoS One Oct 3;9(10):e *Rapamycin (Inh. mtor) González-Mateo et al. Biomed Res Int. 2015;2015: *Rosiglitazone (PPAR-γ agonist) Sandoval et al. Lab Invest Oct;90(10): *Nebivolol (β1-adrenergic blocker) Liappas et al. Oncotarget May 24;7(21): *Bosentan (Inh. ET-1 receptors) *Macitentan (Inh. ET-1 receptors) Busnadiego et al. J Am Soc Nephrol Jan;26(1): Busnadiego et al. J Am Soc Nephrol Jan;26(1):173-82

38 From bench to bed side: Patients

39 Potential therapeutic interventions on MMT to prevent/reverse membrane failure

40 Low-GDPs-containing PD fluids induce less inflammation and fibrosis in vivo in the mouse PD model. High GDP-PDF Low GDP-PDF Inflamación Fibrosis Aroeira et al. J Am Soc Nephrol 20: , 2009.

41 Low-GDPs-containig PD fluids induce less EMT of mesothelial cells in patients (a follow-up study) (1) Mixed models, fluid time P= Time fluid (P=0.017) Fluids (P= ). Bajo MA. et al. Nephrol Dial Transplant (2011) 26:

42 Low-GDPs-containig PD fluids induce less EMT of mesothelial cells in patients (a follow-up study) (2) Bajo MA. et al. Nephrol Dial Transplant (2011) 26:

43 Patients receiving biocompatible solutions showed better mesothelial preservation, less thickness and less vasculopathy when compared to patients treated with conventional fluids. Biocompatible Conventional del Peso G, et al., Perit Dial Int Mar-Apr;36(2):

44 CONCLUSIONS 1. We propose that the combination of several MMTassociated markers, MMT-CHIP, could become a tool for diagnostic/prognostic of peritoneal membrane failure. 2. We propose that MMT or MMT-promoting stimuli could become therapeutic targets in peritoneal dialysis-induced fibrosis and membrane failure.

45 Many thanks to all the collaborators Jesús Loureiro Maria Luisa Pérez Vicente Ruiz-Carpio Angela Rynne Lucía Pascual Guadalupe González Pilar Sandoval Manuel Lopez Cabrera Fernando Rodríguez-Pascual Santiago Lamas Carlos Cabañas Kostas Stamatakis Manuel Fresno Miguel Angel del Pozo Christine-Maria Horejs Timothy Joseph Keane Raffaele Strippoli Abelardo Aguilera José A. Jiménez-Heffernan José A. Sánchez-Tomero Gloria del Peso María A. Bajo Rafael Selgas