Differentiation of mesothelial cells – potential role in fibrosis - eshre

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Differentiation of mesothelial cells – potential role in fibrosis - eshre

Importance of the mesothelium• Mesothelium• single layer of squamousepithelium• mesodermally derived• all three serosal cavities• Subserosal connective tissue20µm• Peritoneal fluid• Functions• Non-adhesive barriersurfactantand microvilli• Solute and fluid transport• Immune function• Pathology5µm


Peritoneal repair scenariosInjuryBOWELBODY WALLClose opposition ofperitoneal surfacesTissue repairNormal repairAdhesionsPeritoneal sclerosis


Abdominal/pelvic surgery often leadsto adhesion formation• Form after a range of peritoneal insults• Complications : Small bowel obstruction infertility in women difficulty of repeat surgery chronic pelvic pain• 5.5% of all abdominal repeat surgerydirectly due to adhesions - SCAR studies• Reform after surgical adhesiolysis• Limited preventative treatment barrierdevices• Pharmaceutical interventions?


Peritoneal dialysis commonly results inperitoneal sclerosis• Peritoneal dialysate high glucose, low pH,high lactate• Peritonitis• Thickening of peritoneal membrane leads toultrafiltration failure• Rare condition of PD - EncapsulatingPeritoneal Sclerosis (EPS)• Cocooning of viscera by mass scar tissueleading to bowel obstruction• Incidence linked to time on dialysis- 2 years 1.9%- 8 years 19.4%• Surgical management• Pathophysiology?van Dellen D et al., BrJ Surg, 2011


How does tissue repair progress to fibrosis?• Coagulation and inflammation• Granulation tissue formation• Re-epithelialisation• Tissue contraction• Remodeling and scar formation -……fibrosis


Important role of the ‘fibroblast’ intissue repair and fibrosisMcAnulty R. Int J Biochem Cell Biol (2007)


Does the mesothelium play a role inperitoneal fibrosis?


Peritoneal repair scenariosInjuryBOWELBODY WALLClose opposition ofperitoneal surfacesTissue repairNormal repairAdhesionsPeritoneal sclerosis


Proposed schemes of mesothelial regeneration5.3.7.2.1.4.6.


Rodent model of surgical injury• Isolate and label rat cellsmesothelial cells• peritoneal lavage cells• peritoneal macrophages• lung fibroblasts• Laparotomy and simple abrasion injury• Intra-peritoneal injection of fluorescentDi-I labelled cells• Assessed distribution of labelled cellspost-injuryFoley-Comer et al., 2002. J Cell Sci 115, 1383


Mesothelial cells in serosal fluid increaseafter injury in ratsHBME+ve7.5Percentage of Mesothelial CellsOver a 3-day periodPercentage5.02.50.0Day 0 Day1 Day 2 Day 3DaysFoley-Comer et al., 2002. J Cell Sci 115, 1383


Incorporation of isolated free-floatingmesothelial cells on denuded surfaceMesothelial cellsday 8Fibroblastsday 8Lavage cellsday 8MacrophagesDay 2MacrophagesDay 5MacrophagesDay 8


Formation of junctional components byincorporated mesothelial cellsDiI-fibroblastsDiI-mesothelial cellsFoley-Comer et al., 2002. J Cell Sci 115, 1383


Di-I labelled mesothelial cells incorporate intomultiple layers


Epithelial-Mesenchymal Transdifferentiation (EMT)of mesothelial cells in vitroEpithelialHGF/TGF-β1/EGFSub-cultureFibrin/collagen gelDialysis fluidMenstrual effluentMesenchymalEMTepithelial fibroblast - myofibroblast - smooth muscle-like phenotype


Pathway to epithelial-mesenchymaltransdifferentiationE-cadherinCytokeratinZO-1LamininEntactinSyndecanMUC 1DesmoplakinCollagen IVmsR200Progressive loss of epithelialmarkers and gain ofmesenchymal markersFSP-1N-cadherinVimentinFibronectinB-cateninSyndecan 1miR10bSnailSlugTwistα SMAFOXC2LEF-1Kalluri R and Weinberg RA, J Clin Invest 199 (2009)


Does mesothelial cell EMT occur in vivo?


Mesothelium EMT contributes to blood vessels of theheart, lung, mesentery and gut during developmentDay 4 Day 7Day 21 Day 28Control AdVNo AdVWilm B et al. Development 2005;132:5317-5328


Mesothelial cell EMT occurs with injury in adultCytokeratin controlControlControl+ TGFβ1Cytokeratin 6 mth dialysisPeritoneal dialysis (CAPD)Mesothelial cell damage/stress(Myo)fibroblast formationTGF-ß1Extracellular matrix deposition- TGFβ1- TGFβ1days7 days Cytokeratin 8mth dialysis.ICAM1 8mth dialysisPeritoneal sclerosisYanez-Mo et al., (2003) NEJM 348:403-413;Yang et al. (2003). Kidney Int 63: 1530-1539


Transforming Growth Factor (TGF)-βFibrogenic cytokine:• Wound healing• Fibrosis• Growth and developmentLatent ligand complex3 mammalian isoforms:• TGF-β1, TGF-β2 and TGF-β3TGF-β ligandTGF-βR2PhosphorylationTGF-βR1Secreted in latent form• ActivationOnce activated can signal through• TGF-βRI• TGF-βRII• SMAD pathwayPhosphorylationR-SmadsPDegradationI-SmadsRegulation ofTranscriptionCo-SmadsFunctions• Induction of myofibroblasts• Increase in ECM production• Upregulates protease inhibitors


Mouse model of peritoneal fibrosis- Peter Margetts, McMaster University, CanadaAdenovirus delivery of TGF-β1 20days transient peritoneal fibrosisExpression extended by helper-dependent adenovirus 70days severe fibrosis/EPSDay 4 Day 7UntreatedAd-ControlDay 21 Day 28Ad-TGF-B1Control AdVNo AdVLiu L et al., Perit Dial Int 29; 508 (2009);Margetts P et al., J Am Soc Nephrol 12; 2029 (2001) and J Am Soc Nephro 16,425 (2005)


Peritoneal expression of mesenchymal markers afteradenovirus infectionα- SMA CollagenSnailAd-TGF-β1Ad-ControlMargetts P J et al. JASN 2005;16:425-436


Is there a genetic predisposition toperitoneal fibrosis?


Mouse strain differences in the fibrotic responseStrain Susceptibility Fibrosis Type ReferenceC57BL/6J Susceptible Pulmonary Haston et al., 1996• It is possible that fibrosis islinked to geneticsusceptibility.• Mice with different geneticbackgrounds have differentsusceptibilities to fibrosis incertain organs• It is also possible thatsusceptibility is linkedResistantto theroute of injury e.g.bleomycin-induced lungfibrosis or radiation inducedlung fibrosisC57BL/6 Susceptible PulmonaryIntestinalHepaticRenalHepatic Hillebrandt et al., 2002Renal Kato et al., 2008Schrier et al., 1983Skwarchuk and Travis, 1998Knight et al., 2007Puri et al., 2010Intermediate Hepatic Shi et al., 1997Resistant Renal Sugimoto et al., 2007Cardiac Faulx et al., 2005BALB/c Susceptible Hepatic Shi et al., 1997PulmonaryHepaticRenalSchrier et al., 1983Knight et al., 2007Puri et al., 2010BALB/cJ Susceptible Hepatic Hillebrandt et al., 2002A/J Susceptible Cardiac Faulx et al., 2005Resistant Hepatic Hillebrandt et al., 2002


Mouse family tree Jackson LabsPetkov P. et al., Genome Res. 2004 14: 1806-1811


Animal model of peritoneal fibrosis- mouse strain differencesC57BL/6J DBA/2J C3H/HeJ SJL/J• Intraperitoneal injection of TGF-β1 expressing adenovirusadministered to mice.• After 4 or 10 days peritoneal tissue and omental tissue samplescollected and analysed for fibrogenic differencesLouise Walkin, PhD student


Mouse stain differences in thedevelopment of peritoneal fibrosisC57BL/6J = fibroticSJL/J = resistantMargetts P et al., Nephrol Dial Transplant. (2012)


Mesothelial cell culture mouse strain differences?The JacksonLaboratory - JAX®- Male , 8 weeksC57BL/6JIsolation & cultureSJL/J+/- 1ng/ml TGF-β1- Collagen production at 0, 24, 48 hours


Mouse strain difference in mesothelial cells collagen productionMesothelial cellsFibroblastsEMT markers?


Inhibition of EMT to prevent peritoneal fibrosis201220122010


Human adhesions show the presence ofmyofibroblasts and clusters of smooth muscleHerrick et al., J Pathology 2000, 192: 67-72; Sulaiman et al., Ann Surg 2001, 234: 256-261


Does mesothelial cell EMT occur in the pathogenesis ofadhesion formation?InjuryCAECUMB ODY WALLClose opposition ofperitoneal surfacesNormal repairReduced fibrinolysisAdhesionformation


In collagen gelOn gel with blood clot30 mins 14 days


Inhibition of TGF-β1 and 2 reduces adhesion formationin murine model• Neutralising antibody to TGF- β1 and 2 giventopically on day of surgery and i.p 4hrs presurgery,post-surgery and every 4hrs for 24hrs• Significant reduction of adhesions by blockingTGF- β1 and -β2 by 7 daysAnti TGF-β(1, 2)Mean No. of adhesions/ animal32.521.510.50* P < 0.05Vehicle Control Anti-TGF-beta (1, 2)Vehicle control100 µm100 µmGorvy et al. Am J Pathol 2005, 1005-1019.


Source and plasticity of mesothelial cells?EMTAdipocyteChrondrocyte


Evidence for a bone marrow source ofregenerating mesothelial cellsGFPmouseIrradiatedmouseChimeraBone marrow donor Bone marrow recipient Bone marrow engraftmentCampbell et al., (2000) J Vasc Res. 37; 364- 371


• GFP- bone marrow transplantinto irradiated mice• Found 2.2% of mesotheliumGFP-labelled after 6 months• Bone-marrow derived cellscontribute to normal mesothelialturnover• After injury?


Stimulated rat mesothelial cells accumulate lipidduring adipogenic differentiation- Steve Mutsaers, UWA, PerthD0D1D3D9Oil Red O StainD15D18Lansley S. et al., J Cell Mol Med 2011;15(10):2095-105.


Stimulated rat mesothelial cells express alkalinephosphatase during chrondrogenic differentiationBone Marrow Mesenchymal Cells (BMMC)D0D6D15D18Mesothelial CellsD0D6D15D18


Stimulated rat mesothelial cells form mineralizedbone nodules Von Kossa stainingBone Marrow Mesenchymal Cells (BMMC)UnstimulatedD18StimulatedMesothelial CellsUnstimulatedStimulatedLansley S. et al., J Cell Mol Med 2011;15(10):2095-105.


Conclusions• Mesothelial cells undergo EMT during peritoneal fibrosis -adhesion formation?• Inhibit EMT prevents peritoneal fibrosis adhesion formation?• Mesothelial cells show multipotential ability in culture inpeople?• Apparent bone marrow source of mesothelial cells do thesecells have stem cell-like properties?• Need to perform cell tracking studies which promoter?• Use primary human mesothelial cells source?• Multidisciplinary approach?


AcknowledgementsManchester UniversitySylwia WilkoszDylan GorvyGrenham IrelandJonathan EpsteinLouise WalkinUCLHassan SulaimanAdam Foley-ComerGeoff LaurentChristie Hospital, ManchesterMalcolm WilsonUniversity of Western Australia, PerthSteve MutsaersMcMaster University, CanadaPeter MargettsManchester Royal InfirmaryAngela SummersPaul BrenchleyTitus Augustine

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