The role of fibroblast in ectopic calcification

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The role of fibroblast in ectopic calcification

  1. 1. The Role of Dermal Fibroblasts inthe Development of EctopicCalcificationsGiulia AnnoviDepartment of Life SciencesUniversity of Modena and Reggio Emilia
  2. 2. CalcificationBiologicalmineralizationEctopiccalcificationPhysiological processBone - TeethCell-mediated processRegulated by proteins, ions,functional moleculesPathological processSoft connective tissuesCell-mediated processInduced by several mechanisms
  3. 3. 5. Loss of “inhibitors”4. Presence of “Bone Proteins”1. Circulating nucleational complexes (high Ca / P)2. Cell death3. Proteolysis and ECM degradation
  4. 4. CellInhibitors:ANKHPC-1MGPOsteopontinPPiStimulators:Matrix vesiclesANKHPC-1AnnexinsApoptotic bodiesTNAPType I, II, X collagenPiMineral formation(Kirsch, 2006)
  5. 5. OsteoarthritisAnkylosing SpondylitisKeutel SyndromeAgingAtherosclerosisAgingChronic renal failureDiabetesBone formationOsteopetrosisAgingOsteoblastsVascular SmoothMuscle CellsChondrocytesFibroblasts?Mesenchymal cells which are resident in a tissue that is only rarely affected bymineralization process.
  6. 6. Angioid streaksHaemorragiesCentral vision lossPapulesSkin laxity and redundancyCardiovascularcomplicationsPseudoxanthoma elasticumas soft connective tissue calcification model
  7. 7. SEManalysis
  8. 8. Calcifying MediumAscorbic Acidβ glicerophosphateDexamethasone
  9. 9. Day10Day40Day30Day20DMEM CMHumanDermalFibroblasts
  10. 10. Day20Day30Day40
  11. 11. The calcification process in a fibroblast cell culturesystem is a slowly progressive event.Fibroblasts phenotype could be different from that ofother mesenchymal cells.
  12. 12. Control PXE CM P CM C CM 20GG 6HPXE CM 20GG 6H01234567ControlPXEANKHgeneexpression6h6h20dDMEM CM CMControl6 PXE6 Control6cm PXE6cm C XXg CM P xxg CM0.00.51.01.52.02.5ControlPXEANKH(Relativebandintensity)DMEM CM CM6h 6h 20d
  13. 13. Control PXE CM P CM C Cm 20 GG 6HPXE CM 6H 20GG01020PXEControlNPP1geneexpressionDMEM CM CM6h 6h20dc 6h P 6h C 6h cm P 6h CM C XXg cm P XXg CM01234ControlPXEPC1(Relativebandintensity)DMEM CM CM6h 6h20d
  14. 14. C CM PXE CM01234ControlPXE4142434ALPactivity(arbitraryunit)DMEM CM CM CM6h6h10d20dTNAP determins higher Pi levels.
  15. 15. DMEM CMCM +100 uMLevamisoleControlPXE
  16. 16. # Genetic studies have implicated PPi metabolism in the development ofcalcification, but this finding sustains the hypothesis that the calcificationprocess could be due to alterations in local PPi metabolism.# Results indicate that NPP1, being capable to produce PPi, is normallyexpressed by human dermal fibroblasts and is significantly upregulated in acalcifying environment.# However, the potent inhibitory action of PPi is abolished by thecontemporary hyperactivation of TNAP, which is consistent with its critical rolein mineralization process.# The higher upregulation of TNAP in PXE could be a condition associatedwith ECM calcification.# Restoration of PPi extracellular pool, by inhibiting TNAP activity, is able tocounteract mineral deposits formation.Conclusions
  17. 17. Federica BoraldiRoberta TiozzoIvonne RonchettiDaniela QuaglinoUniversity of ModenaMaria I. Garcia FernandezUniversity of MalagaThanks toAnne de PaepeOlivier VanakkerUniversity of GhentLeon J.SchurgersCees VermeerUniversity of Maastricht
  18. 18. Is it TNAP sufficient to induce ectopic calcification in PXE?No,it is NECESSARY but NOT SUFFICIENT50kDa37kDaActin Control3-10NL50kDa37kDaActin PXE3-10NLGla-MGP Control15kDa10kDaGla-MGP PXE15kDa10kDaMGP
  19. 19. Control PXE C 20GG 6h PXE 20GG 6H01234ControlPXEBMP2GENEexpression6h20dControl6 PXE60.00.51.01.5 ControlPXEBMP2(Relativebandintensity)6h20dBMP2

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