Genaktivering og immunmodulering ved transplantatrejeksjon: implikasjoner for forbedret diagnostikk og behandling
Prosjekt
- Prosjektnummer
- 3a-128
- Ansvarlig person
- Guttorm Haraldsen, dr.med
- Institusjon
- Oslo universitetssykehus HF
- Prosjektkategori
- Forskningsprosjekt
- Helsekategori
- Inflammatory and Immune System
- Forskningsaktivitet
- 1. Underpinning
Rapporter
Prevention of fibrosis in transplanted organs remains a challenge. We suspect a putative role of matricellular protein deposition as risk factors of fibrosis (Am J Transplantation, pending revision). We are correlating periostin and TnC expression in protocol kidney biopsies with graft outcome and evaluating the activation of possible pathways.We are about to resubmit for publication a microarray study is based on heterotopic cardiac allografts from DA to Lewis rats and isografts from Lewis to Lewis. Three iso- and allografts were harvested before surgery (day 0) and on days 2, 3 and 4 after transplantation (24 samples). Endothelial cells were enriched from the samples using immunomagnetic beads. Statistical analysis enabled us to classify expression profiles of genes significantly affected by transplantation into two categories based on significant differences in transcription patterns between allografts and isografts.
The most strongly changed transcripts showed very similar patterns in allo- and isografts, and were predominantly associated with synthesis of extracellular matrix molecules as well as the mediators that modulate their synthesis.
One set of these mediators are so-called matricellular proteins that modulate cell-matrix interactions and cell functions but do not seem to have a direct structural role. In our model, we have found two such proteins among the most strongly upregulated in our analysis: tenascin C and periostin. We observed that tenascin C is mostly absent in control tissues but induced in all transplants. Positive staining of tenascin C was most notably observed within the perivascular matrix associated with venules and capillaries, but also in areas surrounding some of the larger vessels. Tenascin C expression was more pronounced in areas with infiltrating cells. We observed a strong induction of periostin in the interstitial space between individual muscle fibers that was most pronounced in the marginal areas of tissue lesions on day 3 and 4 post-tx and more prominent in allografts than in isografts. We hypothesized that matricellular proteins deposition may be an risk factor for future fibrosis in transplanted organs. To evaluate this hypothesis we analyzed periostin and tenascin C by immunostaining protocol biopsies living donor kidney transplants (n=30) at 8 weeks postttransplantand correlating these data to kidney function and fibrotic changes at 1 year after transplantation (data currently under statistical analysis). As periostin has been shown to mediate recruitment of activated fibroblasts through FAK-integrin signaling we have also analyzed the presence of activated, phosphorylated FAK (pFAK Y397) in the rat model. pFAK positive infiltrating cells are present in both allo- and isografts starting form day 2 after transplantation and increasing further in allografts up to day five. Accordingly, pFAK-positive cells are also abundant in transplanted human kidney with acute cellular rejection. We are now analyzing the phenotype of pFAK positive cells and correlating their presence with the deposition of periostin. Recently, tenascin C was shown to drive and prolong TLR4-mediated chronic inflammation. To check if this process might be also important for kidney graft outcome we are investigating the possibility of visualizing activation of the TLR4 pathway by immunostaining for second messengers such as IRAK1.
Transplantation is the best treatment for irreversible organ failure but requires immunosuppression. Our microarray screen of endothelial cell activation during rejection has revealed increased synthesis of fibrogenic molecules. Moreover, probes changed in allografts are regulated by IFN-? and involved in regulation of apoptosis.Our microarray study is based on allografts from DA to Lewis rats and isografts from Lewis to Lewis. Three iso- and allografts have been harvested before surgery (day 0) and on days 2, 3 and 4 after transplantation. Samples were enriched in endothelial cells using immunomagnetic beads. Statistical analysis enabled as to classified expression profiles of genes significantly affected by transplantation into two categories basing on significant differences in transcription pattern between allografts and isografts.
The most strongly changed transcripts, showing identical or similar pattern in allo and isografts, are predominantly associated with synthesis of extracellular matrix molecules as well as mediators that modulate their synthesis.
An increase in synthesis or decrease in breakdown results in an accumulation of matrix especially insoluble collagen fibers may lead to fibrosis. Our data has shown significant upregulation of genes coding inhibitors of ECM degradation: plasminogen activator inhibitor 1 and tissue inhibitor of matrix metalloproteinase (TIMP-1). Upregulation of TIPM-1 expression has been verified by immunohisochemistry. We have also observed upregulation of genes encoding collagen type I, III and XII as well as enzymes involved in procollagen processing and formation of insoluble fibrous products (procollagen C-peptidase enhancer, LOX).
Another set of ECM proteins contains the so-called matricellular proteins that modulate cell-matrix interactions and cell functions, and do not seem to have a direct structural role. We have studied two such proteins that are upregulated in our analysis: tenascin C and periostin. In our model we have demonstrated that Tn-C is mostly absent in control tissues and upregulated in all transplants. Positive staining of Tn-C was most notably observed within the perivascular matrix associated with venules and capillaries, but also in some endothelial cells. Periostin was strongly induced in the marginal areas of tissues lesions. Such deposition was also observed in human kidney samples.
We plan to further investigate the pattern of deposition of proteins involved in fibrosis in our rat model as well as human archival biopsy samples.
Many transcripts that are changed only or predominatly in allografts are upregulated by IFN-gamma and some of these probes are involved in regulation of apoptosis.
To confirm role of IFN- gamma signaling we performed immunohistochemical analysis of Stat1 phosphorylation at tyrosine 701 that is induced by IFN- gamma and essential for Stat1 dimerization and translocation to nucleus. Stat1 activation was absent in control tissues and all isograft samples. Positive nuclei could be observed in allograft starting from day 2 and their number show increase for each day with wide distribution of positive vessels on day four.
Our data show upregulation of many of proapoptotic genes like STAT1, Ripk2 or Cyld. We have performed immunostaining for activated form of caspase3 to assess endothelial cells apoptosis rate in our model. We have not observed any significant increase of apoptotic rate in allograft samples compared to isograft. We can speculate that observed upregulation of proapoptotic genes shows rather cell sensitization to apoptotic signals not actual cell death. On the other hand upregulation of proapoptotic genes is might be balanced by increase in transcription of their inhibitors.
Organtransplantasjon er den beste behandling ved organsvikt i nyrer, lever, hjerte og lunger, men krever livslang immunsuppresjon hos resipient for å hindre avstøtnings-reaksjon. Dette er et samlokalisert, multidisiplinært forskningsprosjekt basert på hjertetransplantasjon i rotte og mikroarrayanalyse av karendotelets transkripsjonsprofil.Cytotoksiske lymfocytter vandrer gjennom åreveggen i graftet ved å feste seg til adhesjonsmolekyler på graftets endotellceller og aktiveres av kjemokiner som også sitter på karoverflaten. Vi har nå analysert karendotelets transkripsjonsprofiler i en omfattende studie av transplanterte hjerter fra DA til Lewis rotte (allograft) og med tilsvarende kontroller (hjerter transplantert fra Lewis til Lewis, dvs isograft). Rejeksjonsprosessen er fulgt fra dag 2, 3 og 4, og endotelcellene er isolert fra enzymdigerert hjertevev vha Dynal-kuler.
Vi har funnet flere clustere av genprofiler som er rejeksjonsassosierte (indusert i allo, ikke iso) eller transplantasjonsassosierte (allo og iso er like). Begge clustergrupper innholder kjente og nye aktiveringsmarkører for karendotelet. Vi verifiserer nå flere av disse markørene vha immunhistokjemi på rottehjerter men benytter også vevsprøver fra transplanterte pasienter for å skrive sammen den første artikkelen som beskriver metoden.
Vi har også startet arbeidet med å verifisere endotelcellenes kjemokinprofil. Selv om flere slike kjemokiner er vist å være sentralt involvert i allorejeksjon, er lite kjent mtp hvilke celler som uttrykker kjemokinene og i hvilken grad det handler om presentasjon på karendotelet.
Samlet vil disse studiene sette oss i stand til å forstå hvordan karveggen forandrer sine egenskaper under hjerterejeksjon og gi angrepspunkter for nye legemidler som kan hindre rejeksjon men ha færre bivirkninger enn dagens immunsuppresjon.
Vitenskapelige artikler
Mikalsen B, Lundström M, Fosby B, Hammarström C, Bjærke H, Wang J, Kasprzycka M, Scott H, Line PD and Haraldsen G
Genome-wide transcription profiles of endothelial cells during cardiac allograft rejection
in revision, Am J Transplantation
Bjørg Mikalsen1, Marlene Lundström1, Bjarte Fosby2, Clara Hammarström3, Hilde Bjærke4, Junbai Wang3, Monika Kasprzycka1, Helge Scott3, Pål-Dag Line2 and Guttorm Haraldsen1,3
Genome-wide transcription profiles of endothelial cells during cardiac allograft rejection
Scandinavian Society of Immunology Meeting, Stockholm
Mikalsen B, Lundstrøm M, Fosby B, Hammarstrøm C, Bjærke H, Wang J, Kasprzycka M, Scott H, Line PD, Haraldsen G
Genome-wide transcription profiles of endothelial cells during cardiac allograft rejection
konferanse
eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT
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