Gene activation and immune modulation in transplant rejection
In rodent heterotopic cardiaciso- and allograftsthe majority of the differentially expressed genes in graft endothelial cells are affected by the transplantation procedure whereas relatively few are associated with allograft rejection. Many of the genes upregulated both in iso- and allografts were associated with synthesis of extracellular matrix molecules and mediators that modulate their synthesis, such as the two matricellular proteins tenascin C and periostinthat wereamong the most highly upregulated. To investigate if the early expression of these proteins in renal allografts has an impact on graft outcome, we performed a study in protocol biopsies from kidney transplant recipients (n=31) obtained at 8 weeks and 1 year after transplantation. Intermediate-to-high grade fibrosis in the 1-year biopsies was significantly more frequent in the group of patients who expressed higher levels of tenascin C and periostin 8 weeks after transplantation. Showing that tenascin C and periostin expression in the early post-transplant period may predict and have an impact on long-term graft outcome.Current preliminary data show that tenascin C is induced by alarmins (IL33 and IL1alfa) in cultured human endothelial cells (EC) and both matricellular proteins can increase EC response to the inflammatory cytokine IL1beta.
Despite progress in immunosuppression, chronic allograft dysfunction and loss remain an importantclinical challenge and kidney transplant failure is now a leading cause of end-stage renal disease. A prominent feature of such grafts is the development of interstitial fibrosis and tubular atrophy (IF/TA), thought to either develop as a consequence of chronic alloimmune injury itself but also as a response to other complications after transplantation such as nephrotoxicity of calcineurin inhibitors, hypertension, polyoma virus infection or urinary tract obstruction. Our research shows that early expression tenascin C and periostinis associated with later development of fibrosis and may be important factor in pathogenesis of IF/AT. Identification of factors crucialin the pathogenesis of fibrosis is critical for two reasons. First, it may improve the identification of patients at risk, because it is unclear why certain kidneys maintain a stable function while others develop fibrosis and loss of kidney function. Secondly, we may identify new therapeutic targets. The need for an effective approach is critical because graft loss is not only devastating to patients but also burdens the already lengthy kidney transplantation waiting list. A better understanding of this process is especially important because anti-fibrotic and anti-proliferative agents may also impair wound healing post-operatively.
Gene activation and immune modulation in transplant rejection
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. 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 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.
Gene activation and immune modulation in organ rejection
Transplantation is the best treatment for irreversible organ failure but requires immunosuppression to prevent rejection. Our microarray screen of endothelial cell activation during rejection has revealed synthesis of fibrogenic molecules. Moreover, probes changed in allografts are regulated by IFN-gamma?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 coding collagen type I, III and XII as well as enzymes involved in procollagen processing and formation of insoluble fibrous components (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, 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 upergulated 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.
Genome-wide transcription profile of endothelial cells after cardiac transplantation in the rat.
Am J Transplant 2010 Jul;10(7):1534-44.
Early tenascin C and periostin expression is associated with late fibrosis in renal allografts
included in: Bjørg Mikalsen. Genome-wide endothelial cell activation profiles and matricellular protein expression after solid organ transplantation, Faculty of Medicine, University of Oslo
Early tenascin-C expression is associated with late fibrosis in renal allografts.
Thrombospondins and other Matricellular Proteins in Tissue Organization and Homeostasis, FASEB Summer Research Conference July18-23, 2010 Snowmass Village, Colorado, USA
Early tenascin-C and periostin expression is associated with late fibrosis in renal allografts
NSI General Assembly and Annual General Meeting 2010, 26th November 2010, Oslo
Genome-wide transcription profiles of endothelial cells during cardiac allograft rejection.
in revision, Am J Transplantation
Genome-wide transcription profiles of endothelial cells during cardiac allograft rejection
Scandinavian Society of Immunology Meeting 2008, Stockholm