Final Report Summary - POSAT (Prolong Organ Survival After Transplantation)
Organ transplantation is the treatment of choice for many patients with end-stage diseases. Waiting lists are lengthening but the supply of donor organs has not expanded to meet this demand. Chronic rejection resulting in late loss of graft function now represents the greatest clinical challenge, with many patients re-joining the waiting list years after an initial transplant operation. Indeed, as many as a third of patients joining the kidney transplant waiting list have already rejected one or more previous transplants. Preventing graft loss would have a huge socio-economic impact for patients and transplant medicine. The POSAT scheme was designed to address this by training a cohort of 4 early stage researchers to function at this dynamic interface between academia and industry in order to address specific problems which contribute to chronic graft failure. The project objectives were to 1) develop methods to reduce organ inflammation immediately after transplantation (thereby preserving long-term graft function), and 2) identification of biomarkers which allow transplant patients to be stratified allowing individualized drug treatment. These complementary objectives were addressed by allocation of a trainee-specific project to each of the 4 early stage researchers. Although based at Newcastle University, each trainee spent significant periods working with carefully selected private sector partners (QIAGEN, CELLIX, ALMAC) and academics at another university in the NorthEast of England (Durham University).
The work carried out in POSAT involved:
• Regulation of chemokine function during Ischemia-Reperfusion Injury following Transplantation
• Identification of biomarkers in renal fibrosis.
• Analysis of tissue re-modelling associated with allograft rejection
• Examining the role of HLA class I& II antibodies in kidney rejection
Our results have shown that following transplantation, the stress due to ischemia-reperfusion injury (IRI) results in production of chemokines like CXCL8, and reactive molecules such as peroxynitrite, which modify the chemokines by nitration. We have shown that nitration of chemokines renders the chemokine non-functional. This phenomenon could be a natural form of chemokine regulation to prevent excessive inflammation, preventing its detection by conventional methods and therefore limiting the biological relevance of using unmodified chemokines as biomarkers. We hypothesise that simply measuring the amount of unmodified chemokine in a patient sample gives an incomplete and inaccurate representation of the inflammatory situation, as there is an unknown amount of modified chemokine present. Nitrated CXCL8 could therefore be a biomarker of IRI.
Fibrosis is one of the leading hallmarks of chronic allograft dysfunction. Recent evidence indicates that non-proliferating myofibroblasts, could be derived from other cell types. This specifically includes the process of endothelial-to-mesenchymal transition (EndMT). Lineage tracing in the in vivo mouse model revealed expression of mesenchymal markers in endothelial derived cells, indicating the presence of EndMT in cardiac fibrosis generated post-myocardial infarction. We identified a microRNA signature in an in vitro model of EndMT. On further analysis of these profiles, some miRNAs’ were found to be important in maintaining endothelial cell integrity. Over-expression of these miRNA’s in endothelial cells restored the expression of endothelial markers and repressed the expression of mesenchymal markers induced by TGFβ2 and IL1β treatment. MiRNA target studies allowed identification of common targets using 4 different prediction tools. We conclude that miRNA may have a role and therapeutic potential in pathologic tissue remodelling following allograft injury post transplantation. Overall, this study provides new insights into the role of microRNA in regulation of endothelial phenotype in relation to fibrosis and provides a rationale for therapeutic interventions particularly as we move to clinical trials of treatment that manipulate microRNA function.
Heparan Sulfate (HS) is involved in chronic inflammation and fibrosis development by binding proteins, enhancing their activity and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. HS 3-O-sulfation is a rare but functionally highly relevant modification. However, its role in kidney disease is still unclear. Using a mouse model of renal fibrosis, we found that total HS O-sulfation was increased in damaged kidneys. Interestingly, HS 3-O-sulfotransferase 1 (HS3ST1) expression strongly correlated with the development of fibrosis. HS3ST1 was the most abundantly expressed HS 3-O-sulfotransferase in primary tubular epithelial cells. To determine the implication of HS3ST1 in growth factor binding and signaling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signaling.
Antibody-mediated rejection is one of the major causes of chronic allograft injury. This is mediated by endothelial cell activation, microvascular inflammation and leukocyte recruitment. Despite evidence pointing to the relevance of HLA class II antibody in graft rejection, the role of antibody-endothelium interaction in the absence of complement is not fully understood. We showed that exposure of endothelial cells to HLA class II antibody induces an activation of signalling cascades characteristic of endothelial cell activation. This activation is further responsible for upregulation of adhesion molecules, which facilitate the interaction of donor endothelium and recipient leukocytes. Strategies to block endothelium-leukocyte interaction might reduce the incidence of allograft rejection and improve allograft survival.
In summary this programme of work has gone some way towards increasing our understanding of the mechanisms involved in graft rejection and has suggested strategies to increase the survival of transplanted organs. We have also identified potential biomarkers both of IRI and chronic rejection which could allow transplant patients to be stratified allowing individualized drug treatment.
Impact: This project has generated significant laboratory know-how and expertise around post-translationally modified chemokines, microRNA analysis and Heparan sulphation involved in transplant rejection. We have also identified potential biomarkers of IRI and fibrosis (microRNA & HS-3-O sulphation). Engagement with the Newcastle University Research & Enterprise Services (RES) has been established who are supporting the next phase of translational development.
Furthermore, the ESR’s have developed research skills at the forefront of Immunobiology, with specific relevance for investigation of transplantation and other inflammatory diseases.
Dissemination of information to the general public, the scientific community and stakeholders has been an important part of POSAT networking. More information on POSAT activities can be found at the POSAT’s own website (https://research.ncl.ac.uk/fp7posat/)
Through the website, POSAT has promoted interdisciplinary interaction as well as facilitated the spreading of knowledge. During POSAT training the ESR’s have maintained a high profile in the field of transplantation biology through publications, conferences and were also involved in promoting outreach activities. Further impact is the training of 4 world-class early career scientists who will continue to work in this field to the benefit of the European area.
Project website: https://research.ncl.ac.uk/fp7posat/
The work carried out in POSAT involved:
• Regulation of chemokine function during Ischemia-Reperfusion Injury following Transplantation
• Identification of biomarkers in renal fibrosis.
• Analysis of tissue re-modelling associated with allograft rejection
• Examining the role of HLA class I& II antibodies in kidney rejection
Our results have shown that following transplantation, the stress due to ischemia-reperfusion injury (IRI) results in production of chemokines like CXCL8, and reactive molecules such as peroxynitrite, which modify the chemokines by nitration. We have shown that nitration of chemokines renders the chemokine non-functional. This phenomenon could be a natural form of chemokine regulation to prevent excessive inflammation, preventing its detection by conventional methods and therefore limiting the biological relevance of using unmodified chemokines as biomarkers. We hypothesise that simply measuring the amount of unmodified chemokine in a patient sample gives an incomplete and inaccurate representation of the inflammatory situation, as there is an unknown amount of modified chemokine present. Nitrated CXCL8 could therefore be a biomarker of IRI.
Fibrosis is one of the leading hallmarks of chronic allograft dysfunction. Recent evidence indicates that non-proliferating myofibroblasts, could be derived from other cell types. This specifically includes the process of endothelial-to-mesenchymal transition (EndMT). Lineage tracing in the in vivo mouse model revealed expression of mesenchymal markers in endothelial derived cells, indicating the presence of EndMT in cardiac fibrosis generated post-myocardial infarction. We identified a microRNA signature in an in vitro model of EndMT. On further analysis of these profiles, some miRNAs’ were found to be important in maintaining endothelial cell integrity. Over-expression of these miRNA’s in endothelial cells restored the expression of endothelial markers and repressed the expression of mesenchymal markers induced by TGFβ2 and IL1β treatment. MiRNA target studies allowed identification of common targets using 4 different prediction tools. We conclude that miRNA may have a role and therapeutic potential in pathologic tissue remodelling following allograft injury post transplantation. Overall, this study provides new insights into the role of microRNA in regulation of endothelial phenotype in relation to fibrosis and provides a rationale for therapeutic interventions particularly as we move to clinical trials of treatment that manipulate microRNA function.
Heparan Sulfate (HS) is involved in chronic inflammation and fibrosis development by binding proteins, enhancing their activity and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. HS 3-O-sulfation is a rare but functionally highly relevant modification. However, its role in kidney disease is still unclear. Using a mouse model of renal fibrosis, we found that total HS O-sulfation was increased in damaged kidneys. Interestingly, HS 3-O-sulfotransferase 1 (HS3ST1) expression strongly correlated with the development of fibrosis. HS3ST1 was the most abundantly expressed HS 3-O-sulfotransferase in primary tubular epithelial cells. To determine the implication of HS3ST1 in growth factor binding and signaling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signaling.
Antibody-mediated rejection is one of the major causes of chronic allograft injury. This is mediated by endothelial cell activation, microvascular inflammation and leukocyte recruitment. Despite evidence pointing to the relevance of HLA class II antibody in graft rejection, the role of antibody-endothelium interaction in the absence of complement is not fully understood. We showed that exposure of endothelial cells to HLA class II antibody induces an activation of signalling cascades characteristic of endothelial cell activation. This activation is further responsible for upregulation of adhesion molecules, which facilitate the interaction of donor endothelium and recipient leukocytes. Strategies to block endothelium-leukocyte interaction might reduce the incidence of allograft rejection and improve allograft survival.
In summary this programme of work has gone some way towards increasing our understanding of the mechanisms involved in graft rejection and has suggested strategies to increase the survival of transplanted organs. We have also identified potential biomarkers both of IRI and chronic rejection which could allow transplant patients to be stratified allowing individualized drug treatment.
Impact: This project has generated significant laboratory know-how and expertise around post-translationally modified chemokines, microRNA analysis and Heparan sulphation involved in transplant rejection. We have also identified potential biomarkers of IRI and fibrosis (microRNA & HS-3-O sulphation). Engagement with the Newcastle University Research & Enterprise Services (RES) has been established who are supporting the next phase of translational development.
Furthermore, the ESR’s have developed research skills at the forefront of Immunobiology, with specific relevance for investigation of transplantation and other inflammatory diseases.
Dissemination of information to the general public, the scientific community and stakeholders has been an important part of POSAT networking. More information on POSAT activities can be found at the POSAT’s own website (https://research.ncl.ac.uk/fp7posat/)
Through the website, POSAT has promoted interdisciplinary interaction as well as facilitated the spreading of knowledge. During POSAT training the ESR’s have maintained a high profile in the field of transplantation biology through publications, conferences and were also involved in promoting outreach activities. Further impact is the training of 4 world-class early career scientists who will continue to work in this field to the benefit of the European area.
Project website: https://research.ncl.ac.uk/fp7posat/