Final Report Summary - TRANSVASCED (Celiac Disease autoantibodies targeted against transglutaminase 2: repercussions in vascular biology, disease pathogenesis and treatment)
Summary of the project
Celiac disease (CD) is a common heritable autoimmune disorder triggered by gluten-containing cereals which induce a massive immune reaction and destruction of the small bowel mucosa. The disease is hallmarked by the production of antibodies against transglutaminase 2 (TG2), an enzyme involved also in angiogenesis. Interestingly CD presents an entire disorganization of the intestinal vasculature where the TG2 autoantibodies are deposited. We recently demonstrated that anti-TG2 antibodies can disrupt and affect the vascular functionality in vitro. Therefore we aimed to study whether antibodies against TG2 can affect vasculature in vivo by increasing TG2 enzymatic activity affecting the extracellular matrix composition and signaling intracellularly. In addition, the inhibition of TG2 enzymatic activity and other molecular pathways can ameliorate the anti-angiogenic effect of CD autoantibodies, offering a potential therapeutical target ordered by the primary users of our study, patients. To achieve this question, three main goals were proposed:
To study the in vivo role of celiac type antibodies against transglutaminase 2 on vascular biology (aim 1.A) and functionality (aim 1.B) as well the alterations on vascular permeability and lymphocyte transmigration (aim 1.C). To achieved the present aim, a group of mice were inoculated with celiac type antibodies or controls (provided by Professors Marzari and Sblattero, Trieste University, Italy) and the resulting vascular development were studied together with vascular functionality and permeability by positron emission tomography (in collaboration with Professor Roivainen, Turku Pet Center, Finland). In this context the endothelial joints and vascular integrity of the vessels were studied by electron microscopy (in collaboration with Professor Pelliniemi, Turku University, Finland). Finally the capacity of the lymphocytes to transmigrate across the vessel wall were also evaluated.
To study the molecular mechanisms exerted by celiac type antibodies in TG2 enzymatic activity (aim 2.A) and extracellular matrix composition (aim 2.B) together with molecular pathways and cross talk between extracellular and intracellular compartments using gene array (aim 2.C.i) and peptidomics (aim 2.C.ii). To achieve current aim a group of mice were inoculated with celiac type antibodies (provided by Professors Marzari and Sblattero, Trieste University, Italy) and the enzymatic activity of Transglutaminase 2 was measured. In parallel the extracellular matrix composition was studied by proteomics (in collaboration with Professor Vihinen, Tampere University, Finland) and the cross-talk between the extracellular environment and intercellular was carry out by an array of 116 genes. Twelve candidates were selected for further analysis and one of the proteins found was clearly linked to the changes in vasculature as well as some of the regulatory pathways involved were studied together with some cellular signals.
To study the potential therapeutical effect of the inhibition of the enzymatic activity of TG2 in the presence of CD autoantibodies (aim 3.A) and the change in the composition of the ECM components (aim 3.B) and other downstream molecular pathways (aim 3.C) as mechanisms to improve the vascular response. To achieve the aim a group of mice were inoculated with celiac type antibodies (provided by Professors Marzari and Sblattero, Trieste University, Italy), pro-angiogenic factors together with inhibitors of the enzymatic activity of transglutaminase 2 (provided by Professor Griffin, Aston University, UK). Thus, the enzyme activity was blocked and the downstream effect on vascular formation in the presence of celiac type autoantibodies was studied. Other drugs such as statins were used since they affect some of the proteins and the regulatory pathways found under aim 2. Under this condition, the capacity of the drug was successfully evaluated in vitro.
During the months of the TRANSVASCED project some significant results have been achieved. From a scientific point of view, we had been demonstrated for first time that antibodies derived from celiac patients inhibit angiogenesis in vivo (figure 1 from attached file). In this context, 12 different molecular candidates were characterized and one protein RhoB was selected for further study (for more information see figures 2 and 3 in attached file). Thus, when antibodies agains tranglutaminase 2 are present, the levels of RhoB in endothelial cells are up-regulated. Thus we found a link between the up-regulation of the protein and the anti-angiogenic features found. In addition the silencing of the protein can rescue the anti-angiogenic effect elicited by the antibodies opening a window for therapeutical interventions. In fact similar effects were found by the used of statins, drugs that can modulated the levels of RhoB, exerting similar actions than those described for the silencing of the protein. In other words, we found a potential candidate protein and a group of drugs suitable for the treatment of patients as alternative to gluten free diet. All previous in vitro data were complemented with in vivo, and therefore the presence of sinvastatin could rescue the angiogenic defect found after the treatment with celiac type autoantibodies.
Interestingly, we noticed that celiac type endothelial cells either during active disease or disease remission they expressed an anti-angiogenic phenotype in comparison to controls and this feature was preserved even after a long period of gluten free diet (when patients has no symptoms). Thus, using 10 blood samples from a biobank collected by a collaborative project between Celiac disease study group in Finland and a clinician in Romania (all ethicals as well as consents for secondary use of samples were obtained), we studied a profile of angiogenic proteins and we found that 33 different proteins where similar between patients during active disease or in a gluten free diet but different to healthy controls. This panel of proteins was verified using a bigger population collected by the celiac disease study group (the samples were serum and all ethical approvals as well as the consent for their use were obtained). As a result of this 4 proteins were selected at the most interesting candidates to be used in clinics in order to diagnose the disease in a very early format. As a result the disease biomarkers found may represent a big step forward in the recognition of the disease (currently the only way to detect the disease is when patient eat gluten and has symptoms and therefore elevation of all the markers). Thus the current concept was proof and Kolster Oy (a Finish company experts in property rights) evaluated the case and they recommended to get the property rights upon the utilization of the proteins described. The innovation therefore, was disclosure to the University who gave its approval. Unfortunately we were not succeed to get funding to cover the expenses of the patent either in Finland and in EU.
The present project combines the study of transglutaminase biology and extracellular matrix together with celiac disease merging the knowledge between molecular biology and relevant clinical aspects of the disease. Thus, as result the entire celiac community, especially the patients are benefited directly of the achievements of present project. In fact during the project we found relevant molecular players having a role on disease but also opening a window in therapeutical interventions. More interesting even we found new biomarkers which can be use as early detection biomarkers and therefore the patients will be diagnose faster and more efficient in comparison to current methodologies. On the other hand, and of less importance several publications have been released in scientific journals and congresses, but also on TV. For example the researcher was interviewed by the spanish national TV about celiac disease and rapid methods of detection, giving the information of the disease, but also contributing with the common knowledge of the disease in population. All this actions have a clear impact on patients.
Celiac disease (CD) is a common heritable autoimmune disorder triggered by gluten-containing cereals which induce a massive immune reaction and destruction of the small bowel mucosa. The disease is hallmarked by the production of antibodies against transglutaminase 2 (TG2), an enzyme involved also in angiogenesis. Interestingly CD presents an entire disorganization of the intestinal vasculature where the TG2 autoantibodies are deposited. We recently demonstrated that anti-TG2 antibodies can disrupt and affect the vascular functionality in vitro. Therefore we aimed to study whether antibodies against TG2 can affect vasculature in vivo by increasing TG2 enzymatic activity affecting the extracellular matrix composition and signaling intracellularly. In addition, the inhibition of TG2 enzymatic activity and other molecular pathways can ameliorate the anti-angiogenic effect of CD autoantibodies, offering a potential therapeutical target ordered by the primary users of our study, patients. To achieve this question, three main goals were proposed:
To study the in vivo role of celiac type antibodies against transglutaminase 2 on vascular biology (aim 1.A) and functionality (aim 1.B) as well the alterations on vascular permeability and lymphocyte transmigration (aim 1.C). To achieved the present aim, a group of mice were inoculated with celiac type antibodies or controls (provided by Professors Marzari and Sblattero, Trieste University, Italy) and the resulting vascular development were studied together with vascular functionality and permeability by positron emission tomography (in collaboration with Professor Roivainen, Turku Pet Center, Finland). In this context the endothelial joints and vascular integrity of the vessels were studied by electron microscopy (in collaboration with Professor Pelliniemi, Turku University, Finland). Finally the capacity of the lymphocytes to transmigrate across the vessel wall were also evaluated.
To study the molecular mechanisms exerted by celiac type antibodies in TG2 enzymatic activity (aim 2.A) and extracellular matrix composition (aim 2.B) together with molecular pathways and cross talk between extracellular and intracellular compartments using gene array (aim 2.C.i) and peptidomics (aim 2.C.ii). To achieve current aim a group of mice were inoculated with celiac type antibodies (provided by Professors Marzari and Sblattero, Trieste University, Italy) and the enzymatic activity of Transglutaminase 2 was measured. In parallel the extracellular matrix composition was studied by proteomics (in collaboration with Professor Vihinen, Tampere University, Finland) and the cross-talk between the extracellular environment and intercellular was carry out by an array of 116 genes. Twelve candidates were selected for further analysis and one of the proteins found was clearly linked to the changes in vasculature as well as some of the regulatory pathways involved were studied together with some cellular signals.
To study the potential therapeutical effect of the inhibition of the enzymatic activity of TG2 in the presence of CD autoantibodies (aim 3.A) and the change in the composition of the ECM components (aim 3.B) and other downstream molecular pathways (aim 3.C) as mechanisms to improve the vascular response. To achieve the aim a group of mice were inoculated with celiac type antibodies (provided by Professors Marzari and Sblattero, Trieste University, Italy), pro-angiogenic factors together with inhibitors of the enzymatic activity of transglutaminase 2 (provided by Professor Griffin, Aston University, UK). Thus, the enzyme activity was blocked and the downstream effect on vascular formation in the presence of celiac type autoantibodies was studied. Other drugs such as statins were used since they affect some of the proteins and the regulatory pathways found under aim 2. Under this condition, the capacity of the drug was successfully evaluated in vitro.
During the months of the TRANSVASCED project some significant results have been achieved. From a scientific point of view, we had been demonstrated for first time that antibodies derived from celiac patients inhibit angiogenesis in vivo (figure 1 from attached file). In this context, 12 different molecular candidates were characterized and one protein RhoB was selected for further study (for more information see figures 2 and 3 in attached file). Thus, when antibodies agains tranglutaminase 2 are present, the levels of RhoB in endothelial cells are up-regulated. Thus we found a link between the up-regulation of the protein and the anti-angiogenic features found. In addition the silencing of the protein can rescue the anti-angiogenic effect elicited by the antibodies opening a window for therapeutical interventions. In fact similar effects were found by the used of statins, drugs that can modulated the levels of RhoB, exerting similar actions than those described for the silencing of the protein. In other words, we found a potential candidate protein and a group of drugs suitable for the treatment of patients as alternative to gluten free diet. All previous in vitro data were complemented with in vivo, and therefore the presence of sinvastatin could rescue the angiogenic defect found after the treatment with celiac type autoantibodies.
Interestingly, we noticed that celiac type endothelial cells either during active disease or disease remission they expressed an anti-angiogenic phenotype in comparison to controls and this feature was preserved even after a long period of gluten free diet (when patients has no symptoms). Thus, using 10 blood samples from a biobank collected by a collaborative project between Celiac disease study group in Finland and a clinician in Romania (all ethicals as well as consents for secondary use of samples were obtained), we studied a profile of angiogenic proteins and we found that 33 different proteins where similar between patients during active disease or in a gluten free diet but different to healthy controls. This panel of proteins was verified using a bigger population collected by the celiac disease study group (the samples were serum and all ethical approvals as well as the consent for their use were obtained). As a result of this 4 proteins were selected at the most interesting candidates to be used in clinics in order to diagnose the disease in a very early format. As a result the disease biomarkers found may represent a big step forward in the recognition of the disease (currently the only way to detect the disease is when patient eat gluten and has symptoms and therefore elevation of all the markers). Thus the current concept was proof and Kolster Oy (a Finish company experts in property rights) evaluated the case and they recommended to get the property rights upon the utilization of the proteins described. The innovation therefore, was disclosure to the University who gave its approval. Unfortunately we were not succeed to get funding to cover the expenses of the patent either in Finland and in EU.
The present project combines the study of transglutaminase biology and extracellular matrix together with celiac disease merging the knowledge between molecular biology and relevant clinical aspects of the disease. Thus, as result the entire celiac community, especially the patients are benefited directly of the achievements of present project. In fact during the project we found relevant molecular players having a role on disease but also opening a window in therapeutical interventions. More interesting even we found new biomarkers which can be use as early detection biomarkers and therefore the patients will be diagnose faster and more efficient in comparison to current methodologies. On the other hand, and of less importance several publications have been released in scientific journals and congresses, but also on TV. For example the researcher was interviewed by the spanish national TV about celiac disease and rapid methods of detection, giving the information of the disease, but also contributing with the common knowledge of the disease in population. All this actions have a clear impact on patients.
