Final Report Summary - INFLAMA (Matrix Macromolecules in inflammation)
COVER PAGE
FINAL REPORT
IRSES PROJECT
“Matrix Molecules in Inflammation”
Acronym: INFLAMA”
Proposal Number: 247516
ACTIVITY REPORT
The role of carbohydrates in different aspects of biology is remarkable and increasing body of evidences supports the contribution of these molecules in specific cell and tissue functions. Over the last years the technical achievements in protein and nucleic acid research allowed exceptional results that are not comparable with the level of knowledge in glycobiology. Even if the scientific community introduced a new “omic” in carbohydrates, the glucomic, the level of technologies available is still improving. The main problem is due the extraordinary complexity of the carbohydrates, ranging from simple single molecule to huge polymers of millions of Daltons.
From these starting point this project proposes to support the diffusion of the sophisticated technologies for carbohydrate biochemistry, building a network of researchers of different laboratories which started over the last years collaborations in research and teaching. Our specific interest is in the role of matrix polysaccharides (hyaluronan and proteoglycans) in inflammation. This “gluco-net” is composed by 4 different laboratories leaded by scientists involved in PhD programs as well as in several project grants as PIs and are located in different continents: South America (Universidad Federal de Rio de Janeiro, Brazil), North America (University of Harvard, Boston USA), Europe (Wilhelms Universitaet Muenster Germany and Insubria University Varese, Italy).
The principal aim of this proposal is the preparation of very qualified gluco-biotechnologists and scientists in glucomics, able to address the studies and the analysis of carbohydrates in the academic research or in the development of new molecules of pharmaceutical interest. For these purposes we developed a project which addresses the question concerning the cellular reaction to exposure of mediators of early inflammation and the proteoglycans and glycosaminoglycan metabolism. The rationale of our work is based on the preliminary studies that we have carried out using different in vivo and in vitro models at elucidating inflammation-dependent changes in human primary cell cultures and animal tissues. The research projects carried out in our laboratories was able to shed light on the molecular mechanisms involved in the anti inflammatory, antithrombotic and anti metastatic effect of unique heparin analogs previously isolated and characterized in our labs, and besides these aspects, it was addressed the hyaluronan synthesis control in inflammation using in vitro models. The signaling mediated by proteoglycans was also studied demonstrating how critical are these macromolecules on cell membrane in inflammation triggered stimuli. It was also demonstrated that invertebrate heparin analogs are also potent inhibitors of L- and P-selectins, and as a consequence of this activity they drastically attenuates inflammation and metastasis.
The project started December 1st 2011 and expired November 30th 2014. In the 36 months of the program the scientists involved worked applying the techniques necessary for the glycosaminoglycans purifications and all analyses were correctly carried out sharing techniques procedures. All the experiments were carried out using biochemical and molecular biology approaches and the gene expression pattern of all genes involved in the biological processes studies, have been evaluated. Another important aspect was addressed in this project related to the O-GlcNAcylation. In fact it was demonstrated that several enzymes involved in biological processes are regulated by O-GlcNAcylation and this process seems to be related to hexosamine pathway, one of the metabolic markers of diabetes and a pro inflammatory condition.
An important achievement was obtained by the study on glycans purified from adult specimens of the ascidians Styela plicata, Asidia nigra, collected in Rio de Janeiro at Guanabara Bay.
Experiments on P-selectin KO mice on a C57/BL6 background and control wild-type (WT) mice will be carried out. The care and use of animals, as well as procedures reported in this study were approved by the institutional care committee of the Federal University of Rio de Janeiro and are in accordance with the guidelines of the International Care and Use Committee of the National Institutes of Health, and Guide for the Care and Use of Laboratory Animals. In the animals will be induced colitis and some of them treated with glycans purified from Styela plicata, Asidia nigra. The purified material was used for animal treatments. The deliverables for this package are the purification and characterization of the glycosaminoglycans from Styela plicata and Asidia nigra and their effect on mice colitis are in attached files.
Moreover data on inflammation and matrix synthesis, with particular emphasis on arteriosclerosis, have been achieved. We were able to demonstrate that oxidation of LDL is critical for matrix alterations and hyaluronan production. The data of animal model confirmed the assumption that the increase of arterial wall thickness is due to the cell migration and proliferation coupled with hyaluronan production. The epigenetic control even in diabetes affect this metabolism causing the outcome of arteriosclerosis. The contribution of this project improved the knowledge of this pathology at molecular level opening new perspectives not only in terms of scientific knowledge but also as therapeutically effective strategies.
The importance of heparin from Styela plicata which in the presence and absence of TNFalpha results in complex changes in inflammatory cytokine and adhesion molecule expression. The experiments highlighted the potentially useful polymer in human inflammation. The inflammatory response induced by the strong proinflammatory stimulus in cells and animal models has been considerably reduced by S. plicata heparin treatment. In the same context the role of proteoglycan syndecan 1 has been addressed demonstrating that this molecules is involved in leukocytes adhesion and therefore in inflammation development. The role of syndecan 1 was also demonstrated critical for cell migration and infiltration in vitro and in vivo as well as influencing the FGF stimulatory activity. Additionally Syndecan-1 exhibits a regulatory loop with heparanase, resulting in a differential modulation of colon epithelial cell transformation and invasiveness in vitro dependent on the inflammatory microenvironment. The effect of syndecan 1 on MAPK and FGF2 activity was also clarified and its role discussed. A critical sulphation pattern was identified in heparin sulphate chains of membrane proteoglycans. This is an intriguing aspect in the biomarker research characterizing the cancer invasiveness. The cancer model demonstrated the critical function of this proteoglycan in cancer development including exosome production.
All the deliverables have been collected and are used for publications and several international meeting were these data are presented from platform and discussed. In this context was remarkable the contribution of young scientists who presented in several occasion in international meetings their results from platform obtaining young scientists awards (FEBS-Meeting Kos 2013). Moreover the achievements of the groups have been described in peer reviewed papers and in theperiod the number of papers published by the participants is 54.
As far as the knowledge on biological, psychological, socio-economical and gender factors in concerned, that could promote healthy ageing and postpone the onset of disability, the work in this programme aims at creating critical mass in basic research concerning cellular and molecular mechanisms underlying human inflammation. From a socio-economic point of view, it is important that the cost required by public hospitals, clinics and medical care units could be decreased or strongly reduced as widely as the today suggests. For the time being, the treatment of inflammation related diseases as well as the diagnosis lean on very expensive surgical methods, equipment and devices. Furthermore, nothing or very few data are available for the prediction of the earliest stages of the disease. The present research intends to give answers to the above mentioned points. In order to achieve this goal, however, it is necessary to reinforce the scientific and technological tools, in support of EU policies, at European level, combining the necessary, although very different competences required to gain the deep knowledge as it is expected by our exciting preliminary results
The major point of this project is the training of new biotechnologists with a strong expertise in carbohydrate biochemistry, and these professional figures are very important in a new point of view of matrix biology.
FINAL REPORT
IRSES PROJECT
“Matrix Molecules in Inflammation”
Acronym: INFLAMA”
Proposal Number: 247516
ACTIVITY REPORT
The role of carbohydrates in different aspects of biology is remarkable and increasing body of evidences supports the contribution of these molecules in specific cell and tissue functions. Over the last years the technical achievements in protein and nucleic acid research allowed exceptional results that are not comparable with the level of knowledge in glycobiology. Even if the scientific community introduced a new “omic” in carbohydrates, the glucomic, the level of technologies available is still improving. The main problem is due the extraordinary complexity of the carbohydrates, ranging from simple single molecule to huge polymers of millions of Daltons.
From these starting point this project proposes to support the diffusion of the sophisticated technologies for carbohydrate biochemistry, building a network of researchers of different laboratories which started over the last years collaborations in research and teaching. Our specific interest is in the role of matrix polysaccharides (hyaluronan and proteoglycans) in inflammation. This “gluco-net” is composed by 4 different laboratories leaded by scientists involved in PhD programs as well as in several project grants as PIs and are located in different continents: South America (Universidad Federal de Rio de Janeiro, Brazil), North America (University of Harvard, Boston USA), Europe (Wilhelms Universitaet Muenster Germany and Insubria University Varese, Italy).
The principal aim of this proposal is the preparation of very qualified gluco-biotechnologists and scientists in glucomics, able to address the studies and the analysis of carbohydrates in the academic research or in the development of new molecules of pharmaceutical interest. For these purposes we developed a project which addresses the question concerning the cellular reaction to exposure of mediators of early inflammation and the proteoglycans and glycosaminoglycan metabolism. The rationale of our work is based on the preliminary studies that we have carried out using different in vivo and in vitro models at elucidating inflammation-dependent changes in human primary cell cultures and animal tissues. The research projects carried out in our laboratories was able to shed light on the molecular mechanisms involved in the anti inflammatory, antithrombotic and anti metastatic effect of unique heparin analogs previously isolated and characterized in our labs, and besides these aspects, it was addressed the hyaluronan synthesis control in inflammation using in vitro models. The signaling mediated by proteoglycans was also studied demonstrating how critical are these macromolecules on cell membrane in inflammation triggered stimuli. It was also demonstrated that invertebrate heparin analogs are also potent inhibitors of L- and P-selectins, and as a consequence of this activity they drastically attenuates inflammation and metastasis.
The project started December 1st 2011 and expired November 30th 2014. In the 36 months of the program the scientists involved worked applying the techniques necessary for the glycosaminoglycans purifications and all analyses were correctly carried out sharing techniques procedures. All the experiments were carried out using biochemical and molecular biology approaches and the gene expression pattern of all genes involved in the biological processes studies, have been evaluated. Another important aspect was addressed in this project related to the O-GlcNAcylation. In fact it was demonstrated that several enzymes involved in biological processes are regulated by O-GlcNAcylation and this process seems to be related to hexosamine pathway, one of the metabolic markers of diabetes and a pro inflammatory condition.
An important achievement was obtained by the study on glycans purified from adult specimens of the ascidians Styela plicata, Asidia nigra, collected in Rio de Janeiro at Guanabara Bay.
Experiments on P-selectin KO mice on a C57/BL6 background and control wild-type (WT) mice will be carried out. The care and use of animals, as well as procedures reported in this study were approved by the institutional care committee of the Federal University of Rio de Janeiro and are in accordance with the guidelines of the International Care and Use Committee of the National Institutes of Health, and Guide for the Care and Use of Laboratory Animals. In the animals will be induced colitis and some of them treated with glycans purified from Styela plicata, Asidia nigra. The purified material was used for animal treatments. The deliverables for this package are the purification and characterization of the glycosaminoglycans from Styela plicata and Asidia nigra and their effect on mice colitis are in attached files.
Moreover data on inflammation and matrix synthesis, with particular emphasis on arteriosclerosis, have been achieved. We were able to demonstrate that oxidation of LDL is critical for matrix alterations and hyaluronan production. The data of animal model confirmed the assumption that the increase of arterial wall thickness is due to the cell migration and proliferation coupled with hyaluronan production. The epigenetic control even in diabetes affect this metabolism causing the outcome of arteriosclerosis. The contribution of this project improved the knowledge of this pathology at molecular level opening new perspectives not only in terms of scientific knowledge but also as therapeutically effective strategies.
The importance of heparin from Styela plicata which in the presence and absence of TNFalpha results in complex changes in inflammatory cytokine and adhesion molecule expression. The experiments highlighted the potentially useful polymer in human inflammation. The inflammatory response induced by the strong proinflammatory stimulus in cells and animal models has been considerably reduced by S. plicata heparin treatment. In the same context the role of proteoglycan syndecan 1 has been addressed demonstrating that this molecules is involved in leukocytes adhesion and therefore in inflammation development. The role of syndecan 1 was also demonstrated critical for cell migration and infiltration in vitro and in vivo as well as influencing the FGF stimulatory activity. Additionally Syndecan-1 exhibits a regulatory loop with heparanase, resulting in a differential modulation of colon epithelial cell transformation and invasiveness in vitro dependent on the inflammatory microenvironment. The effect of syndecan 1 on MAPK and FGF2 activity was also clarified and its role discussed. A critical sulphation pattern was identified in heparin sulphate chains of membrane proteoglycans. This is an intriguing aspect in the biomarker research characterizing the cancer invasiveness. The cancer model demonstrated the critical function of this proteoglycan in cancer development including exosome production.
All the deliverables have been collected and are used for publications and several international meeting were these data are presented from platform and discussed. In this context was remarkable the contribution of young scientists who presented in several occasion in international meetings their results from platform obtaining young scientists awards (FEBS-Meeting Kos 2013). Moreover the achievements of the groups have been described in peer reviewed papers and in theperiod the number of papers published by the participants is 54.
As far as the knowledge on biological, psychological, socio-economical and gender factors in concerned, that could promote healthy ageing and postpone the onset of disability, the work in this programme aims at creating critical mass in basic research concerning cellular and molecular mechanisms underlying human inflammation. From a socio-economic point of view, it is important that the cost required by public hospitals, clinics and medical care units could be decreased or strongly reduced as widely as the today suggests. For the time being, the treatment of inflammation related diseases as well as the diagnosis lean on very expensive surgical methods, equipment and devices. Furthermore, nothing or very few data are available for the prediction of the earliest stages of the disease. The present research intends to give answers to the above mentioned points. In order to achieve this goal, however, it is necessary to reinforce the scientific and technological tools, in support of EU policies, at European level, combining the necessary, although very different competences required to gain the deep knowledge as it is expected by our exciting preliminary results
The major point of this project is the training of new biotechnologists with a strong expertise in carbohydrate biochemistry, and these professional figures are very important in a new point of view of matrix biology.
