Marine Polysaccharides: Fucoidan, Alginate and Chitosan – An Investigation of Physical, Chemical and Bioactive Properties

The marine polysaccharides fucoidan, alginate and chitosan are garnering significant research interest in the field of biomedical applications due to their broad range of bioactivities, compatibility with living tissues and their ability to form hydrogels. This thesis presents in-depth investigations of the chemical, physical, and bioactive properties, and potential applications of these marine polysaccharides. The chemical structure of fucoidan from Laminaria hyperborea was elucidated utilizing numerous analytical techniques, including NMR spectroscopy, SEC-MALS, Raman and IR spectroscopy, ICP-MS and glycosidic linkage analysis. Highly purified fucoidan was used to investigate bioactivity in vitro using a lepirudin-based human whole blood model as well as human-derived cell lines from uveal melanoma, retinal pigment epithelium and porcine-derived primary cells from retinal pigment epithelium. Structural features such as the degree of sulfation and molecular weight were tailored to enable elucidation of structure–function relationships. Unmodified fucoidan with a high degree of sulfation and molecular weight was demonstrated to inhibit coagulation at intermediate to high concentrations, showing similar inhibitory effects on the complement system. Experiments on the ocular cell lines unveiled a positive relationship between molecular weight, bioactivity, and potential therapeutic effects for highly sulfated fucoidan. The work underscores the importance of both molecular weight and the degree of sulfation as key parameters for bioactive properties. Alginate hydrogels which are attractive biomaterials for applications such as cell encapsulation, tissue engineering, coatings, and drug delivery, were examined on a fundamental scientific level. Through CD spectroscopy and molecular dynamic simulations, we obtained strong evidence supporting a previously hypothesised zipper-like junction zone formation between alginate M-blocks and chitosan. Furthermore, we developed and investigated a novel alginate-chitosan gelling system. This system relies upon crosslinks between poly-MG-blocks and chitosan oligomers and can enable gel strengths orders of magnitude higher than those observed in poly-M-chitosan hydrogels. Rheological measurements and molecular dynamic simulations suggested a fundamentally different chain–chain interaction mechanism involving localized phase-separation. Gels made from alginates with different block structures were crosslinked with combinations of calcium and chito-oligosaccharides. We found that up to 50% of Ca2+ can be replaced by chitosan oligomers (under specific conditions) without compromising gel strength or the occurrence of syneresis, while simultaneously increasing the rate of gelation. The inclusion of chitosan oligomers in the gelation mechanism allows for customizing gelling kinetics and may enhance hydrogel stability against electrolytes, a factor of great importance in biomedical applications

The Role of mechanical stress in progression of intima hyperplasia associated to vein coronary bypass grafts disease

Coronary artery bypass grafting is a surgical procedure introduced to restore the blood circulation into the myocardium after an ischemic event. Despite progress in the use of arterial conduits, saphenous vein (SV) remains one of the most used vessels for the bypass. A short time after bypass implantation SV undergoes intima hyperplasia (IH) that progressively reduce its patency. One trigger cause of IH is the hemodynamic changes in the blood flow with higher sheer stress on the endothelial layer a radial deformation on the wall of the vein.The aim of this project was to understand the role of saphenous vein progenitors (SVP) in the progression of IH. These cells with high differentiation potential are the pericytes of vasa vasorum in the tunica adventitia. By in vitro and ex vivo models of mechanical stress we demonstrated the susceptibility of SVPs to the strain that causes a cytoskeletal reorganization and the acquisition of a potential migratory phenotype. SVPs showed the stimulus-related up-regulation of Amphoterin-Induced Gene And Open Reading Frame 2 (AMIGO2), that may have a role in the mechanical activation via prosurvival and migratory effects. For the first time has been described the presence of AMIGO2 in SVPs and its relationship with mechanical stress. Migratory phenotype acquisition and AMIGO2 overexpression demonstrate how SVPs are potential targets for further study of IH

Regulated cell death in the pathogenesis of renal ciliopathies

The primary cilium is an evolutionary conserved sensory organelle present on most mammalian cell types. In the kidney, cilia project from the apical surface of tubular epithelial cells. Defects in the structure or function of primary cilia lead to ciliopathies, such as autosomal dominant and recessive Polycystic Kidney Disease (ADPKD/ARPKD), and several genetic syndromes, including Nephronophthisis (NPH), Joubert Syndrome (JBTS) or Bardet-Biedl Syndrome (BBS). These syndromes display overlapping symptoms in different organs and tissues, such as retinopathy, polydactyly, neuronal developmental disorders or obesity, and commonly exhibit development of (poly-)cystic kidney disease. The cystic kidney disease observed in NPH and NPH-related ciliopathies (NPH-RC), such as JBTS or BBS, develops during childhood and adolescence and is accompanied by a massive loss of epithelial cells, as well as inflammation and interstitial fibrosis. This thesis, therefore, follows the hypothesis that regulated cell death (RCD) pathways play a role in the pathogenesis of the kidney phenotype in NPH and NPH-RC, and investigates the bidirectional interconnection between RCD and the primary cilium, as well as the role of RCD in NPH/NPH-RC. In the first part, we studied how loss of primary cilia would affect the RCD response in murine inner medullary collecting duct cells. This revealed increased expression of the necroptosis key regulator receptor-interacting protein kinase 3 (RIPK3) in cells lacking primary cilia, and increased phosphorylation of the mixed lineage kinase domain-like pseudokinase (MLKL) suggesting elevated necroptosis. In summary, cells lacking primary cilia were prone to undergo necroptosis upon induction of cell death, which was not observed in ciliated cells. This resulted in the first conclusion that the absence of primary cilia increases susceptibility to necroptotic cell death. Conversely, the presence of cilia to some extent may offer protection against necroptosis. In the second part, we aimed to understand the contribution of RCD pathways to the pathogenesis of cystic kidney disease in the well-established Nphp9/Nek8jck mouse model, in which a point mutation in the Nphp9/Nek8jck gene leads to a severe and early on-set cystic kidney disease. Crossing this mouse with a conventional knockout of Ripk3 led to an amelioration of cystic kidney disease and kidney function. Notably, this double knockout led to an upregulation of key pyroptotic regulators such as the NLR family pyrin domain containing 3 (NLRP3), Caspase-11 or Gasdermin D (GSDMD). Consistently, the deletion of GsdmD a key regulator of pyroptosis in the Nphp9/Nek8jck mouse model also improved the phenotype and function of the kidney. In summary, the in vivo data demonstrate that necroptosis, and to a certain extent pyroptosis and the inflammasome, contribute to the loss of kidney function in the studied ciliopathy model. In the third part of this thesis, we present a mouse model in which the deletion of the Bbs gene, Bbs8, results in the development of a kidney phenotype, in particular, tubule cystic kidney disease. Our data support the hypothesis that in the kidneys of the Bbs8 deficient mice pyroptosis and fibrosis is expressed, without the regulation of necroptosis. Mechanistically, loss of BBS8 resulted in increased expression and activity of the histone deacetylase 2 (HDAC2), which in turn destabilized ciliary microtubules by deacetylation of acetylated alpha-tubulin. In conclusion, the primary cilium exhibits a protective function to prevent RCD, particularly necroptosis and pyroptosis, and both pathways contribute to cystic kidney disease. Future work will have to address to what extent RIPK3 and GSDMD might serve as potential therapeutic targets in NPH or NPH-RC

The Role of mechanical stress in progression of intima hyperplasia associated to vein coronary bypass grafts disease

Coronary artery bypass grafting is a surgical procedure introduced to restore the blood circulation into the myocardium after an ischemic event. Despite progress in the use of arterial conduits, saphenous vein (SV) remains one of the most used vessels for the bypass. A short time after bypass implantation SV undergoes intima hyperplasia (IH) that progressively reduce its patency. One trigger cause of IH is the hemodynamic changes in the blood flow with higher sheer stress on the endothelial layer a radial deformation on the wall of the vein. The aim of this project was to understand the role of saphenous vein progenitors (SVP) in the progression of IH. These cells with high differentiation potential are the pericytes of vasa vasorum in the tunica adventitia. By in vitro and ex vivo models of mechanical stress we demonstrated the susceptibility of SVPs to the strain that causes a cytoskeletal reorganization and the acquisition of a potential migratory phenotype. SVPs showed the stimulus-related up-regulation of Amphoterin-Induced Gene And Open Reading Frame 2 (AMIGO2), that may have a role in the mechanical activation via prosurvival and migratory effects. For the first time has been described the presence of AMIGO2 in SVPs and its relationship with mechanical stress. Migratory phenotype acquisition and AMIGO2 overexpression demonstrate how SVPs are potential targets for further study of IH

A Study of the Molecular Immunogenetics of Type 1 Diabetes in Man

Type 1 diabetes is caused by immune destruction of pancreatic β cells. There is increasing evidence that genes outside the MHC region contribute to the pathogenesis of type 1 diabetes. Cytokines due to their role in immune regulation seem to play a crucial role in the pathogenesis of the disease. Three hundred and eight patients with type 1 diabetes and 150 normal controls were genotyped for polymorphism in the genes for IFN-γ, IL-4, IL-6, and TGF-β1. All assays employed in this study were PCR based. The IFN-γ CA repeats was an octa-allelic repeat and the 3 / 3 genotype showed a significant association with type 1 diabetes (p=0.0001). The IL-4 C (-590) T polymorphism did not show a significant association with type 1 diabetes. The GG genotype of G (-174) C of the IL-6 gene polymorphism showed a strong association with the susceptibility towards type 1 diabetes (p= 0.002). The TC genotype of the TGF-β1 T (+869) C polymorphism also showed a significant association with type 1 diabetes (p= 0.003). The association of the 3 I 3 genotype of the IFNG CA repeats and no association of IL-4 C (-590) T polymorphism may support the idea of dominance of the TH1 cytokine profile and type 1 diabetes suggesting a cell mediated disease. The IL-6 G (-174) C result attests an existing hypothesis of the important role of IL-6 in the onset of type 1 diabetes and its development. Immunosuppression of the TGF-β1 may have been initiated after deviation of the TH1/TH2 cytokine milieu. The GC of the IL-6 G (-174) C and the TC of the TGF-β1 T (+869) C showed strong association with diabetic nephropathy. Haplotype studies showed that cytokine function might be as a result of a cytokine network rather than individual cytokines. Further, the genetic susceptibility may be influenced not only by genetic composition but by the gender of patients as well as age at onset of type 1 diabetes. In conclusion these results suggest a contribution of the IFNG CA repeats, the TGF-β1 T (+869) C, and the IL-6 G (-174) C to the genetic susceptibility of type l diabetes and may have future therapeutical values

Glycaemic control with Mesenchymal Stem Cells and Endothelial Progenitor Cells in an experimental model of pancreatic islet transplantation

Insulin-Dependent Diabetes Mellitus (IDDM or type 1) is an autoimmune, chronic disease characterised by hyperglycaemia, resulting from an inflammatory infiltration of the islets of Langerhans. The selective destruction of β-cells leads to a lower insulin secretion from the endocrine pancreas. The mainstay treatment for IDDM patients is chronic insulin injection. Although insulin therapy has dramatically reduced mortality from diabetes, patients often incur in complications such as nephropathy, neuropathy, angiopathy and retinopathy. Moreover, patients are risk severe and sometimes fatal hypoglycaemic events. Pancreas transplantation is currently the only therapeutic approach to restore normoglycaemia and maintain long-term glucose homeostasis; moreover, this procedure improves patients’ quality of life. An alternative to the replacement of the whole pancreas is the transplantation of islet of Langherans cells, isolated from donor pancreata and infused into the recipient’s liver via the portal vein. Compared to solid organ transplantation, the advantages of islet transplantation consist in a relatively simple surgical procedure with low incidence of peri-operative risks. Nevertheless, the particular structure of pancreatic islets resulted in being injured after the isolation procedure. However, the recurrence of immune response after transplantation and the diabetogenic and growth-stunting side effects of immunosuppressants are major challenges to the application of islet transplantation. In the last decade many studies have demonstrated the efficacy of cell therapy either with Mesenchymal Stem Cells (MSCs) or Endothelial Progenitor Cells (EPCs) treatment when co-transplanted with pancreatic islets. The first type of cells were reported to modulate the immune response in an allogeneic transplant, preventing the graft-versus-host disease (GVHD) and also improving graft function in the long-term by maintaining glucose homeostasis. The EPCs showed to have strong revascularization properties in several diseases, such as cardiovascular disorders, atherosclerosis and diabetes. This thesis aims to investigate the role of MSCs and EPCs in prolonging graft survival of pancreatic islet transplantation in a chemically induced rat model of type 1 diabetes in order to prolong the graft function and reach normoglycaemic levels in the long-term. We used a rat model to investigate the effect of MSCs and EPCs in combination with islets of Langerhans (700 IE + 500,000 EPCs and 700 IE + 500,000 MSCs) in a syngeneic and an allogeneic diabetic-induced rat model which underwent pancreatic islet transplantation via the portal vein. These types of transplants were compared with islet alone treatment (700 IE), either syngeneic or allogeneic. We obtained the reversal of the diabetic status in animals up to 6 months after the transplant when they had received islets and EPCs, and up to 75 days post transplant when they had received islets and MSC therapy. The glycaemic values were also confirmed by intraperitoneal glucose tolerance test measures for animals transplanted with IE and EPCs either in syngeneic or allogeneic models. From data obtained from molecular biology assays on ex vivo liver tissues deriving from transplanted animals, we observed that a regulation in the revascularization and angiogenesis genes (VEGF-A, ANG-1, PECAM-1, SDF-1) occurred. Thus, EPCs could act through a regulatory mechanism as shown by their angiogenic gene expression. These data suggested that both MSCs and EPCs were able to revascularize pancreatic islets and improve the syngeneic graft survival up to a complete healing in our diabetic animal model