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We examined the cellular responses to various particles injected into the knees and the intramedullary femoral cavities of rats in the presence of polymethyl-methacrylate (PMMA) plugs. The intra-articular particles were mainly ingested by synovial fibroblasts. Increased numbers of macrophages were not detected and there was only a slight increase in synovial thickness. Cellular responses in the intramedullary space were similarly mild and bone resorption around the PMMA plug did not occur. Bone formation was inhibited only by polyethylene particles. In contrast to current views, our study shows that wear particles per se do not initiate bone resorption.

P>Osteopontin (OPN) is a glycophosphoprotein with multiple intracellular and extracellular functions. In vitro, OPN enhances migration of mouse neutrophils and macrophages. In cancer, extracellular OPN facilitates migration of cancer cells via its RGD sequence. The present study was designed to investigate whether osteopontin is responsible for neutrophil and macrophage infiltration in human cancer and in particular in glioblastoma. We found that in vitro mouse neutrophil migration was RGD-dependent. In silico, we found that the OPN gene was one of the 5% most highly expressed genes in 20 out of 35 cancer microarray data sets in comparison with normal tissue in at least 30% of cancer patients. In some types of cancer, such as ovarian cancer, lung cancer and melanoma, the OPN gene was one of those with the highest expression levels in at least 90% of cancer patients. In glioblastoma, the most invasive type of brain tumours/glioma, but not in lower grades of glioma it was one of the 5% highest expressed genes in 90% of patients. In situ, we found increased protein levels of OPN in human glioblastoma versus normal human brain confirming in silico results. OPN protein expression was co-localized with neutrophils and macrophages. In conclusion, OPN in tumours not only induces migration of cancer cells but also of leucocytes

Cancer patients treated for venous thromboembolism with low molecular weight heparin (LMWH) have a better survival rate than patients treated with unfractionated heparin (UFH). Because fibrin-associated angiogenesis is an important determinant in the progression and metastasis of many solid tumors, the effects of heparins on in vitro angiogenesis were investigated. Both UFH and LMWH inhibited bFGF-induced proliferation of human microvascular endothelial cells (hMVECs) to the same the extent (36-60%). VEGF(165)-induced proliferation was inhibited to a to a lesser extent (19-33%). Turbidity measurements and electron microscopy showed that the presence of LMWH during polymerization of the fibrin matrix led to a more transparent rigid network with thin fibrin bundles, whereas the presence of UFA resulted in a more opaque more porous network with thick fibrin fibers, We used a human in vitro angiogenesis model, which consisted of hMVECs seeded on top of a fibrin matrix, and stimulated the cells with basic fibroblast growth factor plus tumor necrosis factor Lu to induce capillary-like tubular structures, The formation of capillary-like tubular structures was retarded with matrices polymerized in the presence of LMWH (46% inhibition compared with a control matrix for both 1.5 and 10 units/ml LMWH), whereas matrices polymerized in the presence of UFH facilitated tubular structure formation (72 and 36% stimulation compared with a control matrix for 1.5 and 10 units/ml UFH, respectively). Similar results were obtained for cells stimulated with vascular endothelial growth factor plus tumor necrosis factor alpha. These data demonstrate the inhibitory effect of heparins on proliferation of hMTECs and provide a novel mechanism by which LMWH may affect tumor progression, namely reduced ingrowth of microvascular structures in a fibrinous stroma matrix by rendering it less permissive for invasion

Cells release small particles, which are called extracellular vesicles (EVs). EVs are present in human body fluids, but their function is unknown. We investigated why normal human body fluids contain EVs, and how such EVs may play a role in physiology. We show that EVs present in human milk and other body fluids expose a protein called tissue factor (TF). TF triggers the clotting of blood, which helps to stop bleeding and reduces the risk of infection. The presence of these TF-exposing EVs in milk explains observations from the 1930-ies that bleeding can be stopped by applying milk-soaked gauzes. The blood clotting activity of milk survives conditions simulating the infants’ gastric and pancreatic digestion, suggesting that milk may possibly contribute to gastrointestinal haemostasis in infants. We also show that measuring the ability of human milk to trigger blood plasma clotting may be useful to evaluate milk pasteurization, a procedure applied when milk is stored in hospital biobanks. Furthermore, we demonstrate that EVs in amniotic fluid, milk, saliva and urine expose not only TF but also its ligand, coagulation factor VII (FVII). In fact, the presence of the complex of TF and FVII on EVs explains why EVs efficiently trigger blood clotting. The complex of TF and FVII also has non-haemostatic functions such as regulation of epithelial permeability, and we provide evidence that FVII, present in amniotic fluid, may interact with TF present on fetal epithelial cells, and we show that this interaction may play a role in regulating epithelial permeability. Taken together, this thesis contributes to our understanding how EVs present in body fluids may contribute to protection by promoting haemostasis and regulation of epithelial permeability.

The specialized endothelial tip cells which lead growing vascular sprouts in angiogenesis may be an attractive target for anti-angiogenesis therapy in cancer and eye disease. These cells initiate the angiogenic process, regulate sprout behavior, and differ in their gene expression from other endothelial cell (EC) phenotypes, such as the proliferating stalk cells that follow tip cells. Although several growth factors and signaling molecules have been identified to play a role in tip cell and stalk cell differentiation, anti-tip cell therapy still remains to be developed. In fact, a lack of appropriate models that allow discrimination between tip cells from non-tip cells has slowed down the development of selective anti-tip cell therapies. In this thesis, the CD34+ tip cell model was used to identify tip cells and to purify tip cell and non-tip cell populations in vitro to study the cellular and molecular regulation as well as the metabolism of tip cells and non-tip cells. Finally, the potential of different EC phenotypes as targets for anti-angiogenesis therapies in cancer was critically evaluated.

Growing blood vessel sprouts are composed of specific subtypes of endothelial cells. In short, a blood vessel sprout is led by a tip cell that is followed by stalk cells and phalanx cells. Because of their leading role in vessel sprouts and their unique protein expression, tip cells form an attractive selective target for anti-angiogenic therapy. Consequently, endothelial tip cells are subject of many pre-clinical studies that aim to identify specific targets for novel therapies to treat retinal diseases. Thus far, most of the research on tip cells has been performed in animal models to identify possible selective molecular therapeutic targets in tip cells. In order to contribute to a reduction in the number of laboratory animals, we have developed an in vitro model to study tip cells. The Ocular Angiogenesis Group AMC has shown for the first time that tip cells can be identified in endothelial cell cultures by expression of the protein CD34. In the present thesis, this in vitro tip cell model was used to further characterize tip cells with respect to the growth factors and pathways involved. The studies presented in this thesis describe 3 approaches to study tip cells in vitro: • Identification of endothelial tip cell-specific proteins, • Effects of angiogenesis inhibitors on endothelial tip cells in vitro, and • Identification of interactions between endothelial tip cells, non-tip cells and extracellular proteins. This thesis shows that tip cells in cultures of human endothelial cells are a versatile model that can be employed for different aspects of tip cells research and has several advantages over the use of animal models.

In the post-genomic era, characterisation of pathways involved in protein turnover has been a major area of research in medicine. The discovery of ubiquitin and thereafter the proteasome has revolutionised the understanding of the mechanisms responsible for protein regulation, also known as proteostasis. The proteasome acts as a nanomachine in eukaryotic and archaeal cells, responsible for proteolysis of soluble proteins that are tagged for degradation. This is achieved by selective ubiquitination of target proteins, a process that involves the covalent attachment of a poly-ubiquitin chain to the protein that is marked for recycling. In recent years, dysfunction of the ubiquitin-proteasome system has been linked to numerous human diseases which has led to the development of novel therapies using proteasome inhibitors. In this dissertation, we explore the contributions of the proteasome to retinal pathology and discuss strategies of therapeutic proteasome modulation in the retinal pigment epithelium.

Overall, the studies described in the present dissertation are aimed at the improvement of personalised medicine strategies for various types of cancer. The studies in parts 1 and 2 describe potential novel therapeutic avenues to treat IDH1/2-mutated cancers and the conditions in which they may have optimal effects. The studies described in part 3 may improve risk/benefit assessments for the application of cytotoxic therapies in an individual patient, and the follow-up after the administration of such cytotoxic therapies. These nuances may ultimately improve personalized treatment and management of cancer.

Bio-imaging using fluorescence microscopy is widely used for the study and visualization of biological processes with a high degree of specificity. However, the illumination used for fluorescence imaging can be invasive enough to affect the imaging process by causing photodamage. At the same time, the quality of the recorded image largely depends on the illumination levels used for imaging resulting in a trade-off between image quality and photodamage. In this study smart illumination strategies are explored to reduce photodamage and improve imaging sensitivity. The study concludes that by adjusting illumination in accordance to the spatial-profile of the biological specimen during real-time imaging, it is possible to improve image quality and reduce photodamage at the same time leading to a win-win scenario.

Our DNA is constantly exposed to endogenous and exogenous factors, including by-products of metabolic processes, chemical compounds, ionizing radiation and ultraviolet light, that threaten its integrity. To restore the various types of resulting DNA damage, cells evolved dedicated repair mechanisms. Double-strand breaks (DSBs) are among the most dangerous DNA lesions and can be repaired by two major DSB repair pathways, called homologous recombination (HR) and non-homologous end joining (NHEJ). Our experiments showed that HR is active in various murine tissues and tumors in vivo and decreases with age. Intriguingly, our results also showed that in vitro not only DSBs, but also nicks can trigger HR. Although mechanisms driving nick-induced HR partially overlap with the classical HR, we concluded that it is a bona-fide pathway for nick repair. DNA repair mechanisms shield cancer cells from the cytotoxic effects of anticancer therapies. Hyperthermia (elevated temperature) is a clinically applied treatment that disrupts various DNA repair pathways, including HR, and can therefore sensitize cancer cells to radio- and chemotherapy. We found that hyperthermia treatment outcomes can be significantly improved by addition of small molecule inhibitors of PARP-1 or HSP90, paving the way for in vivo studies and future clinical trials. In conclusion, this thesis portrays HR as a double-edged sword in cellular metabolism of DNA breaks. On the one hand, HR is essential in genome maintenance in healthy cells. On the other hand, interfering with HR can improve the outcomes of cancer therapies that rely on DNA damaging agents.

Huntington’s disease (HD) is a dominant inherited neurodegenerative disorder caused by a CAG expansion in the Huntingtin gene, which leads to a polyglutamine expansion in the mutant Huntingtin protein (mHTT) and its aggregation in nuclear and cytoplasmic inclusions. mHTT degradation can in principle counteract the toxic effects of mHTT. Intracellular protein degradation occurs via two routes: the macroautophagy pathway and the ubiquitin-proteasome system (UPS). Although the polyglutamine-expanded HTT protein is ubiquitously expressed, neurons appear to be the most sensitive. In contrast, neighbouring glia seem less affected. Glia are essential for proper brain function as they play a role in synaptic communication and have been shown to be involved in several neurodegenerative diseases. In this thesis, we examine whether the apparent selective vulnerability of neurons is due to differences in protein degradation between neurons and glia . We show that there is no impairment of the UPS in HD, as proteasomes and ubiquitin are dynamically recruited into mHTT inclusions and that active enzymes of the UPS are present. We demonstrate that the nuclear inclusion frequency is higher in neurons compared to GFAP-positive astrocytes and microglia in different HD mouse models and that these results correlate with the mHTT half-life in corresponding cell lines. These observed differences between neurons and glia in mHTT half-life may indeed be due to our observed differences in the UPS and autophagy between glial and neuronal cell lines. Together, these findings contribute to a deeper understanding of the dissimilarities in mHTT inclusions between different cell types in the brain.

Periodontium is a specialized tissue that keeps teeth in their position and protects roots of teeth against external noxa. In the present thesis, the 3D structure blood vessels in human gingiva has been investigated using 3D histochemistry and microscopical imaging. For that propose, the tissue has to be cleared, stained and imaged microscopically. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether are the most successful methods for clearing connective tissue-rich gingiva for 3D histochemistry and imaging of fluorescence using light-sheet microscopy. From a clinical point of view, objective measurements of the responses of periodontal tissue to dental treatment are key to determine success or failure of periodontal therapy. The periodontal response to novel dental treatments such as aligner therapy in orthodontics, and to the application of adjunctive tools to support the periodontal health such as photodynamic therapy, herbal tooth paste and a mouth rinses containing aluminium triformate was investigated. It is shown in this thesis that innovations in periodontology benefits from cooperation of clinician and fundamental academic researcher.

Nowadays, approximately 4 million people worldwide experience blindness or severe vision loss caused by diabetic retinopathy. Diabetic retinopathy is a multifactorial disease that can progress from minor changes in vascular permeability, into a proliferative retinal disorder. The increasing incidence of diabetes diagnosis and lack of effective treatments make the development of new therapies an urgent issue. Previous studies have reported increased plasmalemma vesicle-associated protein (PLVAP) expression in the retinal vasculature of diabetic retinopathy patients, which co-localized with vascular permeability. In this thesis, the role of PLVAP in development and progression of diabetic retinopathy is explored. The performed in vitro and in vivo research revealed the key role of PLVAP in regulating Vascular Endothelial Growth Factor (VEGF)-induced angiogenesis and increased vascular permeability. This makes PLVAP an interesting, endothelial cell-specific therapeutic target for diabetic retinopathy.

In this thesis, we investigated the role of peptidases in Alzheimer’s disease (AD). AD is characterized by extracellular aggregates consisting of amyloid-β (Aβ) peptides and accumulation of these peptides starts decades before the onset of the first symptoms. We described which peptidases degrade the Aβ peptide and how their levels and activity change during the progression of AD. We show that in presymptomatic stages of AD Aβ degradation is already decreased. We suggest that especially the diminished levels of IDE contribute to less efficient Aβ degradation in these stages and that it may initiate Aβ accumulation, plaque formation and the progression to AD. Furthermore, we investigated whether changes in peptidase activity in presymptomatic stages of AD can be used to diagnose the disease. We showed that measuring Aβ degradation rates in ante mortem CSF may become a diagnostic tool, but only in combination with other biomarkers. Furthermore, TPP2 activity affects APP levels, processing and trafficking and we suggest a role for TPP2 in AD. This thesis contributes to a better understanding of the role of peptidases in neurodegenerative disease, and we show that these peptidases are important players in the initiation and progression of AD. Understanding of the processes involved in presymptomatic stages may allow for therapeutic intervention before the disease becomes manifest.