Factor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the... Show moreFactor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the extrahepatic production of comple-ment factors, including by cells of the immune system, since this contributes to non-canonical functions of local complement activation and regulation. Here we investigated the production and regulation of factor H and its splice variant factor H-like protein 1 (FHL-1) by human myeloid cells. As validation, we confirmed the pre -dominant presence of intact factor H in serum, despite a strong but comparable mRNA expression of CFH and FHL1 in liver. Comparable levels of CFH and FHL1 were also observed in renal tissue, although a dominant staining for FHL-1 was shown within the proximal tubules. Human in vitro generated pro-and anti-inflammatory macrophages both expressed and produced factor H/FHL-1, but this was strongest in pro-inflammatory macro-phages. Production was not affected by LPS activation, but was increased upon stimulation with IFN-gamma or CD40L. Importantly, in both macrophage subsets mRNA expression of FHL1 was significantly higher than CFH. More -over, production of FHL-1 protein could be confirmed using precipitation and immunoblotting of culture su-pernatants. These data identify macrophages as producers of factor H and FHL-1, thereby potentially contributing to local complement regulation at sites of inflammation. Show less
Background and aims: Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we... Show moreBackground and aims: Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we investigated the role of a c.1769G > T variant in Netrin-1 in (premature) atherosclerosis.Methods: To determine the effect of the genetic variation, purified Netrin-1, either wild type (wtNetrin-1) or the patient observed variation (mutNetrin-1), was used for migration, adhesion, endothelial barrier function and bindings assays. Expression of adhesion molecules and transcription proteins was analyzed by RT-PCR, Western blot or ELISA. To further delineate how mutNetrin-1 mediates its effect on cell migration, lenti-viral knockdown of UNC5B or DCC was used.Results: Bindings assays revealed a decreased binding capacity of mutNetrin-1 to the receptors UNC5B, DCC and beta 3-integrin and an increased binding capacity to neogenin, heparin and heparan sulfate compared to wtNetrin-1. Exposure of endothelial cells to mutNetrin-1 resulted in enhanced monocyte adhesion and expression of IL-6, CCL2 and ICAM-1 compared to wtNetrin-1. In addition, mutNetrin-1 lacks the inhibitory effect on the NF-kappa B pathway that is observed for wtNetrin-1. Moreover, the presence of mutNetrin-1 diminished migration of macrophages and smooth muscle cells. Importantly, UNC5B or DCC specific knockdown showed that mutNetrin-1 is unable to act through DCC resulting in enhanced inhibition of migration.Conclusions: Our data demonstrates that mutNetrin-1 fails to exert anti-inflammatory effects on endothelial cells and more strongly blocks macrophage migration compared to wtNetrin-1, suggesting that the carriers of this genetic molecular variant may well be at risk for premature atherosclerosis. Show less
Hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease, is a rare genetic disorder, known for its endothelial dysplasia causing vessel malformations, severe nose bleeds and... Show moreHereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease, is a rare genetic disorder, known for its endothelial dysplasia causing vessel malformations, severe nose bleeds and internal bleedings. In the majority of patients mutations are found in genes belonging to the TGFβ superfamily, causing a disbalance in the TGFβ signaling pathway by haploinsufficiency of the remaining functional protein. In this thesis we studied different aims and approaches to influence HHT1-MNC homing and differentiation to restore their contribution to tissue repair. In various experimental methods inducing ischemic and/or direct tissue damage, we aimed to improve tissue repair in the Eng+/- mice. Using DPP4 inhibition, we increased the SDF1-CXCR4 homing mechanism, to restore the impaired homing capacity of the HHT1-MNCs. Furthermore, we focused on correcting the M1/M2 differentiation in Eng+/- mice. Via use of the BMP receptor inhibitor LDN we aimed to restore the skewed BMP/TGFβ signaling; stimulating the TGFβ pathway signaling to induce M2 differentiation. We concluded that DPP4 inhibition can be used to improve the HHT1 immune system and tissue repair, and is best used in concert with other drugs or therapies that stimulate cardiac or tissue repair, like anti-coagulants or cell therapy. Show less
Uveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of... Show moreUveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of infiltrating macrophages and T cells is associated with a poor prognosis rather than a good one. The clear link between inflammation and this malignancy provides a paradigm for macrophage plasticity and function. Macrophages in uveal melanoma have an M2-like phenotype and are associated with the loss of one specific chromosome - monosomy 3. The central players involved in this process and discussed include macrophages, T lymphocytes, chemokines and cytokines, including the macrophage-attraction molecules. When a tumor acquires the ability to release significant amounts of macrophage-attraction molecules it causes the expansion of a population of myeloid immature cells that may not only help the tumor to suppress immune reactions but also aid in the construction of new blood vessels for tumor growth. A better understanding of the molecular basis of a local myelomonocytic cell population will bring a better understanding of the immunopathology of this disease and will lead to therapeutic interventions in uveal melanoma. This thesis focuses on the roles of the local inflammatory microenvironment in the development and progression of uveal melanoma. Show less
This thesis describes the role of the immune system as an important phenomenon in the most frequently occurring form of eye cancer in adults, namely in uveal melanoma. We show that the immune... Show moreThis thesis describes the role of the immune system as an important phenomenon in the most frequently occurring form of eye cancer in adults, namely in uveal melanoma. We show that the immune system can be the cause of the tumor, and also plays a role in the development of a tumor, and may be an entry for therapy. In the first chapters of this thesis, we describe the phenomenon of "an inflammatory phenotype" in uveal melanoma. It appears that when this type of cancer shows more inflammation, the survival of patients decreases. A possible explanation for this observation is that one of the key players among the immune cells, the macrophage, plays an essential role in intra-ocular tumor growth. We demonstrate this in patient material, but also in experimental studies; we are able to inhibit tumor growth when we modulate the presence of macrophages. In this thesis, we also show that the immune system can be used effectively to eradicate eye melanomas in experimental models. T cell vaccination in combination with monoclonal antibodies gave promising results for treating eye cancer. Further research has to be performed to translate this into the clinic. Show less
In this thesis the role of several apoptosis regulating proteins in the development of atherosclerosis and atherosclerotic plaque stability is investigated. Apoptosis of different cell types in... Show moreIn this thesis the role of several apoptosis regulating proteins in the development of atherosclerosis and atherosclerotic plaque stability is investigated. Apoptosis of different cell types in atherosclerotic plaques, such as macrophages and smooth muscle cells may inhibit or promote plaque development or stability depending on the stage of atherosclerosis. As many of these apoptosis regulating proteins also display immune-modulating features, we have particularly investigated effects of modulation of apoptosis regulating proteins on plaque and systemic inflammation. We performed a number of studies in mouse models of atherosclerosis. First gene expression profiles of stable and unstable atherosclerotic plaques were compared in order to identify genes or pathways that are associated with plaque vulnerability. We further developed transgenic mice partially or wholly lacking genes involved in apoptosis and/or inflammation such as Bcl-2 family members and focal adhesion kinase, both systemically or in the leukocyte subset. The studies described in this thesis show amongst other things that Bim and Mcl-1, both members of the Bcl-2 family of apoptosis regulators, regulate specific cell death and inflammatory processes relevant to atherosclerosis. Show less
