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- Globisch, Maria Ascención
(författare)
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Inflammation and immunothrombosis in cerebral cavernous malformation : Novel molecular targets for the treatment of an incurable disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cerebral cavernous malformation (CCM) is a vascular disease that causes mulberry-like lesions (cavernomas) in the central nervous system (CNS). Cavernomas are fragile, leaky and prone to rupture which may cause symptoms such as epileptic seizures, focal neurological deficits and hemorrhagic strokes. CCM lesions can appear sporadically in 0.5% of the general population. Alternatively, CCM lesions appear as a consequence of a loss-of-function germline mutation in either CCM1, CCM2, or CCM3 in endothelial cells of the CNS. Inherited CCM is termed familial, and it affects approximately one in ten thousand individuals in an autosomal dominant manner. The aim of this thesis project is to identify novel cell and molecular mechanisms that contribute to the development and progression of cavernous malformations. Additionally, this thesis project aims to identify and validate inhibitors that may reduce lesions and alleviate the side effects in CCM. In this thesis project, two inducible endothelial specific Ccm3 deficient mouse models (acute and chronic) were evaluated with methods such as RNA-sequencing, immunofluorescence, ELISA, scanning and transmission electron microscopy, flow cytometry, and in situ hybridization. Moreover, in vitro cell cultures were used with methods such as immunofluorescence, qPCR, and western blot. Importantly, sporadic and familial human CCM samples were used to show the clinical relevance of our studies. In paper I we focused on the role and kinetics of inflammation in CCM. We analyzed the transcriptome of healthy and Ccm3 deficient mouse brain endothelial cells and found that genes related to inflammation were upregulated in CCM pathology. We identified various inflammatory cytokines in vivo and also identified neutrophils as the most prominent immune cell subtype in CCM. Moreover, we found that neutrophils in CCM produce neutrophil extracellular traps (NETs), that can be inhibited with DNase I. The inhibition of NETs stabilized cavernoma vasculature by reducing fibrinogen and IgG leakage. In paper II we re-used the acute RNA-seq database from paper 1 and focused on endothelial hemostasis and hypoxia. We found that genes related to procoagulation, anticoagulation, and hypoxia were highly upregulated in Ccm3 deficient mice. We validated the findings in vivo and found that the hemostatic system in CCM is dysregulated and that it causes, bleeding, thrombosis, and cerebral hypoxia. In paper III we evaluated the effect of propranolol in CCM. We treated chronic CCM mice with the beta-blocker propranolol and found that propranolol was able to reduce lesions in the brains and retinas of CCM mice as well as reduce cadaverine leakage. Importantly, we identified endothelial plasmalemmal pits and a thick basal membrane between endothelial cells and pericytes, pathological features which reduced upon propranolol treatment. Altogether this thesis significantly contributes to the CCM field as it identified pathological features of cavernomas such as neutrophils with NETs, endothelial plasmalemmal pits, and polyhedrocytes. This thesis work also evaluated pharmacological inhibitors (DNase I and propranolol) in mouse models of CCM and supports the use of anticoagulant therapies in patients with CCM.
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| 2. |
- Herre, Melanie, 1991-
(författare)
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Neutrophil extracellular traps as potential therapeutic targets to prevent tumor-induced organ failure and metastasis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer does not only affect surrounding tissues, but also leads to complications at distant sites. The reason for this is that tumors secrete factors and prime cells that travel through the body, making cancer a systemic disease. In fact, cancer death is mostly caused by systemic complications such as metastasis, organ failure or thrombosis. However, surprisingly little is known about the effect of cancer on distant organs that are not sites of tumor growth.Neutrophil extracellular traps (NETs) are web-like structures consisting of neutrophil DNA and granular proteins that are released from the neutrophil in response to a stimulus. NETs are not only formed in bacterial infections but also in noninfectious inflammatory disorders like cancer. While NETs can be beneficial for the host in severe infections they come with a cost. NETs are pro-thrombotic, cause damage to the vessel-lining endothelial cells, are involved in metastasis formation and impair organ function. The removal of NETs by DNase I an enzyme that breaks the NET backbone can therefore be of use as a therapy for certain conditions.The aim of this thesis was to investigate the mechanisms leading to systemic effects of cancer and potential therapeutic strategies with a special focus on NETs.In paper I we investigated how the presence of a primary tumor affected heart function. We conclude that tumor-induced NETs contribute to inflammation and myocardial strain in malignant disease and identify NETs as potential therapeutic targets in cancer patients to prevent cardiac inflammation and dysfunction.In paper II we investigated if the long-term removal of NETs by the use of a murine DNase I expressing adeno-associated virus vector was possible and how this affected organ function and metastasis formation in tumor-bearing mice. We conclude that long-term, species-specific expression of DNase I is possible. Elevated DNase I expression improved kidney function and reduced the proportion of mice with metastasis.In paper III we investigated how cancer and cancer-associated NETs affect endothelial gene expression in distant organs. We found that the number of differentially expressed genes increased with tumor size and that it was higher in metastatic organs. In response to a tumor, the kidney vasculature showed signs of renal hepatization, a state of kidney stress. In conclusion, this study gives a global overview of the gene expression changes that take place in the vasculature of distant organs in individuals with cancer and the potential role of NETs.
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| 3. |
- Nordling, Sofia, 1985-
(författare)
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Vascular Interactions in Innate Immunity and Immunothrombosis: : Models of Endothelial Protection
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The phenomenon known as immunothrombosis has garnered increased attention over the last few years. Much work has been done to characterize the cross talk between hemostasis and the innate immune system. This thesis outlines the role of the vascular endothelial cells during immunothrombotic events as regulators of coagulation, platelet-, and leukocyte recruitment.A newly developed method for investigating the interaction between endothelial cells and the blood compartment illustrated the procoagulant and proinflammatory effects elicited by tumor necrosis factor α activated endothelial cells upon exposure to whole blood. The method was utilized in evaluating treatment of endothelial dysfunction and disruption with a heparin conjugate. Damaged or hypoxic endothelial cells, in addition to basement membrane collagen, that were pretreated with the heparin conjugate prior to contact with blood were found to have reduced activation of coagulation, platelet-, and leukocyte recruitment; in contrast to unfractionated heparin, which had no effect on the aforementioned parameters. The treatment was then investigated in the setting of ischemia reperfusion injury during kidney transplantation and the heparin conjugate was found to bind cultured endothelial cells with high avidity under cold storage conditions. Furthermore, it was found to bind to the renal vasculature during static cold storage and was subsequently found to be beneficial with regard to early graft function in an experimental mouse model of syngeneic kidney transplantation. Recipients of kidneys treated with the heparin conjugate had reduced serum creatinine compared to controls 24 hours after transplantation. Lastly, the anticoagulant properties of the heparin conjugate were investigated in comparison to unfractionated heparin. While the conjugate exerted reduced capacity with regard to thrombin inhibition, it rapidly inhibited the binding of platelets to exposed collagen. The conjugate was furthermore found to preferentially locate to sites of endothelial cell activation at early stage during endotoxic shock in mice.In conclusion, this thesis demonstrates that disrupted functioning of the vascular endothelial cells actively contributes to immunothrombosis, and that it is possible to model endothelial cell function using whole blood assays. Furthermore, this thesis presents a treatment that enhances the hemocompatibility of damaged endothelial cells and subsequently improves the early renal function after kidney transplantation.
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