Vein grafting is a frequently used surgical intervention for cardiac revascularization. However, vein grafts display regions with intraplaque (IP) angiogenesis, which promotes atherogenesis and... Show moreVein grafting is a frequently used surgical intervention for cardiac revascularization. However, vein grafts display regions with intraplaque (IP) angiogenesis, which promotes atherogenesis and formation of unstable plaques. Graft neovessels are mainly composed of endothelial cells (ECs) that largely depend on glycolysis for migration and proliferation. In the present study, we aimed to investigate whether loss of the glycolytic flux enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) in ECs inhibits IP angiogenesis and as such prevents unstable plaque formation. To this end, apolipoprotein E deficient (ApoE(-/-)) mice were backcrossed to a previously generated PFKFB3(fl/fl) Cdh5(iCre) mouse strain. Animals were injected with either corn oil (ApoE(-/-)PFKFB3(fl/fl)) or tamoxifen (ApoE(-/-)PFKFB3(ECKO)), and were fed a western-type diet for 4 weeks prior to vein grafting. Hereafter, mice received a western diet for an additional 28 days and were then sacrificed for graft assessment. Size and thickness of vein graft lesions decreased by 35 and 32%, respectively, in ApoE(-/-)PFKFB3(ECKO) mice compared to controls, while stenosis diminished by 23%. Moreover, vein graft lesions in ApoE(-/-)PFKFB3(ECKO) mice showed a significant reduction in macrophage infiltration (29%), number of neovessels (62%), and hemorrhages (86%). EC-specific PFKFB3 deletion did not show obvious adverse effects or changes in general metabolism. Interestingly, RT-PCR showed an increased M2 macrophage signature in vein grafts from ApoE(-/-)PFKFB3(ECKO) mice. Altogether, EC-specific PFKFB3 gene deletion leads to a significant reduction in lesion size, IP angiogenesis, and hemorrhagic complications in vein grafts. This study demonstrates that inhibition of endothelial glycolysis is a promising therapeutic strategy to slow down plaque progression. Show less
In recent years, the study of endothelial cell (EC) metabolism has led to the discovery of novel regulatory mechanisms and potential new targets for vascular-related diseases. Despite the fact that... Show moreIn recent years, the study of endothelial cell (EC) metabolism has led to the discovery of novel regulatory mechanisms and potential new targets for vascular-related diseases. Despite the fact that ECs have readily available oxygen in the blood, they mainly generate ATP via anaerobic glycolysis rather than Krebs cycle. In the context of atherosclerosis, there has been growing interest in understanding how EC metabolism affects plaque formation and intraplaque (IP) angiogenesis, which has been identified as a contributing factor for plaque vulnerability in human atherosclerosis.Among the enzymes involved in glycolytic flux modulation, PFKFB3 plays a critical role for the proliferation and migration of ECs. PFKFB3 is upregulated in atheroprone regions of arterial vessels and in carotid plaques of patients with elevated levels of lipoprotein(a). The experimental work of this thesis focuses on PFKFB3 as a modulator of EC glycolysis and its effects on atherosclerosis progression and IP angiogenesis.In the first part of this thesis, a pharmacological study with partial glycolysis inhibitor 3PO in the context of advanced atherosclerotic plaques is described. 3PO treatment restrains IP angiogenesis and plaque frequency but it does not affect plaque size and composition in ApoE-/-Fbn1C1039G+/- mice. In addition, a 3PO-mediated reduction in plaque formation is also observed in ApoE-/- mice that develop plaques without IP neovascularization.Furthermore, EC-specific PFKFB3 deletion leads to a significant reduction in plaque size, IP angiogenesis and hemorrhagic complications in a vein graft model. These findings suggest that endothelial glycolysis inhibition may represent a new therapeutic strategy to slow down plaque progression in vein grafts.A study performed in collaboration with the University of Aberdeen is also presented in this thesis. Here the development of a new PFKFB3-targeted PET radiotracer, [18F]ZCDD083, for in vivo plaque imaging is described. The specificity of the tracer for atherosclerotic plaques is demonstrated by a combination of ex vivo autoradiography and en face Oil Red O staining. This tracer is a promising non-invasive diagnostic tool to detect rupture-prone atherosclerotic plaques,Finally, a novel imaging method for a three-dimensional reconstruction of the IP vessel network is presented. This method is based on iDISCO immunolabeling and confocal microscopy. It may represent a novel tool to investigate the causal relationship between IP angiogenesis and atherogenesis.Overall, the experimental data generated in this thesis strongly argue for a critical role of EC metabolism in the formation and progression of atherosclerosis in addition to IP angiogenesis. Show less
Atherosclerosis, restenosis and cardiac remodeling after myocardial infarction can cause serious clinical problems that greatly contribute to both high morbidity and mortality. In all these... Show moreAtherosclerosis, restenosis and cardiac remodeling after myocardial infarction can cause serious clinical problems that greatly contribute to both high morbidity and mortality. In all these processes, the inflammatory responses caused by activation of the immune system play a very prominent role. This thesis elaborates on the role of specific components of the immune system and the therapeutic possibilities that lay hidden therein. This was done by focussing on the pathophysiological process in which Damage Associated Molecular Patterns (DAMPs) are released upon cell stress and cell death or other tissue damage, and may play an important role via different mechanisms. The data in this thesis illustrates specific involvement of DAMPs recognizing factors such as Toll-like Receptors in vascular remodeling and the therapeutic potential that lies within these findings. We show that endogenous activation of the immune s ystem plays an important role in the post-interventional vascular remodeling process. Multiple DAMPs such as HMGB1 are absent before intervention however they were found highly up regulated locally in the vessel wall after intervention indicating a specific relation with the intervention procedure. The presence and involvement of a variety of Toll Like Receptors in different models of vascular remodeling is interesting since these receptors are considered to be important recognizers of the DAMPs locally found in the vessel wall. Different intracellular signalling pathways and TLR accessory molecules seem to mediate the outcome of the specific DAMP-TLR interactions on vascular remodeling majorly. Protective effects of TLR3 and different outcomes of RP105 deficiency on vascular remodeling processes indicate the complexity of the underlying pathophysiological processes. These results can be partly explained by downstream TLR signalling and involvement of specific cell subtypes. The exploration of these underlying mechanisms offers new opportunities for biomarker selection and therapy development. Show less
Vein graft surgery to treat occlusive arterial disease is a common applied procedure. Each year more than two million vein graft surgeries are performed worldwide. The major drawback of vein... Show moreVein graft surgery to treat occlusive arterial disease is a common applied procedure. Each year more than two million vein graft surgeries are performed worldwide. The major drawback of vein grafting is that within 10 years after vein graft surgery 50-60 % of the vein grafts suffer from patency loss due to thrombosis, intimal hyperplasia formation, accelerated atherosclerosis and rupture. Endogenous factors orchestrate the development and failure of vein grafts. Investigating the role of endogenous constituents on vein graft remodeling can enhance our basic knowledge of the involvement of these factors in vein graft remodeling. By interfering in the function of endogenous factors, as we showed in this thesis, vein graft remodeling can be negatively or positively influenced depending on the factor and strategy used. New therapeutic strategies can be developed based on this knowledge. In this thesis we investigate the role of innate immune components, complement system factors, toll like receptors, mast cells and NK cells and the role of Annexin A5 in vein graft remodeling. Furthermore we explored the role of plaque stability, plaque neovascularization and extracellular matrix remodeling in a hypercholersterolemic mouse vein graft model. Show less
The aim of this thesis was to gain more insight in the involvement of inflammatory processes in vessel wall remodeling seen after PTA or bypass surgery and put these processes in the perspective of... Show moreThe aim of this thesis was to gain more insight in the involvement of inflammatory processes in vessel wall remodeling seen after PTA or bypass surgery and put these processes in the perspective of restenosis, vein graft failure and potential therapeutic preventive strategies. Therefore, we firstly focused on inflammation in general, using the anti-inflammatory agent Dexamethasone, assessing the effects of such a broad approach on restenosis and vein graft remodeling. Then, we further focused on some specific parts of the immune system, namely Interleukin 10 (IL10), chemokines and the complement cascade. Il10 was chosen because it is one of the most studied anti-inflammatory cytokines and this property makes it a potential candidate for ant-restenosis therapy. Furthermore, it was hypothesized that chemokines are involved in vascular remodeling, since they are generally known for their regulatory properties regarding influx of inflammatory cells to tissues and this is one of the first phenomena seen in vascular remodeling. The complement cascade was studied in this context since it contains pro-inflammatory activity and some end-products of the cascade, like chemokines, are potent chemotactic agents. Show less
