Effects of shear stress on NF-κB transcription factors in vascular endothelium

Atherosclerosis, a chronic inflammatory disease of arteries, occurs predominantly at regions of the arterial system that are exposed to disturbed patterns of blood flow. Blood flow influences the atherosclerosis by exerting shear stress on endothelial cells (ECs). Although the signalling pathways that activate pro-inflammatory NF-κB transcription factors are well defined, the regulation and physiological significance of differential NF-κB subunit expression is poorly understood. In this thesis, we demonstrate that RelA NF-κB sub-unit expression is positively regulated in ECs via c-Jun N-terminal kinase (JNK) and the transcription factor ATF2. This pathway promoted focal arterial inflammation as genetic deletion of JNK1 reduced RelA expression and macrophage accumulation at an athero-susceptible site. Furthermore, JNK signalling to RelA is controlled by mechanical forces as en face immunostaining revealed that disturbed flow patterns (generated by a constrictive cuff) elevated RelA expression in murine carotid arteries via JNK1. Positron emission tomography and en face staining revealed that disturbed flow enhanced 18F-fluorodeoxyglucose uptake (a marker of inflammation) and accumulation of CD68-positive inflammatory cells in arteries via JNK1. We conclude that disturbed flow promotes arterial inflammation via a novel JNK-NF-κB cross-talk. The duration of RelA nuclear localisation is an important determinant of the magnitude and specificity of target gene expression. En face staining revealed that RelA rapidly accumulated in the nucleus upon LPS stimulation in ECs at both athero-protected and athero-susceptible sites. RelA was exported from the nucleus to the cytoplasm in response to prolonged stimulation in the athero-protected region but not in the athero-susceptible region. The duration of RelA nuclear localisation was suppressed by histone deacetylases which displayed higher activity at the protected site compared to the susceptible site. Overall, our findings reveal that ECs at athero-susceptible sites are primed for inflammatory activation via complementary mechanisms that enhance both the expression and the activity of NF-κB transcription factors

Gesintertes Polylactid-Polyglycolid-Copolymer als individuell dreidimensional konfigurierbares und bioresorbierbares Knochenersatzmaterial

Ziel der Arbeit war das Anpassen des alloplastischen resorbierbaren Polymers Polyl(DL-lactid-co-glycolid) durch Sintern an den Einsatz als individuell dreidimensional konfektionierbares poröses Knochenersatzmaterial. Es wurden 93 Probekörper aus thermisch vorbehandeltem Copolymer mit 82 herkömmlich gesinterten Probekörpern verglichen. Darüber hinaus wurde das neu entwickelte Verfahren der Doppelsinterung auf das Erreichen von Anforderungen wie guter Porengröße, Verbindung zwischen den Poren, Pufferbarkeit und mechanischer Stabilität überprüft. Im Zellversuch wurde die Kompatibilität des Materials mit osteoblastenähnlichen Zellen getestet. Ein Stereolithographiemodell eines Knochendefektes auf Basis von CT-Daten wurde als Zielform für die dreidimensionale Anpassbarkeit verwendet. Die Versuche ergaben, dass das thermische Vorbehandeln von Poly(DL-lactid-co-glycolid) grundsätzlich die Eigenschaften des Materials zu Lasten der Stabilität optimiert

In vivo mapping of vascular inflammation using the translocator protein tracer 18F-FEDAA1106

YesNon-invasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro-2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5- methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18F- FEDAA1106 and 18F-FDG (a marker of glucose metabolism) for PET imaging of vascular inflammation. This was tested using a murine model where focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18F-FEDAA1106 or 18F-FDG was registered to anatomical data generated by CT/CT angiography. Standardized uptake values (SUV) were significantly increased at cuffed compared to contralateral arteries using either 18F-FEDAA1106 (p<0.01) or FDG (p<0.05). However, the 18F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the non-inflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 correlated with vascular inflammation more specifically than FDG uptake.: This study was funded by the British Heart Foundation and through a grant from the Swiss National Science Foundation (310030_143343/1 to B.R.K.

TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria

The 18-kDa translocator protein (TSPO) localizes in the outer mitochondrial membrane (OMM) of cells and is readily up-regulated under various pathological conditions such as cancer, inflammation, mechanical lesions and neurological diseases. Able to bind with high affinity synthetic and endogenous ligands, its core biochemical function resides in the translocation of cholesterol into the mitochondria influencing the subsequent steps of (neuro-)steroid synthesis and systemic endocrine regulation. Over the years, however, TSPO has also been linked to core cellular processes such as apoptosis and autophagy. It interacts and forms complexes with other mitochondrial proteins such as the voltage-dependent anion channel (VDAC) via which signalling and regulatory transduction of these core cellular events may be influenced. Despite nearly 40 years of study, the precise functional role of TSPO beyond cholesterol trafficking remains elusive even though the recent breakthroughs on its high-resolution crystal structure and contribution to quality-control signalling of mitochondria. All this along with a captivating pharmacological profile provides novel opportunities to investigate and understand the significance of this highly conserved protein as well as contribute the development of specific therapeutics as presented and discussed in the present review

Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo

Intracranial aneurysms are associated with disturbed velocity patterns, and chronic inflammation, but the relevance for these findings are currently unknown. Here, we show that (disturbed) shear stress induced by vortices is a sufficient condition to activate the endothelial NF-kB pathway, possibly through a mechanism of mechanosensor de-activation. We provide evidence for this statement through in-vitro live cell imaging of NF-kB in HUVECs exposed to different flow conditions, stochastic modelling of flow induced NF-kB activation and induction of disturbed flow in mouse carotid arteries. Finally, CFD and immunofluorescence on human intracranial aneurysms showed a correlation similar to the mouse vessels, suggesting that disturbed shear stress may lead to sustained NF-kB activation thereby offering an explanation for the close association between disturbed flow and intracranial aneurysms

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.British Heart Foundation Programme Grant (CS, PE); British Heart Foundation Project Grants PG/09/067/27901 (AB, VR), PG/13/55/30365 (LW, SF), PG/14/38/30862 (CR, VR), PG/16/44/32146 (JJ, EKT, SF); British Heart Foundation Studentship FS/14/8/30605 (BW, VR); MRC Fellowship MR/K023977/1 (RB); and European Union’s Horizon 2020 Marie Skłodowska-Curie Innovative Training Network, TRAIN 721532 (CN)

Expert recommendations on the assessment of wall shear stress in human coronary arteries : existing methodologies, technical considerations, and clinical applications

The aim of this manuscript is to provide guidelines for appropriate use of CFD to obtain reproducible and reliable wall shear stress maps in native and instrumented human coronary arteries. The outcome of CFD heavily depends on the quality of the input data, which include vessel geometrical data, proper boundary conditions, and material models. Available methodologies to reconstruct coronary artery anatomy are discussed in ‘Imaging coronary arteries: a brief review’ section. Computational procedures implemented to simulate blood flow in native coronary arteries are presented in ‘Wall shear stress in native arteries’ section. The effect of including different geometrical scales due to the presence of stent struts in instrumented arteries is highlighted in ‘Wall shear stress in stents’ section. The clinical implications are discussed in ‘Clinical applications’ section, and concluding remarks are presented in ‘Concluding remarks’ section

Is Vasomotion in Cerebral Arteries Impaired in Alzheimer’s Disease?

A substantial body of evidence supports the hypothesis of a vascular component in the pathogenesis of Alzheimer’s disease (AD). Cerebral hypoperfusion and blood-brain barrier dysfunction have been indicated as key elements of this pathway. Cerebral amyloid angiopathy (CAA) is a cerebrovascular disorder, frequent in AD, characterized by the accumulation of amyloid-β (Aβ) peptide in cerebral blood vessel walls. CAA is associated with loss of vascular integrity, resulting in impaired regulation of cerebral circulation, and increased susceptibility to cerebral ischemia, microhemorrhages, and white matter damage. Vasomotion— the spontaneous rhythmic modulation of arterial diameter, typically observed in arteries/arterioles in various vascular beds including the brain— is thought to participate in tissue perfusion and oxygen delivery regulation. Vasomotion is impaired in adverse conditions such as hypoperfusion and hypoxia. The perivascular and glymphatic pathways of Aβ clearance are thought to be driven by the systolic pulse. Vasomotion produces diameter changes of comparable amplitude, however at lower rates, and could contribute to these mechanisms of Aβ clearance. In spite of potential clinical interest, studies addressing cerebral vasomotion in the context of AD/CAA are limited. This study reviews the current literature on vasomotion, and hypothesizes potential paths implicating impaired cerebral vasomotion in AD/CAA. Aβ and oxidative stress cause vascular tone dysregulation through direct effects on vascular cells, and indirect effects mediated by impaired neurovascular coupling. Vascular tone dysregulation is further aggravated by cholinergic deficit and results in depressed cerebrovascular reactivity and (possibly) impaired vasomotion, aggravating regional hypoperfusion and promoting further Aβ and oxidative stress accumulation

Effects of shear stress on NF-κB transcription factors in vascular endothelium

Atherosclerosis, a chronic inflammatory disease of arteries, occurs predominantly at regions of the arterial system that are exposed to disturbed patterns of blood flow. Blood flow influences the atherosclerosis by exerting shear stress on endothelial cells (ECs). Although the signalling pathways that activate pro-inflammatory NF-κB transcription factors are well defined, the regulation and physiological significance of differential NF-κB subunit expression is poorly understood. In this thesis, we demonstrate that RelA NF-κB sub-unit expression is positively regulated in ECs via c-Jun N-terminal kinase (JNK) and the transcription factor ATF2. This pathway promoted focal arterial inflammation as genetic deletion of JNK1 reduced RelA expression and macrophage accumulation at an athero-susceptible site. Furthermore, JNK signalling to RelA is controlled by mechanical forces as en face immunostaining revealed that disturbed flow patterns (generated by a constrictive cuff) elevated RelA expression in murine carotid arteries via JNK1. Positron emission tomography and en face staining revealed that disturbed flow enhanced 18F-fluorodeoxyglucose uptake (a marker of inflammation) and accumulation of CD68-positive inflammatory cells in arteries via JNK1. We conclude that disturbed flow promotes arterial inflammation via a novel JNK-NF-κB cross-talk. The duration of RelA nuclear localisation is an important determinant of the magnitude and specificity of target gene expression. En face staining revealed that RelA rapidly accumulated in the nucleus upon LPS stimulation in ECs at both athero-protected and athero-susceptible sites. RelA was exported from the nucleus to the cytoplasm in response to prolonged stimulation in the athero-protected region but not in the athero-susceptible region. The duration of RelA nuclear localisation was suppressed by histone deacetylases which displayed higher activity at the protected site compared to the susceptible site. Overall, our findings reveal that ECs at athero-susceptible sites are primed for inflammatory activation via complementary mechanisms that enhance both the expression and the activity of NF-κB transcription factors.EThOS - Electronic Theses Online ServiceGBUnited Kingdo