Katharina Urschel

Carotid Plaque Vulnerability A Positive Feedback Between Hemodynamic and Biochemical Mechanisms

Background and Purpose— Rupture of atherosclerotic plaques is one of the main causes of ischemic strokes. The aim of this study was to investigate carotid plaque vulnerability markers in relation to blood flow direction and the mechanisms leading to plaque rupture at the upstream side of carotid stenoses. Methods— Frequency and location of rupture, endothelial erosion, neovascularization, and hemorrhage were determined in longitudinal sections of 80 human carotid specimens. Plaques were immunohistochemically analyzed for markers of vulnerability. Plaque geometry was measured to reconstruct shape profiles of ruptured versus stable plaques and to perform computational fluid dynamics analyses. Results— In 86% of ruptured plaques, rupture was observed upstream. In this region, neovascularization and hemorrhage were increased, along with increased immunoreactivity of vascular endothelial and connective tissue growth factor, whereas endothelial erosion was more frequent downstream. Proteolytic enzymes, mast cell chymase and cathepsin L, and the proapoptotic protein Bax showed significantly higher expression upstream as compared with the downstream shoulder of atherosclerotic lesions. Comparison of geometric profiles for ruptured and stable plaques showed increased longitudinal asymmetry of fibrous cap and lipid core thickness in ruptured plaques. The specific geometry of plaques ruptured upstream induced increased levels of shear stress and increased pressure drop between the upstream and the downstream plaque shoulders. Conclusions— Vulnerability of the upstream plaque region is associated with enhanced neovascularization, hemorrhage, and cap thinning induced by proteolytic and proapoptotic mechanisms. These processes are reflected in structural plaque characteristics, analyses of which could improve the efficacy of vascular diagnostics and prevention.

Shear stress patterns affect the secreted chemokine profile in endothelial cells.

BACKGROUND Atherosclerotic plaques are initiated by pro-inflammatory endothelial activation at arterial regions subjected to non-uniform shear stress. We applied the in vitro flow-through cell culture slides to investigate whether different patterns of shear stress affect the secreted cytokine and chemokine profile in endothelial cells. METHODS Human umbilical vein endothelial cells (ECs) were exposed to 24 h of flow in straight or bifurcating flow-through slides, in some experiments followed by 6 h stimulation with 2.5 ng/mL TNF-α. IL-1β, IL-6, IL-8, IL-12p70, MIP-1α, MCP-1 and sICAM-1 were measured in conditioned medium samples by flow cytometry using antibodies conjugated with fluorescent beads. RESULTS The release of IL-1β, IL-12p70 and MIP-1α from endothelial cells exposed to shear stress was below detectable levels. Strongly increased level of sICAM and significantly increased IL-8 concentration were detected in conditioned medium from endothelial cells exposed to flow in bifurcating slides as compared with cells grown under laminar flow in straight channels. The release of IL-6 and MCP-1 was not significantly induced in bifurcating slides. Treatment with TNF-α for 6 h induced 2-3 fold increase in secreted chemokines and cytokines. In particular, significantly increased MCP-1 and increased IL-8 levels were released from endothelial cells grown in bifurcating slides. This release was partly prevented in cells grown in straight channels, i.e. under exposure to laminar flow only. CONCLUSIONS Although the endothelial monolayer areas exposed to non-uniform shear stress are relatively small, the activation of cells in these regions is strong enough to produce a detectable change in cytokine and chemokine profile, which represents the earliest step in atherogenic endothelial dysfunction.

Impact of telmisartan on the inflammatory state in patients with coronary atherosclerosis – Influence on IP-10, TNF-α and MCP-1

BACKGROUND Hypertension is one of the most prominent risk factors for coronary artery disease (CAD). Treatment of hypertension is therefore important for reducing cardiovascular events and the progression of atherosclerosis. Several treatment strategies are common in clinical practice for example the use of ACE-blockers or angiotensin receptor II blockers (ARBs), so called sartans. Telmisartan, belonging to the class of ARBs, was shown to exert anti-inflammatory effects besides the blood pressure lowering. METHODS In this work, two separate substudy groups of hypertensives were compared. 16 patients with arterial hypertension have been treated with telmisartan (initial 40 mg Kinzalmono®) for 7.3 ± 4.4 months. The telmisartan group was compared to a matched control group including 31 hypertensive patients without telmisartan treatment with a follow up period of 1.9 ± 0.5 years. Serum samples from the beginning and the end of follow up were analyzed with Luminex® technology for 26 cytokines and chemokines. The baseline scores of coronary artery calcification (CAC) were gathered by multislice detector computer tomography. RESULTS After 7 months of telmisartan treatment and 2 years in control patients most of the measured analytes did not change significantly. MCP-1 (P=0.001; P<0.001) was increased significantly in both telmisartan and control group. The relative decrease in IP-10 and TNF-α levels was observed in telmisartan group, as opposed to the increase in control (telmisartan vs. control P=0.048; P=0.01). No linear rank-correlation between measured analytes and the initial CAC was found. CONCLUSION Telmisartan reduced blood pressure in patients with atherosclerosis and arterial hypertension within a short time period, whereas the inflammatory status of these patients remained largely unchanged. An involvement of telmisartan in the regulation of inflammatory and anti-inflammatory mediators in the context of CAD and CAC is possible, but cannot clearly be assumed based on the present findings.

Telmisartan prevents VCAM-1 induction and monocytic cell adhesion to endothelium exposed to non-uniform shear stress and TNF-α.

BACKGROUND Atherosclerotic plaques develop at arterial regions subjected to non-uniform shear stress, and are initiated by increased leukocyte-endothelial interactions. Here we applied the in vitro model of arterial bifurcations to investigate whether telmisartan, an anti-inflammatory angiotensin II receptor blocker with PPAR-gamma activating ability, prevents monocyte recruitment by endothelium. METHODS Human umbilical vein endothelial cells (ECs) were exposed to 18 h non-uniform shear stress in bifurcating flow-through slides, followed by 2 h stimulation with 2.5 ng/mL TNF-alpha. During flow, cells were treated with telmisartan. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells. Adherent THP-1 monocytes were quantified by light microscopy. Endothelial protein expression was determined by immunofluorescence. RESULTS Non-uniform shear stress in combination with TNF-alpha dramatically induced monocytic cell recruitment by endothelial cells. In cells treated with telmisartan (0.5-2.5 μmol/L) during exposure to non-uniform shear stress, dose-dependent inhibition of monocytic cell adhesion was observed, with about 45% reduction at 1 μmol/L. This effect was mediated by a significant reduction of endothelial VCAM-1 expression. On the contrary, the induction of E-selectin by TNF-alpha in ECs exposed to non-uniform shear stress was not affected by telmisartan. The inhibitory effect of telmisartan on monocytic cell recruitment and VCAM-1 induction was prevented in the presence of the PPAR-gamma antagonist GW9662. CONCLUSIONS Treatment with telmisartan decreases the TNF-α-induced recruitment of monocytic cells and endothelial expression of VCAM-1 in regions of non-uniform shear stress in vitro. This mechanism can contribute to the beneficial pleiotropic effects of telmisartan in atherosclerosis-prone arterial regions.

VEGFR2 signalling contributes to increased endothelial susceptibility to TNF-α under chronic non-uniform shear stress

OBJECTIVES Vascular endothelial growth factor receptor 2 (VEGFR2), a tyrosine kinase receptor activated by VEGF and shear stress, is critically involved in endothelial mechanotransduction. We investigated the role of VEGFR2 in non-uniform shear stress-induced endothelial susceptibility to inflammatory stimuli. METHODS Endothelial cells (ECs) were exposed to non-uniform shear stress, followed by stimulation with TNF-α. ECs were transfected with siRNAs against VEGFR2. Alternatively, ECs were treated with blocking antibody against VEGFR2, or with inhibitors of VEGFR2 (ZM 323881), PI3K (LY 294002), or Src-kinase (PP2). THP-1 monocytes were used for dynamic adhesion assays. Endothelial protein expression was determined by immunofluorescence. RESULTS siRNA against VEGFR2 decreased VEGFR2 protein expression by 40% as determined by Western blotting. In endothelial cells exposed to non-uniform shear stress, VEGFR2 knockdown inhibited TNF-α-induced NF-κB translocation to the nucleus, and the upregulation of VCAM-1 and E-selectin. Consequently, monocytic cell recruitment to endothelium under non-uniform shear stress conditions was reduced. Similar effects were observed by blocking VEGFR2 activity using a specific inhibitor ZM 323881, or an antibody against VEGFR2 before TNF-α stimulation. Inhibition of PI3K with LY 294002 significantly reduced non-uniform shear stress-induced endothelial susceptibility to TNF-α, whereas blocking Src-kinase with PP2 was ineffective. CONCLUSION VEGFR2 is critically involved in adhesion molecule induction and monocytic cell recruitment to endothelium in response to non-uniform shear stress and TNF-α. Targeting the mechanosensory cascade can prevent endothelial activation in atherosclerosis-prone regions.

Role of shear stress patterns in the TNF-α-induced atherogenic protein expression and monocytic cell adhesion to endothelium.

BACKGROUND Atherosclerotic plaques develop at arterial regions subjected to non-uniform shear stress, and are initiated by increased leukocyte-endothelial interactions. In this study, we investigated the effects of distinct shear stress patterns on endothelial recruitment of monocytic cells. METHODS Human umbilical vein endothelial cells (ECs) were exposed to laminar or non-uniform shear stress in bifurcating flow-through slides, followed by 2 h stimulation with TNF-α. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells for 1 h. Endothelial protein expression was determined by immunofluorescence. RESULTS Exposure to non-uniform shear stress and TNF-α lead to progressive induction of adhesion molecules and increase in monocytic cell adhesion observed over 0.5-3 h. To investigate the relative role of the shear stress patterns in monocytic cell recruitment, ECs were exposed to reduced levels of shear stress, resulting in a reduced gradient steepness in the non-uniform shear stress regions. Lowering the shear stress from 10 to 5 and 2 dyne/cm2 resulted in increased monocytic cell adhesion under laminar shear stress. However, in these conditions, adherent monocytic cells under non-uniform shear stress were strongly reduced. Moreover, in the region exposed to shear stress gradient parallel to flow direction, monocytic cell adhesion was significantly lower than in the region of non-uniform shear stress, characterized by transversal gradient. CONCLUSION Exposure to non-uniform shear stress results in progressive induction of adhesion molecules and monocytic cell recruitment in response to circulating TNF-α. Enhanced monocytic cell recruitment at bifurcations is affected not only by the magnitude and steepness of shear stress gradient, but also by its direction in relation to the flow.

TNF-α in the cardiovascular system: From physiology to therapy

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php International Journal of Interferon, Cytokine and Mediator Research 2015:7 9–25 International Journal of Interferon, Cytokine and Mediator Research Dovepress