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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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Coronary atherosclerotic plaque characterisation may contribute to risk stratification for future cardiovascular events. The ability of computed tomography to classify plaques as 'fibrous' or 'lipid-rich' based on their average CT attenuation has been investigated but is fraught with substantial limitations. In this study, we evaluated the potential of analysing the distribution of CT attenuation values measured in Hounsfield Units (HU) within coronary atherosclerotic plaques to classify non-calcified plaques into fibrous and lipid-rich subtypes. Intravascular ultrasound (IVUS) served as the gold standard., Patients and Methods: We evaluated the data sets of 40 patients (30 males, 59±10 years) who had been referred for invasive coronary angiography for clinical reasons and in whom IVUS was performed in at least one coronary vessel. Using dual source CT, coronary CT angiography was performed as a part of a research protocol within 24 h previous to invasive coronary angiography. A contrast-enhanced volume dataset was acquired with retrospective ECG gating (120 kV, 400 mAs/rot, collimation 2 mm×64 mm×0.6 mm, 60-80 ml contrast agent i.v). IVUS was performed using a 40-MHz IVUS catheter (Atlantis, Boston Scientific Corporation, Natick, MA) and motorized pullback at 0.5 mm/s. Fifty five corresponding non-calcified plaques within the coronary artery system were identified in both DSCT and IVUS using bifurcation points as fiducial markers. In DSCT data sets, serial parallel cross-sections (1mm slice thickness) were rendered orthogonally to the centre line of the coronary artery for each of the 55 plaques. For each cross section and each plaque, a histogram of CT attenuation values (increments of 10HU) was determined. The percentage of pixels with a density ≤30 HU was calculated. Using IVUS as the gold standard, plaques were classified as predominantly fibrous (hyperechoic) or predominantly lipid-rich (hypoechoic)., Results: 15 predominantly fibrous plaques vs. 40 predominantly lipid-rich plaques were identified in IVUS. The mean CT attenuation in both plaque types was significantly different (67±31 HU vs. 96±40 HU, p=0.006), yet with a wide overlap. For the 15 fibrous plaques identified in IVUS, the mean percentage of pixels ≤30 HU in CT was 6±10%. For lipid-rich plaques it was 16±10% (p<0.0001). ROC curve analysis revealed that a cut-off of 5.5% pixels with an attenuation ≤30 HU identified lipid rich plaques in CT angiography with a sensitivity of 95% (38/40, 95% CI 83-99) and a specificity of 80% (12/15, 95% CI 52-96) [AUC 0.9, 95% CI 0.7-1.0]. Using this threshold, the negative predictive value was 86% (12/14, 95% CI 57-98) and the positive predictive value was 93% (38/41, 95% CI 80-98)., Conclusion: Lipid-rich coronary atherosclerotic plaques contain a significantly higher percentage of pixels with low CT attenuation as compared to fibrous plaques. Histogram analysis may help to differentiate both plaque types. A cut-off of 5.5% of pixels with an attenuation of ≤30 HU allowed identification of lipid-rich plaques with a sensitivity of 95% and a specificity of 80%., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: We analyzed typical morphological features of coronary atherosclerotic plaques in acute coronary syndromes (ACS) using contrast-enhanced coronary Dual-Source CT angiography (CTA) in comparison to stable coronary lesions., Patients and Methods: Fifty-five patients with ACS and 55 controls with stable angina pectoris (SAP) with similar atherosclerotic risk profile were studied. CT angiography was performed using a Dual-Source CT scanner (330 ms rotation, 2 x 64 x 0.6mm collimation, 60-80 mL contrast agent i.v. at 6 mL/s) before invasive catheterization. We analyzed plaque volume (mm(3)), mean and minimal CT density (HU), remodeling index, plaque type (calcified/non-calcified/mixed) and presence of "spotty" calcifications as well as presence of contrast rims., Results: In patients with ACS and SAP, 28 and 10 lesions showed both calcified and non-calcified components, but in a greater proportion of non-calcified material, 6 and 23 lesions showed a greater proportion of calcified material and 21 and 8 lesions were completely non-calcified, respectively. None of the culprit plaques in ACS and 14 of the lesions in SAP were completely calcified. A "spotty" pattern of calcification within the plaque and a central filling defect surrounded by a rim of contrast were present in 11 and 14 of 55 ACS cases, but never in SAP lesions. For culprit lesions in ACS and for lesions in patients with SAP, mean plaque volumes were 192.8 + or - 114.9 mm(3) and 103.8+/-51.8 mm(3) (p=0.001), mean and minimal CT densities were 85.6 + or - 45.1HU and 47.2 + or - 33.7 HU versus 143.8 + or - 104.1 HU and 95.9 + or - 84.0 HU (p<0.01) and mean remodeling indices were 1.6 + or - 0.4 and 0.97 + or - 0.17 (p<0.001), respectively., Conclusion: Plaques of culprit lesions in ACS show specific morphologic characteristics in non-invasive coronary CT angiography. As compared to stable lesions, culprit lesions in ACS display greater proportion of non-calcified material and presence of "spotty" calcifications/contrast rims as well as larger plaque volumes, lower CT attenuation and higher remodeling indices., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: Atherosclerotic plaque formation results from a combination of local shear stress patterns and inflammatory processes. This study investigated the endothelial response to shear stress in combination with the inflammatory cytokine TNF-alpha in a simplified model of arterial bifurcation., Methods: Human umbilical vein endothelial cells (ECs) were exposed to laminar or non-uniform shear stress in bifurcating flow-through slides, followed by stimulation with TNF-alpha. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells. Endothelial protein expression was determined by immunofluorescence., Results: Adhesion of monocytic cells to unstimulated ECs was nearly undetectable under laminar shear stress and was slightly increased under non-uniform shear stress. Exposure of ECs to non-uniform shear stress in combination with TNF-alpha induced a 12-fold increase in monocytic cell recruitment and a significant induction of endothelial E-selectin and VCAM-1 expression. Both these effects were prevented in ECs exposed to laminar shear stress. The significant differences in TNF-alpha-induced monocytic cell recruitment and adhesion molecule expression between laminar and non-uniform shear stress regions were abolished in the absence of shear stress preconditioning. Simvastatin (1 micromol/L) suppressed the non-uniform shear stress- and TNF-alpha-induced increase in monocytic cell adhesion by about 30% via inhibition of VCAM-1 expression. Resveratrol, the active component of red wine, inhibited the expression of both VCAM-1 and E-selectin, and reduced monocytic cell recruitment by 50% at 20 micromol/L., Conclusions: Non-uniform shear stress induces endothelial susceptibility to circulating TNF-alpha and adhesion of monocytic cells. Interference with this process may inhibit inflammatory response in atherosclerosis-prone regions.

Objective: Identification of clinically relevant determinants for acute coronary syndromes (ACS) promises reduction of ACS-associated mortality. C-reactive protein (CRP) has proved to be useful identifying people at risk for cardiovascular events. However, it is unknown whether genetic variants at Fcgamma receptor IIa (FcgammaRIIa), the main receptor for CRP, are involved in CRP-related cardiovascular risk. We evaluated the potential impact of FcgammaRIIa through a genetic association study in patients with ACS., Methods and Results: We conducted a genetic association study among 701 consecutive patients with first event of ACS compared to 467 patients with stable angina pectoris. All patients were genotyped for a frequent functional variant at position 131 of the mature FcgammaRIIa, where the arginine (R) allele results in an increased signal transduction upon CRP binding. In our study, the R/R131 genotype was significantly associated with ACS as the first manifestation of coronary artery disease (P=1.2x10(-9), odds ratio 2.86, 95% CI: 2.06-3.99) compared to the non-R/R131 genotype., Conclusions: Our data show a genetic association of the FcgammaRIIa R/R131 genotype with a more frequent occurrence of ACS as the first manifestation of coronary artery disease, probably mediated via its interaction with CRP. Genotyping of this FcgammaRIIa variant could help to improve risk stratification in the course of coronary disease in the future.

Unlabelled: Multi-detector CT (MDCT) permits non-invasive visualization of the coronary arteries. Coronary plaque can be visualized, and earlier studies have indicated that the CT attenuation measured in atherosclerotic plaques is influenced by plaque composition. Also, MDCT has been shown to permit assessment of remodeling of coronary atherosclerotic lesions. It is assumed that both lipid-rich plaques and those that display positive remodeling are more prone to rupture and erosion. We thus evaluated the relationship between remodeling and CT attenuation of coronary atherosclerotic plaque by MDCT., Methods: Seventy-six patients were investigated by contrast-enhanced 64-slice CT. One-hundred twelve atherosclerotic lesions without substantial calcification and visualized with high image quality were selected. Multiplanar reconstructions orthogonal to the coronary artery were rendered at the lesion and the proximal reference site. Cross-sectional vessel areas were measured to determine the remodeling index (RI: lesion vessel area/reference vessel area) and the CT attenuation of plaque was measured by fitting a region of interest to the plaque area. CT attenuation of plaque was correlated to the presence of positive remodeling index (RI>1.05)., Results: The mean cross-sectional vessel area in the lesion was 0.25+/-0.08 cm(2), the mean reference vessel area was 0.22+/-0.09 cm(2). The mean CT attenuation of the atherosclerotic plaque in the lesions was 71+/-26 HU. CT attenuation of plaque was significantly lower in 72 lesions that displayed positive remodeling (59+/-22 HU) than in 40 lesions with no or with negative remodeling (91+/-20 HU, p<0.001)., Conclusions: Positive remodeling of coronary atherosclerotic lesions correlates to lower CT attenuation of plaque, which has been demonstrated to be associated with lipid-rich plaque. Both characteristics indicate increased risk for plaque rupture and subsequent events and could thus prove useful when the use of CT imaging for the detection of "vulnerable plaque" is considered.

Shear stress changes play an important role in atheroma formation. This study focussed on atherogenic protein expression under non-uniform shear stress and the pharmacological modulation of shear-related endothelial dysfunction. Bifurcating flow-through cell culture slides were used to expose HUVECs to steady laminar or non-uniform shear stress for 18 h at 10 dyn/cm(2). Protein expression was determined by immunofluorescence, and quantified using MetaVue software. Laminar shear stress resulted in cell alignment, reduced F-actin fibers, and significant induction of endothelial nitric oxide synthase expression. Under non-uniform shear stress at bifurcations, minor upregulation of adhesion molecules was observed. Connective tissue growth factor (CTGF) was significantly downregulated by laminar shear stress and induced in cells exposed to non-uniform shear stress. CTGF upregulation by non-uniform shear stress was RhoA-dependent, because it was almost completely inhibited in cells transfected with dominant negative RhoA-N19, and when cells were treated with 1 micromol/L simvastatin during flow. Pre-incubation of HUVECs with inhibitors of Rho-associated kinase before exposure to flow significantly suppressed the CTGF induction in regions of non-uniform shear stress. In conclusion, non-uniform shear stress-dependent CTGF expression requires active RhoA and can be prevented pharmacologically. Interference with shear stress-induced protein expression may inhibit endothelial dysfunction in atheroprone vessel regions.

Unlabelled: Multi-detector row Computed Tomography (MDCT) permits non-invasive visualization of the coronary arteries. The ability to visualize and, with limitations, to characterize non-calcified coronary atherosclerotic plaque has been described. We investigated the CT attenuation of non-calcified plaques as determined by 16-slice MDCT in comparison to intravascular ultrasound (IVUS)., Methods and Results: Thirty-two patients were investigated by contrast-enhanced 16-slice CT. In addition, IVUS of one coronary artery (motorized pullback) was performed (LM+LAD: 22, LM+LCX: 4, RCA: 6). At 252 sites within the coronary system, in which non-calcified atherosclerotic plaque could be identified both in MDCT and IVUS, the CT attenuation within the plaque was measured using a centrally placed region of interest and correlated to the appearance of the plaque in IVUS at the corresponding location. The mean CT attenuation within plaque that corresponded to hyper-echogenic appearance in IVUS was 121+/-34HU (n=76). The mean CT attenuation within plaque that corresponded to hypo-echogenic appearance was 58+/-43HU (n=176, p<0.001). However, there was substantial overlap of the density values measured by MDCT in the two groups., Conclusions: A significant difference of the mean CT attenuation within atherosclerotic lesions of hypo-echogenic and hyper-echogenic appearance in IVUS could be observed. However, we observed substantial overlap of attenuation values between plaque types so that the differentiation of "vulnerable" and "stable" plaques based on their CT attenuation is doubtful.

Objectives: Macrophage migration inhibitory factor 1alpha (MIF), a cytokine with immunoregulatory functions has been suggested to be involved in atherosclerotic plaque development. However, little is known about MIF-inducing conditions in the atherosclerotic process and the association of MIF with plaque instability., Methods and Results: Forty-two carotid endatherectomy samples from 36 patients and 4 aortic samples from young accident victims (as healthy controls) were analyzed for MIF staining. MIF expressing tissues in the atherosclerotic plaques are mainly mononuclear cells (MNCs), but also endothelial cells of intimal microvessels (MVECs). The magnitude and the intensity of their MIF expression was associated with the progression of plaques from early lesions (Stary I-III) to complicated plaque stages (Stary IV-VIII). In highly inflammatory and neovascularized regions of the plaques the colocalization of MIF expressing MNCs with CD40-L+ and angiotensin II (Ang II)-producing MNCs could be established. This finding supports the notion that CD40-L fusion protein and Ang II are able to induce MIF production in the monocytic cell line THP-1. Furthermore hypoxia (< or =1% O2) as a further proinflammatory and especially proangiogenetic factor was able to stimulate MIF secretion by THP-1, human monocytes and HUVECs. Hyperglycemia and insulin remained without effect., Conclusion: MIF is expressed in advanced atherosclerotic lesions in close correlation with signs of instability, such as mononuclear cell inflammation and neointimal microvessel formation. Furthermore, the colocalization of MIF with Ang II-producing MNCs and CD40-L+ cells in these plaques and the finding that proathero- and -angiogenic mediators such as CD40-L, Ang II and hypoxia are able to stimulate MIF expression in vitro suggest an important role of MIF in the modulation of atherosclerotic plaque stability.

Dendritic cells (DC), which are critically involved in various immunological disorders, were detected in atherosclerotic plaques in 1995. Since DC might be related to the immunological processes in atherosclerosis (AS), we analyzed the emergence of DC and other inflammatory cells in different stages of AS. Serial cross-sections of 44 carotid specimens were immunohistochemically analyzed for the presence of DC, T cells, macrophages, and HLA-DR. Atherosclerotic specimens were histologically defined as initial lesions, advanced stable, or vulnerable plaques. In initial lesions significantly lower DC numbers were detected than in advanced plaques (P < 0.001). For advanced plaques, DC numbers were significantly higher in vulnerable than in stable plaques (P = 0.005). In contrast to initial lesions, approximately 70% of DC in advanced plaques exhibited a mature phenotype (CD83+, DC-LAMP+), indicating a functional activity of DC. In plaques of patients with acute ischemic symptoms DC numbers were markedly elevated (P = 0.03), whereas significantly lower DC numbers and more often a stable plaque morphology were detected in statin-treated patients (P = 0.02). DC clusters with a strong HLA-DR expression and frequent DC-T cell contacts were located particularly in the rupture-prone plaque regions and at complications. The results of the present study indicate that DC might contribute to plaque destabilization through an activation of T cells.

The beneficial effects of statins in atherosclerosis have been partly attributed to their immunomodulating functions. Dendritic cells (DC), which are "professional" antigen-presenting cells, were recently detected in atherosclerotic plaques. It is assumed that DC play a critical role in the immunological processes related to atherosclerosis. Thus, we investigated the effects of statins on maturation and antigen-presenting function of DC. Human monocyte-derived DC were incubated with simvastatin or atorvastatin (1-10microM) for different periods (1-48h), and were subsequently stimulated with a cytokine cocktail (1.25ng/ml TNF-alpha, 1ng/ml Il-1beta, and 0.5microg/ml prostaglandin E(2)) to induce maturation. In contrast to untreated DC, statin-preincubated DC exhibited an immature phenotype and a significantly lower expression of the maturation-associated markers CD83, CD40, CD86, HLA-DR, and CCR7. The inhibitory statin effect was completely reversed by mevalonate or geranylgeranyl pyrophosphate. In addition, preincubation with statins significantly reduced the ability of cytokine-stimulated DC to induce T cell proliferation. In the present study, we have shown that statins inhibit the maturation and antigen-presenting function of human myeloid dendritic cells, thus maybe contributing to their beneficial effects in atherosclerosis. Therefore, the use of statins as immunomodulators might also provide a new therapeutic approach to other immunological disorders.

Objectives: The hypothesis was tested that CD40-CD154 interaction is involved in the induction of cyclooxygenase-2 and the release of prostanoids in human endothelial cells., Methods and Results: In a coculture model of human endothelial cells and a transfected CD154 positive cell line, engagement of CD40 on endothelial cells dramatically increased the synthesis of prostacyclin, prostaglandin E(2) and thromboxane A(2). This upregulation was mediated through an induction of cyclooxygenase-2 (Cox-2), as it was blocked by Cox-2-selective inhibitors. Western blot analysis demonstrated that Cox-2 protein was markedly increased in endothelial cells following CD40 engagement, an effect that was inhibited by pretreatment of cells with an anti-CD154 antibody., Conclusion: The data indicate that signaling via CD40 constitutes a major pathway in human endothelial cells for the induction of Cox-2 and release of prostanoids. The CD40-Cox-2 axis thus may represent an important pathway for initiating or maintaining an inflammatory process at the vessel wall.