Dolf Segers

Shear stress–induced changes in atherosclerotic plaque composition are modulated by chemokines

We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-gamma-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.

Effect of shear stress on vascular inflammation and plaque development

Purpose of review This review describes evidence that shear stress acts through modulation of inflammation and by that process affects atherogenesis and plaque composition. Recent findings In low shear stress regions antiatherogenic transcription factors are downregulated and pro-atherogenic transcription factors are upregulated. Consequently, inflammatory cells may home low shear stress regions more easily to the plaques because of increased expression of adhesion factors, a decreased rolling speed and an increased expression of chemokines, thereby changing the composition of the plaques into a more vulnerable phenotype. In contrast, in advanced plaque development vascular lumen decreases and shear stress increases, especially upstream of the plaques. The predominant upstream location of lipids induces a prevalent upstream location of inflammatory cells leading to localized plaque rupture. Summary Shear stress has been shown to play a role in plaque induction, plaque progression and plaque rupture. The mechanism for plaque induction seems to differ from the role of shear stress for plaque rupture, whereby the former mechanism is induced by low shear stress and the latter by high shear stress.

Gelatinolytic Activity in Atherosclerotic Plaques Is Highly Localized and Is Associated With Both Macrophages and Smooth Muscle Cells In Vivo

Background— Atherosclerosis is considered an inflammatory disease. Recent studies provided evidence for a predominant upstream location of plaque inflammation. The present study introduces a novel technique that evaluates the underlying mechanism of this spatial organization. Methods and Results— In hypercholesterolemic rabbits, atherosclerosis of the infrarenal aorta was induced by a combination of endothelial denudation and a high-cholesterol diet (2% cholesterol for 2 months). At the time of death, aortic vessel segments were dissected and reconstructed with a new technique that preserved the original intravascular ultrasound-derived lumen geometry. This enabled us to study the spatial relation of histological markers like macrophages, smooth muscle cells, lipids, gelatinolytic activity, and oxidized low-density lipoprotein. Results showed a predominant upstream localization of macrophages and gelatinase activity. Colocalization studies indicated that gelatinase activity was associated with macrophages and smooth muscle cells. Further analysis revealed that this was caused by subsets of smooth muscle cells and macrophages, which were associated with oxidized low-density lipoprotein accumulation. Conclusions— Upstream localization of a vulnerable plaque phenotype is probably due to an accumulation of oxidized low-density lipoprotein, which activates/induces subsets of smooth muscle cells and macrophages to gelatinase production.

Contrast enhancement by differently sized paramagnetic MRI contrast agents in mice with two phenotypes of atherosclerotic plaque

Interest in the use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of three differently sized gadolinium-based contrast agents to permeate different mouse plaque phenotypes was evaluated with MRI. A tapered cast was implanted around the right carotid artery of apoE(-/-) mice to induce two different plaque phenotypes: a thin cap fibroatheroma (TCFA) and a non-TCFA lesion. Both plaques were allowed to develop over 6 and 9 weeks, leading to an intermediate and advanced lesion, respectively. Signal enhancement in the carotid artery wall, following intravenous injection of Gd-HP-DO3A as well as paramagnetic micelles and liposomes was evaluated. In vivo T(1) -weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy and correlated to lesion phenotype. The two smallest contrast agents, i.e. Gd-HP-DO3A and micelles, were found to enhance contrast in T(1) -weighted MR images of all investigated plaque phenotypes. Maximum contrast enhancement ranged between 53 and 70% at 6 min after injection of Gd-HP-DO3A with highest enhancement and longest retention in the non-TCFA lesion. Twenty-four hours after injection of micelles maximum contrast enhancement ranged between 24 and 35% in all plaque phenotypes. Administration of the larger liposomes did not cause significant contrast enhancement in the atherosclerotic plaques. Confocal fluorescence microscopy confirmed the MRI-based differences in plaque permeation between micelles and liposomes. Plaque permeation of contrast agents was strongly dependent on size. Our results implicate that, when equipped with targeting ligands, liposomes are most suitable for the imaging of plaque-associated endothelial markers due to low background enhancement, whereas micelles, which accumulate extravascularly on a long timescale, are suited for imaging of less abundant markers inside plaques. Low molecular weight compounds may be employed for target-specific imaging of highly abundant extravascular plaque-associated targets.

Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE-/- mice

AIMS Wall shear stress differentially regulates the arginase pathway in carotid arteries perfused ex vivo. Specific patterns of wall shear stress can locally determine atherosclerotic plaque size and composition in vivo. The present work investigates the effects of arginase inhibition on shear stress induced plaque composition. METHODS AND RESULTS Carotid arteries of apolipoprotein E deficient mice were exposed to high (HSS), low (LSS) and oscillatory (OSS) shear stress conditions by the placement of a local shear stress modifier device for 9 weeks with or without the administration of the arginase inhibitor N-ω-Hydroxy-nor-L-arginine (nor-Noha) (10 mg/kg, i.p., 5 days/week). Carotid arginase activity was measured by colorimetric determination of urea. Atherosclerotic plaque size and composition, arginase expression and cellular localization were assessed by immunohistochemistry. Arginase activity was significantly increased in both LSS and OSS regions as compared to HSS. In the lesions, arginase II isoform co-localized preferentially with EC. Inhibition of arginase by nor-Noha decreased arginase activity and reduced plaque size in both LSS and OSS regions. Arginase inhibition affected mainly the composition of plaques developed in LSS regions by decreasing the total vascular ROS, the number of macrophages, apoptosis rate, lipid and collagen contents. CONCLUSIONS Arginase activity is modulated by patterns of wall shear stress in vivo. Chronic inhibition of vascular arginase decreased the size of atherosclerotic lesions in both OSS and LSS regions, whereas changes on plaque composition were more pronounced in plaques induced by LSS. We identified wall shear stress as a key biomechanical regulator of arginase during plaque formation and stability.

Fibromodulin Deficiency Reduces Low-Density Lipoprotein Accumulation in Atherosclerotic Plaques in Apolipoprotein E–Null Mice

Objective—The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques. Methods and Results—Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development. Conclusion—The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability.

The Role of Platelet-Endothelial Cell Adhesion Molecule-1 in Atheroma Formation Varies Depending on the Site-Specific Hemodynamic Environment

Objective—Polymorphisms in the platelet-endothelial cell adhesion molecule (PECAM-1)-1 gene are linked to increased risk of coronary artery disease. Because PECAM-1 has been demonstrated to form a mechanosensory complex that can modulate inflammatory responses in murine arterial endothelial cells, we hypothesized that PECAM-1 contributes to atherogenesis in a shear-dependent and site-specific manner. Approach and Results—ApoE–/– mice that were wild-type, heterozygous, or deficient in PECAM-1 were placed on a high-fat diet. Detailed analysis of the aorta at sites with differing hemodynamics revealed that PECAM-1–deficient mice had reduced disease in areas of disturbed flow, whereas plaque burden was increased in areas of steady, laminar flow. In concordance with these observations, bone marrow chimera experiments revealed that hematopoietic PECAM-1 resulted in accelerated atheroma formation in areas of laminar and disturbed flow, however endothelial PECAM-1 moderated disease progression in areas of high sheer stress. Moreover, using shear stress–modifying carotid cuffs, PECAM-1 was shown to promote macrophage recruitment into lesions developing in areas of low shear stress. Conclusions—PECAM-1 on bone marrow cells is proatherogenic irrespective of the hemodynamic environment, however endothelial cell PECAM-1 is antiatherogenic in high shear environments. Thus, targeting this pathway therapeutically would require a cell-type and context-specific strategy.

Atherosclerotic Plaque Stability Is Affected by the Chemokine CXCL10 in Both Mice and Humans

Background. The chemokine CXCL10 is specifically upregulated during experimental development of plaque with an unstable phenotype. In this study we evaluated the functional consequences of these findings in mice and humans. Methods and Results. In ApoE−/− mice, we induced unstable plaque with using a flow-altering device around the carotid artery. From week 1 to 4, mice were injected with a neutralizing CXCL10 antibody. After 9 weeks, CXCL10 inhibition resulted in a more stable plaque phenotype: collagen increased by 58% (P = 0.002), smooth muscle cell content increased 2-fold (P = 0.03), while macrophage MHC class II expression decreased by 50% (P = 0.005). Also, the size of necrotic cores decreased by 41% (P = 0.01). In 106 human carotid endarterectomy specimens we found that increasing concentrations of CXCL10 strongly associate with an increase in atheromatous plaque phenotype (ANOVA, P = 0.003), with high macrophage, low smooth muscle cell, and low collagen content. Conclusions. In the present study we showed that CXCL10 is associated with the development of vulnerable plaque in human and mice. We conclude that CXCL10 might provide a new lead towards plaque-stabilizing therapy.

A primer on the immune system in the pathogenesis and treatment of atherosclerosis

Atherosclerosis is currently appreciated as a disease with a large inflammatory component. The underlying mechanisms, which are uncovered in a rapid pace, are greatly interconnected and as such very complex. Nevertheless, for clinicians it is important have some degree of insight in these immunologic mechanisms in order to interpret the current research advances. The aim of this review is to supply clinicians with this knowledge, avoiding too much detail. All the relevant immunologic basics will be discussed at first, followed by the immunity related theories of atherosclerosis. Finally, current and new immune-modulatory therapies will be discussed.

Atherosclerosis: cell biology and lipoproteins--shear stress and inflammation in plaque formation: new evidence.

Atherosclerosis is the disease with the highest mortality in the western world. Despite its large socioeconomical impact, the underlying mechanisms are only partially known. Although lipid transport and accumulation has been studied for years, recently two concepts – lipiddriven inflammation and blood flow/shear stress – have undergone a renaissance and gained a lot of interest as complementary explanations for plaque formation and the interaction between lipids, inflammation and shear stress are the topic of some recent publications that will be discussed below.