Novel aspects of atherosclerosis : focusing on new target genes and the effect of cholesterol-lowering

Atherosclerosis is most often the main underlying cause of cardiovascular diseases (CVDs), accounting for more than 31% of all deaths worldwide. It is driven by the uptake of low-density lipoproteins (LDL) by a dysfunctional arterial endothelium. It involves a complex interplay of genetic and cellular factors that result in an uncontrolled inflammatory response which can potentially be fatal. The main treatment for atherosclerosis is cholesterol-lowering drugs, statins. Despite their use, clinical events still occur. In this thesis, three papers regarding CVDs and some of the key events happening during atherosclerosis are discussed. In paper I the role of Lim domain-binding 2 (Ldb2) as a master regulator of transendothelial migration of leukocytes (TEML) during atherosclerosis was investigated. We described its function as a modulator of the leukocyte extravasation process using in vivo mouse models and in vitro systems. By examining Ldb2-deficient mice we found increased atherosclerotic lesions and decreased plaque stability. Their TEML activity was increased, especially regarding monocytes and macrophages, the principal initiators of the atherosclerotic process. Additionally, the role of this gene was reinforced by a functional SNP found in coronary artery disease (CAD) cohorts associated with increased risk of myocardial infarction (MI). In the following publication, paper II, we describe the function of the newly identified cholesterol-responsive gene Poliovirus receptor-related 2 (PVRL2) in atherosclerosis. This gene, as a member of the nectin family, plays a major role during TEML in the extravasation step. Regarding atherosclerosis development, Pvrl2-deficient mice showed fewer lesions and more stable plaques. An increased endothelial expression of Pvrl2 coincided with an increase in leukocyte gene expression, strengthening its potential role during TEML. In fact, we found a significant decrease in leukocyte migration in the Pvrl2-deficient mice using in vivo assays. Moreover, we observed its endothelial expression and cholesterol-responsiveness in humans. The effect of cholesterol-lowering on atherosclerosis is well established, and statins remain the main treatment. Since statins are prescribed to most CVD patients due to the underlying atherosclerosis, their specific effect on single diseases are not well studied. In paper III we aimed to identify the effect of angiotensin II (AngII)-induced AAA on atherosclerosis and the influence of cholesterol-lowering on abdominal aortic aneurysm (AAA) in an atherosclerotic mouse model. We found a low incidence of AAA formation after AngII infusion, possibly because the levels of cholesterol in our mice were not high enough. Nevertheless, AngII was found to enhance atherosclerosis and leukocyte infiltration, stressing the importance of the renin-angiotensin system on atherosclerosis and suggesting a controlling effect of cholesterol in the model. All three papers emphasize the importance of cholesterol during CVDs. Further research in order to elucidate the underlying mechanisms and detailed role of the identified gene targets could have a major impact on the development of new drugs. These could act directly on the TEML process, thus modulating the inflammatory response and attenuating disease complications

Plasma Cholesterol-Induced Lesion Networks Activated before Regression of Early, Mature, and Advanced Atherosclerosis

Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (>= 80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr(-/-)Apob(100/100)Mttp(flox/flox)Mx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions. Author Summary The main underlying cause of heart attacks and strokes is atherosclerosis. One strategy to prevent these often deadly clinical events is therefore either to slow atherosclerosis progression or better, induce regression of atherosclerotic plaques making them more stable. Plasma cholesterol lowering (PCL) is the most efficient way to induce atherosclerosis regression but sometimes fails to do so. In our study, we used a mouse model with elevated LDL cholesterol levels, similar to humans who develop early atherosclerosis, and a genetic switch to lower plasma cholesterol at any time during atherosclerosis progression. In this model, we examined atherosclerosis gene expression and regression in response to PCL at three different stages of atherosclerosis progression. PCL led to complete regression in mice with early lesions but was incomplete in mice with mature and advanced lesions, indicating that early prevention with PCL in individuals with increased risk for heart attack or stroke would be particularly useful. In addition, by inferring PCL-responsive gene networks in early, mature and advanced atherosclerotic lesions, we identified key drivers specific for regression of early (PPARG), mature (MLL5) and advanced (SRSF10/XRN2) atherosclerosis. These key drivers should be interesting therapeutic targets to enhance PCL-mediated regression of atherosclerosis

Induction of the Coxsackievirus and Adenovirus Receptor in Macrophages During the Formation of Atherosclerotic Plaques.

Multiple viruses are implicated in atherosclerosis, but the mechanisms by which they infect cells and contribute to plaque formation in arterial walls are not well understood. Based on reports showing the presence of enterovirus in atherosclerotic plaques we hypothesized that the coxsackievirus and adenovirus receptor (CXADR/CAR), although absent in normal arteries, could be induced during plaque formation. Large-scale microarray and mass spectrometric analyses revealed significant up-regulation of CXADR messenger RNA and protein levels in plaque-invested carotid arteries compared with control arteries. Macrophages were identified as a previously unknown cellular source of CXADR in human plaques and plaques from Ldr-/-Apob100/100 mice. CXADR was specifically associated with M1-polarized macrophages and foam cells and was experimentally induced during macrophage differentiation. Furthermore, it was significantly correlated with receptors for other viruses linked to atherosclerosis. The results show that CXADR is induced in macrophages during plaque formation, suggesting a mechanism by which enterovirus infect cells in atherosclerotic plaques.info:eu-repo/semantics/publishe

Effects of siRNA inhibition of the network top hubs on cholesterol-ester accumulation in a THP-1 foam cell model.

<p>CE, cholesterol ester.</p

Transcriptional profiling during regression of aortic atherosclerotic lesions in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^flox/flox and <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^Δ/Δ mice over time.

<p>Differential expression analyses was used to define sets of genes causally and reactively related to atherosclerosis regression in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^Δ/Δ mice. RNA for the transcriptional profiling was isolated from the atherosclerotic aortic arch. Narrow and bold arrows indicate times of PCL and sacrifice, respectively. Colored horizontal lines indicate time frame of transcriptional profiles used for differential expression analysis to define gene sets. Colors indicate when PCL was started: green, 30 weeks; yellow, 40 weeks; red, 50 weeks. (A) To define the PCL-responsive gene sets, we compared transcriptional profiles (4–6 per time point) of PBS-treated, high-cholesterol littermate controls sacrificed at 30, 40 and 50 weeks with those immediately after PCL. (B) To define the regression-reactive gene sets, we compared transcriptional profiles (3–6 per time point) immediately after PCL with those at 10 weeks after PCL (10 per time point).</p

Plasma cholesterol, triglyceride and glucose concentrations in the study mice at sacrifice.

<p>Values are mean ± SD.</p><p>*<i>P</i><0.05,</p><p>***<i>P</i><0.001 vs. before PCL.</p

Top hubs in the causal TF-regulatory co-expression networks inferred in human macrophages.

<p>TF, transcription factor. Common hubs >5 connections.</p

Atherosclerosis progression in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^flox/flox mice and regression in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^Δ/Δ mice.

<p>(A) Atherosclerosis progression and regression curves. Values are surface lesion area (mean ± SD), assessed by Sudan IV staining, as a percentage of the total area of pinned-out aortas. n = 4–10 per time point. Lesion development in controls without PCL (•) (<i>P</i><0.001 vs. 30 weeks) and in mice after PCL started at week 30 (▴), 40 (▪), or 50 (). Changes in lesion area between 10 and 20 weeks of low plasma cholesterol were significant only in mice with early lesions (PCL at 30 weeks, <i>P</i> = 0.05). *<i>P</i> = 0.05, ***<i>P</i><0.001. (B) Representative aortic trees (above) with magnified arches (below) stained with Sudan IV before and 10 and 20 weeks after PCL at 30, 40 and 50 weeks. Graphs indicate degree of regression at that PCL time-point (red).</p

PCL-responsive and regression reactive gene sets of atherosclerosis regression.

<p>Venn diagrams showing the percentage/number of differentially expressed genes at 30, 40, and 50 weeks. The colors of the circles indicate when PCL was started: green, 30; yellow, 40 weeks; red, 50 weeks. The percentage in the circles to the left represent the percentage of differentially expressed genes for that section and specific time point. The numbers in circles to the right represent numbers of differentially expressed genes. (A) The PCL-responsive gene sets consist of genes that responded immediately to PCL, initiating regression of early (30 weeks), mature (40 weeks), and advanced (50 weeks) atherosclerosis. (B) The regression-reactive gene sets consist of genes altered in lesions between immediately after PCL and 10 weeks of low plasma cholesterol levels.</p

Immunohistochemical characteristics of representative frozen sections of aortic roots from <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^flox/flox and <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^Δ/Δ mice.

<p>(A–C) Average percent stained area of total aortic root area (right) and representative stained aortic roots (left). Bars indicate SD. Original magnification, 50×. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001. (A) Oil-Red-O staining (n = 6–9 per group). (B) CD68 staining (n = 5–8 per group). (C) Sirius Red staining (collagen) (n = 3 per group). (D) Mean plaque stability score (arbitrary units). Bars indicate SD. Average plaque stability scores were divided by total extent of plaque burden to assess stability per mouse (not individual plaques). Inset: magnifications of plaque stability score/mouse at 30, 40, 50, and 60 weeks before regression.</p