Phenotypic Modulation of Smooth Muscle Cells in Atherosclerosis Is Associated With Downregulation of LMOD1, SYNPO2, PDLIM7, PLN, and SYNM.

OBJECTIVE: Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. APPROACH AND RESULTS: Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7, and PLN expression positively correlated to typical SMC markers in plaques (Pearson r>0.6, P<0.0001) and in rat intimal hyperplasia (r>0.8, P<0.0001). By immunohistochemistry, the proteins were expressed in SMCs in normal vessels, but largely absent in human plaques and intimal hyperplasia. Subcellularly, most proteins localized to the cytoskeleton in cultured SMCs and were regulated by active enhancer histone modification H3K27ac by chromatin immunoprecipitation-sequencing. Functionally, the genes were downregulated by PDGFB (platelet-derived growth factor beta) and IFNg (interferron gamma), exposure to shear flow stress, and oxLDL (oxidized low-density lipoprotein) loading. Genetic variants in PDLIM7, PLN, and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. CONCLUSIONS: We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

ATG16L1 Expression in Carotid Atherosclerotic Plaques Is Associated With Plaque Vulnerability.

OBJECTIVE: Autophagy has emerged as a cell survival mechanism critical for cellular homeostasis, which may play a protective role in atherosclerosis. ATG16L1, a protein essential for early stages of autophagy, has been implicated in the pathogenesis of Crohn's disease. However, it is unknown whether ATG16L1 is involved in atherosclerosis. Our aim was to analyze ATG16L1 expression in carotid atherosclerotic plaques in relation to markers of plaque vulnerability. APPROACH AND RESULTS: Histological analysis of 143 endarterectomized human carotid atherosclerotic plaques revealed that ATG16L1 was expressed in areas surrounding the necrotic core and the shoulder regions. Double immunofluorescence labeling revealed that ATG16L1 was abundantly expressed in phagocytic cells (CD68), endothelial cells (CD31), and mast cells (tryptase) in human advanced plaques. ATG16L1 immunogold labeling was predominantly observed in endothelial cells and foamy smooth muscle cells of the plaques. ATG16L1 protein expression correlated with plaque content of proinflammatory cytokines and matrix metalloproteinases. Analysis of Atg16L1 at 2 distinct stages of the atherothrombotic process in a murine model of plaque vulnerability by incomplete ligation and cuff placement in carotid arteries of apolipoprotein-E-deficient mice revealed a strong colocalization of Atg16L1 and smooth muscle cells only in early atherosclerotic lesions. An increase in ATG16L1 expression and autophagy flux was observed during foam cell formation in human macrophages using oxidized-LDL. CONCLUSIONS: Taken together, this study shows that ATG16L1 protein expression is associated with foam cell formation and inflamed plaque phenotype and could contribute to the development of plaque vulnerability at earlier stages of the atherogenic process.

Plasma asymmetric and symmetric dimethylarginine in a rat model of endothelial dysfunction induced by acute hyperhomocysteinemia.

Hyperhomocysteinemia induces vascular endothelial dysfunction, an early hallmark of atherogenesis. While higher levels of circulating asymmetric dimethylarginine (ADMA) and symmetric dimethyl arginine (SDMA), endogenous inhibitors of nitric oxide synthesis, have been associated with increased cardiovascular risk, the role that ADMA and SDMA play in the initiation of hyperhomocysteinemia-induced endothelial dysfunction remains still controversial. In the present study, we studied the changes of circulating ADMA and SDMA in a rat model of acutely hyperhomocysteinemia-induced endothelial dysfunction. In healthy rats, endothelium-related vascular reactivity (measured as acetylcholine-induced transient decrease in mean arterial blood pressure), plasma ADMA and SDMA, total plasma homocysteine (tHcy), cysteine and glutathione were measured before and 2, 4 and 6 h after methionine loading or vehicle. mRNA expression of hepatic dimethylarginine dimethylaminohydrolase-1 (DDAH1), a key protein responsible for ADMA metabolism, was measured 6 h after the methionine loading or the vehicle. Expectedly, methionine load induced a sustained increase in tHcy (up to 54.9 ± 1.9 µM) and a 30 % decrease in vascular reactivity compared to the baseline values. Plasma ADMA and SDMA decreased transiently after the methionine load. Hepatic mRNA expression of DDAH1, cathepsin D, and ubiquitin were significantly lower 6 h after the methionine load than after the vehicle. The absence of an elevation of circulating ADMA and SDMA in this model suggests that endothelial dysfunction induced by acute hyperhomocysteinemia cannot be explained by an up-regulation of protein arginine methyltransferases or a down-regulation of DDAH1. In experimental endothelial dysfunction induced by acute hyperhomocysteinemia, down-regulation of the proteasome is likely to dampen the release of ADMA and SDMA in the circulation.

The minor allele of the missense polymorphism Ser251Pro in perilipin 2 (PLIN2) disrupts an α-helix, affects lipolysis, and is associated with reduced plasma triglyceride concentration in humans.

Perilipin 2 (PLIN2) is the most abundant lipid droplet (LD)-associated protein in nonadipose tissue, and its expression correlates with intracellular lipid accumulation. Here we identified a missense polymorphism, Ser251Pro, that has major effect on protein structure and function, along with an influence on human plasma triglyceride concentration. The evolutionarily conserved Ser251Pro polymorphism was identified with the ClustalW program. Structure modeling using 3D-JigSaw and the Chimera package revealed that the Pro251 allele disrupts a predicted α-helix in PLIN2. Analyses of macrophages from individuals carrying Ser251Pro variants and human embryonic kidney 293 (HEK293) cells stably transfected with either of the alleles demonstrated that the Pro251 variant causes increased lipid accumulation and decreased lipolysis. Analysis of LD size distribution in stably transfected cells showed that the minor Pro251 allele resulted in an increased number of small LDs per cell and increased perilipin 3 protein expression levels as compared with cells carrying the major Ser251 allele. Genotyping of 2113 individuals indicated that the Pro251 variant is associated with decreased plasma triglyceride and very low-density lipoprotein concentrations. Altogether, these data provide the first evidence of a polymorphism in PLIN2 that affects PLIN2 function and may influence the development of metabolic and cardiovascular diseases.

LC3-associated endocytosis and the functions of Rubicon and ATG16L1.

LC3-associated endocytosis (LANDO) is a noncanonical function of the autophagy machinery, in which LC3 (microtubule-associated protein light chain) is conjugated to rab5-positive endosomes, using a portion of the canonical autophagy pathway. LANDO was initially discovered in a murine model of Alzheimer's disease as a critical regulator of amyloid-ß receptor recycling in microglial cells, playing a protective role against neuronal loss and memory impairment. Recent evidence suggests an emerging role of LANDO in cytokine receptor signaling and innate immunity. Here, we discuss the regulation of two crucial effectors of LANDO, Rubicon and ATG16L1, and their impact on endocytosis, autophagy, and phagocytosis.

Rapeseed protein in a high-fat mixed meal alleviates postprandial systemic and vascular oxidative stress and prevents vascular endothelial dysfunction in healthy rats.

High-saturated fat and high-sucrose meals induce vascular endothelial dysfunction, the early hallmark of atherogenesis. The impact of dietary protein on vascular homeostasis remains misunderstood. In this study, we investigated whether rapeseed protein, an emergent arginine- and cysteine-rich protein, can acutely modulate the onset of adverse effects induced by a high-saturated fat meal (HFM). In a series of crossover experiments, healthy rats received 3 HFM (saturated fat: 60%; sucrose: 20%; protein: 20% energy) with the protein source being either total milk protein (MP; control), rapeseed protein (RP), or MP supplemented with cysteine and arginine to the same level as in RP (MP+AA). Endothelium-related vascular reactivity, measured as an acetylcholine-induced transient decrease in blood pressure, and plasma triglycerides, hydroperoxides, cyclic GMP (cGMP), and free 3-nitrotyrosine were measured before and 2, 4, and 6 h after meals. Superoxide anion production, expressed as ethidine fluorescence, was measured in the aorta 6 h after meals. Whereas plasma triglycerides rose similarly in all meals, the decrease in vascular reactivity after MP was attenuated after MP+AA and entirely prevented after RP. The type of meal had no consistent effect on plasma cGMP and free 3-nitrotyrosine over the postprandial period. The postprandial increase in plasma hydroperoxides differed according to the meal, and concentrations were 43% lower 6 h after MP+AA and RP than after MP. Aortic superoxide anion production was 36% lower 6 h after RP than MP. These results show that substituting rapeseed protein for milk protein markedly reduces vascular and oxidative disturbances induced by an HFM and this may be mediated in part by cysteine and arginine.

Whole-body basal nitric oxide production is impaired in postprandial endothelial dysfunction in healthy rats.

In healthy humans, a high-saturated-fat/high-sucrose meal induces vascular endothelial dysfunction, a hallmark of atherogenesis. This transient dysfunction indicates a loss in nitric oxide (NO) production and/or bioactivity in the vasculature but it remains unknown if this is the local manifestation of a general impairment in NO pathway in the postprandial state. Here, we studied whole-body NO production and systemic NO bioactivity in postprandial endothelial dysfunction, as induced by a high-saturated-fat, high-sucrose meal. We first developed a physiological test of endothelial function on conscious rats, based on the transient fall in blood pressure after iv acetylcholine, and showed that this response was NO-dependent. As assessed with this method in healthy rats, endothelial function decreased during the postprandial state, being 60+/-7% lower than baseline at 6h after the meal challenge, associated with important elevations in plasma triglycerides and hydroperoxides. Aortic superoxide anion production, as assessed by oxidative fluorescent detection, was higher 6h after the meal challenge than after the nutrients vehicle (water). During the postprandial period, plasma cGMP, but not plasma ANP, markedly decreased, indicating a general decrease in NO bioavailability, which was numerically maximal 4h after the meal challenge. As determined 4h after ingestion by a tracer-based method using iv [(15)N(2)-(guanido)]-arginine, the whole-body NO production fell by 27+/-9% postprandially. This is the first study evidencing that a meal challenge that impairs the stimulated, NO-mediated, vascular response also reduces whole-body basal NO production and bioavailability. Postprandial pathophysiology may build on this general, fundamental alteration in NO production.

Rapeseed protein inhibits the initiation of insulin resistance by a high-saturated fat, high-sucrose diet in rats.

In contrast to the quality of carbohydrates and lipids, little is known on the influence of the type of dietary protein on the development of the metabolic or insulin resistance syndrome. Cysteine intake has been recently documented to impact insulin sensitivity. The aim of this study was to determine whether rapeseed protein, an emergent cysteine-rich protein, could inhibit the onset of the metabolic syndrome. For 9 weeks, rats were fed a diet rich in saturated fats and sucrose, which also included 20 % protein either as milk protein ('Induction' diet I) or rapeseed protein (diet R). A third, control group received an isoenergetic diet containing milk protein but polyunsaturated fats and starch ('Prudent' diet P). Plasma glucose, insulin, TAG and cholesterol, and blood pressure were monitored during the study, glucose tolerance was tested at week 7 and body composition determined at week 9. Plasma glucose, insulin and TAG increased during the experiment and, at week 9, plasma insulin was significantly 34 % lower in the R group and 56 % lower in P group as compared with the I group. The insulin peak after the glucose load was significantly 28-30 % lower in R and P than in I and the insulin sensitivity index was significantly higher in R than in I. Unexpectedly, peripheral fat deposition was slightly higher in R than in I. In this model, substituting rapeseed protein for milk protein had preventive effects on the early onset of insulin resistance, similar to those achieved by manipulating the types of dietary fat and carbohydrates.

LC3-Associated Phagocytosis and Inflammation.

LC3-associated phagocytosis (LAP) is a novel form of non-canonical autophagy where LC3 (microtubule-associated protein 1A/1B-light chain 3) is conjugated to phagosome membranes using a portion of the canonical autophagy machinery. The impact of LAP to immune regulation is best characterized in professional phagocytes, in particular macrophages, where LAP has instrumental roles in the clearance of extracellular particles including apoptotic cells and pathogens. Binding of dead cells via receptors present on the macrophage surface results in the translocation of the autophagy machinery to the phagosome and ultimately LC3 conjugation. These events promote a rapid form of phagocytosis that produces an "immunologically silent" clearance of the apoptotic cells. Consequences of LAP deficiency include a decreased capacity to clear dying cells and the establishment of a lupus-like autoimmune disease in mice. The ability of LAP to attenuate autoimmunity likely occurs through the dampening of pro-inflammatory signals upon engulfment of dying cells and prevention of autoantigen presentation to other immune cells. However, it remains unclear how LAP shapes both the activation and outcome of the immune response at the molecular level. Herein, we provide a detailed review of LAP and its known roles in the immune response and provide further speculation on the putative mechanisms by which LAP may regulate immune function, perhaps through the metabolic reprogramming and polarization of macrophages.

Mesenchymal state of intimal cells may explain higher propensity to ascending aortic aneurysm in bicuspid aortic valves.

Individuals with a bicuspid aortic valve (BAV) are at significantly higher risk of developing aortic complications than individuals with tricuspid aortic valves (TAV) and defective signaling during the embryonic development and/or life time exposure to abnormal hemodynamic have been proposed as underlying factors. However, an explanation for the molecular mechanisms of aortopathy in BAV has not yet been provided. We combined proteomics, RNA analyses, immunohistochemistry, and electron microscopy to identify molecular differences in samples of non-dilated ascending aortas from BAV (N = 62) and TAV (N = 54) patients. Proteomic analysis was also performed for dilated aortas (N = 6 BAV and N = 5 TAV) to gain further insight into the aortopathy of BAV. Our results collectively showed the molecular signature of an endothelial/epithelial-mesenchymal (EndMT/EMT) transition-like process, associated with instability of intimal cell junctions and activation of RHOA pathway in the intima and media layers of ascending aorta in BAV patients. We propose that an improper regulation of EndMT/EMT during the spatiotemporally related embryogenesis of semilunar valves and ascending aorta in BAV individuals may result in aortic immaturity and instability prior to dilation. Exasperation of EndMT/EMT state in post embryonic life and/or exposure to non-physiological hemodynamic could lead to the aneurysm of ascending aorta in BAV individuals.

Early postprandial low-grade inflammation after high-fat meal in healthy rats: possible involvement of visceral adipose tissue.

In the postprandial period, low-grade inflammation may contribute to vascular endothelial dysfunction, a hallmark of atherogenesis. Little is known about the involvement of the adipose tissue in the initiation of the postprandial inflammatory response such as obtained after a high-saturated fat meal (HFM). In the present study, we first studied the time course of appearance of systemic inflammation after a HFM in healthy rats, and then we investigated whether a HFM activates the inflammatory signaling in the visceral adipose tissue, with a focus on the key component, nuclear factor-kappaB (NF-kappaB). Two hours after the HFM, plasma IL-6 and PAI-1, but not plasma C-reactive protein and soluble intracellular adhesion molecule-1, showed a marked, transient increase. These changes were specific to the postprandial state as not observed after a control water load. Neutrophils count and activation markers CD11B and CD62L, assessed by flow cytometry, also rose significantly 2 h after the HFM, while remaining steady after the control. At the same time, the HFM decreased significantly B-cell count and expression of the activation marker CD62L. Interestingly, at the same early time after the HFM, in the visceral adipose tissue, there was a 2.2-fold increase in the activation of NF-kappaB (p65) in nuclear extract and an increase in IL-6 mRNA. As far as we know, this is the first study evidencing an acute, postprandial activation of inflammation in visceral adipose tissue. This early activation of NF-kappaB pathway after a HFM may play a triggering role in the initiation of the complex postprandial proatherogenic phenotype.