Correction: Proteome-wide analysis of chaperone-mediated autophagy targeting motifs.

[This corrects the article DOI: 10.1371/journal.pbio.3000301.].

Protective role of chaperone-mediated autophagy against atherosclerosis.

Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases.

Colorectal Cancer and Microbiota Modulation for Clinical Use. A Systematic Review.

Colorectal cancer (CRC) is one of the top contributors to the global burden of cancer incidence and mortality, with both genetic and environmental factors contributing to its etiology. Environmental factors may be the cause of up to 60% of the risk of developing CRC, with gut microbiota being a crucial modifiable risk factor. The microbial ecosystem plays a vital role in CRC prevention and antitumoral response through modulation of the immune system and production of short-chain fatty acids. Numerous approaches have been followed to modify the gut microbiota in order to reduce the risk of cancer development, improve treatment efficacy, and reduce side effects. This study aims to perform a systematic analysis of the published literature to elucidate whether microbiota modulation through pre-, pro-, and symbiotic treatment and/or nutritional intervention can be beneficial for patients diagnosed with CRC. Our analysis finds that some prebiotics, mainly in the form of oligo- and polysaccharides, probiotics such as lactic strain producers of short-chain fatty acids, and consumption of a Mediterranean plant-based diet may be beneficial for patients diagnosed with CRC. However, there is a need for clinical data which evaluate the modulation of gut microbiota in a safe and effective manner.

Proteome-wide analysis of chaperone-mediated autophagy targeting motifs.

Chaperone-mediated autophagy (CMA) contributes to the lysosomal degradation of a selective subset of proteins. Selectivity lies in the chaperone heat shock cognate 71 kDa protein (HSC70) recognizing a pentapeptide motif (KFERQ-like motif) in the protein sequence essential for subsequent targeting and degradation of CMA substrates in lysosomes. Interest in CMA is growing due to its recently identified regulatory roles in metabolism, differentiation, cell cycle, and its malfunctioning in aging and conditions such as cancer, neurodegeneration, or diabetes. Identification of the subset of the proteome amenable to CMA degradation could further expand our understanding of the pathophysiological relevance of this form of autophagy. To that effect, we have performed an in silico screen for KFERQ-like motifs across proteomes of several species. We have found that KFERQ-like motifs are more frequently located in solvent-exposed regions of proteins, and that the position of acidic and hydrophobic residues in the motif plays the most important role in motif construction. Cross-species comparison of proteomes revealed higher motif conservation in CMA-proficient species. The tools developed in this work have also allowed us to analyze the enrichment of motif-containing proteins in biological processes on an unprecedented scale and discover a previously unknown association between the type and combination of KFERQ-like motifs in proteins and their participation in specific biological processes. To facilitate further analysis by the scientific community, we have developed a free web-based resource (KFERQ finder) for direct identification of KFERQ-like motifs in any protein sequence. This resource will contribute to accelerating understanding of the physiological relevance of CMA.

Cav-1 (Caveolin-1) Deficiency Increases Autophagy in the Endothelium and Attenuates Vascular Inflammation and Atherosclerosis.

OBJECTIVE: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1-deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr-/- and Cav-1-/-Ldlr-/- mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1-/- mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis. CONCLUSIONS: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1-deficient mice attenuates the atheroprotection observed in Cav-1-/- mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.

Autophagy Is Required for Sortilin-Mediated Degradation of Apolipoprotein B100.

RATIONALE: Genome-wide association studies identified single-nucleotide polymorphisms near the SORT1 locus strongly associated with decreased plasma LDL-C (low-density lipoprotein cholesterol) levels and protection from atherosclerotic cardiovascular disease and myocardial infarction. The minor allele of the causal SORT1 single-nucleotide polymorphism locus creates a putative C/EBPα (CCAAT/enhancer-binding protein α)-binding site in the SORT1 promoter, thereby increasing in homozygotes sortilin expression by 12-fold in liver, which is rich in this transcription factor. Our previous studies in mice have showed reductions in plasma LDL-C and its principal protein component, apoB (apolipoprotein B) with increased SORT1 expression, and in vitro studies suggested that sortilin promoted the presecretory lysosomal degradation of apoB associated with the LDL precursor, VLDL (very-low-density lipoprotein). OBJECTIVE: To determine directly that SORT1 overexpression results in apoB degradation and to identify the mechanisms by which this reduces apoB and VLDL secretion by the liver, thereby contributing to understanding the clinical phenotype of lower LDL-C levels. METHODS AND RESULTS: Pulse-chase studies directly established that SORT1 overexpression results in apoB degradation. As noted above, previous work implicated a role for lysosomes in this degradation. Through in vitro and in vivo studies, we now demonstrate that the sortilin-mediated route of apoB to lysosomes is unconventional and intersects with autophagy. Increased expression of sortilin diverts more apoB away from secretion, with both proteins trafficking to the endosomal compartment in vesicles that fuse with autophagosomes to form amphisomes. The amphisomes then merge with lysosomes. Furthermore, we show that sortilin itself is a regulator of autophagy and that its activity is scaled to the level of apoB synthesis. CONCLUSIONS: These results strongly suggest that an unconventional lysosomal targeting process dependent on autophagy degrades apoB that was diverted from the secretory pathway by sortilin and provides a mechanism contributing to the reduced LDL-C found in individuals with SORT1 overexpression.

Regulation of Liver Metabolism by Autophagy.

Intracellular components must be recycled for cells to maintain energy and ensure quality control of proteins and organelles. Autophagy is a highly conserved recycling process that involves degradation of cellular constituents in lysosomes. Although autophagy regulates a number of cell functions, it was first found to maintain energy balance in liver cells. As our understanding of autophagy has increased, we have found its connections to energy regulation in liver cells to be tight and complex. We review 3 mechanisms by which hepatic autophagy monitors and regulates cellular metabolism. Autophagy provides essential components (amino acids, lipids, and carbohydrates) required to meet the cell's energy needs, and it also regulates energy supply by controlling the number, quality, and dynamics of the mitochondria. Finally, autophagy also modulates levels of enzymes in metabolic pathways. In light of the multiple ways in which autophagy participates to control liver metabolism, it is no surprise that dysregulation of autophagy has been associated with metabolic diseases such as obesity, diabetes, or metabolic syndrome, as well as liver-specific disorders such as fatty liver, nonalcoholic steatohepatitis, and hepatocellular carcinoma. We discuss some of these connections and how hepatic autophagy might serve as a therapeutic target in common metabolic disorders.

RNA binding protein HuR regulates the expression of ABCA1.

ABCA1 is a major regulator of cellular cholesterol efflux and plasma HDL biogenesis. Even though the transcriptional activation of ABCA1 is well established, the posttranscriptional regulation of ABCA1 expression is poorly understood. Here, we investigate the potential contribution of the RNA binding protein (RBP) human antigen R (HuR) on the posttranscriptional regulation of ABCA1 expression. RNA immunoprecipitation assays demonstrate a direct interaction between HuR and ABCA1 mRNA. We found that HuR binds to the 3' untranslated region of ABCA1 and increases ABCA1 translation, while HuR silencing reduces ABCA1 expression and cholesterol efflux to ApoA1 in human hepatic (Huh-7) and monocytic (THP-1) cells. Interestingly, cellular cholesterol levels regulate the expression, intracellular localization, and interaction between HuR and ABCA1 mRNA. Finally, we found that HuR expression was significantly increased in macrophages from human atherosclerotic plaques, suggesting an important role for this RBP in controlling macrophage cholesterol metabolism in vivo. In summary, we have identified HuR as a novel posttranscriptional regulator of ABCA1 expression and cellular cholesterol homeostasis, thereby opening new avenues for increasing cholesterol efflux from atherosclerotic foam macrophages and raising circulat-ing HDL cholesterol levels.

Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice.

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12(-/-) ApoE(-/-) ) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12(+/+) ApoE(-/-) control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) . A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12(+/+) ApoE(-/-) mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treatment in TNFSF12(+/+) ApoE(-/-) mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.

HMGB1 expression and secretion are increased via TWEAK-Fn14 interaction in atherosclerotic plaques and cultured monocytes.

OBJECTIVE: High-mobility group box 1 (HMGB1), a DNA-binding cytokine expressed mainly by macrophages, contributes to lesion progression and chronic inflammation within atherosclerotic plaque. It has been suggested that different cytokines could regulate HMGB1 expression in monocytes. We have analyzed the effect of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) on HMGB1 expression both in vivo and in vitro. METHODS AND RESULTS: Expression of TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) was positively correlated with HMGB1 in human carotid atherosclerotic plaques. TWEAK increased HMGB1 mRNA expression and protein secretion in human acute monocytic leukemia cell line cultured monocytes. TWEAK-mediated HMGB1 increase was only observed in M1 macrophages but not in M2 ones. These effects were reversed using blocking anti-Fn14 antibody or nuclear factor-kappa B and phosphotidylinositol-3 kinase inhibitors. TWEAK also increased monocyte chemoattractant protein-1 secretion in human acute monocytic leukemia cell line cells, an effect blocked with an HMGB1 small interfering RNA. Systemic TWEAK injection in ApoE(-/-) mice increased HMGB1 protein expression in the aortic root and mRNA expression in total aorta of ApoE(-/-) mice. Conversely, TWEAK-blocking antibodies diminished HMGB1 protein and mRNA expression compared with IgG-treated mice. CONCLUSIONS: Our results indicate that TWEAK can regulate expression and secretion of HMGB1 in monocytes/macrophages, participating in the inflammatory response associated with atherosclerotic plaque development.

Proteomic analysis of intraluminal thrombus highlights complement activation in human abdominal aortic aneurysms.

OBJECTIVE: To identify proteins related to intraluminal thrombus biological activities that could help to find novel pathological mechanisms and therapeutic targets for human abdominal aortic aneurysm (AAA). APPROACH AND RESULTS: Tissue-conditioned media from patients with AAA were analyzed by a mass spectrometry-based strategy using liquid chromatography coupled to tandem mass spectrometry. Global pathway analysis by Ingenuity software highlighted the presence of several circulating proteins, among them were proteins from the complement system. Complement C3 concentration and activation were assessed in plasma from AAA patients (small AAA, AAA diameter=3-5 cm and large AAA, AAA diameter >5 cm), showing decreased C3 levels and activation in large AAA patients. No association of a combination of single-nucleotide polymorphisms in complement genes between large and small AAA patients was observed. Intense extracellular C3 inmunostaining, along with C9, was observed in AAA thrombus. Analysis of C3 in AAA tissue homogenates and tissue-conditioned media showed increased levels of C3 in AAA thrombus, as well as proteolytic fragments (C3a/C3c/C3dg), suggesting its local deposition and activation. Finally, the functional role of local complement activation in polymorphonuclear (PMN) cell activation was tested, showing that C3 blockade by anti-C3 antibody was able to decrease thrombus-induced neutrophil chemotaxis and reactive oxygen species production. CONCLUSIONS: A decrease of systemic C3 concentration and activity in the later stages of AAA associated with local complement retention, consumption, and proteolysis in the thrombus could induce PMN chemotaxis and activation, playing a detrimental role in AAA progression.

MicroRNAs and atherosclerosis.

MicroRNAs (miRNAs) are small, ~22 nucleotide (nt) sequences of RNA that regulate gene expression at posttranscriptional level. These endogenous gene expression inhibitors were primarily described in cancer but recent exciting findings have also demonstrated a key role in cardiovascular diseases (CVDs), including atherosclerosis. MiRNAs control endothelial cell (EC), vascular smooth muscle cell (VSMC), and macrophage functions, and thereby regulate the progression of atherosclerosis. MiRNA expression is modulated by different stimuli involved in every stage of atherosclerosis, and conversely miRNAs modulates several pathways implicated in plaque development such as cholesterol metabolism. In the present review, we focus on the importance of miRNAs in atherosclerosis, and we further discuss their potential use as biomarkers and therapeutic targets in CVDs.

Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression.

OBJECTIVE: Polymorphonuclear neutrophils (PMNs) play a main role in abdominal aortic aneurysm (AAA) progression. We have analyzed circulating PMNs isolated from AAA patients and controls by a proteomic approach to identify proteins potentially involved in AAA pathogenesis. METHODS AND RESULTS: PMNs from 8 AAA patients (4 large AAA >5 cm and 4 small AAA 3-5 cm) and 4 controls were analyzed by 2D differential in-gel electrophoresis. Among differentially expressed spots, several proteins involved in redox balance were identified by mass spectrometry (eg, cyclophilin, thioredoxin reductase, catalase). Diminished catalase expression and activity were observed in PMNs from AAA patients compared with controls. In contrast, PMNs from AAA patients displayed higher H(2)O(2) and myeloperoxidase levels than PMNs from controls. Moreover, a significant decrease in catalase mRNA levels was observed in PMNs after phorbol 12-myristate 13-acetate incubation. Catalase plasma levels were also decreased in large (n=47) and small (n=56) AAA patients compared with controls (n=34). We observed catalase expression in AAA thrombus and thrombus-conditioned medium, associated with PMN infiltration. Furthermore, increased H(2)O(2) levels were observed in AAA thrombus-conditioned medium compared with the media layer. CONCLUSIONS: Diminished catalase levels in circulating PMNs and plasma are observed in AAA patients, supporting an important role of oxidative stress in AAA evolution.

Identification of peroxiredoxin-1 as a novel biomarker of abdominal aortic aneurysm.

OBJECTIVE: In the search of novel biomarkers of abdominal aortic aneurysm (AAA) progression, proteins released by intraluminal thrombus (ILT) were analyzed by a differential proteomic approach. METHODS AND RESULTS: Different layers (luminal/abluminal) of the ILT of AAA were incubated, and the proteins released were analyzed by 2-dimensional difference in-gel electrophoresis. Several differentially expressed proteins involved in main AAA pathological mechanisms (proteolysis, oxidative stress, and thrombosis) were identified by mass spectrometry. Among the proteins identified, peroxiredoxin-1 (PRX-1) was more released by the luminal layer compared with the abluminal layer of the ILT, which was further validated by Western blot, ELISA, and immunohistochemistry. We demonstrated increased PRX-1 serum levels in AAA patients compared with healthy subjects and also positive correlation among PRX-1 and AAA diameter, plasmin-antiplasmin, and myeloperoxidase levels. Finally, a prospective study revealed a positive correlation between PRX-1 serum levels and AAA expansion rate. Moreover, the combination of PRX-1 and AAA size had significantly additive value in predicting growth. CONCLUSIONS: Several proteins associated with AAA pathogenesis have been identified by a proteomic approach in ILT-conditioned medium. Among them, PRX-1 serum levels are increased in AAA patients and correlate with AAA size and growth rate, suggesting the potential use of PRX-1 as a biomarker for AAA evolution.

Chaperone-mediated autophagy protects against atherosclerosis.

Atherosclerosis, the leading cause of cardiovascular death, is driven by hyperlipidemia, inflammation and aggravated by aging. As chaperone-mediated autophagy (CMA), a selective type of lysosomal degradation for intracellular proteins, diminishes with age and is inhibited by lipid excess, we studied if the decline in CMA could contribute to atherosclerosis pathogenesis. We found that CMA declines in human and murine vasculature with disease progression. Inhibition and reactivation of CMA using transgenic mouse models establishes a protective effect of CMA against atherogenesis. CMA upregulation ameliorates both systemic metabolic parameters, and vascular cell function. Our work suggests CMA reactivation could be a viable therapeutic strategy to prevent and reduce cardiovascular disease.

The lysosomal proteome of senescent cells contributes to the senescence secretome.

Senescent cells accumulate in tissues over time, favoring the onset and progression of multiple age-related diseases. Senescent cells present a remarkable increase in lysosomal mass and elevated autophagic activity. Here, we report that two main autophagic pathways macroautophagy (MA) and chaperone-mediated autophagy (CMA) are constitutively upregulated in senescent cells. Proteomic analyses of the subpopulations of lysosomes preferentially engaged in each of these types of autophagy revealed profound quantitative and qualitative changes in senescent cells, affecting both lysosomal resident proteins and cargo proteins delivered to lysosomes for degradation. These studies have led us to identify resident lysosomal proteins that are highly augmented in senescent cells and can be used as novel markers of senescence, such as arylsulfatase ARSA. The abundant secretome of senescent cells, known as SASP, is considered their main pathological mediator; however, little is known about the mechanisms of SASP secretion. Some secretory cells, including melanocytes, use the small GTPase RAB27A to perform lysosomal secretion. We found that this process is exacerbated in the case of senescent melanoma cells, as revealed by the exposure of lysosomal membrane integral proteins LAMP1 and LAMP2 in their plasma membrane. Interestingly, a subset of SASP components, including cytokines CCL2, CCL3, CXCL12, cathepsin CTSD, or the protease inhibitor SERPINE1, are secreted in a RAB27A-dependent manner in senescent melanoma cells. Finally, proteins previously identified as plasma biomarkers of aging are highly enriched in the lysosomes of senescent cells, including CTSD. We conclude that the lysosomal proteome of senescent cells is profoundly reconfigured, and that some senescent cells can be highly active in lysosomal exocytosis.

Comprehensive autophagy evaluation in cardiac disease models.

Autophagy is a highly conserved recycling mechanism essential for maintaining cellular homeostasis. The pathophysiological role of autophagy has been explored since its discovery 50 years ago, but interest in autophagy has grown exponentially over the last years. Many researchers around the globe have found that autophagy is a critical pathway involved in the pathogenesis of cardiac diseases. Several groups have created novel and powerful tools for gaining deeper insights into the role of autophagy in the aetiology and development of pathologies affecting the heart. Here, we discuss how established and emerging methods to study autophagy can be used to unravel the precise function of this central recycling mechanism in the cardiac system.

A major role of TWEAK/Fn14 axis as a therapeutic target for post-angioplasty restenosis.

BACKGROUND: Tumor necrosis factor-like weak inducer of apoptosis (Tnfsf12; TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Tnfrsf12a; Fn14) participate in the inflammatory response associated with vascular remodeling. However, the functional effect of TWEAK on vascular smooth muscle cells (VSMCs) is not completely elucidated. METHODS: Next generation sequencing-based methods were performed to identify genes and pathways regulated by TWEAK in VSMCs. Flow-citometry, wound-healing scratch experiments and transwell migration assays were used to analyze VSMCs proliferation and migration. Mouse wire injury model was done to evaluate the role of TWEAK/Fn14 during neointimal hyperplasia. FINDINGS: TWEAK up-regulated 1611 and down-regulated 1091 genes in VSMCs. Using a gene-set enrichment method, we found a functional module involved in cell proliferation defined as the minimal network connecting top TWEAK up-regulated genes. In vitro experiments in wild-type or Tnfrsf12a deficient VSMCs demonstrated that TWEAK increased cell proliferation, VSMCs motility and migration. Mechanistically, TWEAK increased cyclins (cyclinD1), cyclin-dependent kinases (CDK4, CDK6) and decreased cyclin-dependent kinase inhibitors (p15lNK4B) mRNA and protein expression. Downregulation of p15INK4B induced by TWEAK was mediated by mitogen-activated protein kinase ERK and Akt activation. Tnfrsf12a or Tnfsf12 genetic depletion and pharmacological intervention with TWEAK blocking antibody reduced neointimal formation, decreasing cell proliferation, cyclin D1 and CDK4/6 expression, and increasing p15INK4B expression compared with wild type or IgG-treated mice in wire-injured femoral arteries. Finally, immunohistochemistry in human coronary arteries with stenosis or in-stent restenosis revealed high levels of Fn14, TWEAK and PCNA in VSMCs enriched areas of the neointima as compared with healthy coronary arteries. INTERPRETATION: Our data define a major role of TWEAK/Fn14 in the control of VSMCs proliferation and migration during neointimal hyperplasia after wire injury in mice, and identify TWEAK/Fn14 as a potential target for treating in-stent restenosis. FUND: ISCiii-FEDER, CIBERCV and CIBERDEM.

Treatment with amlodipine and atorvastatin has additive effect on blood and plaque inflammation in hypertensive patients with carotid atherosclerosis.

Since previous studies have reported a beneficial effect of amlodipine and atorvastatin treatment in experimental atherosclerosis, we aimed to investigate the effect of the combination of both drugs on blood and plaque inflammation in patients with carotid stenosis. For that purpose, twenty six hypertensive patients undergoing carotid endarterectomy were randomized to receive either atorvastatin 20 mg/day alone (ATV, n=12) or in combination with amlodipine 20 mg/day (ATV+AML, n=14) before scheduled carotid endarterectomy. At the end of follow-up (4-6 weeks), there was a significant decrease in total and LDL-cholesterol levels, but not in blood pressure levels. In contrast, decreased MCP-1 plasma levels, NF-kappaB activation (EMSA) and MCP-1 mRNA expression (quantitative PCR) was only observed in blood from ATV+AML treated-patients. Moreover, carotid atherosclerotic plaques from ATV+AML group demonstrated a significant reduction in macrophage infiltration in relation to ATV group (immunohistochemistry). Our results suggest that combined treatment with atorvastatin and amlodipine decreases inflammatory status of atherosclerotic patients more than atorvastatin treatment alone, suggesting that co-administration of both drugs could have beneficial additive effects.