Retracted: Downregulated microRNA-224 aggravates vulnerable atherosclerotic plaques and vascular remodeling in acute coronary syndrome through activation of the TGF-β/Smad pathway

Recent studies have shown that circulating microRNAs (miRNA) play a critical role in diagnosing acute coronary syndrome (ACS). This study aims to investigate the effect of miR-224 on atherosclerotic plaques forming and vascular remodeling in ACS and its relationship with TGF-β/Smad pathway. Myocardial infarction (MI) rat model was established and lentivirus vector of miR-224 inhibitor was prepared for investigating the effect of downregulated miR-224 on the contents of nitric oxide (NO) and endothelin-1 (ET-1), blood lipid levels and inflammatory factor levels in serum as well as the TGF-β/Smad pathway. The rats suffering from MI had decreased survival rates and exhibited reduced levels of NO, high-density lipoprotein cholesterol, and lumen diameter, and Smad7 messenger RNA (mRNA) and protein expression; while had significantly increased ratio of heart weight or body weight, levels of ET-1, inflammatory factors, blood lipid indexes, vascular remodeling indexes, collagen volume fraction, vulnerable atherosclerotic plaque area, VCAM-1 and MMP-2 protein expression, TGF-β, Smad2, Smad3, and Smad4 mRNA and protein expression. After inhibiting the TGF-β/Smad pathway, the rats suffering from MI showed notably opposite trend. In conclusion, downregulation of miR-224 expression promotes the formation of vulnerable atherosclerotic plaques and vascular remodeling in ACS through activation of the TGF-β/Smad pathway. Therefore, this study provides a new therapeutic target for ACS.     

p38 mitogen-activated protein kinase (MAPK) promotes cholesterol ester accumulation in macrophages through inhibition of macroautophagy

p38 MAPK has been strongly implicated in the development of atherosclerosis, but its role in cholesterol ester accumulation in macrophages and formation of foam cells, an early step in the development of atherosclerosis, has not been investigated. We addressed this issue and made some brand new observations. First, elevated intracellular cholesterol level induced by the exposure to LDL-activated p38 MAPK and activation of p38 MAPK with anisomycin increased the ratio of cholesterol esters over free cholesterol, whereas inhibition of p38 MAPK with SB203580 or siRNA reduced the LDL loading-induced intracellular accumulation of free cholesterol and cholesterol esters in macrophages. Second, exposure to LDL cholesterol inhibited autophagy in macrophages, and inhibition of autophagy with 3-methyladenine increased intracellular accumulation of cholesterol (free cholesterol and cholesterol esters), whereas activation of autophagy with rapamycin decreased intracellular accumulation of free cholesterol and cholesterol esters induced by the exposure to LDL cholesterol. Third, LDL cholesterol loading-induced inhibition of autophagy was prevented by blockade of p38 MAPK with SB203580 or siRNA. Neutral cholesterol ester hydrolase was co-localized with autophagosomes. Finally, LDL cholesterol loading and p38 activation suppressed expression of the key autophagy gene, ulk1, in macrophages. Together, our results provide brand new insight about cholesterol ester accumulation in macrophages and foam cell formation.     

Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9–p38 MAPK pathway

An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1β, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1β, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9–p38 MAPK signal transduction pathway.     

Blockade of NEAT1 represses inflammation response and lipid uptake via modulating miR-342-3p in human macrophages THP-1 cells

Atherosclerosis has been recognized as a chronic inflammation process induced by lipid of the vessel wall. Oxidized low-density lipoprotein (ox-LDL) can drive atherosclerosis progression involving macrophages. Recently, long noncoding RNAs (lncRNAs) have been reported to play critical roles in atherosclerosis development. In our current study, we focused on the biological roles of lncRNA NEAT1 in atherosclerosis progress. Here, we found that ox-LDL was able to trigger human macrophages THP-1 cells, a human monocytic cell line, apoptosis in a dose-dependent and time-dependent course. In addition, we observed that NEAT1 was significantly increased in THP-1 cells incubated with ox-LDL and meanwhile miR-342-3p was greatly decreased. Then, NEAT1 was silenced by transfection of small interfering RNA (siRNA) of NEAT1 into THP-1 cells. As exhibited, CD36, oil-red staining levels, total cholesterol (TC), total cholesterol (TG) levels and THP-1 cell apoptosis were obviously repressed by knockdown of NEAT1. Furthermore, inhibition of NEAT1 contributed to the repression of inflammation in vitro. Interleukin 6 (IL-6), IL-1β, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-α) protein levels were remarkably depressed by NEAT1 siRNA in THP-1 cells. By using bioinformatics analysis, miR-342-3p was predicted as a downstream target of NEAT1 and the correlation between them was confirmed in our study. Moreover, overexpression of miR-342-3p could also greatly suppress inflammation response and lipid uptake in THP-1 cells. Knockdown of NEAT1 and miR-342-3p mimics inhibited lipid uptake in THP-1 cells. In conclusion, we implied that blockade of NEAT1 repressed inflammation response through modulating miR-342-3p in human macrophages THP-1 cells and NEAT1 may offer a promising strategy to treat atherosclerotic cardiovascular diseases.     

MicroRNA-590 Inhibits Lipoprotein Lipase Expression and Prevents Atherosclerosis in apoE Knockout Mice

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE^−/−) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE^−/− mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE^−/− mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion.     

Zinc rescues obesity‐induced cardiac hypertrophy via stimulating metallothionein to suppress oxidative stress‐activated BCL10/CARD9/p38 MAPK pathway

Obesity often leads to obesity‐related cardiac hypertrophy (ORCH), which is suppressed by zinc‐induced inactivation of p38 mitogen‐activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4‐week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B‐cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate‐treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate‐induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate‐induced up‐regulation of BCL10 and phospho‐p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress‐mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress‐activated BCL10 expression and p38 MAPK activation.     

Bone marrow endothelial progenitors in atherosclerotic plaque resolution

Atherosclerosis is a major cause of morbidity and mortality in the United States. Persistently elevated circulating low-density lipoprotein, or hypercholesterolemia, and deposition of low-density lipoprotein in the vascular wall are the main inducers of atherosclerosis, which manifests itself as arterial lesions or plaques. Some plaques become thrombosis-prone and rupture, causing acute myocardial infarction or stroke. Lowering plasma cholesterol through the use of statins is the primary intervention against atherosclerosis. Treatment with statins slows progression of atherosclerosis but can only support limited plaque regression. Partially regressed plaques continue to pose a serious threat due to their remaining potential to rupture. Thus, new interventions inducing complete reversal of atherosclerosis are being sought. Implementation of new therapies will require clear understanding of the mechanisms driving plaque resolution. In this Commentary, we highlight the role of bone marrow endothelial progenitors in atherosclerotic plaque regression and discuss how regenerative cell-based interventions could be used in combination with plasma lipid-lowering to induce plaque reversal in order to prevent and/or reduce adverse cardiovascular events.     

Protein deglycase DJ-1 deficiency induces phenotypic switching in vascular smooth muscle cells and exacerbates atherosclerotic plaque instability

Protein deglycase DJ-1 (DJ-1) is a multifunctional protein involved in various biological processes. However, it is unclear whether DJ-1 influences atherosclerosis development and plaque stability. Accordingly, we evaluated the influence of DJ-1 deletion on the progression of atherosclerosis and elucidate the underlying mechanisms. We examine the expression of DJ-1 in atherosclerotic plaques of human and mouse models which showed that DJ-1 expression was significantly decreased in human plaques compared with that in healthy vessels. Consistent with this, the DJ-1 levels were persistently reduced in atherosclerotic lesions of ApoE^−/− mice with the increasing time fed by western diet. Furthermore, exposure of vascular smooth muscle cells (VSMCs) to oxidized low-density lipoprotein down-regulated DJ-1 in vitro. The canonical markers of plaque stability and VSMC phenotypes were evaluated in vivo and in vitro. DJ-1 deficiency in Apoe^−/− mice promoted the progression of atherosclerosis and exaggerated plaque instability. Moreover, isolated VSMCs from Apoe^−/−DJ-1^−/− mice showed lower expression of contractile markers (α-smooth muscle actin and calponin) and higher expression of synthetic indicators (osteopontin, vimentin and tropoelastin) and Kruppel-like factor 4 (KLF4) by comparison with Apoe^−/−DJ-1^+/+ mice. Furthermore, genetic inhibition of KLF4 counteracted the adverse effects of DJ-1 deletion. Therefore, our results showed that DJ-1 deletion caused phenotype switching of VSMCs and exacerbated atherosclerotic plaque instability in a KLF4-dependent manner.     

Macrophage autophagy regulates mitochondria‐mediated apoptosis and inhibits necrotic core formation in vulnerable plaques

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE^?/? mice. Application of ApoE^?/? mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.     

Plaque Image Characteristics, Hyperhomocysteinemia, and Gene Polymorphism of Homocysteine Metabolism-Related Enzyme (MTHFR C677T) in Acute Coronary Syndrome

This study aims to investigate the correlation between the different characteristics of plaques, plasma level of homocysteine (Hcy), and gene polymorphism of Hcy metabolism-related enzyme. In this consecutive case–control study, we measured the plasma Hcy level using fluorescence biochemistry method and examined the gene polymorphism of Hcy metabolism-related enzyme methylenetetrahydrofolate reductase (MTHFR) C677T using TaqMan probe technology. We also examined these using intravascular ultrasound. We studied the characteristics of the plaque, measured the cross-sectional areas of the external elastic membrane and the lumen, calculated the plaque area, plaque burden, and eccentricity index, and examined the remodeling index. Hard plaques were more dominant in the (SPA) group, whereas soft plaques were more dominant in the acute coronary syndrome (ACS) group (P < 0.001). The risk of plaque rupture and thrombus is higher in the ACS group (P < 0.05). Compared with SPA group, plaque burden was heavier in the ACS group (P < 0.05), but the eccentricity index is significantly higher in SPA group than in the ACS group (P < 0.001). Positive remodeling was more frequent in ACS group, whereas negative remodeling was more frequent in the SPA group (P < 0.001). Plasma Hcy levels were higher in the unstable than in the stable plaque group (P < 0.001). The constituent ratio of MTHFR C677T genotype were different in stable plaque group and vulnerable plaque group (P < 0.05). The T genotype can increase the incidence rate of vulnerable plaque. Hcy and MTHFR C677T gene polymorphism were found to be risk factors for vulnerable plaque. Therefore, these can be used as indices to predict the instability of atherosclerotic plaque.     

Retracted: microRNA-542-5p protects against acute lung injury in mice with severe acute pancreatitis by suppressing the mitogen-activated protein kinase signaling pathway through the negative regulation of P21-activated kinase 1

Severe acute pancreatitis (SAP) is a condition associated with high rates of mortality and lengthy hospital stays. In the current study, SAP mouse models were established in BALB/c wild-type and P21-activated kinase 1 (PAK1) knockdown mice with the objective of determining the expression of microRNA-542-5p (miR-542-5p) and the subsequent elucidation of the mechanism by which it influences acute lung injury (ALI) by mediating mitogen-activated protein kinase (MAPK) signaling and binding to PAK1. The targeting relationship between miR-542-5p and PAK1 was verified using the bioinformatics prediction website and by the means of a dual-luciferase reporter assay. Following the SAP model establishment, the mice were assigned into various groups with the introduction of different mimic and inhibitors in an attempt to investigate the effects involved with miR-542-5p on inflammatory reactions among mice with SAP-associated ALI. Our results indicated that PAK1 was targeted and negatively mediated by miR-542-5p. Mice with SAP-associated ALI exhibited an increased wet-to-dry weight ratio, myeloperoxidase activity, serum amylase activity, TNF-α, interleukin-1 beta (IL-1β), and intercellular adhesion molecule-1 (ICAM-1) contents, p-p38MAPK, p-ERK1/2, and p-JNK protein levels as well as PAK1 positive expression, while decreased miR-542-5p levels were observed. Functionally, overexpression of miR-542-5p improves ALI in mice with SAP via inhibition of the MAPK signaling pathway by binding to PAK1.Based on the evidence from experimental models, miR-542-5p was shown to improve ALI among mice with SAP, while suggesting that the effect may be related to the inactivation of the MAPK signaling pathway and downregulation of PAK1 gene. Thus, miR-542-5p could serve as a promising target for ALI treatment.     

Atorvastatin Improves Plaque Stability in ApoE-Knockout Mice by Regulating Chemokines and Chemokine Receptors

It is well documented that statins protect atherosclerotic patients from inflammatory changes and plaque instability in coronary arteries. However, the underlying mechanisms are not fully understood. Using a previously established mouse model for vulnerable atherosclerotic plaque, we investigated the effect of atorvastatin (10 mg/kg/day) on plaque morphology. Atorvastatin did not lower plasma total cholesterol levels or affect plaque progression at this dosage; however, vulnerable plaque numbers were significantly reduced in the atorvastatin-treated group compared to control. Detailed examinations revealed that atorvastatin significantly decreased macrophage infiltration and subendothelial lipid deposition, reduced intimal collagen content, and elevated collagenase activity and expression of matrix metalloproteinases (MMPs). Because vascular inflammation is largely driven by changes in monocyte/macrophage numbers in the vessel wall, we speculated that the anti-inflammatory effect of atorvastatin may partially result from decreased monocyte recruitment to the endothelium. Further experiments showed that atorvastatin downregulated expression of the chemokines monocyte chemoattractant protein (MCP)-1, chemokine (C-X3-C motif) ligand 1 (CX3CL1) and their receptors CCR2 and, CX3CR1, which are mainly responsible for monocyte recruitment. In addition, levels of the plasma inflammatory markers C-reactive protein (CRP) and tumor necrosis factor (TNF)-α were also significantly decrease in atorvastatin-treated mice. Collectively, our results demonstrate that atorvastatin can improve plaque stability in mice independent of plasma cholesterol levels. Given the profound inhibition of macrophage infiltration into atherosclerotic plaques, we propose that statins may partly exert protective effects by modulating levels of chemokines and their receptors. These findings elucidate yet another atheroprotective mechanism of statins.     

NDRG2 induced by oxidized LDL in macrophages antagonizes growth factor productions via selectively inhibiting ERK activation

During atherogenesis, macrophage foam cells produce prodigious growth factors, cytokines, and chemokines, which play the central roles in inflammatory process in atherosclerotic plaque formation. In the present study, we identified a new protein marker, N-Myc downstream-regulated protein 2 (NDRG2), which is significantly up-regulated in oxidized low density lipoprotein (oxLDL) treated macrophages and in human atherosclerotic plaques. Over-expression and siRNA knockdown studies showed that NDRG2 is a negative regulator of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) productions in macrophages. Furthermore, we investigated the effects of NDRG2 on MAPK signal activation. Our results showed ERK1/2 activation, but not P38 or JNK1/2 activation, is responsible for regulation of NDRG2 on VEGF and PDGF productions. Consistent with the PDGF levels, the vascular smooth muscle cell (VSMC) proliferation was also regulated by the conditional medium of the oxLDL treated macrophages with NDRG2 knockdown or over-expression. Neutralizing anti-PDGF antibody can significantly inhibit the enhanced VSMC proliferation by macrophage medium with NDRG2 knockdown. Our present results demonstrate that NDRG2 participates in oxLDL-induced macrophage activation and modulates ERK1/2-dependent PDGF and VEGF production, which has potential application in atherogenesis.     

Cell type-specific activation of p38 MAPK in the brain regions of hypoxic preconditioned mice

Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly (p < 0.05) in the regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6, n = 6 for each group) when compared to values of the control normoxic group (H0, n = 6) using Western blot analysis. Similar results were also confirmed by an immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38 MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute to the development of cerebral HPC mechanism in mice.     

Role of six single nucleotide polymorphisms,risk factors in coronary disease,in OLR1 alternative splicing

The OLR1 gene encodes the oxidized low-density lipoprotein receptor (LOX-1), which is responsible for the cellular uptake of oxidized LDL (Ox-LDL), foam cell formation in atheroma plaques and atherosclerotic plaque rupture. Alternative splicing (AS) of OLR1 exon 5 generates two protein isoforms with antagonistic functions in Ox-LDL uptake. Previous work identified six single nucleotide polymorphisms (SNPs) in linkage disequilibrium that influence the inclusion levels of OLR1 exon 5 and correlate with the risk of cardiovascular disease. Here we use minigenes to recapitulate the effects of two allelic series (Low- and High-Risk) on OLR1 AS and identify one SNP in intron 4 (rs3736234) as the main contributor to the differences in exon 5 inclusion, while the other SNPs in the allelic series attenuate the drastic effects of this key SNP. Bioinformatic, proteomic, mutational and functional high-throughput analyses allowed us to define regulatory sequence motifs and identify SR protein family members (SRSF1, SRSF2) and HMGA1 as factors involved in the regulation of OLR1 AS. Our results suggest that antagonism between SRSF1 and SRSF2/HMGA1, and differential recognition of their regulatory motifs depending on the identity of the rs3736234 polymorphism, influence OLR1 exon 5 inclusion and the efficiency of Ox-LDL uptake, with potential implications for atherosclerosis and coronary disease.     

Effects of High Fat Feeding and Diabetes on Regression of Atherosclerosis Induced by Low-Density Lipoprotein Receptor Gene Therapy in LDL Receptor-Deficient Mice

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr^-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr^-/- mice in different metabolic states.     

A combination of Lox-1 and Nox1 regulates TLR9-mediated foam cell formation

The formation of foam cells is the hallmark of early atherosclerotic lesions, and the uptake of modified low-density lipoprotein (LDL) by macrophage scavenger receptors is thought to be a key process in their formation. In this study, we examined the role of lectin-like oxLDL receptor-1 (Lox-1) and NADPH oxidase 1 (Nox1) in toll-like receptor 9 (TLR9)-mediated foam cell formation. TLR9 activation of Raw264.7 cells or mouse primary peritoneal macrophages by CpG ODN treatment enhanced Lox-1 gene and protein expression. In addition, CpG ODN-induced Nox1 mRNA expression, which in turn increased foam cell formation. The inhibition of CpG ODN-induced reactive oxygen species (ROS) generation by treatment with antioxidants, as well as with knockdown of Nox1 using siRNA, suppressed the formation of foam cells. The induction of Lox-1 and Nox1 by CpG ODN was regulated via the TLR9-p38 MAPK signaling pathway. CpG ODN also increased NFκB activity, and a potent inhibitor of NFκB that significantly blocked CpG-induced Nox1 expression, suggesting that Nox1 regulation is mediated through an NFκB-dependent mechanism. Taken together, these results suggest that a combination of Lox-1 and Nox1 plays a key role in the TLR9-mediated formation of foam cells via the p38 MAPK pathway.     

Caveolin-1 influences vascular protease activity and is a potential stabilizing factor in human atherosclerotic disease

Caveolin-1 (Cav-1) is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin) reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice. This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target in the prevention of human atherosclerotic disease and the loss of Cav-1 may be a novel biomarker of vulnerable plaque with prognostic value.