Promote Plaque Stability Research Articles

The walking dead: macrophage inflammation and death in atherosclerosis.

To highlight recent studies that describe novel inflammatory and signaling mechanisms that regulate macrophage death in atherosclerosis. Macrophages contribute to all stages of atherosclerosis. The traditional dogma states that in homeostatic conditions, macrophages undergo apoptosis and are efficiently phagocytosed to be cleared by a process called efferocytosis. In advanced atherosclerosis, however, defective efferocytosis results in secondary necrosis of these uncleared apoptotic cells, which ultimately contributes to the formation of the characteristic necrotic core and the vulnerable plaque. Here, we outline the different types of lesional macrophage death: apoptosis, autophagic and the newly defined necroptosis (i.e. a type of programmed necrosis). Recent discoveries demonstrate that macrophage necroptosis directly contributes to necrotic core formation and plaque instability. Further, promoting the resolution of inflammation using preresolving mediators has been shown to enhance efferocytosis and decrease plaque vulnerability. Finally, the canonical 'don't eat me' signal CD47 has recently been described as playing an important role in atherosclerotic lesion progression by impairing efficient efferocytosis. Although we have made significant strides in improving our understanding of cell death and clearance mechanisms in atherosclerosis, there still remains unanswered questions as to how these pathways can be harnessed using therapeutics to promote lesion regression and disease stability. Improving our understanding of the mechanisms that regulate macrophage death in atherosclerosis, in particular apoptosis, necroptosis and efferocytosis, will provide novel therapeutic opportunities to resolve atherosclerosis and promote plaque stability.

Therapeutically inhibiting macrophage necroptosis reduces inflammation‐driven atherosclerosis and promotes plaque stability

Atherosclerosis is a maladaptive disease driven primarily by macrophages. As the atherosclerotic plaque progresses, inflammatory and macrophage cell death pathways drive the formation of complex, unstable and rupture‐prone lesions with large necrotic core(s). Necroptosis is an emerging cell death pathway involving RIP1, RIP3 and MLKL kinases that, in contrast to apoptosis, induces a pro‐inflammatory state. We hypothesize that inhibiting RIP1 and RIP3 reduces macrophage inflammation and necroptosis to reduce atherosclerosis. Oxidized LDL (oxLDL) induces necroptotic cell death in macrophages, which can be blocked by Nec‐1, a RIP1‐RIP3 inhibitor. Additionally, RIP3−/− macrophages are more resistant to oxLDL‐induced cell death. Treatment with oxLDL increased macrophage expression of necroptotic genes RIP1, RIP3 and MLKL through ROS‐dependent activation of the promoter region. ApoE−/− mice fed a western diet (WD) and simultaneously injected with 100mg/kg RIP1 anti‐sense oligonucleotides (ASO) for 8 weeks had a marked reduction in aortic lesion area compared to both control ASO‐ and PBS‐treated controls (58.8% reduction, p<0.001). In addition, serum inflammatory cytokine profiling suggested a marked reduction in circulating inflammatory cytokines (e.g. IL‐1α, IL‐3 and IL‐17A etc) in RIP1 ASO treated mice compared to control ASO. These results are consistent in vitro, where peritoneal macrophages treated with RIP1 ASO had a reduction in the gene expression of inflammatory genes (e.g. IL‐1α, TNFα) relative to control ASO. Next, in the same experimental model of established atherosclerosis (i.e. Apoe−/− mice fed a western diet for 6 weeks), therapeutic intervention with Nec‐1 these mice reduced lesion size (p<0.05) and increased markers of plaque instability, including reduced necrotic core formation (62.6% reduction, p<0.01). In addition, we developed a novel 123I‐Nec‐1 radiotracer that targets this pathway to specifically localize to and tightly correlate with atherosclerotic lesions. Lastly, in humans with unstable carotid atherosclerosis, expression of the necroptotic genes RIP1, RIP3 and MLKL is elevated and MLKL phosphorylation is detected in advanced atheromas, indicating that the necroptotic pathway is highly activated in vulnerable plaques. In conclusion, our findings offer molecular insight into the inflammatory and cell death mechanisms by which atherogenic ligands drives the formation of the vulnerable plaque and identify innovative therapeutic tools to treat atherosclerosis.

Ghrelin inhibits atherosclerotic plaque angiogenesis and promotes plaque stability in a rabbit atherosclerotic model

Intraplaque angiogenesis associates with the instability of atherosclerotic plaques. In the present study, we investigated the effects of ghrelin on intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis. The rabbits were randomly divided into three groups, namely, the control group, atherosclerotic model group, and ghrelin-treated group, with treatments lasting for 4 weeks. We found that the thickness ratio of the intima to media in rabbits of the ghrelin-treated group was significantly lower than that in rabbits of the atherosclerotic model group. The number of neovessels and the levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) decreased dramatically in rabbits of the ghrelin-treated group compared to those of the atherosclerotic model group. Ghrelin significantly decreased the plaque content of macrophages, matrix metalloproteinase (MMP)-2, and MMP-9, in a rabbit model of atherosclerosis. In addition, the level of the pro-inflammatory factor monocyte chemoattractant protein (MCP)-1 was significantly lower in rabbits of the ghrelin-treated group than in rabbits of the atherosclerotic model group. In summary, ghrelin can inhibit intraplaque angiogenesis and promote plaque stability by down-regulating VEGF and VEGFR2 expression, inhibiting the plaque content of macrophages, and reducing MCP-1 expression at an advanced stage of atherosclerosis in rabbits.

A Cure for Coronary Artery Disease

To cure coronary artery disease, complete removal of atheroma from coronary arteries is necessary and may be achieved using pharmacological intervention. This hypothesis requires verification by a trial. The conditions that lead to the formation of atheroma are as follows: Insulin resistance and vascular endothelial dysfunction, Atherogenic dyslipidemia, Risk factors for coronary artery disease, Inflammation of coronary arteries, Drugs that increase insulin resistance. Atheroma formation begins with the formation of fatty streaks on the endothelium, which progress into stable or unstable atheromatous plaques. The unstable plaque has a thin fibrous cap that is prone to rupture. Unstable plaques contain a large lipid pool consisting of oxidized low-density lipoprotein (LDL), apoB and cholesterol that can grow; they are also have heavy inflammatory cell infiltration, including monocytes, macrophages and T cell lymphocytes. Moreover, debris from ruptured macrophages that were overloaded with lipids attracts more macrophages. The plaque can grow and protrude into the coronary artery lumen. Plaques exhibit overgrowth of vasa vasorum and they can rupture, leading to the formation of a large thrombus that may severely or completely obstruct the coronary artery. Hemorrhage may also occur in the plaque. Carvedilol and metformin promote plaque stability. The supply of lipids to the lipid pool in the plaque is reduced by statins and evolocumab, and lipids are removed from the lipid pool by apoA-1 and high-density lipoprotein (HDL) as their levels in the blood are increased. Severe inflammation is treated with carvedilol, metformin, statins and apoA. Growth of vasa vasorum is attributed to insulin resistance and can be reversed by carvedilol and metformin, thereby preventing hemorrhage. The size of the thrombus on a ruptured plaque can also be reduced by carvedilol, metformin and apoA-1, as these three agents prevent platelet aggregation. Thrombi are removed by endothelial thrombolysis. A thrombus may also form in the plaque from intraplaque hemorrhage; if present, the size of this type of thrombus will be small, as vasa vasorum are atrophic. In both cases, thrombi are removed by endothelial thrombolysis. Subsequently, healthy macrophages infiltrate and remove debris, after which the plaque changes to a fibrous nodule that can be removed by endothelial fibrinolysis. The plaque is then completely removed.

Endothelial Cell-Specific Deletion of P2Y2 Receptor Promotes Plaque Stability in Atherosclerosis-Susceptible ApoE-Null Mice.

Nucleotide P2Y2 receptor (P2Y2R) contributes to vascular inflammation by increasing vascular cell adhesion molecule-1 expression in endothelial cells (EC), and global P2Y2R deficiency prevents fatty streak formation in apolipoprotein E null (ApoE-/-) mice. Because P2Y2R is ubiquitously expressed in vascular cells, we investigated the contribution of endothelial P2Y2R in the pathogenesis of atherosclerosis. EC-specific P2Y2R-deficient mice were generated by breeding VEcadherin5-Cre mice with the P2Y2R floxed mice. Endothelial P2Y2R deficiency reduced endothelial nitric oxide synthase activity and significantly altered ATP- and UTP (uridine 5'-triphosphate)-induced vasorelaxation without affecting vasodilatory responses to acetylcholine. Telemetric blood pressure and echocardiography measurements indicated that EC-specific P2Y2R-deficient mice did not develop hypertension. We investigated the role of endothelial P2Y2R in the development of atherosclerotic lesions by crossing the EC-specific P2Y2R knockout mice onto an ApoE-/- background and evaluated lesion development after feeding a standard chow diet for 25 weeks. Histopathologic examination demonstrated reduced atherosclerotic lesions in the aortic sinus and entire aorta, decreased macrophage infiltration, and increased smooth muscle cell and collagen content, leading to the formation of a subendothelial fibrous cap in EC-specific P2Y2R-deficient ApoE-/- mice. Expression and proteolytic activity of matrix metalloproteinase-2 was significantly reduced in atherosclerotic lesions from EC-specific P2Y2R-deficient ApoE-/- mice. Furthermore, EC-specific P2Y2R deficiency inhibited nitric oxide production, leading to significant increase in smooth muscle cell migration out of aortic explants. EC-specific P2Y2R deficiency reduces atherosclerotic burden and promotes plaque stability in ApoE-/- mice through impaired macrophage infiltration acting together with reduced matrix metalloproteinase-2 activity and increased smooth muscle cell migration.

The role of exercise training and the endocannabinoid system in atherosclerotic plaque burden and composition in Apo-E-deficient mice

IntroductionWe investigated the effect of combining exercise training and treatment with an endocannabinoid receptor 1 inhibitor (Rimonabant) on atherosclerosis burden and composition. MethodsForty-eight apolipoprotein E-deficient (ApoE-/-) mice were kept on a 16-week high-fat diet. Mice were then placed on a normal diet and were randomized to the following groups with n=12 mice for 6 more weeks: 1) Control (Co) - no intervention; 2) Exercise (Ex) - exercise training on treadmill; 3) Rimonabant (Ri) - oral administration of rimonabant (10 mg/kg/day); or 4) Rimonabant+Exercise (RiEx) - combination of Ri and Ex groups treatment. At the end, all animals were sacrificed, and blood samples, as well as aortic root specimens, were obtained for histomorphometric analysis and quantification of the serum and plaque content of matrix metalloproteinases (MMPs). ResultsThe mean plaque area was significantly smaller (RiEx: 43.18±1.72%, Ri: 44.66±3.1%, Ex: 49±4.10%, Co: 70.43±2.83%) in all active treatment groups relative to the Co group (p<0.01). Conversely, the relative concentrations of collagen and elastin were increased significantly across all treatment groups compared to Co (p<0.05). Immunohistochemical analysis revealed significantly reduced macrophage content within plaques after all interventions, with the most pronounced effect observed after combined treatment (RiEx: 9.4±3.92%, Ri: 15±2.45%, Ex: 19.78±2.79%, Co: 34.25±4.99%; p<0.05). Within plaques, the TIMP-1 concentration was significantly upregulated in exercise-treated groups. MMP-3 and MMP-9 concentrations were equivalently decreased in all three active treatment groups compared to controls (p<0.001). DiscussionBoth exercise and rimonabant treatments induced plaque regression and promoted plaque stability. The combined treatment failed to show additive or synergistic benefits relative to either intervention alone.

MiRNA-27b modulates endothelial cell angiogenesis by directly targeting Naa15 in atherogenesis

Background and aimsThe CCL20/CCR6 axis has been shown to play a vital role in the pathogenesis of atherosclerosis (AS). However, the regulatory mechanism remains unclear. Here, we studied the miRNA-mediated epigenetic regulation of the CCL20/CCR6 axis in atherogenesis. MethodsCCR6+/+ApoE−/− and CCR6−/−ApoE−/− mice were fed a high-fat diet for 24 weeks. Plaque size was evaluated via ultrasound biomicroscope and hematoxylin and eosin. Protein expression were measured by Western blotting or immunofluorescence/immunohistochemistry or ELISA, and gene mRNA levels were detected by RT-PCR. Seven hundred and sixty miRNAs were screened via miRNA profiling. miRNA-27b target genes were predicted using software and verified with a dual luciferase reporter assay. The tube formation of mouse aortic endothelial cells (MAECs) was performed on Matrigel. ResultsIn contrast to wild-type ApoE−/− mice, CCR6 deficiency led to a significantly decreased plaque size, CD31, CCR6, CCL20 expression and number of CCL20+ macrophages in atherosclerotic plaques. Stimulation of mouse primary peritoneal macrophages (MPPMs) resulted in increased IL-23 release. miRNA-27b was the most highly expressed (5.19-fold increase) miRNA among the 760 miRNAs screened in the vessel. Naa15 was verified as miRNA-27b target gene, which was diminished in the plaques. Transfection of siRNA Naa15 or miRNA-27b mimic into MAECs caused an increase tube formation. ConclusionsCCR6 deletion effectively ameliorates atherosclerosis progression by reducing macrophage accumulation, resulting in reduced secretion of CCL20 and IL-23. Mechanistically, the decreased miRNA-27b regulates the activity of the CCL20/CCR6 axis by targeting Naa15, and promotes plaque stability in atherosclerosis.

An imbalance between specialized pro-resolving lipid mediators and pro-inflammatory leukotrienes promotes instability of atherosclerotic plaques

Chronic unresolved inflammation plays a causal role in the development of advanced atherosclerosis, but the mechanisms that prevent resolution in atherosclerosis remain unclear. Here, we use targeted mass spectrometry to identify specialized pro-resolving lipid mediators (SPM) in histologically-defined stable and vulnerable regions of human carotid atherosclerotic plaques. The levels of SPMs, particularly resolvin D1 (RvD1), and the ratio of SPMs to pro-inflammatory leukotriene B4 (LTB4), are significantly decreased in the vulnerable regions. SPMs are also decreased in advanced plaques of fat-fed Ldlr−/− mice. Administration of RvD1 to these mice during plaque progression restores the RvD1:LTB4 ratio to that of less advanced lesions and promotes plaque stability, including decreased lesional oxidative stress and necrosis, improved lesional efferocytosis, and thicker fibrous caps. These findings provide molecular support for the concept that defective inflammation resolution contributes to the formation of clinically dangerous plaques and offer a mechanistic rationale for SPM therapy to promote plaque stability.

217 Altered Regulation of the Inhibitor IκBα Contributes to Changes in NF-κB Regulated Genes in the TILRR ko Mouse

<h3>Introduction</h3> Inflammatory responses are key drivers in the pathogenesis of atherosclerosis. We have identified a co-receptor, TILRR, a prominent activator of NF-κB controlled responses, which we have demonstrated is highly expressed in atherosclerotic lesions. Using <i>Apoe^–/–</i>(Apo-lipoprotein E knockout) mice we have recently shown that administration of a blocking peptide antibody, which selectively targets amplification of inflammatory responses through TILRR, significantly reduces progression of atherosclerosis and promotes plaque stability. This study examines the molecular mechanisms underlying the reduced NF-κB activation in the TILRR knockout mouse. <h3>Methods and results</h3> Microarray analysis of blood and spleen monocytes from TILRR knockout mice and littermate controls, revealed pronounced reductions in NF-κB controlled inflammatory genes relevant to development of cardiovascular disease including Cathepsin L1, CXCL1 and CXCL13. Western analysis demonstrated a 65% decrease in IκBα expression and a 60% reduction in inhibitor degradation in IL-1 stimulated monocytes from TILRR-/- mice compared to responses in littermate controls. Parallel <i>in vitro</i> studies were carried out to determine the underlying mechanisms and consequences of these changes on interaction of the inhibitor with NF-κB. Deletion of the ANK 2 region of IκBα resulted in loss of function while the impact of ANK deletions 3, 4, and 5 was not significantly different from the wild type protein. 3-D modelling of the ANK2 deleted protein revealed that the modified protein maintained its overall structure. The modified protein lost its ability to interact with NF-κB, identifying a role for this region in IkBa/NF-kB complex formation. Ongoing studies are evaluating alanine-scanning mutants of conserved residues within the ANK2 region to determine their impact on amplified NF-kB responses. <h3>Conclusion</h3> Our results are consistent with a role for TILRR in regulation of NF-κB controlled inflammatory gene activation <i>in vivo</i>, and suggest that functional alterations in IκBα regulation in part are responsible for the reductions in the inflammatory response.

The Bidirectional Relationship between Cholesterol and Macrophage Polarization

Whether an atherosclerotic plaque progresses and eventually ruptures is heavily influenced by the function of macrophages. However, it is clear that a spectrum of macrophage phenotypes is present in the plaque, with some exhibiting stabilizing functions. While macrophages expressing characteristic M1 and M2 markers are evident, the disparate microenvironments of the plaque, such as regions of hemorrhage, promote other distinct macrophage phenotypes. Crucial to plaque development and progression is macrophage exposure to accumulated modified low density lipoproteins that leads to foam cell formation and development of the necrotic core. There are a range of biologically active compounds in low density lipoprotein (LDL) each having some bearing on which macrophage surface receptors are engaged and what cellular response ensues. Understanding the bidirectional interplay between ‘cholesterol’ and macrophage phenotype will provide valuable insight into key pathways to target which may possibly promote plaque stability by modulating macrophage function.

Effects of DNA Damage in Smooth Muscle Cells in Atherosclerosis

DNA damage and the DNA damage response have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double-stranded breaks (DSBs) are hypothesized to promote plaque progression and instability, in part, by promoting cell senescence, apoptosis, and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. Human atherosclerotic plaque VSMCs showed increased expression of multiple DNA damage response proteins in vitro and in vivo, particularly the MRE11/RAD50/NBS1 complex that senses DSB repair. Oxidative stress-induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated 2 novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with apolipoprotein E(-/-) mice. SM22α-NBS1/apolipoprotein E(-/-) VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/apolipoprotein E(-/-) VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. Human atherosclerotic plaque VSMCs show increased DNA damage, including DSBs and DNA damage response activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability.

Abstract 12529: Low Dose Oral Cyclophosphamide Therapy Reduces Atherosclerosis Progression and Promotes Plaque Stability in a Murine Model of Atherosclerosis

Introduction: Atherosclerosis is a chronic inflammatory disease and the primary cause of heart disease and stroke in Western countries. The cytotoxic drug cyclophosphamide (CPA) can modulate immune functions. Extended survival of patients with severe atherosclerosis has been reported after CPA treatment, but the underlying mechanism is still poorly understood. The objective of this study was to examine the antiatherosclerotic effects of CPA and the underlying mechanism in a murine model of atherosclerosis. Hypothesis: CPA treatment can alter inflammatory processes pivotal for the development of atherosclerosis, therefore limiting disease progression. Method: Apolipoprotein E deficient (ApoE-/-) mice fed a high fat diet received CPA resuspended in drinking water (37.5 g/kg /day p.o.) or water for 12 weeks, respectively. In an interventional protocol, mice fed a high fat diet received the same dose of CPA or water from week 14 to 18, respectively. Mice were sacrificed at week 12, 14 and 18, and aorta, peripheral blood cells, spleen cells and peritoneal cells of treated mice were analysed using histological methods and FACS analysis. Result: In a preventive protocol, continuous oral administration of low-dose CPA prevented disease initiation in ApoE-/- mice fed with a high fat diet. Encouraged by these data we treated mice with pre-established atherosclerosis for 4 weeks with CPA, in an interventional protocol. CPA treatment delayed disease progression in mice with advanced atherosclerosis and reduced the macrophage infiltration in plaques. Importantly, improved plaque stability with a thicker fibrous cap, and an increase in collagen deposition and decreased matrix metalloproteinase-2 and -9 expression, and a reduction of classical inflammatory macrophage (M1) numbers was observed. In addition, CPA treatment reduced the numbers of IFN-γ-producing TH1, but not TH2 lymphocytes in vivo. Conclution: Our data demonstrate that oral treatment with CPA inhibits atherosclerosis initiation and progression in ApoE-/- mouse model, through pleiotropic immunomodulatory effects on lymphoid and myeloid cells. Thus, CPA may be a valuable drug for treating advanced atherosclerosis.

Clinical significance of macrophage phenotypes in cardiovascular disease.

The emerging understanding of macrophage subsets and their functions in the atherosclerotic plaque has led to the consensus that M1 macrophages are pro-atherogenic while M2 macrophages may promote plaque stability, primarily though their tissue repair and anti-inflammatory properties. As such, modulating macrophage function to promote plaque stability is an exciting therapeutic prospect. This review will outline the involvement of the different macrophage subsets throughout atherosclerosis progression and in models of regression. It is evident that much of our understanding of macrophage function comes from in vitro or small animal models and, while such knowledge is valuable, we have much to learn about the roles of the macrophage subsets in the clinical setting in order to identify the key pathways to target to possibly promote plaque stability.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-014-0042-1) contains supplementary material, which is available to authorized users.

Biomechanical factors in atherosclerosis: mechanisms and clinical implications.

Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of atherosclerotic lesions, which develop at regions of arteries that are exposed to complex blood flow. In addition, plaque progression and eventually plaque rupture is influenced by a complex interaction between biological and mechanical factors-mechanical forces regulate the cellular and molecular composition of plaques and, conversely, the composition of plaques determines their ability to withstand mechanical load. A deeper understanding of these interactions is essential for designing new therapeutic strategies to prevent lesion development and promote plaque stabilization. Moreover, integrating clinical imaging techniques with finite element modelling techniques allows for detailed examination of local morphological and biomechanical characteristics of atherosclerotic lesions that may be of help in prediction of future events. In this ESC Position Paper on biomechanical factors in atherosclerosis, we summarize the current 'state of the art' on the interface between mechanical forces and atherosclerotic plaque biology and identify potential clinical applications and key questions for future research.

Abstract 257: Smooth Muscle Cell--Derived COL15A1 Increases Indices of Plaque Stability and Mediates Plaque Cellular Content

Introduction: Current dogma states that under conditions that promote atherosclerosis quiescent vascular smooth muscle cells (SMC) phenotypically modulate into migratory, proliferative cells that move into the arterial intima, form a fibrous cap, and secrete extracellular matrix components (ECM) that promote plaque stabilization. However, there is no direct evidence that SMC derived ECM is critical in promoting changes to plaque stability. We identified collagen, type XV, alpha 1 (COL15A1) as a vascular SMC derived ECM protein present in both human and mouse atherosclerotic aortas. COL15A1 links large fibrillar collagens as well as promotes basement membrane cell adhesion and collagen maturation. We have shown in humans that Col15a1 is associated with atherosclerosis and is epigenetically regulated. However, the role for COL15A1 in atherosclerosis had not been explored. To assess the role of SMC derived collagen and COL15A1 in atherosclerosis we developed unique, conditional, tamoxifen inducible SMC specific Col15a1 knockout mice. We hypothesized that SMC production of COL15A1 is essential to promote indices of plaque stability. Methods and Results: At five weeks of age, SMC specific Col15a1fl/flApoE-/-MYH11-CreERT2+/- mice (n=15) and their Col15a1+/+ counterparts (n=12) were injected for 7 days with 1mg tamoxifen to induce SMC specific Col15a1 knockout and were placed on a Western diet (21% milk fat, 0.15% cholesterol) for 18 weeks. We find that SMC specific Col15a1 knockout induces marked detrimental changes in indices of plaque stability including increased necrotic core area (p=0.002), increased lesion size (p=0.03), and decreased plaque collagen maturation (p=0.017). Moreover, these mice showed a decrease in LGALS3+ macrophage content (p=0.008) but no change in SMC marker (ACTA2+) lesion staining. Conclusion: These data provide the first evidence that a SMC derived collagen (COL15A1) can positively affect indices of plaque stability and mediate plaque cellular content as well as identify COL15A1 as a novel player in atherosclerosis.

Abstract 664: The Coronary Heart Disease--Associated Transcription Factor TCF21 Regulates Disease-Related Genes and May Contribute to the Migration of SMC Progenitors to the Fibrous Cap

Introduction: Recent genome-wide association studies have identified a number of genes that contribute to the risk for coronary heart disease, including TCF21, a bHLH transcription factor believed to serve a critical role in the differentiation of epicardial progenitor cells that give rise to coronary smooth muscle. Methods and Results: Results of immunolocalization and in situ hybridization studies showed that TCF21 is not expressed in medial smooth muscle cells (SMC) of adult human coronary arteries but primarily within the adventitia and within cells of unknown origin in atherosclerotic lesions. LacZ reporter animal studies with ApoE null background showed a shift in Tcf21 expression from adventitial and medial cells to the neointima, notably into the area below and within the forming fibrous cap, suggesting that Tcf21 expressing cells migrate into the forming atherosclerotic lesion. To investigate disease-related functions of TCF21 in SMCs, knockdown and overexpression studies were performed in human coronary artery smooth muscle cells, revealing an effect on migration, proliferation and SMC marker gene expression. Genome-wide RNA-seq studies in these cells with siRNA treatment identified putative TCF21 downstream genes and pathways involved in vascular development as well as atherosclerosis. Supporting ChIP-seq data suggests that these - as over half of all differentially expressed genes - are direct transcriptional targets. To elucidate the behavior of Tcf21 expressing cells in the development of disease lesions, we are employing an in vivo Tcf21 lineage tracing model. Preliminary data from these studies suggests that Tcf21 expressing cells give rise to fibrous cap SMCs, and ongoing studies are aimed at the further characterization of molecular pathways that are active in these Tcf21 expressing cells. Conclusions: The gene regulatory and expression profile of Tcf21 during disease progression strongly implies a key role in Coronary Heart Disease. Tcf21 is likely to play a role in the migration of SMC progenitors from the adventitia into the forming atherosclerotic plaque. As a source of differentiated SMCs of the fibrous cap Tcf21 may therefore promote plaque stability and protect from lesion rupture.

Update on the efficacy of statin treatment in acute coronary syndromes

The natural history of atherosclerosis might involve coronary plaque rupture/erosion, thrombus formation and vessel lumen occlusion, clinically recognized as acute coronary syndrome (ACS). International guidelines strongly recommend early statin administration in patients admitted for ACS. In addition to lowering circulating levels of low-density lipoprotein cholesterol (LDL-c), statin treatment was shown to promote plaque stabilization or regression in several ways, including reduction in necrotic lipid core, anti-inflammatory effects and improvement in endothelial function. The aim of this review is to summarize clinical evidence on the role of statins in secondary prevention of ACS. This narrative review is based on the material found on medline and pubmed up to August 2013. We looked for the terms 'statin, acute coronary syndromes' in combination with 'atherosclerosis, acute myocardial infarction, pathophysiology'. This review article emphasizes the relevance of the timing of statin administration to improve the outcomes after ACS. Early and continuous statin administration has emerged as key features to prevent adverse events, especially in patients admitted for ACS undergoing percutaneous coronary intervention. Clinical trials matching the improved clinical outcome with the imaging of atherosclerotic plaque stabilization/regression, further supporting the effectiveness of statin therapy. However, the achievement of these goals requires high dose of statins, thus increasing the risk of adverse events. Although clinical trials and meta-analyses have provided conflicting results, it is likely that in clinical practice, the rate of adverse events is higher, so that many concerns still remain about a statin high-dose approach in ACS patients.

Clinical study on treatment of carotid atherosclerosis with extraction of Polygoni Cuspidati Rhizoma et Radix and Crataegi Fructus：a randomized controlled trial

To observe the effect of detoxifying and blood circulation activating Chinese herb extraction of polygonum cuspidatum and hawthorn on carotid intima-media thickness (IMT), plaque integral and plaque stability related serum indexes of patients with carotid atherosclerosis. Sixty and four cases of carotid artery atherosclerosis patients were assigned randomly to 2 groups: detoxifying and blood circulation activating treatment group (treatment group, 32 cases) and control group (32 cases). Patients in treatment group were treated with capsules of extraction of polygonum cuspidatum and hawthorn, 1 pill po, bid (dosage of administration: polygonum cuspidatum extraction 5.33 mg x kg(-1) x d(-1), hawthorn extraction 5.0 mg x kg(-1) x d(-1)); patients in control group were treated with lovastatin 20 mg po, qd (dosage of administration: 0.33 mg x kg(-1) x d(-1)). The course of treatment was six months. To observe changes of IMT, plaque integral, and detect the level of plaque stability related serum indexes such as Hs-CRP, MMP-1 and TIMP-1. After 6 months of treatment, in control group one patient quit the clinical trial because of liver dysfunction and one patient was rejected because of having not followed the therapeutic regimen. 32 cases in treatment group and 30 cases in control group were analyzed. The results showed that IMT and plaque integral of treatment group decreased significantly after the treatment (P < 0.05, P < 0.01), and there was no significant difference compared with control grope. Serum Hs-CRP, MMP-1 and MMP-1/TIMP-1 decreased after the treatment (P < 0.05, P < 0.01), and the treatment group was superior to control group in decreasing serum Hs-CRP (P < 0.05). Detoxifying and blood circulation activating Chinese herb extraction of polygonum cuspidatum and hawthorn has good effect of anti-atherosclerosis and promoting plaque stability. Its mechanism might be related with anti-inflammation and inhibiting degradation of extracellular matrix, and deserves further studies.

RETRACTED: Probucol via inhibition of NHE1 attenuates LPS-accelerated atherosclerosis and promotes plaque stability in vivo

Activation of Na(+)/H(+) exchanger 1 (NHE1) by lipopolysaccharide (LPS) via Ca(2+)/calpain is responsible in vascular smooth muscle cell (VSMC) apoptosis and to the process of atherosclerosis. Probucol is a lipid-lowering drug which has an anti-atherosclerosis effect. The mechanism remains poorly understood. Here we hypothesized that probucol via inhibition of NHE1 in VSMCs attenuates LPS-accelerated atherosclerosis and promotes plaque stability. Our results revealed that treatment of VSMCs with LPS increased the NHE1 activity in a time-dependent manner, associated with the increased Ca(2+)i. Probucol inhibited the LPS-induced increase of NHE1 activity in a dose-dependent manner in VSMCs for 24-hour co-incubation, as well as the change of Ca(2+)i. In addition, LPS enhanced the calpain activity. Both probucol and calcium chelation of Ca(2+) abolished the LPS-induced increase of calpain activity. Treatment of VSMCs with LPS reduced the expression of Bcl-2 without altering the mRNA level. Probucol inhibited the LPS-reduced expression of Bcl-2 protein in VSMCs. Animal studies indicated administration of probucol suppressed LPS-accelerated apoptosis, atherosclerosis and plaque instability in Apoe(-/-) mice. In conclusion, probucol via inhibition of NHE1 attenuates atherosclerosis lesion growth and promotes plaque stability.

Practical strategies for modulating foam cell formation and behavior.

Although high density lipoprotein (HDL)-mediated reverse cholesterol transport is crucial to the prevention and reversal of atheroma, a recent meta-analysis makes evident that current pharmaceutical strategies for modulating HDL cholesterol levels lower cardiovascular risk only to the extent that they concurrently decrease low density lipoprotein (LDL) cholesterol. This corresponds well with findings of a recent Mendelian randomization analysis, in which genetic polymorphisms associated with HDL cholesterol but no other known cardiovascular risk factors failed to predict risk for myocardial infarction. Although it is still seems appropriate to search for therapies that could improve the efficiency with which HDL particles induce reverse cholesterol transport, targeting HDL cholesterol levels per se with current measures appears to be futile. It may therefore be more promising to promote reverse cholesterol transport with agents that directly target foam cells. Macrophage expression of the cholesterol transport proteins adenosine triphosphate binding cassette transporter A1, adenosine triphosphate binding cassette transporter G1, and scavenger receptor class B member 1 is transcriptionally up-regulated by activated liver X receptors (LXR), whereas nuclear factor (NF)-kappaB antagonizes their expression. Taurine, which inhibits atherogenesis in rodent studies, has just been discovered to act as a weak agonist for LXRalpha. Conversely, it may be possible to oppose NF-kappaB activation in macrophages with a range of measures. Induction of heme oxygenase-1, which can be attained with phase 2 inducer phytochemicals such as lipoic acid and green tea catechins, promotes reverse cholesterol transport in macrophages and inhibits atherogenesis in rodents, likely due to, in large part, NF-kappaB antagonism. Inhibition of macrophage nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity with the spirulina-derived bilirubin-mimetic phycocyanobilin may also oppose NF-kappaB activation, and salicylic acid similarly should be useful for this purpose. The 5' adenosine monophosphate-activated protein kinase activator berberine promotes macrophage reverse cholesterol transport in cell culture; metformin probably shares this property. Many of these measures could also be expected to promote plaque stability by suppressing foam cell production of inflammatory cytokines and matrix metalloproteinases, and to reduce intimal monocyte infiltration by anti-inflammatory effects on vascular endothelium. Direct targeting of foam cells with agents such as phase 2 inducers, spirulina, salicylate, taurine, and berberine or metformin, may hence have considerable potential for preventing and reversing atheroma, and for preventing the plaque rupture that triggers vascular thrombosis.