Stable Atherosclerotic Lesions Research Articles

Enhancement of high-density lipoprotein-associated protease inhibitor activity prevents atherosclerosis progression

Background and aimsInflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. MethodsWe designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis was examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. ResultsHTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe-/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr-/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. ConclusionsThese data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.

Comparative morphometric analysis of contents mononuclear cells in unstable and in stable atherosclerotic lesions

BACKGROUND: Recent research results indicate that inflammatory cellular responses can play a significant role in the pathogenesis of atherosclerosis in humans and experimental animals, with a predominance in infiltrates of lymphocytes and monocytes/macrophages that contribute to destructive processes in the vascular wall. However, no comparative, differentiated morphometric analysis of the content of mononuclear cells in unstable and stable atherosclerotic lesions has been carried out so far.
 AIM: Carrying out comparative histological and morphometric examination of quantitative content of lymphocytes and macrophages in normal vascular wall, in lipid stain, in unstable and in stable atherosclerotic lesions.
 MATERIALS AND METHODS: The study material was samples taken during 15 autopsies aged 56 to 71 years who died from acute cardiovascular failure of atherosclerotic etiology. A comparative histological and morphometric study of the content of mononuclear cells: lymphocytes and macrophages in normal areas of the vascular wall, in lipid stains, in unstable and stable atherosclerotic lesions was carried out a total of 50 tissue samples stained with hematoxylin and eosin.
 RESULTS: A sharp increase in the content of studied cells in intima, atheromatous nucleus and adventition in unstable atherosclerotic plaques was revealed. In normal areas of the vascular wall, in initial atherosclerotic lesions and in stable plaques, the number of macrophages and lymphocytes is small.
 CONCLUSIONS: In general, the results obtained are well consistent with the literature and suggest in favor of the need for lymphocytes and macrophages to form immuno-inflammatory reactions in the formation of unstable atherosclerotic lesions in humans.

Therapeutic inhibition of MPO stabilizes pre-existing high risk atherosclerotic plaque

Currently there are no established therapies to treat high-risk patients with unstable atherosclerotic lesions that are prone to rupture and can result in thrombosis, abrupt arterial occlusion, and a precipitous infarction. Rather than being stenotic, rupture-prone non-occlusive plaques are commonly enriched with inflammatory cells and have a thin fibrous cap. We reported previously that inhibition of the pro-inflammatory enzyme myeloperoxidase (MPO) with the suicide inhibitor AZM198 prevents formation of unstable plaque in the Tandem Stenosis (TS) mouse model of plaque instability. However, in our previous study AZM198 was administered to animals before unstable plaque was present and hence it did not test the significant unmet clinical need present in high-risk patients with vulnerable atherosclerosis. In the present study we therefore asked whether pharmacological inhibition of MPO with AZM198 can stabilize pre-existing unstable lesions in an interventional setting using the mouse model of plaque instability. In vivo molecular magnetic resonance imaging of arterial MPO activity using bis-5-hydroxytryptamide-DTPA-Gd and histological analyses revealed that arterial MPO activity was elevated one week after TS surgery, prior to the presence of unstable lesions observed two weeks after TS surgery. Animals with pre-existing unstable plaque were treated with AZM198 for one or five weeks. Both short- and long-term intervention effectively inhibited arterial MPO activity and increased fibrous cap thickness, indicative of a more stable plaque phenotype. Plaque stabilization was observed without AZM198 affecting the arterial content of Ly6B.2+- and CD68+-cells and MPO protein. These findings demonstrate that inhibition of arterial MPO activity converts unstable into stable atherosclerotic lesions in a preclinical model of plaque instability and highlight the potential therapeutic potency of MPO inhibition for the management of high-risk patients and the development of novel protective strategies against cardiovascular diseases.

Fibrous Cap Thickness Predicts Stable Coronary Plaque Progression: Early Clinical Validation of a Semiautomated OCT Technology.

Imaging-based characteristics associated with the progression of stable coronary atherosclerotic lesions are poorly defined. Utilizing a combination of optical coherence tomography (OCT) and intravascular ultrasound (IVUS) imaging, we aimed to characterize the lesions prone to progression through clinical validation of a semiautomated OCT computational program. Patients with stable coronary artery disease underwent nonculprit vessel imaging with IVUS and OCT at baseline and IVUS at the 12-month follow-up. After coregistration of baseline and follow-up IVUS images, paired 5-mm segments from each patient were identified, demonstrating the greatest plaque progression and regression as measured by the change in plaque burden. Experienced readers identified plaque features on corresponding baseline OCT segments, and predictors of plaque progression were assessed by multivariable analysis. Each segment then underwent volumetric assessment of the fibrous cap (FC) using proprietary software. Among 23 patients (70% men; median age, 67 years), experienced-reader analysis demonstrated that for every 100 μm increase in mean FC thickness, plaques were 87% less likely to progress (P = .01), which persisted on multivariable analysis controlling for baseline plaque burden (P = .05). Automated FC analysis (n = 17 paired segments) confirmed this finding (P = .01) and found thinner minimal FC thickness (P = .01) and larger FC surface area of <65 μm (P = .02) and <100 μm (P = .04) in progressing segments than in regressing segments. No additional imaging features predicted plaque progression. A semiautomated FC analysis tool confirmed the significant association between thinner FC and stable coronary plaque progression along entire vessel segments, illustrating the diffuse nature of FC thinning and suggesting a future clinical role in predicting the progression of stable coronary artery disease.

Plaque Character and Progression According to the Location of Coronary Atherosclerotic Plaque

Although acute coronary syndrome culprit lesions occur more frequently in the proximal coronary artery, whether the proximal clustering of high-risk plaque is reflected in earlier-stage atherosclerosis remains unclarified. We evaluated the longitudinal distribution of stable atherosclerotic lesions on coronary computed tomography angiography (CCTA) in 1,478 patients (mean age, 61 years; men, 58%) enrolled from a prospective multinational registry of consecutive patients undergoing serial CCTA. Of 3,202 coronary artery lesions identified, 2,140 left lesions were classified (based on the minimal lumen diameter location) into left main (LM, n=128), proximal (n=739), and other (n=1,273), and 1,062 right lesions were classified into proximal (n=355) and other (n=707). Plaque volume (PV) was the highest in proximal lesions (median, 26.1 mm3), followed by LM (20.6 mm3) and other lesions (15.0 mm3, p <0.001), for left lesions, and was lager in proximal (25.8 mm3) than in other lesions (15.2 mm3, p <0.001) for right lesions. On both sides, proximally located lesions tended to have greater necrotic core and fibrofatty components than other lesions (left: LM, 10.6%; proximal, 5.8%; other, 3.4% of the total PV, p <0.001; right: proximal, 8.4%; other 3.1%, p <0.001), with less calcified plaque component (left: LM, 18.3%; proximal, 30.3%; other, 37.7%, p <0.001; right: proximal, 23.3%, other, 36.6%, p <0.001), and tended to progress rapidly (adjusted odds ratios: left: LM, reference; proximal, 0.95, p=0.803; other, 0.64, p=0.017; right: proximal, reference; other, 0.52, p <0.001). Proximally located plaques were larger, with more risky composition, and progressed more rapidly.

Sex-dependent gene regulation of human atherosclerotic plaques by DNA methylation and transcriptome integration points to smooth muscle cell involvement in women.

Background and Aims: Sex differences are evident in the clinical presentation and underlying histology of atherosclerotic disease with women developing more stable atherosclerotic lesions than men. It is unknown whether this is explained by sex differences in gene regulation in cellular compartments of atherosclerotic plaques.

Pharmacological treatment with FGF21 strongly improves plasma cholesterol metabolism to reduce atherosclerosis.

Aims Fibroblast growth factor (FGF) 21, a key regulator of energy metabolism, is currently evaluated in humans for treatment of type 2 diabetes and non-alcoholic steatohepatitis. However, the effects of FGF21 on cardiovascular benefit, particularly on lipoprotein metabolism in relation to atherogenesis, remain elusive.Methods and results Here, the role of FGF21 in lipoprotein metabolism in relation to atherosclerosis development was investigated by pharmacological administration of a half-life extended recombinant FGF21 protein to hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model mimicking atherosclerosis initiation and development in humans. FGF21 reduced plasma total cholesterol, explained by a reduction in non-HDL-cholesterol. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into browned WAT, consequently accelerating the clearance of the cholesterol-enriched remnants by the liver. In addition, FGF21 reduced body fat, ameliorated glucose tolerance and markedly reduced hepatic steatosis, related to up-regulated hepatic expression of genes involved in fatty acid oxidation and increased hepatic VLDL-triglyceride secretion. Ultimately, FGF21 largely decreased atherosclerotic lesion area, which was mainly explained by the reduction in non-HDL-cholesterol as shown by linear regression analysis, decreased lesion severity, and increased atherosclerotic plaque stability index.Conclusion FGF21 improves hypercholesterolaemia by accelerating triglyceride-rich lipoprotein turnover as a result of activating BAT and browning of WAT, thereby reducing atherosclerotic lesion severity and increasing atherosclerotic lesion stability index. We have thus provided additional support for the clinical use of FGF21 in the treatment of atherosclerotic cardiovascular disease.

Chitinase 3 like 1 is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability.

Atherosclerotic cerebrovascular disease underlies the majority of ischaemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals. Using two independent human endarterectomy biobanks, we found that the understudied glycoprotein, chitinase 3 like 1 (CHI3L1), is up-regulated in patients with carotid disease compared to healthy controls. Further, CHI3L1 levels were found to stratify individuals based on symptomatology and histopathological evidence of an unstable fibrous cap. Gain- and loss-of-function studies in cultured human carotid artery smooth muscle cells (SMCs) showed that CHI3L1 prevents a number of maladaptive changes in that cell type, including phenotype switching towards a synthetic and hyperproliferative state. Using two murine models of carotid remodelling and lesion vulnerability, we found that knockdown of Chil1 resulted in larger neointimal lesions comprised by de-differentiated SMCs that failed to invest within and stabilize the fibrous cap. Exploratory mechanistic studies identified alterations in potential downstream regulatory genes, including large tumour suppressor kinase 2 (LATS2), which mediates macrophage marker and inflammatory cytokine expression on SMCs, and may explain how CHI3L1 modulates cellular plasticity. CHI3L1 is up-regulated in humans with carotid artery disease and appears to be a strong mediator of plaque vulnerability. Mechanistic studies suggest this change may be a context-dependent adaptive response meant to maintain vascular SMCs in a differentiated state and to prevent rupture of the fibrous cap. Part of this effect may be mediated through downstream suppression of LATS2. Future studies should determine how these changes occur at the molecular level, and whether this gene can be targeted as a novel translational therapy for subjects at risk of stroke.

Non-lethal sonodynamic therapy facilitates the M1-to-M2 transition in advanced atherosclerotic plaques via activating the ROS–AMPK–mTORC1–autophagy pathway

Emerging evidence indicates that macrophage functional polarization is critically involved in the development of atherosclerosis (AS). Here, we examined the role of 5-aminolaevulinic acid (ALA)-mediated non-lethal sonodynamic therapy (NL-SDT) in macrophage-subset polarization and atherosclerotic lesion stability and explored the potential underlying mechanisms. Using Western diet-fed apolipoprotein E (apoE)−/− and green fluorescent protein (GFP)-positive bone marrow (BM) chimeric mouse models, we demonstrated that NL-SDT promoted phenotypic switching of both BM-derived and resident macrophages from M1 to M2 and significantly inhibited AS progression. Further mechanistic studies indicated that NL-SDT enhanced macrophage differentiation toward the M2 phenotype by activating the reactive oxygen species (ROS)–5′ AMP-activated protein kinase (AMPK)–mammalian target of rapamycin complex 1 (mTORC1)–autophagy signaling pathway in murine BM-derived M1 macrophages (BMDM1s). Moreover, NL-SDT drastically reduced lipid droplets, mainly by promoting apoAI-mediated cholesterol efflux in vitro. Specifically, administration of pharmacological inhibitors to the animal model showed a reciprocal effect on NL-SDT-induced macrophage polarization. These findings indicate that NL-SDT engages a virtuous cycle that enhances M1-to-M2 polarization, cholesterol efflux, and anti-inflammatory reactions in advanced plaque in vivo and in BMDM1s in vitro by activating the ROS–AMPK–mTORC1–autophagy pathway. This discovery might help elucidate the mechanism underlying NL-SDT as a potential treatment to prevent atherothrombotic events.

Functional ability of neutrophils in patients with ischemic heart disease

Purpose. The study of protein-producing and lipid-releasing ability of neutrophils and its clinical significance in patients with IHD.Materials and methods. 25 patients with arterial hypertension without clinical and ultrasound manifestations of atherosclerosis were examined; 47 patients with coronary artery disease with stable angina, as well as 19 patients with unstable primary angina. The comparison group — 33 healthy persons. In the serum, enzyme-linked immunosorbent assay was used to evaluate the concentration of C-reactive protein (CRP), lipoprotein a (LPa), VII coagulation factor VII (VIIf), defensins-alpha (1–3), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha). Leukocytes, predominantly represented by neutrophils, which were further cultivated, were excreted from peripheral blood in the presence of dextrans; the levels of CRP, LPa, defensins-alpha (1–3), VIIf, and lipid-releasing ability of leukocytes (LRAL) were assessed according to the method of A. V. Tuev and V. Yu. Mishlanov. An angiographic study, duplex scanning arteries, and echocardiography were performed.Results. It has been established that in patients at the stage of preclinical atherosclerotic lesion, the ability of leukocytes to form protein-lipid complexes (PLC), marked by LRAL, practically does not differ from healthy individuals, but already at this stage of atherogenesis an increase in their ability to produce antimicrobial peptides — defensins-alpha (1–3). In patients with coronary artery disease with stable angina pectoris, an increase in leukocyte production of a wide range of proteins — defensins-alpha (1–3), VIIf, LPA and CRP in combination with signs of a systemic inflammatory reaction, marked by high concentrations of CRP, interleukin-6 and tumor necrosis factor alpha in serum, was noticed. At this stage of atherogenesis, the value of LRAL is significantly different from the values of healthy individuals. It is shown that the higher the LRAL value, the higher the functional class of chronic heart failure in these patients (R = 0.4; p = 0.02). In patients with coronary artery disease with primary unstable angina, the maximum LRAL value was detected, and in patients with ultrasound signs of plaque instability, high concentrations of CRP in neutrophilic supernatants were found. Correlation analysis revealed that, in patients with coronary artery disease, the average percentage of coronary artery stenosis, according to angiography, is directly related to the LRAL value (R = 0.7; p = 0.03) and the serum concentration of TNF-alpha (R = 0.5; p = 0.03), and the number of clinically significant stenoses — with the magnitude of LRAL (R = 0.4; p = 0.04) and the concentration in supernatants VIIf (R = 0.5; p = 0.04).Conclusion. The LRAL value, CRP and defensin-alpha content in leukocyte supernatants in IHD patients are interconnected with the severity and activity of atherosclerotic lesion of arteries, which is confirmed by the results of examination of patients at the stage of preclinical, clinically manifest stable and unstable atherosclerotic lesion.

Myeloperoxidase inhibition in mice alters atherosclerotic lesion composition.

Myeloperoxidase (MPO) is a highly abundant protein within the neutrophil that is associated with lipoprotein oxidation, and increased plasma MPO levels are correlated with poor prognosis after myocardial infarct. Thus, MPO inhibitors have been developed for the treatment of heart failure and acute coronary syndrome in humans. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide PF-06282999 is a recently described selective small molecule mechanism-based inactivator of MPO. Here, utilizing PF-06282999, we investigated the role of MPO to regulate atherosclerotic lesion formation and composition in the Ldlr-/- mouse model of atherosclerosis. Though MPO inhibition did not affect lesion area in Ldlr-/- mice fed a Western diet, reduced necrotic core area was observed in aortic root sections after MPO inhibitor treatment. MPO inhibition did not alter macrophage content in and leukocyte homing to atherosclerotic plaques. To assess non-invasive monitoring of plaque inflammation, [18F]-Fluoro-deoxy-glucose (FDG) was administered to Ldlr-/- mice with established atherosclerosis that had been treated with clinically relevant doses of PF-06282999, and reduced FDG signal was observed in animals treated with a dose of PF-06282999 that corresponded with reduced necrotic core area. These data suggest that MPO inhibition does not alter atherosclerotic plaque area or leukocyte homing, but rather alters the inflammatory tone of atherosclerotic lesions; thus, MPO inhibition could have utility to promote atherosclerotic lesion stabilization and prevent atherosclerotic plaque rupture.

Irradiation abolishes smooth muscle investment into vascular lesions in specific vascular beds.

The long-term adverse effects of radiotherapy on cardiovascular disease are well documented. However, the underlying mechanisms responsible for this increased risk are poorly understood. Previous studies using rigorous smooth muscle cell (SMC) lineage tracing have shown abundant SMC investment into atherosclerotic lesions, where SMCs contribute to the formation of a protective fibrous cap. Studies herein tested whether radiation impairs protective adaptive SMC responses during vascular disease. To do this, we exposed SMC lineage tracing (Myh11-ERT2Cre YFP+) mice to lethal radiation (1,200 cGy) followed by bone marrow transplantation prior to atherosclerosis development or vessel injury. Surprisingly, following irradiation, we observed a complete loss of SMC investment in 100% of brachiocephalic artery (BCA), carotid artery, and aortic arch lesions. Importantly, this was associated with a decrease in multiple indices of atherosclerotic lesion stability within the BCA. Interestingly, we observed anatomic heterogeneity, as SMCs accumulated normally into lesions of the aortic root and abdominal aorta, suggesting that SMC sensitivity to lethal irradiation occurs in blood vessels of neural crest origin. Taken together, these results reveal an undefined and unintended variable in previous studies using lethal irradiation and may help explain why patients exposed to radiation have increased risk for cardiovascular disease.

Green tea polyphenol epigallocatechin-3-gallate increases atherosclerotic plaque stability in apolipoprotein E-deficient mice fed a high-fat diet.

Epigallocatechin-3-gallate (EGCG), which is the principal component of green tea, has been shown to prevent atherosclerosis. However, the effect of EGCG on atherosclerotic plaque stability remains unknown. This study aimed to assess whether EGCG can enhance atherosclerotic plaque stability and to investigate the underlying mechanisms. Apolipoprotein E-deficient mice fed a high-fat diet were injected intraperitoneally with EGCG (10 mg/kg) for 16 weeks. Cross sections of the brachiocephalic arteries were stained with haematoxylin and eosin for morphometric analyses or Masson's trichrome for collagen content analyses. Immunohistochemistry was performed to evaluate the percentage of macrophages and smooth muscle cells (SMCs). Protein expression and matrix metalloproteinase (MMP) activity were assayed by Western blot and gelatin zymography, respectively. Serum inflammatory cytokine levels were quantified by enzyme-linked immunosorbent assays. After 16 weeks of feeding the high-fat diet, there were clear atherosclerotic lesions in the proximal brachiocephalic artery segments according to HE staining. EGCG treatment significantly increased the thickness of the fibrous cap. In the atherosclerotic plaques of the EGCG group, the relative macrophage content was decreased, whereas the relative SMC and collagen contents were increased. The expression levels of MMP-2, MMP-9, and extracellular matrix metalloproteinase inducer (EMMPRIN) were significantly decreased by EGCG treatment. In addition, EGCG treatment decreased the circulat-ing tumour necrosis factor-α, interleukin-6, monocyte chemoattractant protein-1, and interferon-γ levels in apolipoprotein E-deficient mice. EGCG promotes atherosclerotic lesion stability in apolipoprotein E-deficient mice. Potentially, these effects are mediated through the inhibition of inflammatory cytokine, MMPs and EMMPRIN expression.

Structure of Atherosclerotic Plaques in Different Vascular Territories: Clinical Relevance.

Atherosclerosis is a systemic disease with different faces. Despite similar, or even identical, risk factors and pathogenesis, atherosclerotic lesions and their clinical manifestations vary in different parts of the vasculature. Peripheral arterial disease (PAD) in the superficial femoral artery (SFA) represents a frequent clinical manifestation of atherosclerotic disease. The pathohistological characteristics of plaques in PAD differ from lesions in the coronary arteries. Plaques in the SFA have more fibrotic elements with less lipid and degenerative tissue elements; this makes them more stable and less prone to rupture. The density of vasa vasorum, an important determinant of structure and stability of atherosclerotic lesions, is significantly lower in PAD than in coronary arteries. Further, haemodynamic forces and shear stress vary in different segments of the arterial tree and influence the development of atherosclerotic lesions and their stability. It follows that the clinical consequences differ depending on the vascular territory involved. In the coronary arteries, acute thrombotic occlusion with clinical manifestation of myocardial infarction is one of the most frequent manifestations due to unstable atherosclerotic lesions. Atherosclerotic lesions in SFA progress slowly and are more stable; therefore, clinical manifestations develop more gradually. The atherosclerotic process in SFA is frequently asymptomatic or presents as stable intermittent claudication, and in a relatively low percentage, progresses to critical limb ischaemia. Also, remodelling of the arterial wall in peripheral arteries compensates for the reduction of arterial lumen and provides blood flow in spite of relatively large atherosclerotic lesions. However, arterial restenosis after recanalization procedures in SFA reduces the long-term success of recanalization.

5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Alleviates Atherosclerosis via Enhancing Efferocytosis and Facilitating a Shift in the Th1/Th2 Balance Toward Th2 Polarization

Background/Aims: We and other groups have demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (ALA-SDT) induces macrophage and foam cell apoptosis and stabilizes atherosclerosis (AS) plaques in animal models. Lymphocytes also play vital roles in the development of AS. The primary purpose of the present study was to investigate the effects of ALA-SDT on T helper (Th) cell fate and function, Th subset differentiation, and atherosclerotic lesion stability. Methods: We utilized ALA-SDT on Western diet-fed apoE^-/-mice in vivo and human Jurkat cells in vitro. Hematoxylin and eosin staining and TUNEL assays were used to evaluate the atherosclerotic plaque size and apoptosis within the atheroma. ALA induced cytotoxicity on cultured Jurkat cells was determined with CCK-8 assay. To address the mechanisms, levels of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (MPTP) opening were evaluated by staining with fluorescent probes. Western blot analysis and confocal microscopy were used to analyze the protein levels of caspases, Bax and cytochrome c and the release of cytochrome c. Cell apoptosis and necrosis and phagocytosis were examined by flow cytometry. ELISAs and immunofluorescent staining were used to assess the corresponding cytokine levels and Th subset cell numbers within the atheroma. Results: Our studies revealed that ALA-SDT significantly enhanced CD4⁺ cell apoptosis and macrophage-mediated phagocytosis and hence reduced the necrotic core size. ALA-SDT activated the mitochondrial apoptotic signaling pathway with minimal necrosis in Jurkat cells. ALA-SDT inhibited the Th1 response and enhanced the Th2 response. These effects of ALA-SDT were mediated primarily through the generation of ROS. Conclusion: ALA-SDT alleviates AS by enhancing cytotoxic effects on Th cells, subsequently stimulating efferocytosis and facilitating a shift in the Th1/Th2 balance toward Th2 cells, a discovery that might help elucidate the mechanism underlying SDT as a potential treatment to prevent atherothrombotic events.

Ginsenoside Rb1 enhances atherosclerotic plaque stability by skewing macrophages to the M2 phenotype.

Atherosclerosis (AS) is characterized as progressive arterial plaque, which is easy to rupture under low stability. Macrophage polarization and inflammation response plays an important role in regulating plaque stability. Ginsenoside Rb1 (Rb1), one of the main active principles of Panax Ginseng, has been found powerful potential in alleviating inflammatory response. However, whether Rb1 could exert protective effects on AS plaque stability remains unclear. This study investigated the role of Rb1 on macrophage polarization and atherosclerotic plaque stability using primary peritoneal macrophages isolated from C57BL/6 mice and AS model in ApoE−/− mice. In vitro, Rb1 treatment promoted the expression of arginase‐I (Arg‐I) and macrophage mannose receptor (CD206), two classic M2 macrophages markers, while the expression of iNOS (M1 macrophages) was decreased. Rb1 increased interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13) secretion in supernatant and promoted STAT6 phosphorylation. IL‐4 and/or IL‐13 neutralizing antibodies and leflunomide, a STAT6 inhibitor attenuated the up‐regulation of M2 markers induced by Rb1. In vivo, the administration of Rb1 promoted atherosclerotic lesion stability, accompanied by increased M2 macrophage phenotype and reduced MMP‐9 staining. These data suggested that Rb1 enhanced atherosclerotic plaque stability through promoting anti‐inflammatory M2 macrophage polarization, which is achieved partly by increasing the production of IL‐4 and/or IL‐13 and STAT6 phosphorylation. Our study provides new evidence for possibility of Rb1 in prevention and treatment of atherosclerosis.

358 - Pharmacological Inhibition of Myeloperoxidase Improves Endothelial Function and Stabilizes Atherosclerotic Lesions in a Mouse Model of Vulnerable Plaque

The involvement of myeloperoxidase (MPO) in human atherosclerotic plaque progression is well documented, including its potential role in plaque rupture that can cause acute myocardial infarction. Despite this, establishing a causal relationship between MPO and atherosclerosis-associated complications has proven difficult in mouse models. In the present study we employed a mouse model of atherosclerotic plaque instability (Circ Res 113:252) to examine the role of MPO in endothelial dysfunction and atherosclerotic plaque vulnerability. Unstable plaques were introduced in apolipoprotein E gene knockout (Apoe–/–) mice fed a Western diet for 13 weeks by placing a tandem stenosis around the right carotid artery after 6 weeks of diet, and MPO activity was partially blocked by oral administration of a 2-thioxanthine (AZM198). Unstable carotid plaques, but not stable plaques in the aortic root and arch, contained measurable MPO activity as assessed by the in vivo conversion of hydroethidine to 2-chloroethidium and non-invasive magnetic resonance imaging using MPO-gadolinium (bis-5-hydroxytryptamine-DTPA-Gd). Administration of AZM198 did not change circulating white blood cell counts, plasma concentrations of pro-inflammatory cytokines and lipids, nor did it affect atherosclerotic lesion size in the aortic root. In contrast, AZM198 significantly attenuated arterial MPO activity in unstable plaques and endothelial dysfunction in the abdominal aorta. Inhibition of MPO activity was associated with increased plaque stability in the carotid artery, as indicated by the increase in cap thickness and the cap-to-lesion ratio, without affecting necrotic core size and arterial monocytes. Together, these data indicate that vulnerable but not stable atherosclerotic lesions in Apoe–/– mice exhibit MPO activity, and that pharmacological inhibition of MPO stabilizes vulnerable plaques.

Mast cells are increased in the media of coronary lesions in patients with myocardial infarction and may favor atherosclerotic plaque instability

ObjectivesMast cells (MCs) may play an important role in plaque destabilization and atherosclerotic coronary complications. Here, we have studied the presence of MCs in the intima and media of unstable and stable coronary lesions at different time points after myocardial infarction (MI). MethodsCoronary arteries were obtained at autopsy from patients with acute MI (up to 5 days old; n=27) and with chronic MI (5–14 days old; n=18), as well as sections from controls without cardiac disease (n=10). Herein, tryptase-positive MCs were quantified in the intima and media of both unstable and stable atherosclerotic plaques in infarct-related and non-infarct-related coronary arteries. ResultsIn the media of both acute and chronic MI patients, the number of MCs was significantly higher than in controls. This was also found when evaluating unstable and stable plaques separately. In patients with chronic MI, the number of MCs in unstable lesions was significantly higher than in stable lesions. This coincided with a significant increase in the relative number of unstable plaques in patients with chronic MI compared with control and acute MI. No differences in MC density were found between infarct-related and non-infarct-related coronary arteries in patients with MI. ConclusionThe presence of MCs in the media of both stable and unstable atherosclerotic coronary lesions after MI suggests that MCs may be involved in the onset of MI and, on the other hand, that MI triggers intra-plaque infiltration of MCs especially in unstable plaques, possibly increasing the risk of re-infarction.

BIOCHEMICAL MARKERS OF BONE TISSUE METABOLISM INFLUENCE ON VULNERABILITY OF ATHEROSCLEROTIC LESIONS

Aim . To assess some of biochemical markers of bone tissue metabolism, in vessel wall in males with coronary atherosclerosis; to evaluate the influence of these markers on vulnerability of atherosclerotic lesion and to figure out the specifics of their spread among unstable plaques of various kinds. Material and methods. Totally, 65 males included at the age 46-79 y. o., admitted to the Clinics of E. N. Meshalkin Novosibirsk Scientific-Research Institute of Circulation Pathology for coronary bypass surgery. During operation, an endarterectomy was performed of coronary arteries. Each specimen of endarterectomy was symmetrically divided to fragments for histological and biochemical studies. By the results of histological analysis, in 193 specimens we found: 19 fragments of intact intima, 102 stable, 72 unstable plaques. Then the types of unstable plaques were established: 1) lipids’; 2) inflammatory-erosive; 3) distrophic-necrotic. In homogenates of the fragments we assessed: osteoprotegerin, calcitonin, osteocalcin, cholesterol. Statistics was done with SPSS (13.0). Results. By the results of the performed histology, in unstable plaques there was higher level of calcification comparing to stable plaques. Biochemical studies showed increased levels of calcitonin and osteocalcin. If to assess the diverse types of unstable plaques, the highest level of these parameters was found in distrophic-necrotic plaques; lowest amount of calcitonin and osteocalcin was found in inflammatory-erosive type. Content of cholesterol in stable and unstable atherosclerotic lesions was about 4 times higher (p<0,01) in comparison with intact intima. Relation of cholesterol with calcitonin and osteocalcin was found, as well. Conclusion. The data shows that increased osteoprotegerin aids the stabilization of lesion, and calcitonin and osteocalcin might be markers of plaque instability, caused by increased calcification.

Medical applications of nanotechnology.

Nanotechnologies are new areas of research focusing on affecting matter at the atomic and molecular levels. It is beyond doubt that modern medicine can benefit greatly from it; thus nanomedicine has become one of the main branches of nanotechnological research. Currently it focuses on developing new methods of preventing, diagnosing and treating various diseases. Nanomaterials show very high efficiency in destroying cancer cells and are already undergoing clinical trials. The results are so promising that nanomaterials might become an alternative to traditional cancer therapy, mostly due to the fact that they allow cancer cells to be targeted specifically and enable detailed imaging of tissues, making planning further therapy much easier. Nanoscience might also be a source of the needed breakthrough in the fight against atherosclerosis, since nanostructures may be used in both preventing and increasing the stability of atherosclerotic lesions. One area of interest is creating nanomaterials that are not only efficient, but also well tolerated by the human body. Other potential applications of nanotechnology in medicine include: nanoadjuvants with immunomodulatory properties used to deliver vaccine antigens; the nano-knife, an almost non-invasive method of destroying cancer cells with high voltage electricity; and carbon nanotubes, which are already a popular way of repairing damaged tissues and might be used to regenerate nerves in the future. The aim of this article is to outline the potential uses of nanotechnology in medicine. Original articles and reviews have been used to present the new developments and directions of studies.