Early Plaque Research Articles

Abstract 344: Bcl-x Inactivation in Macrophages Accelerates Progression of Advanced Atherosclerotic Lesions in Apoe -/- Mice

Background: Bcl-x is the most abundantly expressed member of the Bcl-2 gene family in macrophages but its role in macrophage apoptosis during atherogenesis is unknown. Methods and results: We previously reported dual pro- and anti-atherogenic effects of macrophage survival in early versus advanced atherosclerotic lesions respectively, potentially reflecting growing impairment of efferocytosis during plaque progression. Here, we specifically inactivated Bcl-x in macrophages and evaluated its impact on atherosclerotic lesion formation in Apoe -/- mice at various stages of the disease. Bcl-x deficiency in macrophages increased susceptibility to apoptosis, resulting in the depletion of tissue macrophages in vivo, including its major pool, Küppfer cells in the liver. We also observed increased cholesterol levels, that was however not associated with any acceleration of early atherosclerotic plaque progression. This observation, suggests that the atheroprotective effect of macrophage apoptosis at that stage of disease was counterbalanced by enhanced cholesterol levels. Bcl-x KOmac/Apoe -/- mice exhibited significantly larger advanced lesions than control mice. These lesions showed vulnerable traits. Such enhanced lesion size may occur as a result not only of apoptotic cell accumulation but also of elevated cholesterol levels. Conclusions: Modulation of macrophage resistance to apoptosis through targeted deletion of Bcl-x has a major impact on the entire macrophage cell population in the body, including Küpffer cells. Macrophage survival may, therefore, not only influence atherosclerotic plaque development and vulnerability but also cholesterol metabolism.

Abstract 497: Cholesterol and Toll-like Receptor Signaling Are Important Regulators of Macrophage Interactions with Atherosclerotic Lipoprotein Aggregates

Macrophages encounter deposits of aggregated low-density lipoproteins (agLDL) in the subendothelial space of blood vessels during the first stages of atherosclerotic plaque formation. Notably, current models for the mechanism of macrophage internalization of cholesterol in early atherosclerotic plaques are incomplete due to the lack of attention paid to the unique cellular mechanisms that are required for macrophages to degrade aggregates of LDL in particular, which can comprise >90% of the LDL in atherosclerotic plaques. In fact, internalization of cholesterol from cholesteryl esters in agLDL involves the development of intriguing cellular processes in which extracellular acidic compartments, lysosomal synapses (LSs), are formed whereby agLDL is partially degraded prior to internalization. This process requires extensive cytoskeletal rearrangements and secretion of lysosomal enzymes responsible for hydrolysis of cholesteryl esters from the agLDL. Subsequent delivery of free cholesterol from agLDL to the macrophage plasma membrane is central for development of the LS. Nonetheless, the molecular mechanism underlying initiation and propagation of the LS are currently largely unknown. This research proposal aims to elucidate the molecular mechanisms of LS formation and the role that cholesterol plays in eliciting these morphological responses to agLDL. Fluorescence microscopy assays were used to identify activation of TLR4 and downstream signaling involving PI3K and Akt as important events leading to LS formation. Furthermore, morphological responses of macrophages to cholesterol overloading require overlapping signaling pathways, indicating the role of interplay of cholesterol and TLR4 signaling in development of this novel macrophage interaction with aggregated LDL found in plaques. Identification of specific molecular pathways involved in this process will not only contribute to the basic understanding of one of the primary cellular processes contributing to atherosclerosis, one of the primary causes of heart disease, but also provide tangible molecular targets for the ultimate development of therapies.

Fatty liver index, gamma-glutamyltransferase, and early carotid plaques

An association between fatty liver and carotid atherosclerosis has been established; however, it is not clear whether this relationship is a consequence of shared conventional risk factors or whether it is determined by specific circulating factors originating from liver or adipose tissue. To identify the factors possibly linking fatty liver and atherosclerosis, we assessed, in 1,012 subjects free of confounding diseases (e.g., hypertension, diabetes, cardiovascular diseases, and dyslipidemia) and metabolic syndrome, the relationship between the presence of early plaques at carotid bifurcation and fatty liver index (FLI; a validated surrogate marker of fatty liver), as well as the associations between carotid plaque presence and established atherosclerotic risk factors, family history of cardiovascular disease (FH-CVD) or diabetes, insulin sensitivity, serum liver enzymes, adipokines, fatty free acids, and high-sensitivity C-reactive protein (hsCRP). A total of 55 of 1,012 subjects (5.4%) had small plaque at carotid bifurcation. Subjects with plaque were older and had higher prevalence of FLI ≥60 and FH-CVD, higher blood pressure, plasma low-density lipoprotein cholesterol, glucose, gamma-glutamyltransferase (GGT), and hsCRP, as compared to subjects without plaques (P < 0.05). In a logistic regression model, adjusted for sex, liver transaminase, and alcohol consumption, the independent predictors of plaque presence were age (P < 0.0005), FLI ≥60 (P < 0.0005), and current smoking (P < 0.05). When FLI in the model was replaced by variables used in its equation (e.g., body mass index, waist circumference, plasma triglycerides, and GGT), the independent determinants of plaque presence were age (P < 0.001), GGT (P = 0.001), and current smoking (P < 0.05). Our cross-sectional study suggests that subjects with FLI ≥60 are at higher risk of atherosclerotic lesions, independently of established risk factors, and that serum GGT may represent a link between fatty liver and the development of early atherosclerosis.

C-type Natriuretic Peptide and its Receptors in Atherosclerotic Plaques of the Carotid Artery of Clinically Asymptomatic Patients

ObjectivesC-type natriuretic peptide (CNP) has anti-inflammatory, anti-proliferative and anti-migratory properties. No data exist on the presence of CNP in human atherosclerotic plaques of the carotid artery. Therefore, this study aimed to analyse qualitatively the distribution pattern and characteristics of CNP and its receptors in both, early and advanced human carotid plaques, as well as in stable and unstable lesions.In addition, the aim of this study was to evaluate CNP and its receptors as possible biomarkers to predict plaque stability in advanced lesions. MethodsAdvanced carotid artery plaques of 40 asymptomatic patients (20 histologically stable and 20 histologically unstable) and early arteriosclerotic lesions of three patients were analysed. ResultsSerum level of CNP was similar in patients with stable and unstable plaques (196 ± 19 pg ml−1 vs. 198 ± 25 pg ml−1, p = 0.948). Expression level of natriuretic peptide receptor 3 (NPR3) was significantly higher in unstable plaques compared to stable plaques (5.6 ± 1.8% vs. 1.7 ± 0.5%, p = 0.045). Expression levels of CNP and NPR2 were higher in unstable plaques but the differences were not statistically significant. The distribution pattern of CNP, NPR2 and NPR3 varied qualitatively between early and advanced carotid plaques. No relevant histological differences were observed with respect to plaque stability. ConclusionsThis study shows the presence of CNP and its receptors in atherosclerotic plaques of human carotid artery, with increased expression of NPR3 in histologically unstable plaques. In this study, serum CNP was not associated with histological plaque stability. In future, larger studies are required to further evaluate whether proteins of the CNP axis would be useful as biomarkers.

ADAMTS13 reduces vascular inflammation and the development of early atherosclerosis in mice

AbstractADAMTS13, a metalloprotease, plays a pivotal role in preventing spontaneous microvascular thrombosis by cleaving hyperactive ultra large von Willebrand factor multimers into smaller, less active multimers. Reduced ADAMTS13 activity in plasma has been described in many diseases associated with systemic inflammation. It remains uncertain, however, whether ADAMTS13 contributes to disease pathogenesis or rather simply serves as an inflammation-associated marker. We hypothesized that, by decreasing vascular inflammation, ADAMTS13 reduces the development of early atherosclerotic plaques. Using intravital fluorescence microscopy, we observed excessive leukocyte adhesion and accelerated atherosclerotic plaque formation at the carotid sinus of Adamts13−/−/ApoE−/− mice compared with ApoE−/− mice fed a high-fat Western diet. At 4 months of age, there was a significant increase in atherosclerosis in the aorta and aortic sinus of Adamts13−/−/ApoE−/− mice compared with ApoE−/− mice. Interestingly, we detected a 2-fold increase in macrophage recruitment to the atherosclerotic plaque of the Adamts13−/−/ApoE−/− mice compared with ApoE−/− mice, suggesting that the atherosclerotic lesions in these mice were not only larger but also more inflammatory. These findings reveal a new functional role for the antithrombotic enzyme ADAMTS13 in reducing excessive vascular inflammation and plaque formation during early atherosclerosis.

Bcl-x Inactivation in Macrophages Accelerates Progression of Advanced Atherosclerotic Lesions in Apoe −/− Mice

Bcl-x is the most abundantly expressed member of the Bcl-2 gene family in macrophages, but its role in macrophage apoptosis during atherogenesis is unknown. We previously reported dual pro- and antiatherogenic effects of macrophage survival in early versus advanced atherosclerotic lesions, respectively, potentially reflecting growing impairment of efferocytosis during plaque progression. Here, we specifically inactivated Bcl-x in macrophages and evaluated its impact on atherosclerotic lesion formation in Apoe(-/-) mice at various stages of the disease. Bcl-x deficiency in macrophages increased their susceptibility to apoptosis, resulting in the depletion of tissue macrophages in vivo, including its major pool, Küppfer cells in the liver. We also observed increased cholesterol levels that were, however, not associated with any acceleration of early atherosclerotic plaque progression. This observation suggests that the atheroprotective effect of macrophage apoptosis at that stage of disease was counterbalanced by enhanced cholesterol levels. Bcl-x KO(mac)/Apoe(-/-) mice exhibited significantly larger advanced lesions than control mice. These lesions showed vulnerable traits. Such enhanced lesion size may occur as a result not only of apoptotic cell accumulation but also of elevated cholesterol levels. Modulation of macrophage resistance to apoptosis through targeted deletion of Bcl-x has a major impact on the entire macrophage cell population in the body, including Küpffer cells. Macrophage survival may, therefore, not only influence atherosclerotic plaque development and vulnerability but also cholesterol metabolism.

Ultrasonic Imaging of Endothelial CD81 Expression Using CD81-Targeted Contrast Agents in In Vitro and In Vivo Studies

This study is designed to investigate the feasibility for molecular imaging of endothelial CD81 expression in vitro and in vivo using the CD81-targeted ultrasound contrast agents (UCA). In the in vitro study, murine bEnd.3 cells were stimulated with phenazine methosulfate (PMS), an oxidative stress inducer. Changes in CD81 expression after stimulation were confirmed by Western blotting, tracked by using the targeted UCA and further imaged under ultrasound imaging system with 5 MHz transmit frequency. In the in vivo study, expression of endothelial CD81 proteins in murine carotid artery vessels was studied using high-frequency ultrasound system with 40 MHz transmit frequency. Our results showed that endothelial CD81 expression was gradually up-regulated with the increase of PMS concentration. Correspondingly, the accumulation of targeted UCA was gradually improved and could be inhibited significantly upon addition of free anti-CD81 antibodies. The mean video intensity (grey-level) of stimulated cells and vessels from backscatter of the CD81-targeted UCA was 17.2 (interquartile range [IQR] 15.4–19.8) and 27.2 (IQR 22.4–29.8), significantly greater than that of non-stimulated cells with 9.0 (IQR 8.6–10.8) (p < 0.01) and non-stimulated vessels with 11.3 (IQR 10.4–13.2) (p < 0.01), respectively. In conclusion, CD81-targeted UCA allows noninvasive assessment of the expression levels of CD81 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.

Advanced glycation end-product Nɛ-carboxymethyl-Lysine accelerates progression of atherosclerotic calcification in diabetes

ObjectiveVascular calcification is an active deposition process of calcium phosphate which resembles bone formation and is highly regulated by osteoblast-like cells. Existing studies demonstrate that advanced glycation end-products (AGEs) may play a pathogenic role in the vascular calcification process. However, their mechanism remains poorly understood. The aim of our current study is to investigate how non-cross-link and non-fluorescent Nɛ-carboxymethyl-Lysine (CML), a major immunogen of AGEs, affect the progression of atherosclerotic calcification in diabetes. MethodsThe present study consisted of an in vivo investigation and two in vitro investigations. In study I, male apoE−/− mice were first rendered diabetic by the administration of 5 daily intraperitoneal injections of streptozotocin (STZ, 40mg/kg), and then given a semi-synthetic high-fat diet (HFD) plus daily injections of CML (10mg/kg/day). The mice were euthanized and analyzed at 0 month (group 0M, n=10), 2 months (group 2M, n=10), and 4 months (group 4M, n=10) after the triple administrations of STZ–CML–HFD. In study II, the effects of CML on the apoptosis in macrophages were investigated. RAW264.7 cells were incubated with or without 50μg/mL oxLDL plus various concentrations of CML for 48h. In study III, we investigated whether A7r5 aortic smooth muscle cells were induced into osteoblast-like phenotypes by incubation with or without 80μg/mL of RAW264.7-derived-apoptotic bodies and 50μg/mL of oxLDL plus various concentrations of CML (or high-glucose) for 7 days. Related analyses (i.e., H&E staining, Masson staining, von Kossa staining, TUNEL staining, immunohistochemical staining, calcium content assay, annexin V-FITC/PI double-staining, and Western blot) were performed. ResultsMorphological analysis showed that early atherosclerotic plaques appeared 2 months after the triple administrations of STZ–CML–HFD, and that typically advanced plaques with extensive calcification lesions, abundant cholesterol crystals, and proliferative collagen were formed 4 months after the triple administrations of STZ–CML–HFD. Furthermore, CML deposition signals and the expression of receptor for advanced glycation end-products (RAGE) in the aortic wall were mainly restricted in the atherosclerotic plaques. After the incubation of A7r5 smooth muscle cells with 10μmol/L CML plus 50μg/mL oxLDL, and 80μg/mL apoptotic bodies (ABs) for 7 days, semi-quantitative analysis of bone morphogenetic protein 2 (BMP-2), core-binding factor α1 (cbfα1), and alkaline phosphatase (ALP) expression showed 5.0-, 2.0-, and 2.9-fold increases, respectively, compared with those in 50μg/mL oxLDL and 80μg/mL ABs. Subsequently, a similar trend was observed in the calcium deposition of the cell layer. However, high-glucose had no effects on the ALP activity and calcium deposition of A7r5 cell layer under high-lipid, apoptosis-coexisting conditions. Both animal and cell studies consistently demonstrated that the CML/RAGE axis may first initiate the apoptosis of macrophages in atherosclerotic lesions and then induce BMP-2-cbfα1-ALP-calcification cascade in a high-lipid, apoptosis-coexisting environment. ConclusionThe CML/RAGE axis may play an important role in atherosclerotic calcification of diabetes through the mechanism that induces the apoptosis of macrophages followed by the osteogenic differentiation of aortic smooth muscle cells.

Pellicle and early dental plaque in periodontitis patients before and after surgical pocket elimination

Objective. Gingival inflammation may affect the composition of the dental pellicle and initial acquisition of bacteria, which in turn could affect the healing of the periodontal pocket. The aim of this study was to examine the dental pellicle and early supragingival biofilms in periodontitis patients with an established subgingival infiltrate before and after surgical pocket elimination. Materials and methods. Eleven patients with remaining pockets were selected. Samples were taken before and after surgical pocket elimination and after subsequent experimental gingivitis. Pellicle proteins were analyzed by SDS-PAGE, immunoblotting and image analysis and 4-h supragingival plaque by culturing. Results. The inflammatory status affected to a greater extent the concentration of plasma proteins than salivary proteins in the dental pellicle. The highest plasma protein concentrations were observed at remaining periodontal pockets where also the highest bacterial counts were found. The TVC was reduced on the gingival tooth surfaces (p < 0.05) after pocket elimination and increased slightly during experimental gingivitis. The finding of streptococci was highest on the incisal tooth surfaces and increased after surgery. Gram-negative anaerobes were sparse but seen more often before than after pocket elimination and on gingival than on incisal surfaces. Conclusions. This study suggests that increased amounts of plasma proteins in the pellicle formed in the presence of remaining periodontal pockets may foster the acquisition of bacteria, including proteolytic Gram-negative species. This, in turn, results in an increased de novo plaque formation rate.

Early atherosclerotic plaques show evidence of infection by Chlamydia pneumoniae

Chlamydia pneumoniae (Cpn) could play an important role in the development of atherosclerosis. Cpn interferes with HIF-1alpha regulation in infected host cells during intracellular replication in hypoxia. We obtained carotid artery specimens with low (n=38), high (n=25) levels of stenosis and 10 middle cerebral arteries. Fifty eight percent of the carotids with low levels of stenosis showed evidence of the viable organism. Ninety one percent of the positive results were derived from pre-atheromatous lesions. Only 12 percent of plaques removed at endarterectomy showed the presence of Cpn DNA. All middle cerebral arteries failed to show evidence of live Chlamydia. Ninety one percent of sera from 22 endarterectomy patients failed to show the presence of Cpn antibodies. Immunohistology of carotid arteries with low levels of stenosis was used to confirm the presence of HIF-1alpha in infected specimens and showed a correlation between the over-expression of HIF-1alpha and Cpn in the plaque (p less than 0.05). Cpn might play an important role in activation and development of the initial stages of atherosclerotic lesions.

Inverse Association between Circulating Levels of Soluble Receptor for Advanced Glycation End-Products and Coronary Plaque Burden

Low levels of soluble receptor for advanced glycation end-products (sRAGE) have been reported to be associated with coronary artery disease (CAD) and peripheral atherosclerosis. This study explored the relationship between circulating levels of sRAGE and the characteristics of coronary vessels detected by 64-slice computed tomography angiography (CTA). In this cross-sectional study we included 127 consecutive patients with CAD but without acute coronary syndrome. Quantitative volumetric analysis of the lumen and plaque burden of the vessel wall (soft and calcific components) was performed for the three major coronary vessels. Each component was expressed as a percentage of vessel volume and utilized in per-patient analysis. The patients were classified into two groups according to the presence of calcium volume: non-calcified plaque (NCP) group (calcium volume %=0) and calcified plaque (CP) group (calcium volume % >0). In the NCP group, but not in the CP group, simple regression analysis revealed a negative association of total plaque burden % with sRAGE (β=-0.378, p=0.0019) and HDL cholesterol (β=-0.368, p=0.003) and a positive association with creatinine (β=0.258, p=0.041) and male gender (β=0.317, p=0.01). After adjusting for confounding factors, the total plaque burden % remained significantly associated only with sRAGE (β=-0.358, p=0.011). Circulating sRAGE levels are associated in an inverse manner with non-calcified plaque burden, suggesting that it may be related with early atherosclerosis and plaque progression.

18FDG PET-CT imaging detects arterial inflammation and early atherosclerosis in HIV-infected adults with cardiovascular disease risk factors.

BackgroundPersistent vascular inflammation has been implicated as an important cause for a higher prevalence of cardiovascular disease (CVD) in HIV-infected adults. In several populations at high risk for CVD, vascular 18Fluorodeoxyglucose (18FDG) uptake quantified using 3D-positron emission-computed tomography (PET-CT) has been used as a molecular level biomarker for the presence of metabolically active proinflammatory macrophages in rupture-prone early atherosclerotic plaques. We hypothesized that 18FDG PET-CT imaging would detect arterial inflammation and early atherosclerosis in HIV-infected adults with modest CVD risk.MethodsWe studied 9 HIV-infected participants with fully suppressed HIV viremia on antiretroviral therapy (8 men, median age 52 yrs, median BMI 29 kg/m2, median CD4 count 655 cells/μL, 33% current smokers) and 5 HIV-negative participants (4 men, median age 44 yrs, median BMI 25 kg/m2, no current smokers). Mean Framingham Risk Scores were higher for HIV-infected persons (9% vs. 2%, p < 0.01). 18FDG (370 MBq) was administered intravenously. 3D-PET-CT images were obtained 3.5 hrs later. 18FDG uptake into both carotid arteries and the aorta was compared between the two groups.ResultsRight and left carotid 18FDG uptake was greater (P < 0.03) in the HIV group (1.77 ±0.26, 1.33 ±0.09 target to background ratio (TBR)) than the control group (1.05 ± 0.10, 1.03 ± 0.05 TBR). 18FDG uptake in the aorta was greater in HIV (1.50 ±0.16 TBR) vs control group (1.24 ± 0.05 TBR), but did not reach statistical significance (P = 0.18).ConclusionsCarotid artery 18FDG PET-CT imaging detected differences in vascular inflammation and early atherosclerosis between HIV-infected adults with CVD risk factors and healthy HIV-seronegative controls. These findings confirm the utility of this molecular level imaging approach for detecting and quantifying glucose uptake into inflammatory macrophages present in metabolically active, rupture-prone atherosclerotic plaques in HIV infected adults; a population with increased CVD risk.

The Anti-Thrombotic Enzyme ADAMTS13 Reduces Inflammation and the Development of Atherosclerosis In Mice

Abstract 367 Background and objective:ADAMTS13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13) plays a pivotal role in preventing spontaneous thrombosis in the microvasculature by cleaving hyperactive ultra large von Willebrand factor (ULVWF) multimers into smaller, less active multimers. Severe deficiency of ADAMTS13 in humans causes thrombotic thrombocytopenic purpura (TTP) and numerous epidemiological studies have demonstrated associations between decreased ADAMTS13 activity and adverse disease outcome in patients with systemic inflammation. It remains unknown, however, whether reduced ADAMTS13 activity plays a direct pathogenic role in inflammatory diseases or rather simply serves as an inflammation-associated marker. We hypothesized that deficiency of ADAMTS13 enhances inflammation and accelerates the development of early atherosclerotic plaques. Results:Using intravital fluorescence microscopy, we show that the number of adherent leukocytes (adherent for > 60 s) was increased approximately four-fold at the carotid sinus, a lesion prone site, of Adamts13−/−/ApoE−/− mice (Mean ± SEM = 37 ± 6) as compared to ApoE−/− mice (Mean ± SEM = 9 ± 4, P <0.01) fed a high-fat Western diet. Interestingly, intravital microscopy showed that 100% (10/10) of the Adamts13−/−/ApoE−/− mice had plaque that occluded the carotid sinus by approximately 70–80%, whereas only 20% (2/10) of the ApoE−/− mice had plaque at the carotid sinus, and the plaques were smaller in size than those in Adamts13−/−/ApoE−/− mice (P=0.0003). Next, we determined the effects of ADAMTS13 deficiency on atherosclerotic plaque formation in the aorta and aortic sinus. We compared the extent of atherosclerosis in whole aortae stained with Oil Red O and en face lesion area measured by morphometry. Both Adamts13−/−/ApoE−/− male and female mice demonstrated significantly larger lesions in the descending aorta (P<0.01), arch of the aorta (P<0.001), and total aorta (P<0.0001) compared to ApoE−/− mice fed a high-fat Western diet. Next, we quantified the corss-sectional area of lesions in the aortic sinus using the VerHoeffs/Van Gieson method. We observed a two-fold increase in the mean lesion area in the aortic sinus of both male and female Adamts13−/−/ApoE−/− mice (P<0.01) compared to ApoE−/− mice. Macrophage content (% of total lesion area), as quantitated by immunohistochemistry, was significantly elevated in the aortic root lesions of Adamts13−/−/ApoE−/− mice compared to ApoE−/− mice, suggesting that exacerbated atherosclerosis was due to increased inflammation. Adamts13−/−/ApoE−/− mice fed a normal chow diet also demonstrated accelerated atherosclerotic plaque formation compared to ApoE−/− mice. Total cholesterol and triglyceride levels were similar in Adamts13−/−/ApoE−/− and ApoE−/− mice fed a high-fat Western diet or normal chow diet. Conclusions:These findings unravel a new functional role for the anti-thrombotic enzyme ADAMTS13 in reducing excessive inflammation and plaque formation during atherosclerosis. Disclosures:Lentz:Celgene: Ownership interest; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Inflammation, lipid metabolism dysfunction, and hypertension: Active research fields in atherosclerosis-related cardiovascular disease in China

Atherosclerosis-related cardiovascular disease is one of the leading causes of death in China [1]. With advances in our understanding of the molecular mechanisms of atherosclerosis vascular inflammation, lipid metabolism dysfunction, and hypertension are regarded as the main pathogenetic pathways of both early atherogenesis and advanced plaque rupture [2,3]. Currently, much attention is being paid to the control of these pathways, which offers the potential for development of novel therapeutic approaches in the treatment of cardiovascular disease in China.

Early arterial abnormalities in young adulthood

The conclusion of the paper by Koivistinen and coworkers on young adults, published on Atherosclerosis [1] is that pulse wave velocity (PWV), a vascular marker related to the elastic properties of the arterial wall reflects a different aspect of sublinical damage than brachial flow mediated dilation (FMD, an estimate of a functional disturbance of vasodilatory capacity of the vessel) or carotid intima media thickness (CIMT, a marker of early atherosclerotic plaques).

Evaluation of texture parameters for the quantitative description of multimodal nonlinear optical images from atherosclerotic rabbit arteries

The composition and structure of atherosclerotic lesions can be directly related to the risk they pose to the patient. Multimodal nonlinear optical (NLO) microscopy provides a powerful means to visualize the major extracellular components of the plaque that critically determine its structure. Textural features extracted from NLO images were investigated for their utility in providing quantitative descriptors of structural and compositional changes associated with plaque development. Ten texture parameters derived from the image histogram and gray level co-occurrence matrix were examined that highlight specific structural and compositional motifs that distinguish early and late stage plaques. Tonal–texture parameters could be linked to key histological features that characterize vulnerable plaque: the thickness and density of the fibrous cap, size of the atheroma, and the level of inflammation indicated through lipid deposition. Tonal and texture parameters from NLO images provide objective metrics that correspond to structural and biochemical changes that occur within the vessel wall in early and late stage atherosclerosis.

Serum cystatin C is associated with early stage coronary atherosclerotic plaque morphology on multidetector computed tomography

ObjectiveCystatin C, a novel marker of kidney function, has been reported to be a predictor of adverse cardiovascular outcomes in patients without established chronic kidney disease. However, the relationship between serum cystatin C concentrations and early stage coronary atherosclerotic plaque morphology among patients with preserved kidney function has not been fully evaluated. Methods and results405 outpatients with early coronary artery disease with estimated glomerular filtration rate (eGFR) ≥60ml/min/1.73m2 and <50% stenosis on 64-slice CT coronary angiography were enrolled. Subjects were categorized into quartiles by serum cystatin C (quartile I: ≤0.88mg/L – quartile IV: ≥1.16mg/L). Plaques in coronary segments were categorized as calcified or noncalcified. Multiple linear regression analysis revealed that lower eGFR, higher age, increasing numbers of noncalcified and calcified plaques, lower high-density lipoprotein cholesterol, and female gender were statistically significant predictors of increased cystatin C concentrations. The risk for presence of noncalcified plaques increased significantly with increasing quartiles of cystatin C. Compared with those in the lowest quartile, patients in each subsequent quartile were at steadily increased risk of having noncalcified plaque (quartile IV: OR 5.6; 95% CI 2.3–13.9, p-value <0.001). Both number of segments with calcified plaque and Agatston score were highly correlated with cystatin C concentrations (both p<0.001), but when adjusted for segments with noncalcified plaque and other risk factors, calcified plaque segments were no longer independently predictive. ConclusionHigher serum cystatin C concentrations were correlated with early stage coronary atherosclerotic plaques among patients without established chronic kidney dysfunction. Noncalcified plaques increased with serum cystatin C concentrations, an association independent of eGFR and other cardiovascular risk factors.

Associations of Framingham Risk Score Profile and Coronary Artery Calcification with Sleep Characteristics in Middle-aged Men and Women: Pittsburgh SleepSCORE Study

Short and less efficient sleep may be risk factors for atherosclerosis. Few studies have investigated the associations between sleep characteristics and early cardiovascular disease (CVD) risk. Evaluate the associations between coronary artery calcification (CAC) and Framingham risk score profile with sleep characteristics in middle-aged men and women with no history of diagnosed myocardial infarction, interventional cardiology procedures, stroke, diabetes, or sleep disorders. 224 participants enrolled in an epidemiological study of disparities in CVD risk were recruited for a 9-night assessment of sleep, with 2 nights of polysomnography (PSG) and 9 nights of actigraphy and sleep diaries. Of the 224 participants, 110 had high/moderate Framingham risk scores and 114 had low scores; 195 had computed tomography measures of CAC. Individuals who had any CAC or higher Framingham risk scores had elevated apnea/hypopnea index (AHI) values, independent of age, race, and gender. The AHI association with CAC was nonsignificant in analyses adjusting for body mass index (BMI). Those with higher Framingham risk score profiles had shorter PSG sleep duration and less percent stage 3-4 and delta power sleep. High blood pressure and left ventricular hypertrophy were related to AHI and sleep duration, independent of BMI. Neither sleep duration nor efficiency was associated with CAC. CAC was not associated with AHI, independent of BMI in a community-based sample of middle-aged men and women. Framingham risk score profiles were related to poor sleep. Sleep duration may not be related to early plaque burden in relatively healthy individuals.

Naturally Occurring Autoantibodies against β-Amyloid: Investigating Their Role in Transgenic Animal andIn VitroModels of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disorder primarily affecting regions of the brain responsible for higher cognitive functions. Immunization against β-amyloid (Aβ) in animal models of AD has been shown to be effective on the molecular level but also on the behavioral level. Recently, we reported naturally occurring autoantibodies against Aβ (NAbs-Aβ) being reduced in Alzheimer's disease patients. Here, we further investigated their physiological role: in epitope mapping studies, NAbs-Aβ recognized the mid-/C-terminal end of Aβ and preferentially bound to oligomers but failed to bind to monomers/fibrils. NAbs-Aβ were able to interfere with Aβ peptide toxicity, but NAbs-Aβ did not readily clear senile plaques although early fleecy-like plaques were reduced. Administration of NAbs-Aβ in transgenic mice improved the object location memory significantly, almost reaching performance levels of wild-type control mice. These findings suggest a novel physiological mechanism involving NAbs-Aβ to dispose of proteins or peptides that are prone to forming toxic aggregates.

Bone marrow-derived smooth muscle-like cells are infrequent in advanced primary atherosclerotic plaques but promote atherosclerosis.

Although vascular smooth muscle cells (VSMCs) provide the major structural integrity of atherosclerotic plaques, their origin has been questioned. In particular, although some studies identified plaque VSMCs originating from bone marrow or peripheral blood, their frequency is controversial and their function unknown. We used genetic tracking of cell fate through smooth muscle cell (SMC)-specific LacZ reporter activity and VSMC-selective apoptosis to investigate the frequency, distribution, and role of marrow-derived VSMCs in atherogenesis. Cultured mouse bone marrow-derived smooth muscle-like cells expressed SMC markers and functional SMC promoter-driven transgenes over time. Transplantation of apolipoprotein E (ApoE)(-/-) mice with smooth muscle myosin heavy chain-Cre/ROSA26R/ApoE(-/-) marrow showed that 0.7±0.14% cells expressed LacZ in atherosclerotic plaques, located superficially in early plaques, and in necrotic cores but not fibrous caps of advanced lesions. Cells expressing both progenitor and SMC markers showed a similar distribution and frequency. Apoptosis of marrow-derived SMC-like cells transplanted from SM22α-human diphtheria toxin receptor/ApoE(-/-) mice retarded atherogenesis, with reduced plaque macrophage content. Cultured marrow-derived SMC-like cells secreted proinflammatory cytokines and promoted macrophage migration, VSMC proliferation, and collagen synthesis. Bone marrow-derived SMC-like cells are infrequent in advanced primary atherosclerotic plaques and absent in fibrous caps. However, these cells secrete proinflammatory cytokines and mitogens and promote atherosclerosis.