Atherosclerotic Plaque Instability Research Articles

The IL-33/ST2 pathway, inflammation and atherosclerosis: Trigger and target?

The “inflammatory hypothesis” of atherosclerosis postulates that inflammatory cell signalling drives the formation, growth and ultimately the instability of atherosclerotic plaques, setting up the substrate for the thrombotic response that causes myocardial damage or infarction. The recent Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial has been hailed as the first demonstration, ex iuvantibus, of the inflammatory hypothesis. Indeed, interleukin (IL)-1β inhibition was found to reduce cardiovascular events in patients with previous myocardial infarction and raised high-sensitivity C-reactive protein, despite no effects on the lipid profile. These results prompt a dissection of inflammatory mechanisms of atherosclerosis in order to search for specific biomarkers with prognostic value and/or therapeutic targets.Under this respect, the IL-33/suppression of tumorigenesis 2 (ST2) pathway deserves consideration. Indeed, its elements are particularly expressed in the endothelium of arterial vessels, and the interaction between IL-33 and the ST2 receptor blunts the immune response characteristic of atherosclerosis. By contrast, soluble ST2 (sST2) acts as a decoy receptor for IL-33, thus blocking its protective effects. Despite a solid theoretical framework, no definite demonstration of an involvement of the IL-33/ST2 pathway in atherosclerosis has been provided. Therefore, further studies are warranted to verify if elements of the IL-33/ST2 pathway may be proposed as markers of plaque burden and predictors of future cardiovascular events, and to explore the potential clinical benefit of enhanced IL-33/ST2 signalling in atherosclerosis.

Abstract 125: Intensive Statin Therapy Negatively Modulates AdipoR Activity in the Monocyte-macrophage Lineage in vivo and in vitro

Introduction: Statins are widely used for primary and secondary prevention of cardiovascular disease and its complications through cholesterol lowering and anti-inflammatory effects. Adiponectin, an anti-inflammatory adipokine, acts via 2 receptors, AdipoR1 and AdipoR2, to exert protective effects on the vasculature. Recently, we demonstrated that AdipoR are highly expressed on macrophages and that decreased AdipoR2 activity is associated with atherosclerotic plaque instability. We aimed to investigate whether statins can modulate AdipoR activity in the monocyte-macrophage lineage. Methods: Blood monocytes were isolated from whole blood of patients (statin-naïve vs statin users) with severe carotid atherosclerosis by a Magnetic Cell-Sorting technique. AdipoR1 and AdipoR2 mRNA expression on circulating monocytes was determined using qRT-PCR. In vitro , THP-1 macrophages were treated for 24 or 72 hours with varying doses of atorvastatin or rosuvastatin (1μM, 10μM, 60μM) to determine the effect of statins on AdipoR activity (AMPK, PPAR-α) by qRT-PCR and Western Blot analyses. Macrophage cytokine secretion (IL-1β, IL-10, IL-6, TNF-α) was assessed by electrochemiluminescence. Results: AdipoR1 and AdipoR2 mRNA expression on circulating monocytes was significantly lower by 1.36- and 1.17-fold (P<0.05), respectively, in statin users (n=157) compared to statin-naïve patients (n=37). When analyzing by statin type and dose, only patients on high doses of atorvastatin (40-80 mg) or rosuvastatin (20-40 mg) had significantly lower AdipoR expression compared to statin-naïve patients (P<0.05). Statins effects on lowering AdipoR expression was only apparent in patients with unstable but not stable plaques. In vitro , higher doses and longer exposure of macrophages to atorvastatin or rosuvastatin resulted in a greater decrease in AdipoR mRNA expression and signalling through AMPK and PPAR-α, and greater secretion of pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α. Conclusion: In our study, intensive statin therapy resulted in negative modulation of AdipoR expression and function. Despite their known protective properties, statins may also elicit severe adverse effects, which are often associated with long-term treatment and high doses.

Abstract 605: Inhibition of the Key Glycolytic Enzyme PFKFB3 with Novel Compounds Suppresses Angiogenesis

Aim: Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of theglycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compoundswith a potential to efficiently modulate (inhibit) angiogenesis. Methods: Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. Moreover, gene expression of important markers related to angiogenesis were measured at mRNA levelby real-time PCR. Results: PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2suppressedcapillary tube formation, 3PO did not have any effect. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions: These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeuticapproach to promote plaque stability.

Abstract 435: Inhibition of the Key Glycolytic Enzyme PFKFB3 with Novel Compounds Suppresses Angiogenesis

Aim: Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of the glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compounds with a potential to efficiently modulate (inhibit) angiogenesis. Methods: Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. Moreover, gene expression of important markers related to angiogenesis were measured at mRNA level by real-time PCR. Results: PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2 suppressed capillary tube formation, 3PO did not have any effect. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions: These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeutic approach to promote plaque stability.

Abstract 338: Statin Therapy is Associated with Decreased Plaque Instability in Symptomatic Carotid Atherosclerotic Plaque: a Clinicopathological Analysis

Background: Statin therapy has been shown to deplete atherosclerotic plaque instability in human carotid arteries by various imaging modalities or in animal models by pathological analysis. However, whether statin therapy attenuates plaque instability in human carotid plaque has not been fully described in a pathological analysis. The aim of this study was to analyze pathologically the possible relationships between statin therapy and plaque stability in patients undergoing carotid endarterectomy (CEA). Methods: Among consecutive 79 patients with advanced carotid artery stenosis (>70%) between May 2015 and February 2017, 66 patients without statin therapy (group 1) and 13 patients with statin therapy (group 2) were analyzed. The specimens were stained with hematoxylin/eosin and elastica-Masson. Immunohistochemistry was performed, using an endothelial specific antibody to CD31, CD34 and PDGFRβ. Results: Plaques from group 2 had significantly less plaque rupture (P=0.009), lumen thrombus (P=0.009), inflammatory cells (P=0.008), intraplaque hemorrhage (P=0.030) and intraplaque microvessels (P<0.001), compared with those from group 1. Among stroke patients with infarct size >1.0 cm 3 in MRI, the mean infarct volume in group 2 (4.2 ± 2.5 cm 3 ) was significantly smaller than that in group 1 (8.2 ± 7.1 cm 3 , p = 0.031). There was no significant difference in the mean concentration of low-density lipoprotein cholesterol between group 1 and group 2 (121 ± 32 mg/dl vs. 105 ± 37 mg/dl, P=0.118). Conclusions: This clinicopathological analysis of carotid atherosclerotic plaque suggests that statin therapy is associated with decreased plaque instability, which might result in smaller infarct volume in stroke patients undergoing CEA. This plaque stability might be derived from pleiotropic effect beyond lipid lowering of statin therapy.

Lipid derivatives of arachidonic acid used as markers of atherosclerotic plaque instability: a pilot study.

To compare the results of computer estimation of atherosclerotic plaque with biochemical data and ascertain any relationship with the occurrence of stroke. The study involved 20 atherosclerotic plaques causing 70-99% stenosis of internal carotid arteries (ICA). Ultrasonographic examination (USG) images of plaques were analyzed using a computer program. A histogram was obtained for each plaque and a gray scale median (GSM) was determined for each histogram in order to measure the echogenicity of an examined plaque. Then the plaques, collected during endarterectomy, were examined with regard to the concentration of prostaglandins E2 (PGE2), thromboxane A2 (TXA2), and 8 - epi-prostaglandin F2α. This data was compared with GSM and the occurrence of stroke. The statistical analysis showed significant correlations between low GSM and the occurrence of strokes. Out of 10 plaques with GSM<35, 6 (60.0%) were associated with a stroke. In contrast, out of 10 plaques with GSM>35, only 1 (10.0%) had a stroke. In addition, there were significant differences in the plaque content of PGE 2, (P<0.05) and (TXA2, P<0.011) between groups. High levels of PGE2 and TXA2, correlated with the low GSM values, may be the features of unstable plaques and that may be associated with a risk for stroke.

Noncanonical NF-κB signaling in microvessels of atherosclerotic lesions is associated with inflammation, atheromatous plaque morphology and myocardial infarction

Background and aimsNeovascularization is associated with atherosclerotic plaque instability and increased chance of myocardial infarction (MI). Patients with chronic inflammatory diseases (CID) have increased risk of atherosclerosis, and evidence demonstrates that NF-κB inducing kinase (NIK)-mediated noncanonical NF-κB signaling in endothelial cells (EC) is linked to inflammation and angiogenesis. Here, we hypothesized NIK may also be activated in EC of atherosclerotic lesion microvessels. MethodsUsing cohorts of atherosclerotic lesions from coronary and carotid arteries, we quantified NIK expression in plaque microvessels and compared it to pathological markers, including inflammatory cell content, plaque characteristics and MI. Differences in gene transcripts were evaluated between stable and ruptured lesions. ResultsNIK+EC were present in both coronary and carotid lesions. In CID patients, plaques with stenosis >40% had an increased number of NIK+EC and higher content of immune cells (p < .05) as compared to controls. Immune cells per NIK+EC were also greater in CID patients (p < .05), with pronounced differences as stenosis increased. In unstable lesions, NIK+EC were elevated as were EC expressing CXCL12 (p < .05). NIK+EC were increased in lesions with lipid content >40% (p < .05) and more abundant in coronary artery lesions implicated in MI (p < .05). These vessels also associated with atheromatous rather than fibrous plaque morphology (p < .05). Transcriptomic profiling demonstrated components of noncanonical NF-κB pathway were also upregulated in ruptured plaques (p < .05). ConclusionsNIK+EC associate with chronic inflammation in advanced lesions and are linked to markers of local inflammation, lipid content, unstable plaque phenotype and development of MI. Therefore, targeting noncanonical NF-κB signaling may hold therapeutic potential for patients with atherosclerosis and cardiovascular disease.

Порівняння вмісту біомаркерів запалення у пацієнтів при стенозі сонних артерій та вивчення його змін залежно від прогресування атеросклерозу та виникнення атеросклеротичних ускладнень

Мета. Вивчити рівень гомоцистеїну та ліпопротеїн-асоційованої фосфоліпази А2 (Lp-PLA2) у пацієнтів при атеросклеротичному ураженні сонних артерій, оцінити його зв’язок з морфологічними змінами атеросклеротичної бляшки, ступенем стенозу та вираженістю симптомів захворювання.&#x0D; Матеріали і методи. Проаналізовані зміни біомаркерів у 130 пацієнтів за симптомного та асимптомного перебігу стенозу сонних артерій залежно від його ступеня та структури атеросклеротичної бляшки.&#x0D; Результати. Виявлене достовірне збільшення рівня Lp-PLА2 у пацієнтів за симптомного стенозу внутрішньої сонної артерії (ВСА) та за його асимптомного перебігу за наявності м’якої атеросклеротичної бляшки. Концентрація гомоцистеїну не залежала від структури атеросклеротичної бляшки.&#x0D; Висновки. При порівнянні вмісту гомоцистеїну та Lp-PLA2 у пацієнтів при атеросклеротичному ураженні сонних артерій доведено, що вміст Lp-PLA2 є більш інформативним діагностичним критерієм для виявлення пацієнтів з можливим виникненням гострого порушення кровообігу головного мозку. Підвищення рівня Lp-PLA2 у пацієнтів при симптомному стенозі ВСА є свідченням нестабільності атеросклеротичної бляшки.

Imaging of neoangiogenesis of internal carotid artery's atherosclerotic plaque by contrast-enhanced sonography and histological examination

Previously, atherosclerosis was considered a disease accompanied exclusively by lipids accumulation. At present time success of fundamental and experimental science confirmed that atherosclerotic process is also associated with neovascularization and prolonged inflammatory response at all stages of atherogenesis from initial manifestations to thrombotic complications. The cause of atherosclerotic plaque instability is neovascularization, which is accompanied by intra-plaque hemorrhage and damage. Complications of carotid arteries atherosclerosis are strokes and transient ischemic attacks. The use of a wide range of diagnostic and pathohistological techniques is required for assessing this pathology. The most promising diagnostic technique is Contrast Enhanced Ultrasonography (CEUS) which allows to assess neovascularization degree in atherosclerotic plaque through the injection of a contrast agents.

Role of microRNA in Development of Instability of Atherosclerotic Plaques.

MicroRNAs are small noncoding single-stranded RNAs that regulate gene expression. Today, we see an increasing number of studies highlighting the important role of microRNAs in the development and progression of cardiovascular diseases caused by atherosclerotic lesions of arteries. We review the available scientific data on association of the expression of these biomolecules with instability of atherosclerotic plaques in animal models and humans. We made special emphasis on miR-21, -100, -127, -133, -143/145, -221/222, and -494 because they were analyzed in more than one study. We discuss the possibility of microRNAs using in the diagnosis and therapy of atherosclerosis and its complications.

Attenuated Superoxide Dismutase 2 Activity Induces Atherosclerotic Plaque Instability During Aging in Hyperlipidemic Mice.

BackgroundAtherosclerosis progression during aging culminates in the development of vulnerable plaques, which may increase the risk of cardiovascular events. Increased generation and/or decreased scavenging of reactive oxygen species in the vascular wall are major contributors to atherogenesis. We previously showed that superoxide dismutase 2 deficiency increased vascular oxidative stress and reduced aortic compliance in aged wild‐type mice and that young Apoe −/−/Sod2 +/− had increased mitochondrial DNA damage and atherosclerosis versus young Apoe −/− mice. Here we investigated the effects of superoxide dismutase 2 deficiency on atherosclerosis progression and plaque morphology in middle‐aged Apoe −/− mice.Methods and ResultsCompared with Apoe −/−, middle‐aged Apoe −/−/Sod2 +/− mice had increased vascular wall reactive oxygen species (P<0.05) and higher atherosclerotic lesion area (P<0.001). The atherosclerotic plaques in middle‐aged Apoe −/−/Sod2 +/− mice had an increased necrotic core with higher inflammatory cell infiltration, a thinned fibrous cap with depleted smooth muscle content, and intraplaque hemorrhage. In addition, the plaque shoulder area had higher levels of calpain‐2, caspase‐3, and matrix metalloproteinase‐2 in intimal smooth muscle cells and depleted fibrous cap collagen. Targeting mitochondrial reactive oxygen species with MitoTEMPO attenuated features of atherosclerotic plaque vulnerability in middle‐aged Apoe −/−/Sod2 +/− mice by lowering expression of calpain‐2, caspase‐3, and matrix metalloproteinase‐2 and decreasing smooth muscle cell apoptosis and matrix degradation.ConclusionsEnhanced mitochondrial oxidative stress under hyperlipidemic conditions in aging induces plaque instability, in part by increasing smooth muscle cell apoptosis, necrotic core expansion, and matrix degradation. Targeting mitochondrial reactive oxygen species or its effectors may be a viable therapeutic strategy to prevent aging‐associated and oxidative stress–related atherosclerosis complications.

COMPARISON STUDY OF THE QUANTITATIVE AND SEMI-QUANTITATIVE ANALYSIS OF PATHOLOGICAL FEATURES OF CAROTID ATHEROSCLEROTIC PLAQUE INSTABILITY

Comparison study of the quantitative and semi-quantitative analysis of pathological features of carotid atherosclerotic plaque instability Huaien Zheng1, Karina Gasbarrino1, Chi Lai2, John Veinot2, Stella S. Daskalopoulou1; 1) Research Institute of the McGill University Health Centre, Montréal, Québec; 2) Department of Pathology and Laboratory Medicine, University of Ottawa, Ontario, Canada Atherosclerosis and its thrombotic complications are a major cause of morbidity and mortality worldwide. Plaque instability and rupture is an important causative factor of these complications. To estimate plaque instability level, Lovett et al has proposed a histological classification that ranges from definitely stable to definitely unstable plaques based on semi-quantitative assessment of 10 plaque pathological features. However, semi-quantitative analysis can be subject to variability. Thus, herein, we aimed to develop a method to quantitatively measure plaque pathological features and validate it with established semi-quantitative results. Carotid surgical specimens were obtained from patients who underwent carotid endarterectomy (CEA). The atherosclerotic specimen sections were taken from the site of maximum stenosis, and stained with hematoxylin & eosin, smooth muscle cell actin, CD68 (for macrophages), and CD3 (for lymphocytes). Plaque features and stability were assessed by two vascular pathologists following Lovett’s semi-quantitative classification. Whole plaque specimen sections were scanned digitally. Pathological characteristics listed in the semi-quantitative classification were quantitatively measured using ImagePro Primer software. The quantitative image analysis results were analyzed and compared with the semi-quantitative pathological scores. The quantitative results of major pathologic features are significantly different between stable and unstable plaques and they are in accordance with the semi-quantitative analysis, performed by the pathologists. The quantitative results show that fibrous tissue occupies a major part of stable plaques (35-45%), while the lipid core occupies a large part of unstable plaques (40-47%). Furthermore, inflammatory cell infiltration is much greater in unstable plaques (P < 0.01). On the other hand, plaque calcification, neovascularization, and hemorrhage have no obvious differences between stable and unstable plaques. Our results demonstrate that quantitative image analysis can provide new insight into plaque morphology and composition: 1) the amount of fibrosis, necrotic core, fibrous cap, and inflammatory cell infiltration are major decisive factors of plaque instability; 2) Calcification and neovascularization are not closely related with plaque instability. Furthermore, quantitative image analysis of plaque features is a promising method for accurate classification of plaque instability. This new quantitative method needs to be externally validated in a larger subset of CEA plaques for future clinical practice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Protease-Activated Receptor-2 Deficiency Attenuates Atherosclerotic Lesion Progression and Instability in Apolipoprotein E-Deficient Mice.

Inflammatory mechanisms are involved in the process of atherosclerotic plaque formation and rupture. Accumulating evidence suggests that protease-activated receptor (PAR)-2 contributes to the pathophysiology of chronic inflammation on the vasculature. To directly examine the role of PAR-2 in atherosclerosis, we generated apolipoprotein E/PAR-2 double-deficient mice. Mice were fed with high-fat diet for 12 weeks starting at ages of 6 weeks. PAR-2 deficiency attenuated atherosclerotic lesion progression with reduced total lesion area, reduced percentage of stenosis and reduced total necrotic core area. PAR-2 deficiency increased fibrous cap thickness and collagen content of plaque. Moreover, PAR-2 deficiency decreased smooth muscle cell content, macrophage accumulation, matrix metallopeptidase-9 expression and neovascularization in plaque. Relative quantitative PCR assay using thoracic aorta revealed that PAR-2 deficiency reduced mRNA expression of inflammatory molecules, such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. In vitro experiment, we found that PAR-2 deficiency reduced mRNA expression of interferon-γ, interleukin-6, TNF-α and MCP-1 in macrophage under unstimulated and lipopolysaccharide-stimulated conditions. These results suggest that PAR-2 deficiency attenuates the progression and instability of atherosclerotic plaque.

Metalloproteinases in atherosclerosis

Atherosclerosis underlies most cardiovascular diseases, and is accepted as a primary cause of mortality worldwide. Proteases have been implicated in the development and progression of atherosclerosis, due to their ability to provoke focal destruction of the vascular extracellular matrix. Members of the metalloproteinase family, especially matrix metalloproteinases (MMPs), and their endogenous tissue inhibitors (TIMPs) have been suggested to perform complex dual roles during late-stage progression and rupture of atherosclerotic plaques. Proposed favourable actions of metalloproteinases include the promotion of vascular smooth muscle growth and survival which stabilises plaques, while conversely extracellular matrix destruction alongside interminable monocyte/macrophage accumulation can encourage plaque rupture. This review provides a summary of the cogent evidence connecting the contribution of individual metalloproteinases to atherosclerotic plaque development, progression, and instability. Topics discussed include structural, functional and cell-specific diversity of MMP members; evidence from animal models of atherosclerosis and comparisons with findings in humans; the dual role of MMPs and the requirement to selectively target individual MMPs; and the need for efficient surrogate markers of MMP inhibition. Accordingly, as our knowledge of the complex roles individual MMPs play especially during the progression and rupture of atherosclerotic plaques expands, new impetus is required for clinical trials evaluating the therapeutic potential of selective MMP inhibition, which could limit cardiovascular morbidity and mortality worldwide.

Decreased cathepsin K levels in human atherosclerotic plaques are associated with plaque instability.

Investigating the determinants and dynamics of atherosclerotic plaque instability is a key area of current cardiovascular research. Extracellular matrix degradation from excessive proteolysis induced by enzymes such as cathepsin K (Cat K) is implicated in the pathogenesis of unstable plaques. The current study assessed the expression of Cat K in human unstable atherosclerotic plaques. Specimens of popliteal arteries with atherosclerotic plaques were classified as stable (<40% lipid core plaque area; n=6) or unstable (≥40% lipid core plaque area; n=14) based on histopathological examinations of hematoxylin and eosin stained sections. The expression of Cat K and cystatin C (Cys C) were assessed by immunohistochemical examination and levels of Cat K mRNA were detected by semi-quantitative reverse transcriptase polymerase chain reaction. Morphological changes including a larger lipid core, endothelial proliferation with foam cells and destruction of internal elastic lamina were observed in unstable atherosclerotic plaques. In unstable plaques, the expression of Cat K protein and mRNA was upregulated, whereas Cys C protein expression was downregulated. The interplay between Cat K and Cys C may underlie the progression of plaques from stable to unstable and the current study indicated that Cat K and Cys C are potential targets for preventing and treating vulnerable atherosclerotic plaque ruptures.

Impact of physical activity and sedentary behavior on biological risk factors of carotid atherosclerotic plaque instability

Impact of physical activity and sedentary behavior on biological risk factors of carotid atherosclerotic plaque instability

DNA repair measuring DNA ligase activity in non ST-elevation myocardial infarction determines atherosclerotic plaque instability

DNA repair measuring DNA ligase activity in non ST-elevation myocardial infarction determines atherosclerotic plaque instability

Novel approaches to assessing the signs of atherosclerotic plaque instability in the carotid arteries

Introduction. Application of contrast agents in vascular untrasonography has become a new direction in noninvasive assessment of signs of atherosclerotic plaque (ASP) instability; the type of plaque neovascularization being the major sign. However, questions regarding the accuracy of the methods for quantitative evaluation of plaque neovascularization are still to be answered. Objective. To evaluate signs of ASP instability in the carotid arteries according to the contrast-enhanced duplex scanning data and to elaborate our own approach to quantitative evaluation of neovascularization. Materials and methods. Twenty-six patients with carotid atherosclerosis who had been subjected to carotid endarterectomy (n=27) followed by morphological plaque verification were enrolled in this study. All patients underwent standard duplex scanning and scanning using contrast agent SonoVue. Results. Neovascularization was revealed in all 27 patients with ASP according to the pathomorphological and contrast-enhanced ultrasonography data. The total number of vessels per cm2 of plaque was 6–51 [21±14/cm2] according to the ultrasonography data and 19–1224 [236±249/cm2] according to the pathomorphological examination. According to the ultrasonography data, the absolute values were close to the density of plaque vessels ≥ 30 μm in diameter determined during pathomorphological examination and did not differ significantly from this value (p = 0.67). The morphology data show that vessels <20 μm in diameter, which constituted up to 96% of all microvessels in ASP, cannot be detected by ultrasonography. In one case, ulceration of the ASP surface was detected only by contrast medium injection. Calcified plaques with different degrees of calcination imposed the greatest difficulties when performing ultrasonic assessment of neovascularization. Conclusions. Contrast-enhanced untrasonography can be used as an informative method to noninvasively detect signs of ASP instability enabling rather accurate assessment of neovascularization at microvessel diameter ≥30 μm. Calcification of ASPs may significantly affect the study results.

Abstract 324: Pathological Quantitative Assessment of Plaque Instability in Patients Undergoing Carotid Endarterectomy

Introduction: Instability of carotid atherosclerotic plaques leads to cerebral thromboemboli and ischemic symptoms. However, there has been no specific pathological quantification for the instability of carotid atherosclerotic plaque. The purpose of this study was to quantify atherosclerotic plaque instability in patients undergoing carotid endarterectomy (CEA). Methods: Carotid plaques were collected after CEA from 67 symptomatic and 15 asymptomatic patients between May 2015 and August 2016. Samples were stained with hematoxylin/eosin and Elastica-Masson (E-Masson). Immunohistochemistry was performed by using an endothelial specific antibody to CD31, CD 34 and PDGFRβ. Plaques were assessed for histopathological characteristics. Results: Multivariable logistic regression analysis demonstrated that plaque instability was independently associated with the presence of plaque rupture (odds ratio [OR], 9.75, 95% confidence interval [CI]: 1.62 to 58.6, p = 0.013), the minimal fibrous cap thickness (FCT) (OR per 10 μm 0.70, 95% CI: 0.51 to 0.96, p = 0.025), the presence of microcalcification in the fibrous cap (OR 7.82, 95% CI: 1.35 to 45.4, p = 0.022) and the intraplaque microvessels (OR 1.91, 95% CI: 1.02 to 3.57, p = 0.043). If these four independent parameters were combined to a score using the equation derived from the multivariable logistic regression model (Logit(Score) = 0.179 + 2.277 * (insert 1 if plaque rupture present; else 0) - 0.355 * (insert minimal FCT in multiples of 10 μm) + 2.057 * (insert 1 if microcalcification in the fibrous cap present; else 0) + 0.646 * (insert intraplaque microvessels /mm 2 ), the diagnostic efficiency could be improved to an AUC 0.92 (95% CI: 0.85-0.99, optimal cut-off value 0.814, sensitivity 89.6%, specificity 86.7%, PPV 96.8%, NPV 65.0%, diagnostic accuracy 89.0%). Conclusions: This study suggested the diagnostic scoring was useful for the quantification of carotid plaque instability in patients undergoing CEA.

Lysophosphatidic Acid Is Associated with Atherosclerotic Plaque Instability by Regulating NF-κB Dependent Matrix Metalloproteinase-9 Expression via LPA2 in Macrophages.

Lysophosphatidic acid (LPA), one of the simplest phospholipid signaling molecules, participates in formation and disruption of atherosclerotic plaque. Matrix metalloproteinases (MMPs) contribute to atherosclerotic plaque rupture by involving in extracellular matrix (ECM) degradation and then thinning fibrous cap. Our previous study demonstrated that macrophage-derived MMP-9 was associated with coronary plaque instability, but the relationship between LPA and MMP-9 remains unclear. The present work therefore aimed at elucidating association between LPA and MMP-9 and the regulation mechanism of LPA on MMP-9 in macrophages. We found that plasma LPA and MMP-9 levels were correlated positively (r = 0.31, P < 0.05) and both elevated significantly in patients with acute myocardial infarct (AMI). Consistent with peripheral blood levels, histochemical staining indicated that autotaxin (ATX), LPA-producing ectoenzyme, and MMP-9 were expressed frequently in the necrotic core and fibrous cap of human unstable plaques, which might increase the instability of plaque. Experiments in vitro were done with THP-1-derived macrophages and showed that LPA enhanced the expression, secretion and activity of MMP-9 in a time- and dose-dependent manner. Induction of LPA on pro-MMP-9 and active-MMP-9 was confirmed in human peripheral blood monocyte-derived macrophages. PDTC, NF-κB inhibitor, but not inhibitor of AP-1 and PPARγ, effectively prevented LPA-induced MMP-9 expression and NF-κB p65 siRNA decreased MMP-9 transcription, confirming that LPA might induce MMP-9 elevation by activating NF-κB pathway. In addition, knockdown of LPA2 attenuated LPA-induced MMP-9 expression and nucleus p65 levels. These findings revealed that LPA upregulated the expression of MMP-9 through activating NF-κB pathway in the LPA2 dependent manner, hence blocking LPA receptors signaling may provide therapeutic strategy to target plaque destabilization.