Fungal rDNA signatures in coronary atherosclerotic plaques

Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology.     

Apoptosis in human atherosclerotic plaques

Intimal cell death has been a recognized feature of advanced atherosclerotic disease. With the advent of DNA in situ end labelling and/or ultrastructural techniques, recent findings suggest that cells of an atheroma undergo programmed cell death or apoptosis. The pathophysiologic relevance of apoptosis in atherosclerotic disease is debatable. Apoptotic cell death may influence lesion progression and thus reduce overall plaque burden. Alternatively, apoptosis may prove a means of quenching the inflammation, converting cellular-rich lesions to so-called stable fibrous hypocellular plaques or conversely weaken the fibrous cap causing plaque rupture, a major cause of acute coronary syndromes. Apoptotic cells within plaques are typically macrophages, smooth muscle cells and T-cells and the frequency of death varies in the different regions of the lesion. The precise signalling pathways of apoptosis in plaques are unknown. There is however, some evidence that production of immune cytokines may promote apoptosis through activation of the Fas ligand-mediated death pathway. Genetic signals that regulate apoptosis in the atheroma, at least in smooth muscle cells, may involve the tumour suppressor genes p105 ^RB and p53. Further studies as to the relevance of apoptosis in acute coronary syndromes and potential mechanisms are emerging.     

High frequency of cytokeratin-producing smooth muscle cells in human atherosclerotic plaques

Using immunofluorescence microscopy we show that cells expressing cytokeratins 8 and 18 are frequently enriched in human vascular wall tissue pathologically altered by the appearance of intimal thickenings and atherosclerotic plaques. These cytokeratins occur in cells which also synthesize IFs containing vimentin and/or desmin, and a considerable proportion of the cytokeratin-positive cells has been identified as smooth muscle cells by colocalization of desmin and/or smooth muscle type alpha-actin. The presence of extremely low concentrations of these cytokeratins in such vascular tissues has been confirmed by gel electrophoresis with immunoblotting as well as by Northern blot hybridization using specific cytokeratin cRNA probes. The results are discussed in relation to the recent demonstration that low-level synthesis of cytokeratins 8 and 18 occurs in other muscular tissues and to the specific proliferative activity of these cells.     

Electrochemical impedance spectroscopy to characterize inflammatory atherosclerotic plaques

Despite advances in diagnosis and therapy, atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in the Western world. Predicting metabolically active atherosclerotic lesions has remained an unmet clinical need. We hereby developed an electrochemical strategy to characterize the inflammatory states of high-risk atherosclerotic plaques. Using the concentric bipolar microelectrodes, we sought to demonstrate distinct Electrochemical Impedance Spectroscopic (EIS) measurements for unstable atherosclerotic plaques that harbored active lipids and inflammatory cells. Using equivalent circuits to simulate vessel impedance at the electrode–endoluminal tissue interface, we demonstrated specific electric elements to model working and counter electrode interfaces as well as the tissue impedance. Using explants of human coronary, carotid, and femoral arteries at various Stary stages of atherosclerotic lesions (n = 15), we performed endoluminal EIS measurements (n = 147) and validated with histology and immunohistochemistry. We computed the vascular tissue resistance using the equivalent circuit model and normalized the resistance to the lesion-free regions. Tissue resistance was significantly elevated in the oxLDL-rich thin-cap atheromas (1.57 ± 0.40, n = 14, p < 0.001) and fatty streaks (1.36 ± 0.28, n = 33, p < 0.001) as compared with lesion-free region (1.00 ± 0.18, n = 82) or oxLDL-absent fibrous atheromas (0.86 ± 0.30, n = 12). Tissue resistance was also elevated in the calcified core of fibrous atheroma (2.37 ± 0.60, n = 6, p < 0.001). Despite presence of fibrous structures, tissue resistance between ox-LDL-absent fibroatheroma and the lesion-free regions was statistically insignificant (0.86 ± 0.30, n = 12, p > 0.05). Hence, we demonstrate that the application of EIS strategy was sensitive to detect fibrous cap oxLDL-rich lesions and specific to distinguish oxLDL-absent fibroatheroma.     

Embryonic stem cells markers are present within rabbit atherosclerotic plaques

Recent evidence suggests that smooth muscle cells within atherosclerotic plaques originate from vascular progenitor cells. We have previously shown that smooth muscle cells and macrophages present within rabbit atherosclerotic plaques are positive for factors of the renin angiotensin and nitric oxide systems as well as the hematopoietic stem-cell marker CD34 and the pan-leukocyte marker CD45. To explore the idea that these cells are of primitive types, immunohistochemistry was used to identify pluripotent embryonic stem cells (ESC) markers (Oct-4, SSEA1,3,4, TRA1-60, 81) in these plaques and to compare these to intimal thickening. Objective: To immunolocalise ESC markers in rabbit aortic intimal thickening and atherosclerotic plaques. Design: New Zealand White rabbits were fed either a control (Con) diet, 0.5% cholesterol (Chol) or 1% methionine (Meth) for 12 weeks. Animals were perfusion fixed, aortae excised and processed for paraffin. Immunohistochemistry was performed by standard techniques. Results: Oct-4, SSEA 1, 3 and 4, TRA-1-60 and TRA-1-81 were all present within in atherosclerotic plaques. However, some cells were not positive for TRA-1-60 and TRA-1-81. In fact, positive TRA-1-81 macrophages were uncommon, and positive TRA-1-81 smooth muscle cells were rare. Intimal thickening in Meth did not show any TRA-1-81 positive cells Conclusions: Macrophages and smooth muscle cells within atherosclerotic plaques express markers of ESC. These results suggest that cells within these plaques are primitive and might differentiate into other types of cells.     

Cytogenetic analysis reveals clonal proliferation of smooth muscle cells in atherosclerotic plaques

Summary Cytogenetic analysis of primary cell cultures from human atherosclerotic fibrous plaques revealed clonal chromosome abnormalities in 13 of the 18 cases studied. Loss of the Y chromosome and del(13)(q14) were present as single clonal abnormalities in eight cases; in five cases separate clones were found involving loss of the Y and a XXY karyotype, trisomy 10 and 18, loss of the Y and trisomy 7. A variety of single numerical and structural abnormalities were present in all but two of the 18 cases. Immunocytochemical studies were performed on cells from the same cultures used for cytogenetic analysis using monoclonal antibodies to human leucocyte common antigen, to human vimentin and to muscle actin. The immunoreactivity was positive for actin in 70–80% of the cells; 100% of the cells were positive for vimentin and all cells were ALC negative. These results indicated that the chromosomal abnormalities are present in the smooth muscle cells of the plaque. The hypothesis is proposed that the proliferation leading to the atherosclerotic lesion may primarily represent a hyperplastic response to mechanical and biological injuries and that this reactive proliferation is, in turn, associated with a tendency to chromosome instability.     

Participation of smooth muscle cells in the formation of atherosclerotic plaques]

A study of the participation of the smooth muscle cells in the formation of atherosclerotic lesions was made on the autopsy material with the use of specific antiserum to the smooth muscle actomyosin and of indirect Coons' method. Typical forms of atherosclerotic lesions in the aorta, cerebral vessels and coronary arteries were studied. Smooth muscle cells were detected in the thickened intima alongside the atherosclerotic lesions, in fatty streaks, in the fibrous tissue of the atherosclerotic plaque, but they were not found in the atheromatous masses. The proliferation and migration of the smooth muscle cells is regarded as an essential factor in the pathogenetic mechanisms of atherosclersis.     

Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding

During the last decade not only multicolor fluorescence in situ hybridization (FISH) using whole chromosome paints as probes, but also numerous chromosome banding techniques based on FISH have been developed for the human and for the murine genome. This review focuses on such FISH-banding techniques, which were recently defined as 'any kind of FISH technique, which provide the possibility to characterize simultaneously several chromosomal subregions smaller than a chromosome arm. FISH-banding methods fitting that definition may have quite different characteristics, but share the ability to produce a DNA-specific chromosomal banding'. While the standard chromosome banding techniques like GTG lead to a protein-related black and white banding pattern, FISH-banding techniques are DNA-specific, more colorful and, thus, more informative. For some, even high-resolution FISH-banding techniques the development is complete and they can be used for whole genome hybridizations in one step. Other FISH-banding methods are only available for selected chromosomes and/or are still under development. FISH-banding methods have successfully been applied in research in evolution- and radiation-biology, as well as in studies on the nuclear architecture. Moreover, their suitability for diagnostic purposes has been proven in prenatal, postnatal and tumor cytogenetics, indicating that they are an important tool with the potential to partly replace the conventional banding techniques in the future.     

荧光定量PCR 法检测肿瘤细胞端粒酶活性的变化

目的：利用荧光定量PCR法检测端粒酶抑制剂作用于人肝癌细胞SMMC-7721后端粒酶活性的变化,探讨其抑制端粒酶活性的可能机制,为端粒酶抑制剂的临床应用提供理论依据。方法：利用荧光染料SYBR—Green I建立一种新的端粒酶活性检测方法：FQ—TRAP法。利用FQ—TRAP法检测端粒酶抑制剂作用后肿瘤细胞端粒酶活性变化。结果：端粒酶抑制剂作用后,肝癌细胞端粒酶活性都有变化,其中以ASODN,EGCG,AZT抑制效果较明显。结论：端粒酶FQ—TRAP法是一种特异性、灵敏度、重复性都较好,可快速、简便及定量检测人端粒酶活性的方法,端粒酶抑制剂作用后癌细胞端粒酶活性的变化,为端粒酶抑制剂的临床应用提供理论依据。     

Sex-related differences in the association between plasma fibrinogen and non-calcified or mixed coronary atherosclerotic plaques

Background

Plasma fibrinogen (FIB) has been demonstrated to be a risk factor for cardiovascular disease. Patients with non-calcified plaque (NCP) or mix plaque (MP) have a higher risk of poor outcomes. However, the association between FIB and the presence of NCP or MP (NCP/MP) remains unclear, and if present, whether sex has any impact on this association remains unknown. The aim of this study was to investigate the role of FIB in predicting the presence of NCP/MP and evaluate whether sex has any impact on this association.

Methods

A total of 329 subjects were recruited, and the clinical and laboratory data were collected. Plasma FIB was detected by enzyme-linked immunosorbent assay. According to whether they had coronary atherosclerotic plaques and the characteristics of the most stenotic plaque, we divided them into three groups: no plaque (NP), calcified plaque (CP), and NCP/MP.

Results

Patients with NCP/MP had significantly higher FIB level in females, but not in males. Multiple logistic regression analysis showed that FIB was an independent risk factor for the presence of NCP/MP (odds ratio [OR]?=?3.677, 95% CI 1.539–8.785, P?=?0.003) in females. Receiver operating characteristic (ROC) curve analysis showed that the optimal cut-off value FIB for predicting the presence of NCP/MP was 3.41?g/L (area under curve [AUC]?=?0.73, 95% CI 0.63–0.82, P?<? 0.001) in females.

Conclusions

FIB is independently associated with the presence of NCP/MP in females, but not in males. These results suggest that the potential significance of FIB-lowering regimens in females with NCP/MP.

     

Phenotype commitment in vascular smooth muscle cells derived from coronary atherosclerotic plaques: differential gene expression of endothelial nitric oxide synthase

Unstable angina and myocardial infarction are the clinical manifestations of the abrupt thrombotic occlusion of an epicardial coronary artery as a result of spontaneous atherosclerotic plaque rupture or fissuring, and the exposure of highly thrombogenic material to blood. It has been demonstrated that the proliferation of vascular smooth muscle cells (VSMCs) and impaired bioavailabilty of nitric oxide (NO) are among the most important mechanisms involved in the progression of atherosclerosis. It has also been suggested that a NO imbalance in coronary arteries may be involved in myocardial ischemia as a result of vasomotor dysfunction triggering plaque rupture and the thrombotic response. We used 5' nuclease assays (TaqMan PCRs) to study gene expression in coronary plaques collected by means of therapeutic directional coronary atherectomy from 15 patients with stable angina (SA) and 15 with acute coronary syndromes (ACS) without ST elevation. Total RNA was extracted from the 30 plaques and the cDNA was amplified in order to determine endothelial nitric oxide synthase (eNOS) gene expression. Analysis of the results showed that the expression of eNOS was significantly higher (p<0.001) in the plaques from the ACS patients. Furthermore, isolated VSMCs from ACS and SA plaques confirmed the above pattern even after 25 plating passages. In situ RT-PCR was also carried out to co-localize the eNOS messengers and the VSMC phenotype. The eNOS gene was more expressed in ACS plaques and VSMCs cultured from them, thus indicating that: a) the expression of the most important differentiation markers is retained under in vitro conditions; and b) NO may play a pivotal role in coronary artery disease. Our findings suggest a new cell system model for studying the pathophysiology of unstable angina and myocardial infarction.     

Human smooth muscle cells cultured from atherosclerotic plaques and uninvolved vessel wall

Summary Smooth muscle cells (SMC) were cultured from atherosclerotic plaques and uninvolved arteries to determine if differences exist between growth characteristics or ultrastructure of the cultured cells. Eighteen aortic punch biopsies provided the uninvolved tissue, and 58 carotid plaques provided the atherosclerotic tissue. Eighty percent of the sample yielded viable cultured cells, which reached a maximum population doubling time during log phase growth of 72 h (seeding density=1.0×10⁴ cells/cm², 2nd passage). Growth characteristics of both normal and plaque-derived cells were the same in vitro. Growth rate declined with time in culture, and cell division ceased by the 5th or 6th passage. In culture, spindle shaped cells formed the “hill and valley” configuration typical of SMC. Plaquederived SMC were ultrastructurally similar to SMC from uninvolved vessel wall. Proliferative potential did not vary with age of sex, with method of culture, or with whether the cells were plaque derived or not. This study was supported in part by National Institutes of Health Grant HL-17269     

Proteomic analysis of human vessels: application to atherosclerotic plaques

Atherosclerosis is a chronic disease that affects medium and large arteries. This process originates from the interaction between cells of the arterial wall, lipoproteins and inflammatory cells, leading to the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in acute clinical complications such as myocardial infarction and stroke. Owing to the heterogeneous cellular composition of the plaques, a proteomic analysis of the whole lesion is not appropriate. Therefore, we have studied the proteins secreted by human carotid atherosclerotic plaques, obtained by endarterectomy. Normal artery segments and different regions of the surgical pieces (noncomplicated plaque, complicated plaque with thrombus) were cultured in protein-free medium and the secreted proteins (supernatants) analyzed by two-dimensional gel electrophoresis. Normal artery segments secreted a moderate number of proteins (42 spots). However in the two-dimensional (2-D) gels (pH 3-10) of segments bearing a plaque, the number of spots increased markedly (154). The number of spots also increased (202) in the 2-D gels of artery segments with a ruptured plaque and thrombus. Thus, the more complicated the lesion, the higher the number of secreted proteins, suggesting the production of specific proteins relating to the complexity of the atherosclerotic lesion.     

Detection of nanobacteria-like particles in human atherosclerotic plaques

Recent and historical evidence is consistent with the view that atherosclerosis is an infectious disease or microbial toxicosis impacted by genetics and behavior. Because small bacterial-like particles, also known as nanobacteria have been detected in kidney stones, kidney and liver cyst fluids, and can form a calcium apatite coat we posited that this agent is present in calcified human atherosclerotic plaques. Carotid and aortic atherosclerotic plaques and blood samples collected at autopsy were examined for nanobacteria-like structures by light microscopy (hematoxylin-eosin and a calcium-specific von Kossa staining), immuno-gold labeling for transmission electron microscopy (TEM) for specific nanobacterial antigens, and propagation from homogenized, filtered specimens in culture medium. Nanobacterial antigens were identified in situ by immuno-TEM in 9 of 14 plaque specimens, but none of the normal carotid or aortic tissue (5 specimens). Nanobacteria-like particles were propagated from 26 of 42 sclerotic aorta and carotid samples and were confirmed by dot immunoblot, light microscopy and TEM. [3H]L-aspartic acid was incorporated into high molecular weight compounds of demineralized particles. PCR amplification of 16S rDNA sequences from the particles was unsuccessful by traditional protocols. Identification of nanobacteria-like particles at the lesion supports, but does not by itself prove the hypothesis that these agents contribute to the pathogenesis of atherosclerosis, especially vascular calcifications.     

Initial stress in biomechanical models of atherosclerotic plaques

Rupture of atherosclerotic plaques is the underlying cause for the majority of acute strokes and myocardial infarctions. Rupture of the plaque occurs when the stress in the plaque exceeds the strength of the material locally. Biomechanical stress analyses are commonly based on pressurized geometries, in most cases measured by in-vivo MRI. The geometry is therefore not stress-free. The aim of this study is to identify the effect of neglecting the initial stress state on the plaque stress distribution. Fifty 2D histological sections (7 patients, 9 diseased coronary artery segments), perfusion fixed at 100 mmHg, were segmented and finite element models were created. The Backward Incremental method was applied to determine the initial stress state and the zero-pressure state. Peak plaque and cap stresses were compared with and without initial stress. The effect of initial stress on the peak stress was related to the minimum cap thickness, maximum necrotic core thickness, and necrotic core angle. When accounting for initial stress, the general relations between geometrical features and peak cap stress remain intact. However, on a patient-specific basis, accounting for initial stress has a different effect on the absolute cap stress for each plaque. Incorporating initial stress may therefore improve the accuracy of future stress based rupture risk analyses for atherosclerotic plaques.     

Co-accumulation of dendritic cells and natural killer T cells within rupture-prone regions in human atherosclerotic plaques.

We previously reported that CD1d, a molecule responsible for the presentation of lipid antigens, is expressed in atherosclerotic lesions and that its expression is restricted to dendritic cells. Recent studies demonstrating that CD1d-restricted natural killer T (NKT) cells are involved in atherogenesis prompted the present study investigating whether NKT cells are present in human atherosclerotic lesions and, if so, whether there is an association between NKT cells and dendritic cells. We found that NKT cells do accumulate in rupture-prone shoulders of atherosclerotic plaques and observed direct contacts of dendritic cells with NKT cells in rupture-prone regions of plaque.     

Neutrophil infiltration and oxidant-production in human atherosclerotic carotid plaques

To clarify the clinical implications of neutrophils in vulnerable plaques we evaluated the function and activity of infiltrated neutrophils in an atherosclerotic plaque, focusing on oxidant production. A histopathological investigation was performed using carotid arterial samples obtained from seven patients. The atherosclerotic plaques were examined cytochemically for naphthol-ASD-chloroacetate esterase activity and oxidant-production, and immunohistochemically using N-formyl peptide receptor-like 1 (fPRL1)-, CD66b-, CD68- or p22phox-specific antibodies. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque was estimated using an activity index. Naphthol-ASD-chloroacetate esterase activity was found in cells located in the atherosclerotic plaque, indicating that the cells were neutrophils. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque decreased to approximately 60% of the intensity observed in the capillary vessels. Oxidant-production was also detected in the plaques, and both neutrophils and macrophages were observed at the corresponding oxidant-production sites. p22phox expression was also located in the same areas in which oxidant-production was observed in these plaques. We could not directly evaluate how much ROS generated from the infiltrated neutrophils contributed the plaque vulnerability followed by its rupture. However, the infiltrated neutrophils in the atherosclerotic plaques morphologically appeared activated and were actively generating oxidant, implying that neutrophils, together with macrophages, infiltrate into atherosclerotic plaques and contribute to plaque vulnerability.     

Calcifications in atherosclerotic plaques and impact on plaque biomechanics

The catastrophic mechanical rupture of an atherosclerotic plaque is the underlying cause of the majority of cardiovascular events. The infestation of vascular calcification in the plaques creates a mechanically complex tissue composite. Local stress concentrations and plaque tissue strength properties are the governing parameters required to predict plaque ruptures. Advanced imaging techniques have permitted insight into fundamental mechanisms driving the initiating inflammatory-driven vascular calcification of the diseased intima at the (sub-) micron scale and up to the macroscale. Clinical studies have potentiated the biomechanical relevance of calcification through the derivation of links between local plaque rupture and specific macrocalcification geometrical features. The clinical implications of the data presented in this review indicate that the combination of imaging, experimental testing, and computational modelling efforts are crucial to predict the rupture risk for atherosclerotic plaques. Specialised experimental tests and modelling efforts have further enhanced the knowledge base for calcified plaque tissue mechanical properties. However, capturing the temporal instability and rupture causality in the plaque fibrous caps remains elusive. Is it necessary to move our experimental efforts down in scale towards the fundamental (sub-) micron scales in order to interpret the true mechanical behaviour of calcified plaque tissue interactions that is presented on a macroscale in the clinic and to further optimally assess calcified plaques in the context of biomechanical modelling.     

Artery buckling affects the mechanical stress in atherosclerotic plaques

Background

Tortuous arteries are often seen in patients with hypertension and atherosclerosis. While the mechanical stress in atherosclerotic plaque under lumen pressure has been studied extensively, the mechanical stability of atherosclerotic arteries and subsequent effect on the plaque stress remain unknown. To this end, we investigated the buckling and post-buckling behavior of model stenotic coronary arteries with symmetric and asymmetric plaque.

Methods

Buckling analysis for a model coronary artery with symmetric and asymmetric plaque was conducted using finite element analysis based on the dimensions and nonlinear anisotropic materials properties reported in the literature.

Results

Artery with asymmetric plaque had lower critical buckling pressure compared to the artery with symmetric plaque and control artery. Buckling increased the peak stress in the plaque and led to the development of a high stress concentration in artery with asymmetric plaque. Stiffer calcified tissue and severe stenosis increased the critical buckling pressure of the artery with asymmetric plaque.

Conclusions

Arteries with atherosclerotic plaques are prone to mechanical buckling which leads to a high stress concentration in the plaques that can possibly make the plaques prone to rupture.

     

Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength

Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.