Alterations of endothelin-converting enzyme expression in early and advanced stages of human coronary atherosclerosis

Endothelin-1 is a potent vasoconstrictor and exhibits a mitogenic activity on vascular smooth muscle cells (SMCs). Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin-1 processing. We studied the immunolocalization of ECE in human coronary atherosclerotic lesions with different disease stages. Frozen sections of normal coronary arteries with diffuse intimal thickening (n=13) and those of coronary arteries with early (n=10) or advanced atherosclerotic plaques (n=13) were studied. Monoclonal antibodies used were directed against SMCs, macrophages, endothelial cells, and ECE. For the identification of cell types that express ECE, double immunostaining analysis was also used. In normal coronary arteries, ECE immunoreactivity was observed in luminal endothelial cells and medial SMCs. Early atherosclerotic plaques, which consisted predominantly of SMCs, showed enhanced ECE expression in luminal endothelial cells and intimal SMCs. In advanced atherosclerotic plaques, distinct ECE expression was found in accumulated macrophages and in endothelial cells of intraplaque microvessels, while luminal endothelial cells showed relatively weak immunoreactivity for ECE. In conclusion, the present study demonstrates that the major cell types expressing ECE within the plaques are different between early and advanced stages of human coronary atherosclerosis. Enhanced ECE expression and possible endothelin-1 generation may contribute to SMC proliferation and vasoconstriction in early atherosclerotic stages, and may promote plaque destabilization in advanced atherosclerotic stages.     

MicroRNA-21 is a unique signature associated with coronary plaque instability in humans by regulating matrix metalloproteinase-9 via reversion-inducing cysteine-rich protein with Kazal motifs

BackgroundCoronary atherosclerotic unstable plaque is one of the leading causes of cardiovascular death. Macrophage-derived matrix metalloproteinase (MMP) 9 is considered for degrading extracellular matrix and collagen, thereby thinning the fibrous cap in plaques. miR-21 is implicated to play an important role in the progression of atherosclerosis. Nevertheless, miR-21 as the biomarker for coronary atherosclerotic unstable plaque remains unknown. We aimed to investigate the prediction role of miR-21 for unstable plaque by pathway study of miR-21 on MMPs and its inhibitor RECK in macrophages.MethodsExpression of miR-21 in macrophages and serum miR-21 as well as MMP-9 was measured in patients with coronary non-calcified plaque, calcified plaque and controls. In vitro experiment was done in human macrophages by over-expressing miR-21 or down-regulating RECK. The regulation of RECK and MMP-9 by miR-21 was evaluated by western blotting and siRNA strategy.ResultsPatients with non-calcified coronary artery lesions had significantly higher miR-21 in macrophages and lower miR-21 serum levels compared to the control and calcified plaque patients. At the same time, the serum levels of MMP-9 were significantly elevated in non-calcified patients. Experiments in vitro indicated that over-expressing miR-21 could induce the expression and secretion of pro-MMP-9 and active-MMP-9 in human macrophages via targeting gene RECK, and knocking down RECK expression by specific siRNA can resemble that of miR-21 over-expression.ConclusionsmiR-21 might be a biomarker for plaque instability by suppressing target gene RECK to promote the expression and secretion of MMP-9 in macrophages.     

Type I collagen gene expression in human atherosclerosis. Localization to specific plaque regions.

Because collagen is a major component of the human atherosclerotic plaque, factors controlling collagen synthesis may have a profound influence on the volume growth of these intimal lesions. In human arteries, we compared normal vs atherosclerotic media vs intimas for type I collagen gene expression using immunocytochemistry and in situ messenger RNA hybridization with subsequent correlations with plaque topographical features. We also determined the associations of such collagen gene expression with proximity to monocyte/macrophages and T lymphocytes. Type I collagen synthesis appears to be upregulated in atherosclerotic plaques compared with their underlying medias and normal internal mammary arteries and coronary diffuse intimal thickenings. At least in established and advanced coronary and carotid plaques, type I collagen gene expression is focal and especially prevalent in fibrous cap and vascularized regions. Although macrophages and type I procollagen messenger RNA and protein are both found in atherosclerotic plaques, no apparent spatial correlation between macrophage presence and type I procollagen presence was found within these atherosclerotic intimas. Type I procollagen presence appears to be negatively associated with the spatial presence of T cells. Thus, human atherosclerotic plaques exhibit nonuniform patterns of type I collagen gene expression. Although the biochemical determinants of this focal gene expression have yet to be determined, it is conceivable that stimulatory/inhibitory cytokines and other factors (eg hemodynamics) play important roles in determining the focal nature of collagen synthesis in atherosclerosis.     

IGF-1 has plaque-stabilizing effects in atherosclerosis by altering vascular smooth muscle cell phenotype

Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype.     

Dual effects of miR-155 on macrophages at different stages of atherosclerosis: LDL is the key?

Atherosclerosis is a chronic, maladaptive, nonresolving inflammatory response which underlies the leading cause of death in the world today. During the process, macrophages play a central role in both the initiation and development stages of disease pathogenesis. MicroRNAs are a class of small non-coding RNAs that regulate almost all biological processes. MiR-155 is multi-target molecule specifically expressed in atherosclerotic plaques and pro-inflammatory macrophages. However, the effects of miR-155 on atherogenesis have been controversial. Several lines of evidence collectively indicated that, both as inducers and carriers of miR-155, LDL and its oxidized derivatives could modulate miR-155-mediated inflammatory and apoptotic responses in lesional macrophages at different stages of atherosclerosis. During early lesion formation, both native and mildly-oxidized LDL facilitated endogenous miR-155-mediated macrophage activation and recruitment. In the meantime, they may also increase the accumulation of exogenous LDL-bound miR-155, along with lipid intake and foam cell formation. During advanced stages, the levels of exogenous miR-155 and extensively-oxidized LDL could gradually increase and become highly enough to synergistically induce macrophage apoptosis and atheroma formation. Taken together, we hypothesized that native LDL and oxidized LDL played a key role in modulating the effects of miR-155 on macrophages at different stages of atherosclerosis.     

Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis

The growth arrest-specific gene 6 (Gas6) plays a role in pro-atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE(-/-) mice, but alters its plaque composition. Compared to Gas6(+/+): ApoE(-/-) mice, initial and advanced plaques of Gas6(-/-): ApoE(-/-) mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFbeta was increased. In addition, fewer macrophages were found in advanced plaques of Gas6(-/-): ApoE(-/-) mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability.     

Proteomic analysis of differential protein expression in human atherosclerotic plaque progression

In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two-dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole-mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis identified vinexin-beta and alpha1-antitrypsin (AAT) in the same spot that was differentially expressed in plaques with a thrombus. Immunohistochemistry and western blotting showed limited expression of both vinexin-beta and AAT in early lesions, whereas high expression of both proteins was found in advanced lesions. Differential expression of vinexin-beta in lesions with a thrombus compared to stable plaques could not be confirmed, indicating the importance of validation of proteomic analysis. For AAT, western blotting of 2D gels revealed expression of six isoforms in advanced plaques, one of which was confirmed to be solely expressed in thrombus-containing plaques. In conclusion, vinexin-beta is expressed in advanced human atherosclerotic plaques, but differential expression of this protein in lesions with a thrombus versus stable plaques could not be confirmed. However, this analysis revealed expression of six isoforms of AAT in advanced plaques, one of which was uniquely expressed in thrombus-containing plaques.     

Micro-RNA Profiling of Exosomes from Marrow-Derived Mesenchymal Stromal Cells in Patients with Acute Myeloid Leukemia: Implications in Leukemogenesis

Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (n?=?3) and from healthy controls (n?=?3; not age-matched). Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified. Of the five candidate miRs identified by differential packaging in exosomes, only miR-26a-5p and miR-101-3p were significantly increased in AML-derived samples while miR-23b-5p, miR-339-3p and miR-425-5p were significantly decreased. Validation of the predicted change in gene expression of the potential targets was investigated by interrogating gene expression levels from public datasets of marrow-derived CD34-selected cells from patients with AML (n?=?69) and healthy donors (n?=?40). Two molecules with decreased gene expression in AML (EZH2 and GSK3β) were predicted by the miR profiling and have been previously implicated in AML while three molecules were increased in AML-derived cells and have not been previously associated with leukemogenesis (KRBA2, RRBP1 and HIST2H 2BE). In summary, profiling miRs in exosomes from AML-derived MSCs allowed us to identify candidate miRs with potential relevance in AML that could yield new insights regarding leukemogenesis or new treatment strategies.     

VAP-1, Eotaxin3 and MIG as potential atherosclerotic triggers of severe calcified and stenotic human aortic valves: effects of statins

Sclerotic calcification of the aortic valve is a common disease in advanced age. Its pathophysiology is unclear. However, pathobiological similarities to atherosclerosis have been shown in several studies. The current study assesses gene profiling of severe calcified stenotic human aortic valves identifying transforming growth factor (TGF)-beta, Eotaxin3, vascular adhesion protein-1 (VAP-1) and monokine induced by interferon-gamma (MIG) as potential atherosclerotic target genes in severe calcified and stenotic aortic valves, and analyzes the effects of statins on their expression as part of an anti-inflammatory treatment strategy. We collected human severe calcified and stenotic aortic valves with (CSAV+) or without (CSAV-) statin pre-treatment prior to valve replacement and investigated gene profiling by using micro-array technique and real-time PCR for the TGF-beta, Eotaxin3, VAP-1 and MIG expression. In comparison to atherosclerotic plaques of carotid arteries, immunohistochemical staining was investigated. Results were contrasted to human normal non-calcified aortic valves as controls (C). As compared to C, TGF-beta, Eotaxin3, MIG or VAP-1 was significantly upregulated in CSAV-. In CSAV+ no significant change in gene expression was found for Eotaxin3 and MIG. In contrast, VAP-1 and TGF-beta were still upregulated. Corresponding gene expression was confirmed on atherosclerotic plaque formations of carotid arteries. Monocyte/Macrophage infiltration (presence of CD68) on aortic valves (CSAV+, CSAV-, or C) confirmed inflammatory nature of the disease. Our data support further evidence for atherosclerotic inflammation as a trigger for sclerosis in end-stage calcified stenotic aortic valves by showing upregulation of gene expression for TGF-beta, VAP-1, MIG and Eotaxin3, which is only partially inhibited by previous statin therapy. Potent benefits of statin treatment on early stages of valve disease are still propagated.     

Accurate magnetic resonance imaging of atherosclerotic plaques: change future strategies for the diagnosis and therapy of atherosclerotic disease

In recent years, magnetic resonance imaging (MRI) have been developed to image atherosclerosis and is emerging as a useful tool to assess the burden of atherosclerosis, whereas the potential influence on the diagnosis and therapy of atherosclerotic disease have not been fully determined. MRI allows for three-dimensional evaluation of vascular structures and outstanding depiction of various components of the atherosclerotic plaque. The self-contained intravascular MRI probe appears to hold promise in the identification of high-risk coronary and peripheral atherosclerotic lesions. Molecular and targeted contrast MRI can offer exciting possibilities of direct visualization of biologic processes within atherosclerotic tissue. The addition of quantitative hydrogen 1 magnetic resonance spectroscopy and diffusion weighted imaging within atherosclerotic plaques can provide important data on the biological activity of potentially vulnerable lesions. Therefore, we hypothesized that accurate magnetic resonance imaging of atherosclerotic plaques maybe further affect and change future strategies for the diagnosis and therapy of atherosclerotic disease.     

Microvascular endoglin (CD105) expression correlates with tissue markers for atherosclerotic plaque vulnerability in an ageing population with multivessel coronary artery disease

Li X, van der Meer J J, van der Loos C M, Ploegmakers H J P, de Boer O J, de Winter R J & van der Wal A C
(2012) Histopathology  61, 88–97 Microvascular endoglin (CD105) expression correlates with tissue markers for atherosclerotic plaque vulnerability in an ageing population with multivessel coronary artery disease Aims: Vulnerable atherosclerotic plaques are lesions with a high propensity to develop plaque disruption and superimposed thrombosis. No systematic studies have been carried out on tissue markers for plaque vulnerability throughout the entire coronary artery system at the end stages of coronary atherosclerosis. Methods and results: Nine autopsied patients (mean age 77 years) with angiographically severe trivascular coronary atherosclerosis were selected for this study. All visible lesions in postmortem coronary angiograms (n = 125) were histologically and immunohistochemically screened for the presence of intraplaque haemorrhages (activated) microvessels and inflammatory infiltrates. Intraplaque haemorrhages were observed in 76/125 plaques (61%). Chronic inflammation was found superficially in 59/125 plaques (47%) and deeply inside the plaque tissue in 103/125 plaques (83%). Microvessels were found in 100/125 lesions (80%), of which 58% showed endothelial expression of the vascular activation marker CD105. Moreover, microvascular CD105 positivity correlated positively with plaque haemorrhage and deeply seated plaque inflammation. Conclusions: Plaque inflammation and haemorrhages can be found at a high frequency throughout the coronary artery system of elderly patients with multivessel coronary atherosclerosis. Microvascular expression of endoglin (CD105), which correlates positively with both of these features of plaque vulnerability, can serve as a marker of the risk of developing coronary thrombotic complications.     

MICA/B expression in macrophage foam cells infiltrating atherosclerotic plaques

Infiltrating macrophages accumulate in fatty streak lesions and transform into foam cells, leading to the formation of atherosclerotic plaques. Inflammatory mechanisms underlying the plaque formation mediated by NKG2D-positive lymphocytes such as CD8⁺ T cells, natural killer cells and natural killer T cells have been extensively investigated. Yet, the involvement of the NKG2D system itself remains poorly understood. Recent work in mouse models has shown that blockade of an NKG2D receptor–ligand interaction reduces plaque formation and suppresses inflammation in aortae. In this study, we conducted immunohistochemical analysis of NKG2D ligand expression in autopsy-derived aortic specimens. Foam cells expressing NKG2D ligands MICA/B were found in advanced atherosclerotic lesions accompanied by a large necrotic core or hemorrhage. Human monocyte-derived macrophages treated in vitro with acetylated low-density lipoproteins enhanced expression of MICA/B and scavenger receptor A, thus accounting for NKG2D ligand expression in foam cells infiltrating atherosclerotic plaques. Our results suggest that, as in mice, the NKG2D system might be involved in the development of atherosclerosis in humans.     

The expression of vascular dendritic cells in human atherosclerotic carotid plaques

Atherosclerosis is currently considered a chronic inflammatory disease, and evidence is accumulating for a role of the immune system in the progression of atherosclerosis. Dendritic cells are specialized antigen-presenting cells with the unique ability to initiate a primary immune response to certain antigens by the activation of naive T-lymphocytes. Although dendritic cells are well known to be important in the development of different diseases, studies of vascular dendritic cells in atherosclerosis are rare, and their role is not clearly understood. Therefore, we investigated the immunohistochemical expression of vascular dendritic cells in atherosclerotic plaques. Between April 2003 and December 2005, carotid endarterectomy was performed in 26 consecutive patients, and 27 carotid plaque specimens were analyzed. We investigated the immunohistochemical expression of vascular dendritic cells in human carotid plaques by measuring the signal intensity of fascin-positive cells using an image analyzer. In addition, these immunohistochemical results were related to clinical data. The highest signal intensity of dendritic cells was found in plaque shoulders, and the mean signal intensity of dendritic cells was significantly higher in complicated than in uncomplicated plaques (P = .0029). Moreover, the mean signal intensity of dendritic cells in plaques from symptomatic patients was significantly elevated compared with plaques from asymptomatic patients (P = .0004). Although atherosclerotic plaque instability is determined by multiple factors, the immune and inflammatory pathways play a particularly important role. Dendritic cells play a role in atherosclerosis, and the present study suggests that the expression of dendritic cells in human carotid arteries may be strongly associated with the occurrence of ischemic stroke.     

Biology of the smooth muscle cells in human atherosclerosis

The aim of this study was to reconstruct dynamic biological steps of human atherosclerosis at different ages of life and, in particular, to clarify the role of the smooth muscle cells (SMCs) by means of evaluation of several markers implicated in proliferative diseases (c-fos, proliferating cell nuclear antigen: PCNA, apoptosis, chromosome 7). We examined the biological features of 67 atherosclerotic arterial lesions obtained from fetuses, infants, young people and adults. From each case serial sections were stained for histological examination, PCNA, c-fos and apoptosis detection by immunohistochemical methods and for chromosome 7 number evaluation by fluorescence in situ hybridization. In coronary specimens of fetuses we observed SMCs with c-fos positivity. In infant lesions the predominant result was positivity for PCNA. Similar results were obtained from the plaques from young subjects with a greater presence of PCNA-positive cells. In adult subjects numerous apoptotic cells were present in the stable plaques, whereas in the unstable plaques we frequently detected joint positivity for both PCNA and c-fos gene and supernumerary chromosomes 7. During the evolution of the atherosclerotic process we observed a biological modulation of SMC proliferation, which begins after activation of the c-fos gene, increases during progression of the lesion and declines in stable plaques, when apoptosis increases. In unstable plaques, the same early steps observed in fetus and infant arteries occur. The observation in some cases of chromosome 7 alterations, markers of tumorigenesis, suggests the possible transformation of an advanced atherosclerotic plaque into a neoplastic-like process.     

兔动脉粥样硬化易损斑块模型的建立

目的利用血管内超声(IVUS)的影像学特点分析建立的兔模型的易损斑块。方法16只雄性新西兰纯种兔,通过球囊损伤腹主动脉和高脂(1%胆固醇)饲料喂养8周,建立动脉粥样硬化易损斑块的兔模型。应用IVUS分别测量腹主动脉同一血管段中多个病变部位及其参考部位的各项指标,然后给予中国斑点蝰蛇毒(CRVV)和组胺药物触发,造成实验性斑块破裂和血栓形成。结果破裂与未破裂斑块相比较,破裂斑块具有较大的偏心性(P<0.05),破裂斑块呈现明显的正性重构,而未破裂斑块主要表现为负性重构。结论IVUS应用于已建立的动脉粥样硬化易损斑块的动物模型上,能够准确地识别动脉粥样硬化易损斑块,为以后实验性大样本研究药物稳定易损斑块的治疗奠定了基础。     

Increased IL‐37 in Atherosclerotic Disease could be Suppressed by Atorvastatin Therapy

Recently, the evidence showed that interleukin‐37 (IL‐37) was expressed in the foam‐like cells of atherosclerotic coronary and carotid artery plaques in IL‐37‐transgenic mice, suggesting that interleukin‐37 is involved in atherosclerosis‐related diseases. The purpose of this study was to determine the change of IL‐37 in atherosclerotic plaque, the effect of atorvastatin on IL‐37 and the association between IL‐37 and Smad3 in atherosclerotic disease. Rabbits were subjected to atherosclerosis by the immunologic injury composite with balloon injury (BI). Some rabbits received atorvastatin treatment from 6 weeks to 12 weeks. Serum levels of IL‐37 were assessed at baseline, 6 weeks and 12 weeks in normal, atherosclerotic and atorvastatin groups. Protein and RNA levels of IL‐37 atherosclerotic plaque from abdominal aorta were processed at 12 weeks. Abdominal aorta including atherosclerotic plaque was immunostained with IL‐37 and Smad3. Serum IL‐37 significantly increased in atherosclerotic disease, and this increase could be reduced by the atorvastatin treatment. IL‐37 and Smad3 were accumulated in the macrophage‐derived foam cells in the plaque and significantly increased in protein and RNA levels. Atorvastatin treatment could significantly suppress the increase of both IL‐37 and Smad3. Plasma level of IL‐37 and the IL‐37 expression of the plaque were significantly increased in atherosclerotic disease. This increase could be suppressed by the atorvastatin treatment. In addition, Smad3 might be required for IL‐37 activity during the atherosclerotic physiologic process.     

Chemokine-like functions of MIF in atherosclerosis

The cytokine macrophage migration inhibitory factor (MIF) is a unique pro-inflammatory regulator of many acute and chronic inflammatory diseases. In the pathogenesis of atherosclerosis, chronic inflammation of the arterial wall characterized by chemokine-mediated influx of leukocytes plays a central role. The contribution of MIF to atherosclerotic vascular disease has come into focus of many studies in recent years. MIF is highly expressed in macrophages and endothelial cells of different types of atherosclerotic plaques, and functional studies established the contribution of MIF to lesion progression and plaque inflammation. This proatherogenic effect may partly be explained by the finding that MIF regulates inflammatory cell recruitment to lesion areas. Similar to chemokines, MIF induces integrin-dependent arrest and transmigration of monocytes and T cells. These chemokine-like functions are mediated through interaction of MIF with the chemokine receptors CXCR2 and CXCR4 as a non-canonical ligand. In atherogenic monocyte recruitment, MIF-induced monocyte adhesion involves CD74 and CXCR2, which form a signaling receptor complex. In addition to lesion progression, MIF has been implicated in plaque destabilization, since MIF is predominantly expressed in vulnerable plaques and can induce collagen-degrading matrix metalloproteinases. The latter could be a relevant mechanism in atherosclerotic abdominal aneurysm formation, where MIF expression is correlated with aneurysmal expansion. In summary, MIF has been identified as an important regulator of atherosclerotic vascular disease with exceptional chemokine-like functions. Detailed analysis of the interaction of MIF with its receptors could provide valuable information for drug development for the anti-inflammatory treatment of established and unstable atherosclerosis.     

Morphological and morphometric analysis of coronary atherosclerosis--an autopsy study

Coronary atherosclerosis is the most frequent cause of ischemic heart disease. The composition and vulnerability of the atherosclerotic plaque determines the development of acute coronary syndromes. In this study, 224 advanced atherosclerotic plaques were identified from the main coronary arterial branches of 10 autopsy heart specimens. The plaques were classified by American heart association (AHA) and modified American heart association classification. Vulnerability of the plaques and factors influencing vulnerability were assessed. Vulnerable plaques were mostly of Type IV category of AHA classification and of thin cap fibroatheroma type by modified American heart association classification. Inflammation was more frequent and was of a higher grade in vulnerable plaques. Calcification was predominantly of mild grade.