Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells.

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.     

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1.

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis.     

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.     

家鸽实验性主动脉粥样硬化的电镜和组化研究

应用扫描电镜以及非特异性酯酶的组织化学染色,观察喂胆固醇和猪油的家鸽的主动脉斑块的形态发生。结果表明,斑块处内皮细胞收缩、细胞间隙增宽、内皮细胞局部脱落和增生;单核细胞粘附增多;早期斑块主要由巨噬细胞源性泡沫细胞组成,典型斑块则由巨噬细胞源性和平滑肌源性两种泡沫细胞组成。本文对这些变化的意义进行了讨论。     

Comparative Analysis of the Expression Patterns of Various TNFSF/TNFRSF in Atherosclerotic Plaques

Members of the TNFSF/TNFRSF are involved in the immunoregulation of various immune reactions and diseases. Recently, LIGHT/TR2, GITRL/GITR, and TL1A/DR3 have been reported as playing roles in the inflammatory reactions in atherosclerosis, but a comparative analysis of these molecules has not been conducted. In order to compare their expression patterns, immunohistochemical analyses were performed using six human carotid endoarterectomy samples. The expression of these molecules was detected in the various cell types that constitute atherosclerotic plaques. The expression of all analyzed molecules was detected, albeit at various levels, mainly in foamy macrophages in all tested samples. The strong expression of these molecules in endothelial and smooth muscle cells was also detected in 2 and 1 plaque samples, respectively, while others express only some of the tested molecules. Flow cytometry analyses of human monocyte/macrophage cell lines, U937 and THP-1, detected the expression of the tested molecules while a relatively undifferentiated monocytic cell line, TF-1A, failed to express them. These data indicate that activated and differentiated macrophages are the main cell type expressing tested molecules in atherosclerotic plaques while endothelial and smooth muscle cells can express them in limited cases. Pro-inflammatory activities of the tested molecules may contribute to the atherogenesis by stimulating the cells expressing them in atherosclerotic plaques and the successful treatment of atherosclerosis may require cooperative regulation of these activities.     

Comparative analysis of the expression patterns of various TNFSF/TNFRSF in atherosclerotic plaques

Members of the TNFSF/TNFRSF are involved in the immunoregulation of various immune reactions and diseases. Recently, LIGHT/TR2, GITRL/GITR, and TL1A/DR3 have been reported as playing roles in the inflammatory reactions in atherosclerosis, but a comparative analysis of these molecules has not been conducted. In order to compare their expression patterns, immunohistochemical analyses were performed using six human carotid endoarterectomy samples. The expression of these molecules was detected in the various cell types that constitute atherosclerotic plaques. The expression of all analyzed molecules was detected, albeit at various levels, mainly in foamy macrophages in all tested samples. The strong expression of these molecules in endothelial and smooth muscle cells was also detected in 2 and 1 plaque samples, respectively, while others express only some of the tested molecules. Flow cytometry analyses of human monocyte/macrophage cell lines, U937 and THP-1, detected the expression of the tested molecules while a relatively undifferentiated monocytic cell line, TF-1A, failed to express them. These data indicate that activated and differentiated macrophages are the main cell type expressing tested molecules in atherosclerotic plaques while endothelial and smooth muscle cells can express them in limited cases. Pro-inflammatory activities of the tested molecules may contribute to the atherogenesis by stimulating the cells expressing them in atherosclerotic plaques and the successful treatment of atherosclerosis may require cooperative regulation of these activities.     

The distribution of adhesion molecules in human atherosclerosis

Chronic inflammatory cells are a recognized component of atherosclerotic plaques at all stages of development. As adhesion molecules play a fundamental role in inflammatory processes, we have carried out an immunohistochemical investigation of the distribution of endothelial leucocyte adhesion molecule-1 (ELAM-1)*, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human atherosclerotic lesions. Autopsy specimens from abdominal aorta and coronary arteries were obtained from 21 cases within 24 h of death. ELAM-1 and ICAM-1 were consistently expressed by the entire intimal endothelium of normal coronary arteries and also by the intimal endothelium overlying aortic fatty streaks. Both coronary artery and aortic lesions showed strong staining for ICAM-1 on and around macrophages. VCAM-1 was not detected on intimal endothelial cells, but strong staining of adventitial lymphoid aggregates for this molecule was seen. This work suggests a role for ELAM-1 and ICAM-1 in mononuclear cell recruitment during atherogenesis.     

Implication and mode of action of infectious agents in the formation of atheromatous plaques. Infection and atherosclerosis]

Different kinds of infectious agents seem to be implied in the atherosclerotic process. Indeed, some bacteria and viruses have been identified in atherosclerotic lesions: Chlamydia pneumoniae, Helicobacter pylori, Streptococcus sanguis, Porphyromonas gingivalis, herpes simplex viruses type 1 and 2, coxsakievirus B and hepatitis virus A. By their ability to induce antigenic and functional changes in the cells of the vascular wall (endothelial cells, smooth muscle cells, monocytes/macrophages, and fibroblasts) and to stimulate immune response as well as oxidative stress, it is more and more tempting to speculate that infectious agents probably play key roles in the initiation and in the formation of atherosclerotic plaques in certain patients.     

溶血磷脂酰胆碱对内皮细胞表面粘附分子表达的影响

大量的单核细胞募集是动脉粥样损伤形成的早期表现之一,相关的内皮细胞粘附分子在其中具有积极作用。本文研究了溶血磷脂酰胆碱（Ｌｙｓｏｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ,Ｌｙｓｏ－ＰＣ）对培养的人脐静脉内皮细胞（ＨＵＶＥＣｓ）膜上细胞间粘附分子－（Ｉｎｔｅｒｃｅｌｌｕｌａｒ ａｄｈｅｓｉｏｎ ｍｏｌｅｃｕｌｅ－１,ＩＣＡＭ－１）、Ｅ选择素（Ｅｎｄｏｔｈｅｌｉａｌ－ｌｅｕｋｏｃｙｔｅ ａｄｈｅｓｉｏｎ     

Progress in computer-generated three-dimensional reconstruction

The expression of PECAM, ICAM-1, VCAM-1, and E-selectin was studied in 64 samples of human coronary arteries taken from 15 explanted hearts obtained within 5 min of transplantation. Normal artery (n=12), predominantly fibrous plaques (n=23), and plaques containing extracellular lipid (n=26) and three segments showing recanalization channels were studied. All endothelial cells strongly and equally expressed PECAM; positive staining was used to check that artefactual denudation of the endothelial surface had not occurred. PECAM was also present in some lipid-filled macrophages. Normal arteries showed no VCAM-1 staining but focal segments of the endothelium were positive for ICAM-1 and E-selectin. ICAM-1 was strongly and constantly expressed by the endothelium over all types of plaques and in macrophages. E-selectin expression was confined to endothelial cells and occurred on the surface in 35 per cent of fibrous and 22 per cent of lipid-containing plaques. VCAM-1 staining of surface endothelium occurred in 39 per cent of fibrous and 20 per cent of lipid-containing plaques. A population of spindle-shaped cells of macrophage type (positive for EMB11 antigen) expressed VCAM-1 in lipid-containing plaques. Adventitial vessels adjacent to plaques showed endothelial expression of ICAM-1 and E-selectin. VCAM-1 staining of adventitial vessel endothelium was associated with local lymphoid aggregation. In conclusion, the expression of cell adhesion molecules is an important element in the inflammatory component of atherosclerosis and contributes to both monocyte and lymphocyte activation and recruitment from advential vessels and the arterial lumen.     

Expression of pentraxin 3 (PTX3) in human atherosclerotic lesions

Pentraxin 3 (PTX3) and C-reactive protein (CRP) are members of the pentraxin superfamily. PTX3 expression is induced in response to inflammatory signals, and is produced at sites of inflammation by several types of cell, primarily monocytes/macrophages, dendritic cells (DCs), endothelial cells, smooth muscle cells (SMCs), and fibroblasts, but is not produced by hepatocytes, which are a major source of CRP. The aim of our study was to investigate the expression pattern of PTX3 in human atherosclerotic lesions using a novel monoclonal antibody against PTX3. We examined coronary arterial thrombi containing an atherosclerotic plaque component removed from patients with acute myocardial infarction and human aortic tissues with various degrees of atherosclerosis sampled from autopsy cases. Immunohistochemical study of paraffin and frozen sections indicated that macrophages, mainly foam cells, expressed PTX3 in advanced atherosclerotic lesions. Interestingly, we also clearly observed PTX3-positive neutrophils infiltrating into atherosclerotic plaques, suggesting that PTX3 derived from neutrophils as well as macrophages plays an important role in atherogenesis.     

Cannabinoid receptor and N-acyl phosphatidylethanolamine phospholipase D--evidence for altered expression in multiple sclerosis

Cannabinoids have been shown to have a beneficial effect in both animal models of multiple sclerosis (MS) and human disease, although the mechanisms of action are unclear. We examined expression of the major cannabinoid receptors [(CBRs) cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2)] and a key enzyme involved in synthesis of the endocannabinoid anandamide [N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] in autopsy brain samples from patients with MS. CB1 was expressed in neurons, injured axons, oligodendrocytes, macrophages/microglia, some astrocytes, endothelial cells, smooth muscle cells and pericytes. CB2 and NAPE-PLD were localized to cerebral endothelial cells, pericytes, smooth muscle cells, astrocytes and macrophages/microglia. NAPE-PLD immunoreactivity was also seen in neurons. Endothelial CB2 expression was greatest in chronic inactive plaques, and in areas was seen in segments of endothelium where the endothelial expression of adhesion molecules (VCAM-1 and ICAM-1) was focally undetectable, and was often expressed in areas of blood-brain barrier damage. Vascular density was increased in chronic active plaques and normal-appearing white matter compared with controls. These data support findings from animal models which suggest a role for the endocannabinoid system in the MS, particularly in the regulation of endothelial leukocyte adhesion and the cellular response to injury.     

Vascular oxidant stress and inflammation in hyperhomocysteinemia

Elevated plasma levels of homocysteine are a metabolic risk factor for atherosclerotic vascular disease, as shown in numerous clinical studies that linked elevated homocysteine levels to de novo and recurrent cardiovascular events. High levels of homocysteine promote oxidant stress in vascular cells and tissue because of the formation of reactive oxygen species (ROS), which have been strongly implicated in the development of atherosclerosis. In particular, ROS have been shown to cause endothelial injury, dysfunction, and activation. Elevated homocysteine stimulates proinflammatory pathways in vascular cells, resulting in leukocyte recruitment to the vessel wall, mediated by the expression of adhesion molecules on endothelial cells and circulating monocytes and neutrophils, in the infiltration of leukocytes into the arterial wall mediated by increased secretion of chemokines, and in the differentiation of monocytes into cholesterol-scavenging macrophages. Furthermore, it stimulates the proliferation of vascular smooth muscle cells followed by the production of extracellular matrix. Many of these events involve redox-sensitive signaling events, which are promoted by elevated homocysteine, and result in the formation of atherosclerotic lesions. In this article, we review current knowledge about the role of homocysteine on oxidant stress-mediated vascular inflammation during the development of atherosclerosis.     

Various cell types in human atherosclerotic lesions express ICAM-1. Further immunocytochemical and immunochemical studies employing monoclonal antibody 10F3.

The specificity of monoclonal antibody 10F3, generated to smooth muscle cells isolated from fetal human aorta, has been further explored in a series of biological, biochemical, and immunocytochemical studies. In the first assay, it was found that 10F3 could inhibit aggregation of phytohemagglutinin (PHA)-induced lymphocytes in a manner comparable to that of antibody RR1/1, an anti-intercellular adhesion molecule 1 (ICAM-1) monoclonal antibody. In immunoprecipitation experiments followed by one-dimensional gel electrophoresis, both 10F3 and RR1/1 immunoprecipitated 90 kd proteins, with results suggesting that the two antibodies recognized different epitopes of the same molecule. A series of immunocytochemical studies on human atherosclerotic lesions was performed; using single-labeling techniques, 10F3-positive cells were found in the vessel wall and in lesions of virtually all specimens of fatty streaks and fibrous plaques. Using double-labeling techniques, 10F3-positive macrophages and 10F3-positive smooth muscle cells were found; however, there were also a significant number of non-smooth muscle, nonmacrophage 10F3-positive cells. These studies demonstrate that 10F3 identifies ICAM-1, and that this protein is expressed on a variety of cell types in human atherosclerotic lesions. ICAM-1 may represent a developmentally regulated protein that is expressed in fetal but not adult mesenchymal cells, but can be re-expressed in pathologic processes such as atherosclerosis.     

Localized adhesion of monocytes to human atherosclerotic plaques demonstrated in vitro: implications for atherogenesis.

Blood-derived macrophages in the arterial intima are a characteristic feature of active atherosclerotic plaques. Adherent monocytes on the luminal surface and increased adhesion molecules on the endothelium have suggested that specific molecular mechanisms are involved in monocyte/macrophage traffic into the arterial wall. Adhesion of human monocytes and related cell lines was therefore studied in vitro to histological sections of human plaques. At 37 degrees C, these cells bound selectively to the plaques. Binding to the endothelium occurred and was also present extensively in the diseased intima. Inhibition studies showed that the endothelial and general intimal binding had largely similar molecular properties. Strong inhibition was produced by antibodies to the monocyte-specific adhesion molecule CD14, to beta2 integrins, and to ICAM-1. Likewise, a peptide containing the Arg-Gly-Asp sequence was strongly inhibitory, suggesting that binding of leukocyte integrins to arterial extracellular matrix was synergistic with cell-cell interactions. A P-selectin antibody was exceptional in giving selective inhibition of endothelial adhesion, which correlates with the specific endothelial localization of this adhesion molecule. These results show that monocytes adhere to atherosclerotic plaques through the focal activation of multiple arterial wall adhesion molecules, confirming the adhesion hypothesis. A positive feedback theory for the pathogenesis of atherosclerosis can be suggested, based on the ability of macrophages in the wall to activate the endothelium, induce adhesion molecules, and facilitate additional monocyte entry. The adhesion assay provides a means for the identification of adhesion inhibitors with therapeutic potential.     

Adhesion molecules on the endothelium and mononuclear cells in human atherosclerotic lesions.

Atherosclerotic lesions show features of a cell-mediated immune inflammatory process. From this viewpoint, the potential role of arterial endothelium in the recruitment of mononuclear cells (T lymphocytes and macrophages) was studied. The endothelium of diffuse intimal thickening (DIT) and atheromatous plaques (AP) in human coronary arteries and abdominal aortas was characterized for the expression of adhesion molecules ELAM-1, ICAM-1, and the major histocompatibility complex (MHC) class II antigens HLA-DR/DP. A marked increase in expression of ICAM-1 and ELAM-1, and to a lesser extent HLA-DR/DP was observed on endothelial cells that were adjacent to subendothelial infiltrates of T lymphocytes (CD3+, CD11a+, HLA-DR/DP+) and macrophages (CD14+, CD11a+, CD11c+, HLA-DR/DP+). This contrasted with a lower or absent expression of these activation markers at sites without prominent inflammatory cell infiltrates. These findings could be demonstrated in DIT as well as in AP. The observations suggest that cytokines produced by the subintimal infiltrates may activate the endothelium in a similar way as is observed in the microvasculature at sites of immune inflammation. The expression of these activation markers in the microvasculature is associated with enhanced leukocyte adhesion, permeability for macromolecules, and procoagulant activity, features known to occur also in early experimental atherosclerosis. The findings therefore support the concept that arterial endothelium plays an active role in the recruitment of mononuclear cells in atherosclerotic lesions.     

Differential expression of interleukin‐17 family cytokines in intact and complicated human atherosclerotic plaques

In addition to the classical TH1 and TH2 cytokines, members of the recently identified IL‐17 cytokine family play an important role in regulating cellular and humoral immune responses. At present nothing is known about the role of these cytokines in atherosclerosis. Expression of IL‐17A, ‐E and ‐F was investigated in atherosclerotic tissue by rtPCR and immunohistochemistry. IL‐17E and its receptor were further studied in cultured smooth muscle cells and endothelial cells, using rtPCR and western blot. rtPCR showed that IL‐17A, ‐E and ‐F were expressed in the majority of plaques under investigation. IL‐17A/F was expressed by mast cells in all stages of plaque development. IL‐17A/F⁺ neutrophils were always observed in complicated plaques, but hardly in intact lesions. IL‐17A/F⁺ Tcells (‘TH17’) were never observed. IL‐17E was expressed by smooth muscle cells and endothelial cells in both normal and atherosclerotic arteries, and in advanced plaques also extensively by mature B cells. Cultured smooth muscle cells and endothelial cells were found to express both IL‐17E and its functional receptor (IL‐17RB). The constitutive expression of IL‐17E by resident plaque cells, and the additional presence of IL‐17E⁺ B cells and IL‐17A/F⁺ neutrophils in advanced and complicated plaques indicates a complex contribution of IL‐17 family cytokines in human atherosclerosis, depending on the stage and activity of the disease. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Identification of macrophages and smooth muscle cells with monoclonal antibodies in the human atherosclerotic plaque

Summary Sections of human atherosclerotic plaques, obtained from 21 autopsy cases with various degrees of atherosclerosis, were stained with the indirect immunoperoxidase technique using specific monoclonal antibodies against macrophages and smooth muscle cells. Distinctive results were found in differing stages: Single blood monocytes were observed in diffuse intimal thickening and the foam cells seen in fatty streaks were mostly identified as mature tissue macrophages, while only very few blood monocytes were present. The spindle cells observed in fibroelastic plaques showed positive reactions to antibodies against desmin, which points to their derivation from smooth muscle cells, whereas only a few macrophage-derived foam cells were seen in these lesions. In the complicated lesions the majority of foam cells were macrophage-derived, but there was also a small number of foam cells positive to antibodies against desmin, suggesting a smooth muscle cell derivation. - Our results confirm that in human atherosclerotic plaques the majority of the foam cells are obviously macrophage-derived, which emphasizes the important role of macrophages in the morphogenesis of these lesions.Supported by Landesamt für Forschung Nordrhein-Westfalen     

切应力和溶血磷脂酰胆碱对内皮细胞表面黏附分子表达的影响

在体内 ,内皮细胞的功能不仅受化学因子的调节 ,而且还受力学因素的影响。为探讨流体切应力和溶血磷脂酰胆碱 ( L ysophosphatidylcholine,L yso- PC)的双重作用对培养的人脐静脉内皮细胞 ( Hum an um bilical veinendothelial cells,HU VECs)表面黏附分子 ICAM- 1、VCAM- 1、E- selectin表达的影响 ,采用流式细胞仪技术检测了L yso- PC( 3 0 μg/m l)和流体切应力 ( 2 .2 3、4.2 0 dyne/cm2 )的协同作用下内皮细胞黏附分子表达的变化。结果显示 :在受剪切作用之前 ,用 L yso- PC孵育激活内皮细胞 ,或预先剪切后再用 L yso- PC孵育 ,内皮细胞的 ICAM- 1和VCAM- 1表达与两种刺激同时作用相比 ,显著增加 ( P<0 .0 5 ) ;切应力或 L yso- PC的单独作用 ,以及两种刺激同时存在对 HU VEC的 E- selectin表达无显著影响。而在受剪切作用之前 ,用 L yso- PC孵育激活内皮细胞 ,或预先剪切后再用 L yso- PC孵育 ,内皮细胞的 E- selectin表达与两种刺激同时作用相比 ,显著增加 ( P<0 .0 5 )。结论认为 :即使在不利于细胞黏附的力学环境中 ,流体切应力与 L yso- PC的协同作用 ,也可能是在炎症部位单核细胞对内皮细胞募集增加的重要原因之一