Low molecular weight hyaluronan increases the Uptaking of oxidized LDL into monocytes

Since accumulation and interaction of immune cells including T cells and monocytes/macrophages are involved in the processes of atherosclerosis, atherosclerosis is currently understood as an inflammatory disorder. Entrapment of extracellular matrices components such as hyaluronan by monocytes and macrophages, as well as uptake of oxidized low-density lipoprotein (ox-LDL) by these cells, plays a central role in foam cell formation and the pathogenesis of atherosclerosis. We investigated the role of CD44, the principal receptor for hyaluronic acid, and ox-LDL in scavenger receptor expression on resting monocytes prepared by counterflow centrifugal elutriation from the endothelium. Our results showed that the low-molecular weight (6.9 kDa) form of hyaluronan increased the expression of CD36 scavenger receptor; the incorporation of (125) I-labeled ox-LDL, and the transendothelial migration of monocytes, which were mediated at least in part via tyrosine kinase and the PKC pathway. Our results imply that low molecular weight hyaluronan produced in large amounts in atherosclerotic lesions induces differentiation of circulating monocytes to macrophages/foam cells and enhances the progression of atherosclerosis via the PKC pathway. Furthermore, low molecular weight hyaluronan also amplifies the migration of monocytes into inflamed atherosclerotic plaques. Thus, we propose that engagement of CD44 with low molecular weight hyaluronan is centrally involved in the inflammatory pathogenesis of athelosclerotic plaques through migration of monocytes and foamed macrophage differentiation.     

Extracellular matrix metalloproteinase inducer (CD147) is a novel receptor on platelets, activates platelets, and augments nuclear factor kappaB-dependent inflammation in monocytes

In atherosclerosis, circulating platelets interact with endothelial cells and monocytes, leading to cell activation and enhanced recruitment of leukocytes into the vascular wall. The invasion of monocytes is accompanied by overexpression of matrix metalloproteinases (MMPs), which are thought to promote atherosclerosis and trigger plaque rupture. Following interaction with itself, the extracellular matrix metalloproteinase inducer (EMMPRIN) induces MMP synthesis via a little-known intracellular pathway. Recently, we showed upregulation of EMMPRIN on monocytes during acute myocardial infarction. EMMPRIN also stimulates secretion of MMP-9 by monocytes and of MMP-2 by smooth muscle cells, indicating that it may be an important regulator of MMP activity. Expression of EMMPRIN on platelets has not been described until now. Here, we demonstrate that resting platelets show low surface expression of EMMPRIN, which is upregulated by various platelet stimulators (flow cytometry). EMMPRIN is located in the open canalicular system and in alpha granules of platelets (according to electron microscopy and sucrose gradient ultracentrifugation). Platelet stimulation with recombinant EMMPRIN-Fc induced surface expression of CD40L and P-selectin (according to flow cytometry), suggesting that EMMPRIN-EMMPRIN interaction activates platelets. Coincubation of platelets with monocytes induced EMMPRIN-mediated nuclear factor kappaB activation (according to Western blot) in monocytes with increased MMP-9 (zymography), interleukin-6, and tumor necrosis factor-alpha secretion (according to ELISA) by monocytes. In conclusion, EMMPRIN displays a new platelet receptor that is upregulated on activated platelets. Binding of EMMPRIN to platelets fosters platelet degranulation. Platelet-monocyte interactions via EMMPRIN stimulate nuclear factor kappaB-driven inflammatory pathways in monocytes, such as MMP and cytokine induction. Thus, EMMPRIN may represent a novel target to diminish the burden of protease activity and inflammation in atherosclerosis.     

Effects of OX40–OX40 ligand interaction on the levels of ROS and Cyclophilin A in C57BL/6J mice atherogenesis

Background

An increasing amount of evidence shows that the OX40–OX40L interaction serves an important function in atherosclerosis. However, the mechanism of the OX40 signaling pathway remains unclear. This study investigates the effect of OX40–OX40L interaction on the levels of intracellular reactive oxygen species (ROS) and the secretion of Cyclophilin A (CyPA) in C57BL/6J mice atherogenesis.

Methods

The atherosclerotic plaque model was established by placing a rapid perivascular carotid collar on C57BL/6J mice fed with a western-type diet. In vivo, the expressions of CyPA in mouse plaque and lymphocytes were detected by immunohistochemical and Western blot analyses, respectively. In vitro, the expression of CyPA protein in cultured lymphocytes of C57BL/6J mice was assessed by using Western blot analysis. The level of ROS was detected through flow cytometry.

Results

CyPA expression was significantly increased in the atherosclerotic lesions and lymphocytes from C57BL/6J mice. The ROS levels in OX40⁺-lymphocytes were increased in vitro and in vivo. After stimulating the OX40–OX40L interaction, the ROS and CyPA levels in lymphocytes were obviously increased in vitro, whereas anti-OX40L mAb significantly down-regulated the anti-OX40 mAb-induced ROS generation and inhibited CyPA secretion in lymphocytes.

Conclusion

The OX40–OX40L interaction up-regulates intracellular levels of ROS in C57BL/6J mice and increases CyPA secretion in lymphocytes. Increased CyPA secretion may serve an important function in atherosclerotic plaque formation.     

铁离子螯合剂去铁胺对巨噬细胞、泡沫细胞细胞外基质金属蛋白酶诱导因子表达的影响

目的:观察铁负荷过低对巨噬细胞、泡沫细胞细胞外基质金属蛋白酶诱导因子(EMMPRIN)表达的影响。方法: 体外诱导THP-1单核细胞转化为巨噬细胞、泡沫细胞。实验细胞分为3组:对照组（正常巨噬细胞、泡沫细胞）、铁离子螯合剂去铁胺(DFO)刺激组、柠檬酸铁和DFO共刺激组。应用RT-PCR和Western blot测定巨噬细胞、泡沫细胞中EMMPRIN基因和蛋白的表达。用Western blot测定MMP-9蛋白的表达。用明胶酶谱法测定MMP-9的活性。结果: DFO刺激组中EMMPRIN基因及蛋白的水平、MMP-9蛋白表达的水平及活性均明显高于对照组（P<0.05,P<0.01）。柠檬酸铁逆转了DFO对EMMPRIN表达的上调作用。结论: 铁负荷过低可增加巨噬细胞及泡沫细胞中炎症因子的表达及活性,可能会促进心血管事件的发生。     

神经导向因子Netrin-1调节单核巨噬细胞迁移与动脉粥样斑块关系的研究进展

动脉粥样硬化是动脉壁的一种慢性炎症性疾病,单核巨噬细胞在其发生发展中起着关键作用。动脉粥样斑块中单核巨噬细胞迁移能力受损,滞留于斑块内,增加了斑块不稳定性,加速动脉粥样硬化病变的进展。目前研究表明动脉粥样斑块中巨噬细胞分泌的神经导向因子Netrin-1通过与巨噬细胞表面相应受体结合,可以抑制巨噬细胞迁出斑块,促进动脉粥样硬化的进展。但在动脉粥样硬化形成初期,血管内皮细胞表达的Netrin-1却被发现对动脉粥样硬化起到保护作用。     

Cyclophilin A is secreted by a vesicular pathway in vascular smooth muscle cells

Reactive oxygen species (ROS) contribute to the pathogenesis of atherosclerosis in part by promoting vascular smooth muscle cell (VSMC) growth. Previously we demonstrated that cyclophilin A (CyPA) is a secreted oxidative stress-induced factor (SOXF) that promotes inflammation, VSMC growth, and endothelial cell apoptosis. However, the mechanisms that regulate CyPA secretion are unknown. In this study, we hypothesized that ROS-induced CyPA secretion from VSMC requires a highly regulated process of vesicle transport, docking, and fusion at the plasma membrane. Conditioned medium and plasma membrane sheets were prepared by exposing VSMC to 1 micromol/L LY83583, which generates intracellular superoxide. A vesicular transport mechanism was confirmed by colocalization at the plasma membrane with vesicle-associated membrane protein (VAMP). CyPA transport to the plasma membrane and secretion were significantly increased by LY83583. Reduction of VAMP-2 expression by small interfering RNA inhibited LY83583-induced CyPA secretion. Pretreatment with 3 micromol/L cytochalasin D, an actin depolymerizing agent, abrogated CyPA secretion. Infection with dominant-negative RhoA and Cdc42 adenovirus inhibited CyPA secretion by 72% and 63%, respectively, whereas dominant-negative Rac1 had a small effect (11%). Pretreatment with the Rho kinase inhibitor Y27632 (3 to 30 micromol/L) and myosin II inhibitor blebbistatin (1 to 10 micromol/L) inhibited CyPA secretion in a dose-dependent manner. Simvastatin (3 to 30 micromol/L) also dose-dependently inhibited LY83583-induced CyPA secretion likely via decreased isoprenylation of small GTPases. Our findings define a novel VSMC vesicular secretory pathway for CyPA that involves actin remodeling and myosin II activation via RhoA-, Cdc42-, and Rho kinase-dependent signaling events.     

细胞外基质金属蛋白酶诱导因子通过Toll样受体-4信号通路调节巨噬细胞自噬水平

目的 探讨细胞外基质金属蛋白酶诱导因子(EMMPRIN)是否通过Toll样受体-4(TLR4)通路调节巨噬细胞自噬水平.方法 用终浓度为5ng/ml的佛波酯(PMA)诱导人单核细胞白血病细胞系(THP-1)THP-1单核细胞48h,采用分化成功的巨噬细胞进行实验.实验分为空白对照组、EMMPRIN组和TAK-242组....     

Cyclophilin A affects inflammation, virus elimination and myocardial fibrosis in coxsackievirus B3-induced myocarditis

Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Recently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA(-/-) mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8 days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8 days p.i. At 28 days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811-treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis.     

Advanced protein glycosylation induces transendothelial human monocyte chemotaxis and secretion of platelet-derived growth factor: role in vascular disease of diabetes and aging.

Diabetes and aging are commonly accompanied by arterio- and atherosclerosis. Infiltration of the arterial subendothelial intima by macrophages/monocytes is an important early event preceding the development of atheromatous lesions; these macrophages are known to produce mitogenic factors in early atherosclerotic lesions. It has been previously shown that, over time, vascular matrix accumulates proteins nonenzymatically modified by advanced glycosylation end products (AGEs). In view of the fact that macrophages/monocytes have AGE-specific receptors associated with the expression of several growth factors, we investigated the possibility that AGEs mediate initial monocyte-vessel wall interactions that occur before overt formation of vascular lesions. This study demonstrates that (i) in vitro- and in vivo-formed AGEs are chemotactic for human blood monocytes, (ii) sub-endothelial AGEs can selectively induce monocyte migration across an intact endothelial cell monolayer, and (iii) subsequent monocyte interaction with AGE-containing matrix results in the expression of platelet-derived growth factor. These results support the existing hypothesis that in vivo-forming glucose-derived protein adducts can act as signals for the normal turnover of senescent tissue protein by means of the AGE-specific receptor system. Time-dependent glucose-induced deposition of AGEs on matrix proteins may promote monocyte infiltration into the subendothelium. Subsequent AGE-triggered macrophage activation and consequent elaboration of proliferative factors may normally coordinate remodeling but may also lead to the diverse pathogenic changes typical of arterio- and atherosclerosis in diabetic or aging populations.     

Human monocytes are severely impaired in base and DNA double-strand break repair that renders them vulnerable to oxidative stress

Monocytes are key players in the immune system. Crossing the blood barrier, they infiltrate tissues and differentiate into (i) macrophages that fight off pathogens and (ii) dendritic cells (DCs) that activate the immune response. A hallmark of monocyte/macrophage activation is the generation of reactive oxygen species (ROS) as a defense against invading microorganisms. How monocytes, macrophages, and DCs in particular respond to ROS is largely unknown. Here we studied the sensitivity of primary human monocytes isolated from peripheral blood and compared them with macrophages and DCs derived from them by cytokine maturation following DNA damage induced by ROS. We show that monocytes are hypersensitive to ROS, undergoing excessive apoptosis. These cells exhibited a high yield of ROS-induced DNA single- and double-strand breaks and activation of the ATR-Chk1-ATM-Chk2-p53 pathway that led to Fas and caspase-8, -3, and -7 activation, whereas macrophages and DCs derived from them were protected. Monocytes are also hypersensitive to ionizing radiation and oxidized low-density lipoprotein. The remarkable sensitivity of monocytes to oxidative stress is caused by a lack of expression of the DNA repair proteins XRCC1, ligase IIIα, poly(ADP-ribose) polymerase-1, and catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs)), causing a severe DNA repair defect that impacts base excision repair and double-strand break repair by nonhomologous end-joining. During maturation of monocytes into macrophages and DCs triggered by the cytokines GM-CSF and IL-4, these proteins become up-regulated, making macrophages and DCs repair-competent and ROS-resistant. We propose that impaired DNA repair in monocytes plays a role in the regulation of the monocyte/macrophage/DC system following ROS exposure.     

Cyclophilin A is a secreted growth factor induced by oxidative stress

Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. Many VSMC growth factors are secreted by VSMC and act in an autocrine manner. Here we demonstrate that cyclophilin A (CyPA), a member of the immunophilin family, is secreted by VSMCs in response to oxidative stress and mediates extracellular signal-regulated kinase (ERK1/2) activation and VSMC growth by reactive oxygen species. Human recombinant CyPA can mimic the effects of secreted CyPA to stimulate ERK1/2 and cell growth. The peptidyl-prolyl isomerase activity is required for ERK1/2 activation by CyPA. In vivo, CyPA expression and secretion are increased by oxidative stress and vascular injury. These findings are the first to identify CyPA as a secreted redox-sensitive mediator, establish CyPA as a VSMC growth factor, and suggest an important role for CyPA and enzymes with peptidyl-prolyl isomerase activity in the pathogenesis of vascular diseases.     

铁离子螯合剂去铁胺上调巨噬细胞、泡沫细胞细胞外基质金属蛋白酶诱导因子表达的机制探讨

目的: 探讨铁负荷过低上调巨噬细胞、泡沫细胞细胞外基质金属蛋白酶诱导因子(EMMPRIN)表达的机制。研究促分裂原活化的蛋白激酶(MAPK)信号通路、视黄醛x受体（RXR）及过氧化物酶体增殖剂活化受体γ（PPARγ）在此过程中的作用。方法: 将巨噬细胞和泡沫细胞给予MAPK（p38,ERK1/2）信号通路抑制剂及RXR的天然配体预处理,加入或不加入去铁胺继续培养24 h,用Western blot测定细胞中EMMPRIN蛋白的表达。于巨噬细胞和泡沫细胞中加入铁离子鳌合剂去铁胺刺激,用Western blot检测MAPK（p38,ERK1/2）的磷酸化及PPARγ的水平。结果: p38 MAPK通路抑制剂及RXR和配体在本身不影响EMMPRIN表达的同时,可抑制去铁胺对EMMPRIN表达的上调。去铁胺可促进p38 MAPK磷酸化,但不影响PPARγ蛋白表达。结论: p38 MAPK 参与了铁负荷过低上调巨噬细胞和泡沫细胞中EMMPRIN表达的过程;RXR可能参与了该过程。     

EMMPRIN (CD147) regulation of MMP-9 in bronchial epithelial cells in COPD

Background and objective: Extracellular matrix metalloproteinase inducer (EMMPRIN or CD147) induces the production of matrix metalloproteinases (MMP) such as MMP‐9, which plays an important role in COPD. We determined its cellular origin and role in MMP‐9 production in COPD. Methods: Bronchial biopsies, alveolar macrophages (AM) and blood monocytes (BM) from patients with COPD, healthy smokers and non‐smokers, and bronchial epithelial cells (EC) from surgically resected airways from patients with COPD were stimulated with LPS or CRP in the presence and absence of an anti‐EMMPRIN blocking antibody. EMMPRIN in BAL, plasma, conditioned media and cell lysates was quantified and immunohistochemical localization of EMMPRIN was determined in bronchial biopsies. MMP‐9 activity and mRNA was also determined. Results: EMMPRIN levels in BAL fluid were higher in patients with COPD compared with non‐smokers and smokers. There was greater EMMPRIN expression in EC from patients with COPD compared with smokers and non‐smokers. EC secreted and expressed more EMMPRIN protein than BM and AM. Blocking EMMPRIN decreased MMP‐9 activity in supernatant of EC, but not in those from AM and BM, and decreased MMP‐9 mRNA expression in EC. Conclusions: The increased EMMPRIN expression in COPD is reflected by an increased release from bronchial EC, which are one of the main source of EMMPRIN. EMMPRIN regulates MMP‐9 expression in COPD.     

cIAP1-dependent TRAF2 degradation regulates the differentiation of monocytes into macrophages and their response to CD40 ligand

Peripheral blood monocytes are plastic cells that migrate to tissues and differentiate into various cell types, including macrophages, dendritic cells, and osteoclasts. We have described the migration of cellular inhibitor of apoptosis protein 1 (cIAP1), a member of the IAP family of proteins, from the nucleus to the Golgi apparatus in monocytes undergoing differentiation into macrophages. Here we show that, once in the cytoplasm, cIAP1 is involved in the degradation of the adaptor protein tumor necrosis factor receptor-associated factor 2 (TRAF2) by the proteosomal machinery. Inhibition of cIAP1 prevents the decrease in TRAF2 expression that characterizes macrophage formation. We demonstrate that TRAF2 is initially required for macrophage differentiation as its silencing prevents Ikappa-Balpha degradation, nuclear factor-kappaB (NF-kappaB) p65 nuclear translocation, and the differentiation process. Then, we show that cIAP1-mediated degradation of TRAF2 allows the differentiation process to progress. This degradation is required for the macrophages to be fully functional as TRAF2 overexpression in differentiated cells decreases the c-Jun N-terminal kinase-mediated synthesis and the secretion of proinflammatory cytokines, such as interleukin-8 and monocyte chemoattractant protein 1 (MCP-1) in response to CD40 ligand. We conclude that TRAF2 expression and subsequent degradation are required for the differentiation of monocytes into fully functional macrophages.     

Oxidized LDL,LOX-1 and Atherosclerosis

An elevated level of low density lipoprotein (LDL) cholesterol constitutes a major risk factor for genesis of atherosclerosis. Ox-LDL plays a more important role in the genesis and progression of atherosclerosis than the native LDL. Ox-LDL leads to endothelial dysfunction leading to expression of adhesion molecules and recruitment of monocyte in subendothelial space. Ox-LDL is taken up by macrophages via scavenger receptors, such as SR-A1, SR-A2 and LOX-1. Lately, LOX-1, a type II membrane protein receptor of ox-LDL, has gained much importance in relation to effects of ox-LDL on endothelial biology. Endothelial cells primarily express LOX-1 as receptor for ox-LDL and ox-LDL has been shown to upregulate expression of LOX-1. In addition, ox-LDL promotes the growth and migration of smooth muscle cells, monocytes/macrophages and fibroblasts. In this review we discuss the role of ox-LDL and LOX-1 in genesis and progression of atherosclerosis.     

Pigment epithelium-derived factor inhibits TNF-alpha-induced interleukin-6 expression in endothelial cells by suppressing NADPH oxidase-mediated reactive oxygen species generation

Pigment epithelium-derived factor (PEDF) has recently been shown to be involved in the pathogenesis of proliferative diabetic retinopathy. Atherosclerosis is an inflammatory-fibroproliferative disease as well. Oxidative stress plays a major role in retinopathy and atherosclerosis. Accordingly, we investigated effects of PEDF on reactive oxygen species (ROS) generation, NF-kappaB activation and interleukin (IL)-6 expression in TNF-alpha-exposed HUVEC. TNF-alpha significantly increased intracellular ROS generation, which was completely blocked by PEDF or diphenylene iodonium, an inhibitor of NADPH oxidase. Further, PEDF completely prevented the TNF-alpha-induced increase in NADPH oxidase activity. PEDF or an antioxidant, N-acetylcysteine, significantly inhibited the TNF-alpha-induced NF-kappaB activation. PEDF inhibited TNF-alpha-induced expression of IL-6 at both mRNA and protein levels. Moreover, TNF-alpha downregulated PEDF mRNA levels. Ligand blot analysis revealed that HUVEC possessed a membrane protein with binding affinity for PEDF. The results demonstrated that PEDF inhibited TNF-alpha-induced NF-kappaB activation and subsequent IL-6 overexpression in HUVEC by suppressing NADPH oxidase-mediated ROS generation. Our present study suggests that PEDF may play an important role in the development and progression of atherosclerosis.     

Upstream regulation of matrix metalloproteinase by EMMPRIN; extracellular matrix metalloproteinase inducer in advanced atherosclerotic plaque

From experimental and clinical studies it is known that matrix conservation and degradation by matrix metalloproteinases (MMPs) plays a major role in plaque progression and destabilization with related onset of acute vascular events such as acute coronary syndromes or cerebrovascular accidents. Recently, extracellular MMPs inducer (EMMPRIN) has been reported to induce and activate the expression of MMPs in myocardium and plays an important role in the ventricular remodeling in human heart failure. Similarly to heart failure myocardium, EMMPRIN may be expressed in human atheroma and play a role in the extracellular matrix (ECM) remodeling and atherogenic cell differentiation. This study was designed to investigate the possible biological role of EMMPRIN in human atheroma. Immunohistochemical analysis for MMPs and EMMPRIN was performed on human carotid endarterectomy specimens and control aortas. EMMPRIN showed significant immunoreactivity in human atherosclerotic carotid lesions, and was colocalized with macrophage/monocyte infiltrates in atherosclerotic intima, plaque itself and vascular smooth muscle cells (VSMCs). Zymography and Western blot analysis revealed EMMPRIN expression in the carotid atheromas, but not in the control aortas. Human bone marrow monocytes, which were cultured with atherogenic proinflammatory cytokine stimulation revealed increased EMMPRIN and MMPs expressions. ECM remodeling is under the control of induction and inhibition of matrix degrading protease and the novel MMP inducer, EMMPRIN may play a role in influx and differentiation of monocytes and destabilizing atheroma.