Thrombotic phenotype of mice with a combined deficiency in plasminogen activator inhibitor 1 and vitronectin

OBJECTIVE: The role of vitronectin (VN) in thrombosis is not fully understood, primarily because this adhesive glycoprotein not only stabilizes plasminogen activator inhibitor 1 (PAI-1) and thus protects fibrin from premature lysis, but also because it binds to platelet integrins and may influence platelet aggregation. The absence of quantitative approaches to characterize the thrombi formed in animal models under different conditions further complicates this analysis. Methods: In this report, we describe a more comprehensive approach to assess the stability of thrombi formed in mice deficient in PAI-1 (PAI-1(-/-)), VN (VN(-/-)) or both (PAI-1(-/-)/VN(-/-)). RESULTS: We observed that all deficient mice developed unstable thrombi compared with wild type (WT) mice. Thus, only 31% of the thrombi formed in WT mice were unstable compared with 74% of PAI-1(-/-), 80% of VN(-/-), and 87% of PAI-1(-/-)/VN(-/-) mice. In this regard, the average number of emboli per WT mouse was significantly lower (0.55) compared with VN(-/-) (2.66), PAI-1(-/-) (2.1), and VN(-/-)/PAI-1(-/-) (2.35) mice. Finally, the total duration of complete vascular occlusion was higher and the rate of vascular patency was lower in the WT mice compared with the deficient mice. CONCLUSIONS: Taken together, these observations indicate that the thrombotic phenotype of mice with a combined deficiency in PAI-1 and VN does not differ significantly from the phenotype of mice with deficiencies in only PAI-1 or VN. This observation suggests that PAI-1 and VN may influence thrombus stability by regulating a common pathway.     

IL-33 reduces the development of atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the vasculature commonly leading to myocardial infarction and stroke. We show that IL-33, which is a novel IL-1-like cytokine that signals via ST2, can reduce atherosclerosis development in ApoE(-/-) mice on a high-fat diet. IL-33 and ST2 are present in the normal and atherosclerotic vasculature of mice and humans. Although control PBS-treated mice developed severe and inflamed atherosclerotic plaques in the aortic sinus, lesion development was profoundly reduced in IL-33-treated animals. IL-33 also markedly increased levels of IL-4, -5, and -13, but decreased levels of IFNgamma in serum and lymph node cells. IL-33 treatment also elevated levels of total serum IgA, IgE, and IgG(1), but decreased IgG(2a), which is consistent with a Th1-to-Th2 switch. IL-33-treated mice also produced significantly elevated antioxidized low-density lipoprotein (ox-LDL) antibodies. Conversely, mice treated with soluble ST2, a decoy receptor that neutralizes IL-33, developed significantly larger atherosclerotic plaques in the aortic sinus of the ApoE(-/-) mice compared with control IgG-treated mice. Furthermore, coadministration of an anti-IL-5 mAb with IL-33 prevented the reduction in plaque size and reduced the amount of ox-LDL antibodies induced by IL-33. In conclusion, IL-33 may play a protective role in the development of atherosclerosis via the induction of IL-5 and ox-LDL antibodies.     

A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation

The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE(-)(/)(-) mice before the development of manifest atherosclerotic lesions. Platelet-endothelial cell interaction involved both platelet glycoprotein (GP)Ibalpha and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE(-)(/)(-) mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process.     

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.     

载脂蛋白E基因敲除模型小鼠转化生长因子β1表达与尼古丁的刺激

背景：研究表明尼古丁可以诱导动脉粥样硬化,但其具体作用机制尚不明确。目的：探讨不同剂量尼古丁刺激下,载脂蛋白E基因敲除小鼠主动脉粥样斑块形成与外周血转化生长因子β1水平的关系。方法：载脂蛋白E基因敲除小鼠腹膜下注射2种不同剂量的尼古丁[2mg/（kg·d）及0.5mg/（kg·d）]12周,对照组注射等量生理盐水。用ELISA法检测外周血转化生长因子β1水平,苏木精-伊红染色观察动脉粥样斑块的病理变化。结果与结论：尼古丁干预12周后,低剂量尼古丁组的外周血转化生长因子β1水平低于对照组（P〈0.05）,高剂量尼古丁组的外周血转化生长因子β1水平低于对照组及低剂量尼古丁组（P〈0.05）。高剂量尼古丁组的血管狭窄程度最严重,低剂量尼古丁组次之,对照组最低,各组间比较差异均具有显著性意义（P〈0.05）。低剂量尼古丁组及高剂量尼古丁组的斑块个数多于对照组（P〈0.05）,高剂量尼古丁组的斑块个数与低剂量尼古丁组比较差异无显著性意义（P〉0.05）;血管狭窄率与转化生长因子β1水平呈负相关（r=-0.920,P=0.000）。结果提示尼古丁刺激增加载脂蛋白E基因敲除小鼠动脉粥样硬化斑块数量,这种作用可能与尼古丁抑制转化生长因子β1水平相关。     

Overexpression of PAI-1 prevents the development of abdominal aortic aneurysm in mice

Vessel wall inflammation and matrix destruction are critical to abdominal aortic aneurysm (AAA) formation and rupture. We have previously shown that urokinase plasminogen activator (uPA) is highly expressed in experimental AAA and is essential for AAA formation and expansion. In this study, we examined the effects of overexpression of a natural inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), on the development of angiotensin (Ang) II-induced AAA in ApoE-deficient (ApoE(-/-)) mice. Mice were treated with recombinant adenovirus containing either the human PAI-1 gene (Ad5.CMV.PAI-1) or the luciferase gene (Ad5.CMV.Luc) delivered either locally by intra-adventitial injection or systemically by tail vein injection. Our results show that local delivery of the PAI-1 gene completely prevented AAA formation (0 vs 55.6% in Ad5.CMV.Luc controls, P<0.05). In contrast, systemic delivery of the PAI-1 gene did not affect AAA incidence (78 vs 90% in Ad5.CMV.Luc controls, P=0.125). Local delivery of the PAI-1 gene 2 weeks after Ang II infusion prevented further expansion of small aneurysms, but had no significant effect on the progression of larger aneurysms. These data suggest that local PAI-1 gene transfer could be used to stabilize small AAA and reduce the rate of expansion and risk of rupture.     

纤溶酶原激活物抑制物-1与动脉粥样硬化关系的研究

目的 通过对动脉粥样硬化（AS）兔血清和粥样硬化斑块中纤溶酶原激活物抑制物-1（PAI-1）表达的研究,探讨PAI-1在AS中的作用.方法 16只雄性大耳白兔随机分为正常饮食组和高脂饮食组,每组8只,饲养16周.两组白兔均于0周、16周取耳缘静脉血,检测血清中PAI-1 的水平;16周后处死,应用免疫组化方法检测PAI-1在主动脉粥样硬化斑块中的表达.结果 正常饮食组和高脂饮食组0周血清中PAI-1水平差异无显著性（P＞0.05）;高脂饮食组16周后血清PAI-1水平较0周显著增加（P＜0.01）;免疫组化结果显示高脂饮食组主动脉壁PAI-1 的表达明显高于对照组（P＜0.01）.结论 动脉粥样硬化的发生伴有血清和粥样斑块中PAI-1表达增加,PAI-1可能参与了AS的形成.     

Aggregated low‐density lipoprotein induce impairment of the cytoskeleton dynamics through urokinase‐type plasminogen activator/urokinase‐type plasminogen activator receptor in human vascular smooth muscle cell

Summary. Background: Urokinase‐type plasminogen activator (UPA) regulates vascular smooth muscle cell (VSMC) functions relevant in vascular remodeling by facilitating proteolysis at the cell surface and inducing cell signaling pathways. Our previous results demonstrated that aggregated low‐density lipoprotein (agLDL) impair cytoskeleton dynamics, a key event contributing to VSMC behavior during progression of atherosclerotic plaques. Objectives: To investigate whether mechanisms underlying inhibition of cytoskeleton dynamics in lipid‐loaded VSMC occurs through a UPA‐mediated process. Methods: Adhesion assay was performed in lipid‐loaded human VSMC after 16‐h exposition to agLDL (100 μg mL^?1). Protein subcellular localization and actin‐fiber formation were assessed by confocal microscopy. For analysis of protein expression western blots were carried out. Co‐immunoprecipitates of UPAR were examined by one‐dimensional‐ or two‐dimensional electrophoresis (1‐DE or 2‐DE), mass spectrometry MALDI‐TOF and western blot. Results: agLDL induced UPA subcellular delocalization and significantly decreased UPA levels during attachment of VSMC. UPA (enhanced endogenous‐expression or exogenous added) acting as a urokinase‐type plasminogen activator receptor (UPAR)‐ligand restored actin‐cytoskeleton organization and adhesion capacity of lipid‐loaded cells to control levels. UPAR co‐immunoprecipitated with the unphosphorylated form of myosin regulatory light chain (MRLC) in lipid‐loaded cells. The detrimental effects of agLDL on MRLC phosphorylation were reversed by high levels of UPA. The UPA effects on VSMC exposed to agLDL involved FAK phosphorylation. Conclusions: The detrimental effects of atherogenic LDL on VSMC are mediated by a decrease and delocalization of the UPA–UPAR interaction that result in an impairment of cytoskeleton dynamics and adhesion capacity affecting cell phenotype and function.     

TRAIL-expressing T cells induce apoptosis of vascular smooth muscle cells in the atherosclerotic plaque

Acute coronary syndromes (ACS) are precipitated by a rupture of the atherosclerotic plaque, often at the site of T cell and macrophage infiltration. Here, we show that plaque-infiltrating CD4 T cells effectively kill vascular smooth muscle cells (VSMC). VSMCs sensitive to T cell-mediated killing express the death receptor DR5 (TNF-related apoptosis-inducing ligand [TRAIL] receptor 2), and anti-TRAIL and anti-DR5 antibodies block T cell-mediated apoptosis. CD4 T cells that express TRAIL upon stimulation are expanded in patients with ACS and more effectively induce VSMC apoptosis. Adoptive transfer of plaque-derived CD4 T cells into immunodeficient mice that are engrafted with human atherosclerotic plaque results in apoptosis of VSMCs, which was prevented by coadministration of anti-TRAIL antibody. These data identify that the death pathway is triggered by TRAIL-producing CD4 T cells as a direct mechanism of VSMC apoptosis, a process which may lead to plaque destabilization.     

Oxidized LDL activates fas-mediated endothelial cell apoptosis.

Oxidized low density lipoproteins (OxLDL) promote chronic inflammatory responses in the vasculature that give rise to atherosclerotic plaques. Fas ligand (FasL) is naturally expressed on the vascular endothelium where it can induce apoptosis in Fas-expressing immune cells as they enter the vessel wall. Although vascular endothelial cells are normally resistant to Fas-mediated cell death, OxLDL were shown to induce apoptosis in cultured endothelial cells and endothelium of arterial explants by a process that could be inhibited with Fas L neutralizing antibodies. OxLDL-induced cell death was also reduced in the aortic endothelium cultured from gld (FasL-/-) and lpr (Fas-/-) mice as compared with wild-type mice. OxLDL acted by sensitizing endothelial cells to death signals from the Fas receptor. Thus, the ability of OxLDL to promote Fas-mediated endothelial cell suicide may be a feature that contributes to their atherogenicity.     

Pathophysiology of the plasminogen/plasmin system

Circumstantial evidence has been provided for a role of the human fibrinolytic or plasminogen/plasmin system in a variety of biological phenomena. Recently, generation of mice with single or combined deficiencies of main components of the fibrinolytic system, including plasminogen, tissue-type and urokinase-type plasminogen activator, plasminogen activator inhibitor-1, and the cellular receptor for urokinase-type plasminogen activator has allowed the role of the fibrinolytic system in vivo to be established more conclusively. Plasminogen-deficient mice survive embryonic development, but develop spontaneous fibrin deposition due to an impaired thrombolytic potential, and suffer retarded growth and reduced fertility and survival. Plasminogen deficiency in man is extremely rare, but viable, although it is associated with thrombolic complications. Disruption of the plasminogen activator inhibitor-1 gene in mice induces a mild hyperfibrinolytic state and a greater resistance to venous thrombosis, but does not impair hemostasis. In contrast, in man, the inhibitor deficiency results in delayed rebleeding. Tissue-type plasminogen activator-deficient mice have a reduced thrombolytic potential, whereas mice deficient in the urokinase-type plasminogen activator occasionally develop spontaneous fibrin deposits in tissues and display deficient plasmin-mediated macrophage function. Mice deficient in both types of activator develop normally but are significantly less fertile, have retarded growth and shorter survival, and display a severe thrombotic phenotype in normal and inflamed tissues. At present, genetic deficiencies of either type activator have not been reported in man. The urokinase-type plasminogen activator/receptor system has been implicated in localized extracellular proteolytic activity. Receptor-deficient mice are, however, healthy, and fertile, and have a normal endogenous thrombolytic capacity; their macrophages have, however, an impaired (urokinase-type plasminogen activator mediated) plasminogen activating potential. Mice with single or combined inactivation of components of the plasminogen/plasmin system thus may be valuable models for studying physiological and pathophysiological processes in vivo.     

Activator protein-1 (AP-1) signalling in human atherosclerosis: results of a systematic evaluation and intervention study

Animal studies implicate the AP-1 (activator protein-1) pro-inflammatory pathway as a promising target in the treatment of atherosclerotic disease. It is, however, unclear whether these observations apply to human atherosclerosis. Therefore we evaluated the profile of AP-1 activation through histological analysis and tested the potential benefit of AP-1 inhibition in a clinical trial. AP-1 activation was quantified by phospho-c-Jun nuclear translocation (immunohistochemistry) on a biobank of aortic wall samples from organ donors. The effect of AP-1 inhibition on vascular parameters was tested through a double blind placebo-controlled cross-over study of 28?days doxycycline or placebo in patients with symptomatic peripheral artery disease. Vascular function was assessed by brachial dilation as well as by plasma samples analysed for hs-CRP (high-sensitivity C-reactive protein), IL-6 (interleukin-6), IL-8, ICAM-1 (intercellular adhesion molecule-1), vWF (von Willebrand factor), MCP-1 (monocyte chemoattractant protein-1), PAI-1 (plasminogen activator inhibitor-1) and fibrinogen. Histological evaluation of human atherosclerosis showed minimal AP-1 activation in non-diseased arterial wall (i.e. vessel wall without any signs of atherosclerotic disease). A gradual increase of AP-1 activation was found in non-progressive and progressive phases of atherosclerosis respectively (P<0.044). No significant difference was found between progressive and vulnerable lesions. The expression of phospho-c-Jun diminished as the lesion stabilized (P<0.016) and does not significantly differ from the normal aortic wall (P<0.33). Evaluation of the doxycycline intervention only revealed a borderline-significant reduction of circulating hs-CRP levels (-0.51 μg/ml, P=0.05) and did not affect any of the other markers of systemic inflammation and vascular function. Our studies do not characterize AP-1 as a therapeutic target for progressive human atherosclerotic disease.     

CLOCK is involved in obesity-induced disordered fibrinolysis in ob/ob mice by regulating PAI-1 gene expression

BACKGROUND: An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. AIM: The present study investigates whether the circadian clock component CLOCK is involved in obesity-induced PAI-1 elevation. METHODS: We examined plasma PAI-1 and mRNA expression levels in tissues from leptin-deficient obese and diabetic ob/ob mice lacking functional CLOCK protein. RESULTS: Our results demonstrated that plasma PAI-1 levels were augmented in a circadian manner in accordance with the mRNA expression levels in ob/ob mice. Surprisingly, a Clock mutation normalized the plasma PAI-1 concentrations in accordance with the mRNA levels in the heart, lung and liver of ob/ob mice, but significantly increased PAI-1 mRNA levels in adipose tissue by inducing adipocyte hypertrophy in ob/ob mice. The Clock mutation also normalized tissue PAI-1 antigen levels in the liver but not in the adipose tissue of ob/ob mice. CONCLUSION: These observations suggest that CLOCK is involved in obesity-induced disordered fibrinolysis by regulating PAI-1 gene expression in a tissue-dependent manner. Furthermore, it appears that obesity-induced PAI-1 production in adipose tissue is not closely related to systemic PAI-1 increases in vivo.     

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.     

Emodin promotes atherosclerotic plaque stability in fat-fed apolipoprotein E-deficient mice

Increasing evidence indicated that plaque stabilization is attributed to the composition of the atherosclerotic plaque, and inflammation plays an important role in the formation and progress of vulnerable atherosclerotic plaque (VAP), which is prone to rupture. Emodin, an important component of traditional Chinese herb rhubarb, has obvious anti-inflammatory effect, although its effect on atherosclerotic plaque stabilization is unknown. Apolipoprotein E (ApoE) is an important component of plasma lipoprotein with anti-atherosclerosis function, and the plaque in the aorta of ApoE-deficient mice has been demonstrated with characteristics of VAP. Therefore, this study was designed to determine whether emodin can stabilize the VAP in the ApoE-deficient mice and explain the possible mechanism. After fat-fed for 13 weeks, mice were randomized into three groups (11 animals/group) and intragastrically administrated with emodin, simvastatin or distilled water for 13 weeks, respectively. The plaque stability was evaluated by the morphology and composition of atherosclerotic plaques. Additionally, the expression of peroxisomal proliferator-activated receptor-gamma (PPAR-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 9 (MMP-9) in plaques was determined by the immunohistochemistry method. We showed that emodin could decrease the lipid core area and the ratio of lipid to collagen content in plaques. In addition, emodin significantly inhibited the expression of GM-CSF and MMP-9, whereas it induced the expression of PPAR-gamma in plaques. In conclusion, these results suggest that emodin can stabilize the VAP in the aortic root of ApoE-knockout mice, which is probably due to its anti-inflammatory effect.     

No functional role of plasminogen activator inhibitor-1 in murine adipogenesis or adipocyte differentiation

OBJECTIVE: To substantiate a potential role of plasminogen activator inhibitor-1 (PAI-1) in adipogenesis, we have studied its effects on in vitro adipocyte differentiation and on in vivo adipose tissue formation. RESULTS: Our in vitro data do not support a functional role of PAI-1, as substantiated by our findings that: (i) inhibition of PAI-1 with a neutralizing antibody did not affect differentiation of 3T3-F442A preadipocytes; (ii) overexpression of murine PAI-1 in 3T3-F442A cells had no effect on differentiation; and (iii) differentiation of PAI-1-deficient murine embryonic fibroblasts into mature adipocytes was comparable to wild-type (WT) cells. Furthermore, our in vivo studies did not reveal an important role for PAI-1, as suggested by our findings that: (i) de novo fat pad formation in NUDE mice following injection of 3T3-F442A cells was not affected by a PAI-1 neutralizing antibody; and (ii) adipose tissue formation following combined injection of Matrigel and basic fibroblast growth factor was comparable in WT and in PAI-1 deficient mice. CONCLUSION: Taken together, these in vitro and in vivo studies in murine model systems do not support an important functional role of PAI-1 in adipogenesis.     

Distribution and regulation of plasminogen activator inhibitor-1 in murine adipose tissue in vivo. Induction by tumor necrosis factor-alpha and lipopolysaccharide.

Although elevated plasma plasminogen activator inhibitor 1 (PAI-1) is associated with obesity, very little is known about its tissue or cellular origin, or about the events that lead to increased PAI-1 levels under obese conditions. Since TNF-alpha is increased in rodents both during obesity and in response to endotoxin treatment, we examined the effects of these agents on PAI-1 gene expression in the adipose tissue of CB6 mice. In untreated mice, PAI-1 mRNA was detected in both mature adipocytes and in stromal vascular cells. Both TNF-alpha and endotoxin significantly increased PAI-1 mRNA in the adipose tissue, peaking at 3-8 h. In situ hybridization analysis of adipose tissue from untreated mice revealed a weak signal for PAI-1 mRNA only in the smooth muscle cells within the vascular wall. In contrast, after endotoxin or TNF-alpha treatment, PAI-1 mRNA also was detected in adipocytes and in adventitial cells of vessels. Endotoxin also induced PAI-1 in endothelial cells, while TNF-alpha additionally induced it in smooth muscle cells. Mature 3T3-L1 adipocytes in culture also expressed PAI-1 mRNA, and its rate of synthesis was also upregulated by TNF-alpha. These studies suggest that the adipose tissue itself may be an important contributor to the elevated PAI-1 levels observed in the plasma under obese conditions.     

Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.

Angiotensin II (AII)- and Arg8-vasopressin (AVP)-regulated gene expression in vascular cells has been reported to contribute to vascular homeostasis and hypertrophy. In this report, AVP-induced expression of plasminogen activator inhibitor (PAI)-2 mRNA in rat microvessel endothelial (RME) cells was identified using differential mRNA display. Further characterization of vasoactive peptide effects on PAI expression revealed that AII stimulated a 44.8 +/- 25.2-fold and a 12.4 +/- 3.2-fold increase in PAI-2 mRNA in RME cells and rat aortic smooth muscle cells (RASMC), respectively. AII also stimulated a 10- and 48-fold increase in PAI-1 mRNA in RME cells and RASMC, respectively. These AII effects were inhibited by either Sar1, Ile8-angiotensin or the AT1 antagonist DuP 735, but were not significantly altered in the presence of the AT2 antagonist PD123319. AII stimulation of RASMC and RME cells also significantly increased both PAI-1 protein and PAI activity released to the culture medium. Inhibition of protein kinase C completely blocked PMA-stimulated induction of PAI-2 mRNA in both cell types and inhibited the AII-stimulated increase in RASMC by 98.6 +/- 2.8%. In contrast, protein kinase C inhibition only partially decreased the AII-stimulated PAI-2 expression in RME cells by 68.8 +/- 11.1%, suggesting that a protein kinase C-independent mechanism contributes to a 6.9 +/- 1.5-fold AII induction of PAI-2 expression in endothelial cells. AII and PMA also stimulated protein tyrosine phosphorylation in RME cells, and the tyrosine kinase inhibitor genistein partially blocked their induction of PAI-2 mRNA. These findings suggest that AII may regulate plasminogen activation in the vasculature by inducing both PAI-1 and PAI-2 expression.     

The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-κB activation

BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory disorder. Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Complement components were identified in atherosclerotic plaques, and a correlation between adverse events and C5a plasma levels was found. These findings support the notion that complement activation contributes to development and progression of atherosclerotic lesions. OBJECTIVES: We investigated whether complement components C3a and C5a regulate plasminogen activator inhibitor (PAI-1) in human macrophages. METHODS: Human monocyte-derived macrophages (MDM) and human plaque macrophages were cultured and incubated with the complement component C5a. RESULTS: C5a increased PAI-1 up to 11-fold in human MDM and up to 2.7-fold in human plaque macrophages. These results were confirmed at the mRNA level using real time-polymerase chain reaction. Pertussis toxin or anti-C5aR/CD88 antibody completely abolished the effect of recombinant human C5a on PAI-1 production, suggesting a role of the C5a receptor. Experiments with antitumor necrosis factor (TNF)-alpha antibodies and tiron showed that the effect of C5a was not mediated by TNF-alpha or oxidative burst. Furthermore C5a induced NF-kappaB binding to the cis element in human macrophages and the C5a-induced increase in PAI-1 was completely abolished by an NF-kappaB inhibitor. CONCLUSIONS: We conclude that C5a upregulates PAI-1 in macrophages via NF-kappaB activation. We hypothesize that - if operative in vivo- this effect could favor thrombus development and thrombus stabilization in the lesion area. On the other hand one could speculate that C5a-induced upregulation of PAI-1 in plaque macrophages could act as a defense mechanism against plaque destabilization and rupture.     

Life style-related disease

Diabetes mellitus, hyperlipidemia, hypertension and atherosclerotic diseases have recently defined as typical life style-related diseases. A common background of these life style-related diseases is overnutrition and its consequence, obesity. Recent advances in the biology of adipose tissue have revealed that adipose is not simply an energy storage organ but it also secretes a variety of molecules which affect the metabolism of the whole body. Through a systematic search of active genes in adipose tissue, we found that adipose tissue, especially visceral fat expressed numerous genes for secretory proteins. Among them, plasminogen activator inhibitor-1(PAI-1) was over expressed in the visceral fat in an animal model of obesity. Plasma level of PAI-1 was closely correlated with visceral adiposity in human. Thus, PAI-1 secreted from visceral fat may play an important role in vascular disease in visceral obesity. Adiponectin, a novel adipose-specific gene product, is abundantly presented in human plasma. This molecule has been shown to have protective roles against atherosclerotic vascular changes and its plasma level is negatively correlated with visceral adiposity. In conclusion, dysregulated secretion of these adipose-specific secretory proteins(adipocytokines) may have important roles in the development of life style-related diseases, especially atherosclerotic diseases.