Glycation amplifies lipoprotein(a)-induced alterations in the generation of fibrinolytic regulators from human vascular endothelial cells

Increased lipoprotein(a) [Lp(a)] in plasma is an independent risk factor for premature cardiovascular diseases. The levels of glycated Lp(a) are elevated in diabetic patients. The present study demonstrated that glycation enhanced Lp(a)-induced production of plasminogen activator inhibitor-1 (PAI-1), and further decreased the generation of tissue-type plasminogen activator (t-PA) from human umbilical vein endothelial cells (HUVEC) and human coronary artery EC. The levels of PAI-1 mRNA and its antigen in the media of HUVEC were significantly increased following treatments with 5 μg/ml of glycated Lp(a) compared to equal amounts of native Lp(a). The secretion and de novo synthesis of t-PA, but not its mRNA level, in EC were reduced by glycated Lp(a) compared to native Lp(a). Treatment with aminoguanidine, an inhibitor for the formation of advanced glycation end products (AGEs), during glycation normalized the generation of PAI-1 and t-PA induced by glycated Lp(a). Butylated hydroxytoluene, a potent antioxidant, inhibited native and glycated Lp(a)-induced changes in PAI-1 and t-PA generation in EC. The results indicate that glycation amplifies Lp(a)-induced changes in the generation of PAI-1 and t-PA from venous and arterial EC. This may attenuate fibrinolytic activity in blood circulation and potentially contributes to the increased incidence of cardiovascular complications in diabetic patients with hyperlipoprotein(a). EC-mediated oxidative modification and the formation of AGEs may be implicated in glycated Lp(a)-induced alterations in the generation of fibrinolytic regulators from vascular EC.     

Thrombin modulates synthesis of plasminogen activator inhibitor type 2 by human peripheral blood monocytes

Fibrin deposition is characteristic of inflammatory diseases. The monocytes is central to the inflammatory response and can affect fibrinolysis by expression of urokinase (u-PA) and plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2, respectively). This study examines whether thrombin, which promotes fibrin deposition, can contribute to fibrin persistence by modulating expression of proteins of the fibrinolytic system. Monocytes were isolated from human peripheral blood and analyzed for PAI-2, PAI-1, and u-PA antigens by enzyme-linked immunosorbent assay (ELISA). Monocytes responded to thrombin by increased expression of PAI-2 in a dose- and time-dependent manner, with maximal synthesis at a concentration of 1 U/mL to 10 U/mL. This trend was also evident for PAI-1, which was present at much lower levels. Thrombin and lipopolysaccharide (LPS) stimulated comparable levels of PAI-2, studied at the antigen and mRNA level. The dose effet of LPS on PAI-2 and PAI-1 was found to differ from that of thrombin. The level of u-PA was undetectable by ELISA and zymography in all samples. Thrombin stimulates PAI-2 synthesis by human monocytes, therefore creating an imbalance in the fibrinolytic system. This may contribute to persistence of fibrin, deposited during inflammation.     

Plasminogen activator inhibitor-1 messenger RNA expression is induced in rat hepatocytes in vivo by dexamethasone.

Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue plasminogen activator (tPA), plays a crucial role in the regulation of fibrinolysis. Both hepatocytes and endothelial cells have been implicated as major sources of plasma PAI-1. To study the relative contribution of these cell types to hepatic PAI-1 production, we have separated hepatocytes and hepatic sinusoidal endothelial cells by fractionation of freshly isolated rat livers using metrizamide density gradients and centrifugal elutriation. In untreated animals, PAI-1 messenger RNA (mRNA) was detected only in the purified endothelial cell fraction, and not in the hepatocyte fraction or in unfractionated liver. However, when the animals were treated with dexamethasone, PAI-1 mRNA expression was transiently induced in the liver. This induction paralleled the appearance of PAI-1 mRNA in purified hepatocytes, while PAI-1 expression in sinusoidal endothelial cells was unchanged. Four hours after dexamethasone treatment, plasma PAI-1 levels were increased approximately twofold over levels measured in animals treated with the diluent alone. These data suggest that PAI-1 production by hepatocytes may contribute to elevated plasma PAI-1 levels in the setting of acute injury and stress.     

Endotoxin-induced production of plasminogen activator inhibitor by human monocytes is autonomous and can be inhibited by lipid X

Peripheral blood mononuclear cells (PBMs) produce both tissue factor and plasminogen activator inhibitor type 2 (PAI-2) in response to gram-negative bacterial lipopolysaccharide (LPS). The cellular roles in the tissue factor response have been previously elucidated, and we now report those roles in PAI-2 production. Monocytes are the only cells among LPS-stimulated PBMs that produce PAI-2 as assessed by measurement of PAI-2 activity and antigen. Concomitant immunohistochemistry demonstrated that monocytes contain PAI-2, with a greater number staining positively and more intensely after exposure to LPS. LPS-stimulated monocytes produced increased amounts of PAI-2 with or without addition of lymphocytes. Lymphocytes prestimulated with LPS and then washed did not induce PAI-2 production in monocytes to which they were added. Lipid X, a precursor in the biosynthetic pathway of lipid A and LPS, was able to inhibit LPS induction of monocyte PAI-2 in a dose-dependent manner. This inhibition was not due to cellular toxicity, the phospholipidlike nature of lipid X, interference with the PAI-2 assay, or monocyte production of a substance interfering with PAI-2. Lipid X was an effective inhibitor of PAI-2 production even when added up to 30 minutes after LPS.     

普伐他丁对兔脂肪肝肝脏1型纤溶酶原激活物抑制物mRNA表达的 …

目的 探讨普伐他丁对亲兔脂肪肝肝脏１型纤溶酶原激活物抑制物（ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｌｏｒ ｉｎｈｉｂｉｔｏｒ １．ＰＡＩ－１）ｍＲＮＡ表达的影响。方法 观察普伐他丁（治疗组,ｎ＝１０）作对照。结果 与正常组相比,模型组家兔肝脏呈重度肝细胞脂肪变性,血浆脂质和ＰＡＩ－１活性显著增高,肝组织ＰＡＩ－１ｍＲＮＡ呈过度表达。与模型组相比,治疗组肝组织学脂肪变性无明显改善,但血脂和ＰＡＩ－１ｍ     

Differential effects of gonadotropin-releasing hormone I and II on the urokinase-type plasminogen activator/plasminogen activator inhibitor system in human decidual stromal cells in vitro

To date, the factors capable of regulating the coordinate expression of the urokinase-type plasminogen activator (uPA) and its endogenous inhibitor, plasminogen activator inhibitor (PAI-1), at the maternal-fetal interface remain poorly characterized. In these studies we examined the ability of the classical form of gonadotropin-releasing hormone (GnRH) I and the second, mammalian form of this hormone, GnRH II, to regulate uPA and PAI-1 mRNA and protein expression levels in cultures of stromal cells isolated from first trimester decidual tissues using quantitative competitive-PCR and ELISA, respectively. GnRH I and GnRH II increased uPA mRNA and protein expression levels in these primary cell cultures in a dose- and time-dependent manner. In contrast, GnRH I increased, whereas GnRH II decreased PAI-1 mRNA and protein expression levels in these cells. Cetrorelix, a GnRH receptor antagonist, inhibited the regulatory effects of GnRH I, but not GnRH II, on uPA and PAI-1 expression levels in these decidual stromal cell cultures. Taken together, these observations suggest that GnRH I and GnRH II differentially regulate the balance between uPA and PAI-1 expression levels in the human decidua, possibly via distinct receptor-mediated signaling pathways.     

Circadian mRNA expression of coagulation and fibrinolytic factors is organ-dependently disrupted in aged mice

To evaluate the effects of aging on the circadian gene expression of coagulation and fibrinolytic factors in the mouse tissues, we examined temporal mRNA expression profiles of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), tissue factor (TF), and thrombomodulin (TM) genes together with circadian clock genes in the brains, hearts and livers of young (5weeks old) and aged (15months old) mice. Cardiac mRNA expression of β-myosin heavy chain (β-MHC), a molecular marker of cardiac hypertrophy, was obviously increased in the aged mice. Rhythmic expression of the clock genes mPer2 and BMAL1 in these organs was almost identical between young and aged mice, whereas that of PAI-1, TF and TM mRNAs and of clock-controlled genes such as DBP and Dec1 were damped to low levels in the livers of aged mice. Expression levels of tPA mRNA were significantly decreased and those of TF were significantly elevated throughout the day in the brain of aged mice. Expression levels of PAI-1 in the heart of aged mice were continuously elevated over 2-fold the peak levels of young mice throughout the day. However, day/night fluctuations in plasma PAI-1 levels were unaffected by aging. Aging tissue- and time-dependently affects the mRNA expression of coagulation and fibrinolytic factors. Aging-dependent constitutive PAI-1 induction in the heart might be a risk factor for cardiovascular diseases that is independent of plasma PAI-1 levels.     

Interleukin-4 suppresses plasminogen activator inhibitor-2 formation in stimulated human monocytes.

Using a specific enzyme-linked immunosorbent assay, plasminogen activator inhibitor-2 (PAI-2) was quantitated in cultures of human monocytes. Lipopolysaccharide (LPS) increased both extracellular and cell-associated PAI-2 levels, as well as PAI-2 mRNA measured by Northern analysis. Both the lymphokine, interleukin-4 (IL-4) (greater than or equal to 10 pmol/L), and the glucocorticoid, dexamethasone (100 nmol/L), inhibited PAI-2 formation and PAI-2 mRNA induction. Another lymphokine, interferon-gamma (IFN-gamma) (100 U/mL), as for IL-4 alone, did not stimulate PAI-2 formation; however, in contrast to IL-4, IFN-gamma did not reverse the LPS effect but could potentiate it. The suppression of PAI-2 formation by IL-4 and glucocorticoid in stimulated human monocytes extends the list of monocyte products whose synthesis can be downregulated in these cells by the two agents. The findings could have relevance to the control by monocytes/macrophages of connective tissue resorption, including that of fibrin, at sites of inflammation.     

Fibrinolytic parameters and lipoprotein(a) in young women with myocardial infarction

Impaired fibrinolysis and elevated lipoprotein(a) (Lp[a]) are possible nonclassic risk factors for myocardial infarction (MI) at young age. The fibrinolytic system in young women with MI has not been evaluated yet and the role of Lp(a) is still controversial. The authors determined fibrinolytic parameters and Lp(a) in premenopausal women (mean age 42+/-3 years, n = 22) 0.5 to 6 (mean 3.5) years after MI, who were all without severe classic risk factors and had an otherwise low risk for MI. Elevated levels of tissue type plasminogen activator (t-PA) (p< 0.05) were measured in comparison to 52 age-matched controls; no difference was found in plasminogen activator inhibitor, plasminogen, fibrinogen, euglobulin clotting time and D-dimers. Significantly more MI patients had Lp(a) levels greater than 300 mg/L compared to controls (36% vs 13.5%, p< 0.05). The combination of elevated Lp(a), mild hyperlipidemia, and nonsevere smoking was found in 62.5% of MI patients who had elevated levels of Lp(a), in 23% of all women with MI, and in none of the controls. Elevated t-PA is probably only a marker of increased risk of MI, whereas elevated Lp(a) probably has a causative role. A combination of elevated Lp(a), hyperlipidemia, and nonsevere smoking seems to be a high-risk profile, relatively common in young women with MI.     

Glucagon-like peptide-1 attenuates tumour necrosis factor-alpha-mediated induction of plasminogen [corrected] activator inhibitor-1 expression

Glucagon-like peptide-1 (GLP-1) has been proposed as a target for treatment of type 2 diabetes. GLP-1 has also been demonstrated to improve endothelial cell dysfunction in diabetic patients. Elevated plasminogen activator inhibitor type-1 [corrected] (PAI-1) levels have been implicated in endothelial cell dysfunction. The effect of GLP-1 on PAI-1 expression in vascular endothelial cells has not been explored. In a spontaneously transformed human umbilical vein endothelial cell (HUVEC) line, C11-spontaneously transformed HUVEC (STH) and primary HUVEC cells, GLP-1 treatment, in the presence of a dipeptidyl peptidase IV inhibitor, attenuated induction of PAI-1 protein and mRNA expression by tumour necrosis factor-alpha (TNF-alpha). GLP-1 also inhibited the effect of TNF-alpha on a reporter gene construct harbouring the proximal PAI-1 promoter. In addition, GLP-1 attenuated TNF-alpha-mediated induction of Nur77 mRNA and TNF-alpha-mediated binding of nuclear proteins (NPs) to the PAI-1, Nur77, cis-acting response element nerve growth factor induced clone B response element (NBRE). GLP-1 treatment also inhibited TNF-alpha-mediated induction of Akt phosphorylation. Taken together, these observations suggest that GLP-1 inhibits TNF-alpha-mediated PAI-1 induction in vascular endothelial cells, and this effect may involve Akt-mediated signalling events and the modulation of Nur77 expression and NP binding to the PAI-1 NBRE.     

C反应蛋白和肿瘤坏死因子-α对单核细胞妊娠相关血浆蛋白-A mRNA表达影响的实验研究

目的 研究C反应蛋白(CRP)和肿瘤坏死因子-α(TNF-α)对人外周血单核细胞妊娠相关血浆蛋白-A(PAPP-A) mRNA表达的影响.方法 采用密度梯度离心法分离人外周血单核细胞,用RT-PCR方法分别观察CRP和TNF-α刺激单核细胞PAPP-A mRNA表达的时间及剂量效应.结果 与空白对照组PAPP-A的mRNA表达(0.1842±0.0101)相比,CRP(20 mg/L)刺激单核细胞2 h后,PAPP-A mRNA表达开始显著增加(0.2128±0.0136),于24 h达最高值(0.6837±0.1360),呈时间依赖性.rhTNF-α(100 ng/ml)刺激后,PAPP-A mRNA表达在2 h迅速升高并达峰值(1.2546±0.0866),24 h仍高于空白对照组(0.8203±0.0413).CRP和rhTNF-α均可呈剂量依赖性诱导PAPP-A的mRNA表达,其中CRP(1、5、10和20 mg/L)刺激组PAPP-A 的mRNA表达分别为0.2544±0.0611、0.4177±0.1200、0.5828±0.0152和0.6837±0.1360,rhTNF-α(5、10、25、50和100 ng/ml)刺激组分别为0.2424±0.1378、0.3335±0.0196、0.5742±0.0131、0.6913±0.0219和0.8203±0.0413.放线菌素D(1 μg/ml)能抑制CRP和rhTNF-α对单核细胞PAPP-A mRNA表达的诱导作用.结论 促炎因子CRP和rhTNF-α可在转录水平直接调控人外周血单核细胞PAPP-A的基因表达,这可能是急性冠状动脉综合征患者循环中PAPP-A水平升高的机制之一.     

Does smoking status influence the effect of physical exercise on fibrinolytic function in healthy volunteers?

Exercise has been reported to simultaneously trigger and protect against sudden death, the so-called “The Paradox of Exercise”. Differences in fibrinolytic function appear to exist between chronic and acute exercise. The aim of the present study was to assess the fibrinolytic system after strenuous exercise in healthy people and explored the influence of smoking habit. Methods: 23 healthy male volunteers were studied (14 non-smokers; 9 current smokers). Citrated plasma blood samples were taken before and 30 minutes after a maximal exercise treadmill test, and levels of tissue type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor (PAI-1) antigen and lipoprotein-a, Lp(a), [all ELISA] were measured as indices of fibrinolytic function. Results: Smokers had higher body mass index and higher heart rate at baseline than non smokers (p = 0.046 and p = 0.001, respectively). At baseline, smokers showed increased plasma Lp(a) levels than non smokers (p = 0.04), with no differences in t-PA and PAI-1 antigen levels. Following the exercise treadmill test, smokers had a shorter exercise duration and lower exercise capacity than non smokers (p = 0.008 and p = 0.004, respectively). This was associated with a reduction in t-PA antigen levels in the whole study population, (p = 0.048) without differences in PAI-1 levels, with no significant differences between smokers and non smokers. Lp(a) levels were also significantly reduced (p = 0.0001). Conclusions: Acute exercise alters plasma tPA antigen and Lp(a) levels, but there was no significant effect of smoking status in healthy subjects.     

The renin-angiotensin system is involved in the production of plasminogen activator inhibitor type 1 by cultured endothelial cells in response to chylomicron remnants.

Triglyceride-rich lipoproteins have been suggested to promote atherosclerosis. Plasminogen activator inhibitor type 1 (PAI-1) plays an important role in the events of cardiovascular pathophysiology. The renin-angiotensin system influences various vascular functions, including PAI-1 production. We examined whether or not chylomicron remnants increased PAI-1 mRNA and protein production in endothelial cells and whether or not an inhibition of the renin-angiotensin system interfered with this effect. Chylomicron remnants were isolated from functionally hepatectomized rats injected with chylomicrons. Human umbilical vein endothelial cell cultures (HUVECs) were incubated with chylomicron remnants with or without an angiotensin-converting enzyme inhibitor (temocaprilat), an angiotensin II receptor type 1 antagonist (RNH-6270), or an angiotensin II receptor type 2 antagonist (PD123319). Chylomicron remnants increased PAI-1 secretion in HUVECs (0.5 microg/ml; 128.3 +/- 6.1%, the mean +/- SEM) as well as angiotensin II (10 nmol/l; 130.7 +/- 9.5%) in 18 h, as compared with the controls, as well as stimulated PAI-1 mRNA expression to a maximum level at 4 h. Temocaprilat and RNH-6270, but not PD123319, attenuated all of these effects. Chylomicron remnants enhanced nuclear extract binding to a very low-density lipoprotein response element in the PAI-1 promoter region and activated nuclear factor-kappaB. Extracellular signal-regulated kinase (ERK 1/2) was phosphorylated in response to chylomicron remnants. These effects were inhibited by temocaprilat or RNH-6270. In conclusion, chylomicron remnants increased protein secretion and mRNA expression of PAI-1 in HUVECs. Inhibition of the renin-angiotensin system reduced this stimulation.