Analgesic, antiulcer, antithrombotic drugs and organ damage: a population-based case-control study.

AIM: Oral medication is of paramount importance for pain treatment. Analgesics, antiulcer (AUDs) and antithrombotic drugs (ATDs) are often coprescribed in elderly people. Non-steroidal anti-inflammatory drugs (NSAIDs) require AUDs to lower the risk of peptic ulcer, and potentially interfere with ATDs. The aim of this study was to quantify the prevalence of NSAID use in patients with gastrointestinal, cardiac or kidney damage in the year 2013, compared to the general population. METHODS: We performed a population-based case-control study in the Republic of San Marino to evaluate the Odds-Ratios for upper gastrointestinal damage (gastroduodenal ulcers and/or erosions, GUE), ischemic heart disease (IHD), heart failure (HF), and renal function impairment (assessed using the CKD-EPI formula), in people who had taken AUDs, ATDs, or NSAIDs in the previous 90 days, versus people who had not taken such drugs in the same period of time. RESULTS: We found that AUDs decreased the OR for GUE (OR: 0.762; CI:0.598-0.972), while ATDs and NSAIDs increased the risk (OR: 1.238 and CI: 0.935-1.683; OR:1.203 and CI:0.909-1.592, respectively). NSAIDs seemed to increase the risk of IHD, although this was not statistically significant (OR=1.464; CI=0.592-3.621). AUDs and ATDs significantly increased the risk of renal function impairment (OR=1.369 and CI=1.187-1.579; OR=1.818 and CI=1.578-2.095, respectively), while this effect was not observed for NSAIDs. CONCLUSION: NSAIDs may induce gastrointestinal and cardiovascular damage, not only by themselves, but also when used concomitantly with common medications such as AUDs or ATDs, due to additive and/or synergistic effects. We performed a "pragmatic" analysis of the association of organ damage with use of NSAIDs/AUDs/ATDs, including patient age, treatment duration and dose, to allow for an immediate application of our findings to everyday clinical practice.

Statins prevent tissue factor induction by protease-activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro.

BACKGROUND: Protease-activated receptors (PARs) are G-protein-coupled receptors that function in hemostasis and thrombosis, as well as in the inflammatory and proliferative responses triggered by tissue injury. We have previously shown that PAR1 or PAR2 occupancy by specific PAR-agonist peptides (PAR-APs) induces tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs), where TF regulation by PAR1 (but not by PAR2) requires intact endothelial caveolin-enriched membrane microdomains in which PAR1 and caveolin-1 associate. OBJECTIVES: The aim of this study was to determine the effects of cholesterol-lowering agents (statins) and cholesterol-loading lipoprotein on PAR1-AP-mediated and PAR2-AP-mediated TF induction in HUVECs. RESULTS: Statins completely prevented TF induction by PAR-APs in an isoprenoid-independent manner, induced the delocalization of PAR1 from caveolin-enriched membrane microdomains without affecting PAR1 mRNA, and decreased PAR2 mRNA and protein levels. Statins also prevented PAR-AP-mediated extracellular signal-related kinase 1/2 activation, which is crucial for TF induction. The redistribution of PAR1 is accompanied by the relocation of the membrane microdomain-associated G-protein α, caveolin-1, and Src, which we previously showed to play a key role in signal transduction and TF induction. Conversely, cholesterol loading potently amplified PAR1-AP-induced TF, probably as a result of the increased abundance of PAR1 and the Src and G-protein α signaling molecules in the caveolin-1-enriched fraction, without affecting PAR1 mRNA. CONCLUSIONS: As PARs have important functions in hemostasis, cancer, thrombosis, and inflammatory processes, our findings that statins prevent TF induction by PAR-APs altering the membrane localization of PAR1 and the expression of PAR2 suggest that they may provide health benefits other than reducing atherosclerosis.

Mitochondrial reactive oxygen species: a common pathway for PAR1- and PAR2-mediated tissue factor induction in human endothelial cells.

BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique proteolytic activation mechanism. PARs regulate a broad range of cellular functions and are involved in the pathogenesis of inflammatory disorders. Moreover, PAR1 and PAR2 activation in the endothelium shifts it toward a prothrombotic condition. OBJECTIVES: To assess the relevance of intracellular reactive oxygen species (ROS) in the signaling events underlying tissue factor (TF) expression elicited by PAR1 and PAR2 occupancy in endothelial cells, and to investigate their source. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to specific PAR1 and PAR2 agonist peptides. TF expression was determined by real-time reverse transcription polymerase chain reaction analysis and measurement of procoagulant activity. ROS generation was determined by a fluorometric assay after cell loading with 2'-7'-dichlorofluorescein diacetate. RESULTS: ROS generated by the mitochondrial chain, mostly from complex III, provide a pathway through which PAR1 and PAR2 occupancy induces TF. Other sources of ROS do not participate in TF induction. Activation of both ERK1/2 and p38 MAPK is critical for mitochondrial ROS generation. In addition to these pathways shared by the two PARs, mechanisms downstream from PAR1 and PAR2 activation, different for the two receptors, also induced TF. A module that sensitively regulates PAR1 signaling and ultimately involves NF-kappaB activation has been identified. CONCLUSIONS: Our data identify ROS originating in mitochondria as key mediators of the signaling pathways triggered by PAR1 and PAR2 engagement in endothelial cells and show that downstream from receptor activation occur cascades that are mechanistically coupled to procoagulant activity.

Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells.

BACKGROUND: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. OBJECTIVES: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. METHODS: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. RESULTS: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively). CONCLUSIONS: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.

Distinct roles for PAR1- and PAR2-mediated vasomotor modulation in human arterial and venous conduits.

BACKGROUND: Patency rates after coronary artery bypass grafting (CABG) are better if the internal mammary artery (IMA) is used rather than the greater saphenous vein (GSV), and may be related to the endothelial release of vasodilators antagonizing vascular contraction. It has recently been shown that a family of protease-activated receptors (PARs) modulate endothelium-dependent vasodilatation. OBJECTIVE AND METHODS: The aim of this study was to evaluate the presence and functional role of protease-activated receptor 1 (PAR1) and protease-activated receptor 2 (PAR2) in mediating vascular tone in IMAs and GSVs from patients undergoing CABG by means of real time-PCR and isometric tension measurements. RESULTS: PAR1 mRNA levels were higher than those of PAR2 mRNA in both vessels. A selective PAR2-activating peptide (PAR2-AP), SLIGKV-NH(2) (0.01-100 micromol L(-1)), failed to induce vasorelaxation in precontracted IMA and GSV rings, whereas the selective PAR1-AP, TFLLR-NH(2) (0.001 to 10 micromol L(-1)), caused greater endothelium-dependent relaxation in the IMAs (pD(2) values 7.25 +/- 0.6 vs. 7.86 +/- 0.42, P < 0.05; E(max) values 56.2 +/- 17.3% vs. 29.7 +/- 13.4%, P < 0.001). Preincubation with TNFalpha (3 nmol L(-1)) induced vasorelaxation in IMAs in response to PAR2-AP (P < 0.05 vs. non-stimulated vessels); the response to PAR1-AP was unchanged. The relaxation induced by both PAR-APs was NO- and endothelium-dependent. CONCLUSION: These data show that functionally active PAR1 and PAR2 are present in IMAs and GSVs, and that inflammatory stimuli selectively enhance endothelium-dependent relaxation to PAR2-AP in IMAs.

Oxidized LDLs influence thrombotic response and cyclooxygenase 2.

Oxidative modification of low-density lipoproteins (LDLs) plays a key role in the development of atherosclerosis and the onset of coronary artery disease. LDL oxidation alters the antithrombotic balance of human endothelial cells inducing surface tissue factor (TF) pathway activity, which results in enhanced fibrin deposition. Fibrinolysis, which is strictly regulated by plasminogen activator inhibitor-1 (PAL-1) and tissue-type plasminogen activator (tPA). Is also dysregulated by LDL oxidation with a net increase in the inhibitory rate. Oxidized LDLs (oxLDLs) also affect many aspects of macrophage function linked to the inflammatory response of these cells, In particular, oxLDLs downregulate inducible cyclooxigenase (Cox-2) in human monocyte-derived macrophages exposed to bacterial lipopolysaccharide. This observation may support the hypothesis that, within atheromata, the transformation macrophages into foam cells results in the attenuation of the inflammatory response, thus contributing to the progression of athrogenesis. Among lipid constituents of oxLDLs, Ox-PAPC, a mixture of oxidized arachidonic acid-containing phospholipids, prevents Cox-2 expression, suggesting that it could be considered responsible for the biological activity of oxLDLs.

[PAI-1, the primary plasmatic inhibitor of fibrinolysis. Physiopathologic role and molecular mechanisms]. / PAI-1, il principale inibitore plasmatico della fibrinolisi. Ruolo fisiopatologico e meccanismi molecolari.

Plasminogen activator inhibitor 1 (PAI-1) is the primary physiological inhibitor of plasminogen activation in blood. PAI-1 is known to contribute to thrombus formation and to the development and the clinical course of acute and chronic cardiovascular diseases. Plasma levels of PAI-1 are regulated on a genetic basis but, more important, is the dependence on a series of other atherosclerotic risk factors like hypertriglyceridemia, diabetes and insulin resistance. The insulin resistance syndrome, which is characterized partly by obesity with visceral fat accumulation, is considered as a major regulator of PAI-1 expression. At least in vitro, insulin is a potent inducer of PAI-1 synthesis by human hepatic cells, and, we have recently disentangled the molecular mechanisms responsible for enhanced PAI-1 gene expression by insulin. However, clinical data fail to support a direct acute contribution of insulin in regulating circulating PAI-1 levels. Recently, it has been proposed that adipose tissue could be responsible for the elevated plasma PAI-1 level observed in insulin resistance. It now seems reasonable to view PAI-1 as one of the factors contributing to the complex gene-environment interactions involved in the formation and dissolution of thrombi.

Transcriptional regulation of plasminogen activator inhibitor type 1 gene by insulin: insights into the signaling pathway.

Impairment of the fibrinolytic system, caused primarily by increases in the plasma levels of plasminogen activator inhibitor (PAI) type 1, are frequently found in diabetes and the insulin-resistance syndrome. Among the factors responsible for the increases of PAI-1, insulin has recently attracted attention. In this study, we analyzed the effects of insulin on PAI-1 biosynthesis in HepG2 cells, paying particular attention to the signaling network evoked by this hormone. Experiments performed in CHO cells overexpressing the insulin receptor indicate that insulin increases PAI-1 gene transcription through interaction with its receptor. By using inhibitors of the different signaling pathways evoked by insulin-receptor binding, it has been shown that the biosynthesis of PAI-1 is due to phosphatidylinositol (PI) 3-kinase activation, followed by protein kinase C and ultimately by mitogen-activated protein (MAP) kinase activation and extracellular signal-regulated kinase 2 phosphorylation. We also showed that this pathway is Ras-independent. Transfection of HepG2 cells with several truncations of the PAI-1 promoter coupled to a CAT gene allowed us to recognize two major response elements located in the regions between -804 and -708 and between -211 and -54. Electrophoretic mobility shift assay identified three binding sites for insulin-induced factors, all colocalized with putative Sp1 binding sites. Using supershifting antibodies, the binding of Sp1 could only be confirmed at the binding site located just upstream from the transcription start site of the PAI-1 promoter. A construct comprising four tandem repeat copies of the -93/-62 region of the PAI-1 promoter linked to CAT was transcriptionally activated in HepG2 cells by insulin. These results outline the central role of MAP kinase activation in the regulation of PAI-1 induced by insulin.

Effect of valsartan on angiotensin II-induced plasminogen activator inhibitor-1 biosynthesis in arterial smooth muscle cells.

Previous studies have shown that angiotensin II stimulates the synthesis of plasminogen activator inhibitor-1 in cultured vascular cells, which suggests that activation of the renin-angiotensin system may impair fibrinolysis. We have investigated the effects of angiotensin II and of valsartan, a recently developed angiotensin II antagonist that is highly specific and selective for the angiotensin II subtype 1 receptor, on plasminogen activator inhibitor-1 secretion by smooth muscle cells isolated from rat and human vessels. Angiotensin II induced a time- and concentration-dependent increase of plasminogen activator inhibitor activity in supernatants of rat aortic cells, which reached a plateau after 6 hours of incubation with 100 nmol/L angiotensin II (2.4+/-0.6-fold over control value; P:<0.001). The angiotensin II-induced plasminogen activator inhibitor activity was inhibited, in a concentration-dependent manner, by valsartan with an IC(50) value of 21 nmol/L. Valsartan fully prevented the angiotensin II-induced increase in plasminogen activator inhibitor-1 protein and mRNA. Furthermore, angiotensin II doubled the secretion of plasminogen activator inhibitor-1 by smooth muscle cells obtained from human umbilical and internal mammary arteries, and valsartan fully prevented it. Angiotensin II did not affect the secretion of tissue plasminogen activator antigen by any of the cell systems tested. Thus, valsartan effectively inhibits angiotensin II-induced plasminogen activator inhibitor-1 secretion without affecting that of tissue plasminogen activator in arterial rat and human smooth muscle cells.

Magnesium inhibits arterial thrombi after vascular injury in rat: in vivo impairment of coagulation.

Magnesium deficiency is associated with a high frequency of cardiac arrhythmia, hypertension and sudden ischemic death. We investigated the if vivo effects of intravenous magnesium administration in a rat model of chemically induced (FeCl3) carotid thrombosis. The infusion of magnesium sulfate (MgSO4) before the topical application of FeCl5 prevented thrombus formation at concentrations of 0.3 M and 0.6 M, and delayed it even at 0.15 M. Similar results were obtained with MgCl2. The infusion of MgSO4 0.6 M seven minutes after FeCl3 application delayed but did not prevent thrombus formation. MgSO4 slightly reduced platelet aggregation ex vivo without affecting plasma clotting tests, but in vivo blood clotting time was markedly prolonged (tail transection method), thus indicating profoundly impaired coagulation. These data provide a rationale for the use of magnesium as an antithrombotic agent. but its pharmacological effect critically depends on the timing of administration.

Fluvastatin inhibits basal and stimulated plasminogen activator inhibitor 1, but induces tissue type plasminogen activator in cultured human endothelial cells.

The effects of fluvastatin, a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitor, on the biosynthesis of tissue plasminogen activator (t-PA) and of its major physiological inhibitor (plasminogen activator inhibitor type 1, PAI-1) were investigated in cultured human umbilical vein endothelial cells (HUVEC). Fluvastatin (0.1 to 2.5 microM), concentration-dependently reduced the release of PAI-1 antigen by unstimulated HUVEC, subsequent to a reduction in PAI-1 steady-state mRNA levels and de novo protein synthesis. In contrast, it increased t-PA secretion. The drug also reduced PAI-1 antigen secreted in response to 10 microg/ml bacterial lipopolysaccharide (LPS), 100 U/ml tumour necrosis factor alpha (TNFalpha) or 0.1 microM phorbol myristate acetate (PMA). Mevalonate (100 microM), a precursor of isoprenoids, added to cells simultaneously with fluvastatin, suppressed the effect of the drug on PAI-1 both in unstimulated and stimulated cells as well as on t-PA antigen. Among intermediates of the isoprenoid pathway, all-transgeranylgeraniol (5 microM) but not farnesol (10 microM) prevented the effect of 2.5 microM fluvastatin on PAI-1 antigen, which suggests that the former intermediate of the isoprenoid synthesis is responsible for the observed effects.

Relationship between fibrinolytic and metabolic variables: a study in patients attending a lipid clinic.

We investigated the relationship between plasma levels of metabolic and fibrinolytic variables in 163 fasted patients attending a lipid clinic. Of these patients, 118 had hypertriglyceridaemia (HTG) and 45 had normotriglyceridaemia (NTG). In HTG, basal fibrinolytic activity, ie tissue plasminogen activator (t-PA) activity, was impaired as a result of high plasminogen activator inhibitor type 1 (PAI-1) antigen and activity. Multiple stepwise regression analysis identified insulin and triglyceride levels as independent determinants of plasma PAI-1 levels (R2 = 0.18; P = 0.0001). When the patients were stratified into tertiles according to their levels of triglyceride and insulin, PAI-1 antigen levels were found to increase with rising levels of triglyceride in each insulin tertile. In contrast, the increase of PAI-1 with rising insulin levels was evident in the highest triglyceride tertile. In addition, subjects in the lowest tertile of both triglyceride and insulin had the lowest PAI-1 antigen levels, and subjects in the highest tertile of both triglyceride and insulin had the highest levels of PAI-1. Both basal and stimulated levels of t-PA antigen were significantly higher in HTG than in NTG. Multiple stepwise regression analysis identified triglyceride level as the sole major determinant of t-PA antigen levels (R2 = 0.13; P = 0.00003). The observation that both insulin and triglycerides correlate with PAI-1, whereas triglycerides were involved only in the increased secretion of t-PA, suggests that these two proteins are regulated by different mechanisms.

Effects of gemfibrozil on insulin sensitivity and on haemostatic variables in hypertriglyceridemic patients.

In order to assess the efficacy of gemfibrozil on lipid and haemostatic parameters in patients with plurimetabolic syndrome, a multicenter double-blind placebo controlled, parallel study was carried out in 56 patients with primary hypertriglyceridemia and glucose intolerance. These patients had elevated PAI activity and antigen and t-PA antigen levels at rest and after venous occlusion. Gemfibrozil reduced plasma triglyceride levels (P<0.001), whereas it increased free fatty acids (P<0.05) and high density lipoprotein cholesterol levels (P<0.05). In those patients reaching normalization of plasma triglyceride levels (triglyceride reduction > or =50%) (n=15), insulin levels (P<0.05) as well as the insulin resistance index were reduced by gemfibrozil treatment, suggesting an improvement of the insulin resistance index in this patient subgroup. Gemfibrozil treatment did not affect plasma fibrinolysis or fibrinogen levels, despite marked reduction of plasma triglycerides and improvement of the insulin sensitivity associated with triglyceride normalization.

Very low density lipoprotein-mediated signal transduction and plasminogen activator inhibitor type 1 in cultured HepG2 cells.

In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[(3)H]inositol-labeled HepG2 cells, VLDL (100 microg/mL) caused a time-dependent increase in [(3)H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all P<0.01 versus VLDL-treated cells). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)-octyl ester, which prevents Ca2+ release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% (P<0.05), and the MAP kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 completely suppressed both basal and VLDL-induced PAI-1 secretion. These data demonstrate that VLDL-induced PAI-1 biosynthesis results from a principal signaling pathway involving PKC-mediated MAP kinase activation.

Increased thrombogenic potential of human monocyte-derived macrophages spontaneously transformed into foam cells.

This study investigated whether spontaneous lipid enrichment of human macrophages affects their thrombogenic potential as measured by increased production of tissue factor (TF) and plasminogen activation inhibitor types 1 and 2 (PAI-1 and PAI-2). Macrophages were obtained following a 7-day culture period of monocytes, isolated from the same donor, in autologous serum (HS) or in fetal bovine serum (FBS). Those cultured in HS underwent marked lipid accumulation relative to those cultured in FBS that was accompanied by increased production of TF and PAI-1, but not of PAI-2, and decreased production of interleukin-1beta. They also contained more arachidonic and linoleic acid and lower amounts of n-3 polyunsaturated fatty acids, particularly docosahexaenoic acid (22: 6). These data indicate that the transformation of macrophages into foam cells results in an increase in their thrombogenic and antifibrinolytic potential and provide a possible explanation of the thrombotic sequelae frequently consequent on plaque fissuring and disruption.

Endothelial tissue factor induction by T lymphocytes in systemic sclerosis.

We evaluated the ability of circulating T lymphocytes obtained from patients with systemic sclerosis (SSc) to induce the expression of tissue factor (TF) by human umbilical vein endothelial cells (HUVECs), and compared the results with those obtained from healthy controls. Nine patients with SSc and 10 sex- and age-matched healthy subjects were studied. Peripheral T lymphocytes obtained from SSc patients induced TF activity from HUVECs in a dose-dependent manner. A significant induction of endothelial TF was observed when 2 x 10(6) lymphocytes per well (TF values, mean+/-SD: 0.34+0.21U/microg of cell protein vs 0.04+/-0.03; n = 9, p = 0.001) or 1 x 10(6) lymphocytes per well (0.13+/-0.06 vs 0.04+/-0.04; n = 8, p<0.001) were added to HUVEC cultures. Lower concentrations of T lymphocytes were ineffective. Similar results were obtained with control lymphocytes. There were no differences in endothelial TF induction between patients and controls at any lymphocyte concentration tested. Within the SSc group, there were no correlations between TF activity and clinical features or disease duration.

The effect of synthetic malaria pigment (beta-haematin) on adhesion molecule expression and interleukin-6 production by human endothelial cells.

The effects of synthetic malaria pigment (beta-haematin, BH) on the expression of the intercellular adhesion molecule 1 (ICAM-1) and platelet endothelial cell adhesion molecule 1 (PECAM-1) and the production of interleukin-6 (IL-6) by human microvascular endothelial cells were measured using flow cytometry analysis and immunoenzymatic assay. BH alone did not affect basal levels of ICAM-1, PECAM-1 or IL-6. When added to cell cultures before or with, but not after, lipopolysaccharide or tumour necrosis factor alpha, BH at 1-100 micrograms/mL induced a dose-dependent inhibition of ICAM-1 and PECAM-1 expression and IL-6 production. Cell viability and human leucocyte antigen A,B,C expression remained unaffected. Similar, though more variable, results were obtained using human umbilical vein endothelial cells. These results suggested that accumulation of pigment within endothelial cells following repeated malaria infection reduces local inflammation and parasite sequestration through inhibition of either cytokine production or parasitized erythrocyte receptors on endothelial cells.

Platelet activation supports the development of venous thrombosis in hyperlipidemic rats.

This investigation sought to determine how different components of the hemostatic system affect the development of venous thrombosis in rats displaying hyperlipidemia, either on a genetic basis or secondary to metabolic disorders. On employing an experimental model of collagen-triggered venous thrombosis, both spontaneously hyperlipidemic (Yoshida strain) and streptozotocin-induced diabetic rats generated about 2.3-fold greater thrombi than normolipidemic controls. This was associated with significant platelet activation, as revealed by increased levels of serum thromboxane B2 in diabetics (1.5-fold) as well as in Yoshida (8-fold) rats, in comparison with controls. In contrast, ex vivo total fibrinolytic activity, as measured by euglobulin lysis time, did not differ between normo- and hyperlipidemic or diabetic animals. Plasminogen activator inhibitor activity was lower in both Yoshida and diabetic rats than in controls. However, tissue-type plasminogen activator activity was differently affected by the genetic or the diabetes-related hyperlipidemia, showing significantly lower values in Yoshida (-26%), but significantly higher values in diabetic rats (+29%) than in normolipidemic controls. We conclude that platelet activation, rather than consistent modifications of the fibrinolytic system, is likely to influence the enhanced thrombus development associated with primary or secondary forms of hyperlipidemia.

Linoleic acid enhances the secretion of plasminogen activator inhibitor type 1 by HepG2 cells.

This study was undertaken in order to assess whether triglycerides and/or their fatty acids directly influence the secretion of plasminogen activator inhibitor type 1 (PAI-1) in HepG2 cells. To this end, subconfluent HepG2 cells were incubated with triglyceride-rich particles (TGRP) isolated from Intralipid for 16 h, and PAI-1 levels were determined in conditioned medium using a specific ELISA. TGRP (1 to 6 mg triglycerides/ml) concentration-dependently increased PAI-1 secretion by cells, concomitantly with significant increases in intracellular triglyceride (TG) levels. Fatty acid analysis indicated that the incubation of cells with 3 mg of TG per ml of TGRP induced significant accumulation of 18:2 n-6 (linoleic acid, LA) and 18:3 n-3 (linolenic acid) reflecting the fatty acid composition at the added triglycerides. We then tested the comparative effects on PAI-1 secretion by HepG2 cells of LA and 18:1 n-9 (oleic acid, OA). LA, as a bovine serum albumin (BSA) complex, concentration-dependently (1 to 35 mumol/L) increased the secretion of PAI-1 by cells, whereas OA-BSA only minimally affected it at the highest concentration used (35 mumol/L). Incorporation of LA into cell pools, in the presence of increasing concentration of the FA in the medium, was studied by the use of a preparation containing [14C]LA. LA accumulated in all lipid classes including diacylglycerol, the incorporated LA being converted into arachidonic acid (AA) as assessed by HPLC radiochromatography of the fatty acid methyl esters. It is concluded that PAI-1 secretion in HepG2 cells is modulated by triacylglycerols and by linoleic acid and/or its metabolic products.