Metalloproteases, vascular remodeling and atherothrombotic syndromes

Defects in the synthesis and breakdown of the extracellular matrix (ECM) are now seen as key processes in the development of atherosclerosis and its thrombotic complications. Correlations have been observed between circulating levels of ECM biomarkers and the clinical manifestations of and risk factors for atherosclerosis. Several matrix metalloproteinases (MMPs), endopeptidases that can degrade the ECM, such as MMP-9 and MMP-10, play important roles in the pathophysiology of atherothrombosis and contribute to the expansion of abdominal aortic aneurysms. Moreover, they may also be useful biomarkers of atherosclerotic risk and serve as predictors of coronary and cerebrovascular disease recurrence. Although at present the effect of tissue inhibitors of MMPs (TIMPs) on cardiovascular disease prognosis is still uncertain, the ECM could be a promising therapeutic target in atherothrombotic disease, and several MMP inhibitors are currently undergoing clinical trials

Validation of plasma fibrinogen as a marker of carotid atherosclerosis in subjects free of clinical cardiovascular disease

BACKGROUND AND OBJECTIVES: Fibrinogen has been found to be an independent risk factor for cardiovascular disease. The aim of this study was to validate the measurement of plasma fibrinogen as a marker of subclinical atherosclerosis in a series of asymptomatic subjects (n=519, median age 55.5 years, 80% men). DESIGN AND METHODS: All individuals had a complete clinical examination, lipid profile (cholesterol and its high and low density lipoprotein fractions and triglycerides), global vascular risk assessment (PROCAM), and B-mode ultrasonography of the carotid arteries to determine the intima-media thickness (IMT) and the presence of atheroma plaques. C-reactive protein (CRP), and von Willebrand factor (vWF) were also measured in all subjects as markers of inflammation/endothelial damage. RESULTS: In the univariate model, a positive relationship was found between plasma fibrinogen concentration and carotid IMT (p<0.001). Fibrinogen concentration also correlated positively with age (p<0.001), systolic blood pressure (p<0.001), smoking (p<0.05), diabetes (p<0.05), PROCAM (p<0.001), CRP and vWF (p<0.001). In the multivariate analysis, the association of fibrinogen with carotid IMT remained significant (p=0.008) after adjustment for all parameters analyzed. INTERPRETATION AND CONCLUSIONS: In a population sample of adults without clinically overt atherosclerotic disease, elevated fibrinogen levels was related to carotid IMT independently of a wide range of important confounding variables. Plasma fibrinogen may represent a systemic marker of carotid atherosclerosis

The 4G/5G PAI-1 polymorphism influences the endothelial response to IL-1 and the modulatory effect of pravastatin

BACKGROUND: Increased plasminogen activator inhibitor (PAI-1) levels lead to impaired fibrinolytic function associated with higher cardiovascular risk. PAI-1 expression may be regulated by different inflammatory cytokines such as interleukin-1alpha (IL-1). Several polymorphisms have been described in the PAI-1 gene. AIM: We examined the influence of the 4G/5G polymorphism in the promoter region on IL-1alpha-induced PAI-1 expression by human umbilical vein endothelial cells (HUVEC) in presence or absence of pravastatin. METHODS AND RESULTS: Genotyped HUVEC were incubated with IL-1alpha (500 U mL(-1)) in presence or absence of pravastatin (1-10 microm). PAI-1 expression was analyzed by real time polymerase chain reaction (PCR), and PAI-1 antigen measured in supernatants by ELISA. IL-1alpha increased PAI-1 secretion in a genotype-dependent manner, and higher values were observed for 4G/4G compared with both 4G/5G and 5G/5G cultures (P < 0.05). Preincubation of HUVEC with 10 microm pravastatin significantly reduced IL-1-induced PAI-1 expression in 4G/4G HUVEC compared with untreated cultures (177.5% +/- 24.5% vs. 257.9% +/- 39.0%, P < 0.05). Pravastatin also attenuated the amount of secreted PAI-1 by 4G/4G HUVEC after IL-1 stimulation (5020.6 +/- 165.7 ng mL(-1) vs. 4261.1 +/- 309.8 ng mL(-1), P < 0.05). This effect was prevented by coincubation with mevalonate, indicating a dependence on HMG-CoA reductase inhibition. CONCLUSIONS: The endothelial 4G/5G PAI-1 genotype influences the PAI-1 response to IL-1alpha and the modulatory effect of pravastatin. As increased PAI-1 levels have been linked to cardiovascular disease the observed endothelial modulation by pravastatin may have potential clinical implications

Evidence that heparin but not hirudin reduces PAI-1 expression in cultured human endothelial cells

Heparin and other antithrombotic drugs besides their anticoagulant action could have a profibrinolytic effect. We have analyzed the effect of unfractionated heparin (UFH) and hirudin on PAI-1 gene expression in human umbilical vein endothelial cells (HUVEC). Cells were stimulated with UFH (1 and 10 IU/ml) and hirudin (20 and 100 TIU/ml). Samples were obtained before and 2, 6, and 24 hours after stimulation. mRNA analysis was conducted by reverse transcription followed by polymerase chain reaction, and PAI-1 antigen was determined by ELISA. Addition of UFH (10 IU/ml) to HUVEC resulted in a decrease of PAI-1 mRNA at 6 hours (40% reduction) and 24 hours (60% reduction) and PAI-1 antigen. Hirudin, however, did not modify significantly the PAI-1 mRNA nor the inhibitor secretion. The addition of UFH (10 or 100 IU/ml) to endotoxin-stimulated HUVEC also reduced the increased PAI-1 mRNA and antigen secretion (45%), whereas no effect could be observed with hirudin. Our results suggest that UFH, but not hirudin, by reducing the endothelial expression of PAI-1 might have a profibrinolytic effect

Effects of low molecular weight heparin, alone or combined with antithrombin III, on mortality, fibrin deposits and hemostatic parameters in endotoxin-induced disseminated intravascular coagulation in rabbits

The effect of low molecular weight heparin (LMWH) with or without antithrombin III (AT III) has been studied in a rabbit model of disseminated intravascular coagulation (DIC) induced by continuous infusion of 100 microg/kg/hr of Escherichia coli endotoxin for 6 hr. LMWH (5 and 10 IU/kg/hr/6 hr), alone or in combination with AT III (20 U/kg/hr/6 hr), or saline were administered simultaneously with endotoxin. Hemostatic markers at 0, 2, and 6 hr as well as kidney fibrin deposits and the mortality rate at 24 hr were determined. Rabbits receiving only endotoxin showed an impairment in hemostasis, as well as high kidney fibrin deposits and a high mortality rate. LMWH alone did not exert any effect. The simultaneous infusion of LMWH and AT III exerted a beneficial effect on the hemostatic markers and reduced the kidney fibrin deposits as well as the mortality rate in a LMWH dose-dependent manner. Fibrinogen and protein C consumption were significantly higher and renal fibrin deposits more intense in the rabbits that had died in the first 24 hr. There was also a significant positive correlation between kidney fibrin deposits and platelets, fibrinogen, and protein C consumption, taking the whole rabbit population. It is concluded that the simultaneous infusion of LMWH and AT III is useful in this DIC model and would make it possible to reduce significantly the AT III doses used when AT III is given alone

Effect of the administration of recombinant hirudin and/or tissue-plasminogen activator (t-PA) on endotoxin-induced disseminated intravascular coagulation model in rabbits

We evaluated the effect of r-hirudin and/or tissue-plasminogen activator (t-PA) in a model of DIC in rabbits induced by i.v. infusion of 100 micrograms/kg/h/6 h endotoxin. Rabbits were treated with saline (endotoxin control group), r-hirudin at 0.3 mg/kg/h/6 h, t-PA at 0.3 mg/kg for 90 min and r-hirudin plus t-PA at the doses described above. The best results were achieved when r-hirudin and t-PA were infused together. This treatment reduced the consumption of platelets and protein C and attenuated the increase of PAI-1 more efficiently than r-hirudin or t-PA alone. r-Hirudin plus t-PA also resulted in the lowest formation of fibrin deposits in the kidneys. Finally, mortality at 24 h dropped from 70% in the endotoxin control group to 40%, 10% and 0% in the t-PA, r-hirudin and r-hirudin plus t-PA groups respectively. None of the t-PA-infused rabbits which had died by 24 h showed macroscopic signs of haemorrhage. r-Hirudin alone was better than t-PA alone, as was shown by fibrin deposits and mortality. We conclude that r-hirudin and t-PA given simultaneously were more efficient than either given alone in this model of DIC. Effective thrombin inhibition, which could influence other pathophysiological mechanisms apart from coagulation, together with the improvement in fibrinolysis, would explain these results

Protective effect of the G-765C COX-2 polymorphism on subclinical atherosclerosis and inflammatory markers in asymptomatic subjects with cardiovascular risk factors

BACKGROUND: Cyclooxygenase (COX)-2, a key regulatory enzyme in prostanoid synthesis, plays an important role in inflammatory processes. The -765G>C COX-2 polymorphism has been associated with lower promoter activity in vitro and reduced levels of C-reactive protein (CRP) in atherosclerotic carriers of the C allele. However, its pathophysiological relevance in vivo has not been fully elucidated. METHODS AND RESULTS: We assessed the -765G>C polymorphism and COX-2 expression in 220 asymptomatic subjects free of cardiovascular disease, in relation to global vascular risk, carotid intima-media thickness (IMT), and inflammatory markers (fibrinogen, C-reactive protein [CRP], von Willebrand factor [vWF] and interleukin-6 [IL-6]). Genotype frequencies were: CC (7.7%), CG (34.5%), GG (57.7%). Among hypercholesterolemic subjects (n=140), C allele carriers had lower COX-2 expression (p<0.05), reduced carotid IMT (p<0.01) and diminished levels of inflammatory markers CRP, vWF and IL-6 (p<0.05), as compared to GG homozygous subjects. The association between carotid IMT and COX-2 polymorphism remained significant after adjusting for cardiovascular risk factors and inflammatory markers (p=0.008). CONCLUSIONS: In asymptomatic hypercholesterolemic subjects the C allele of -765G>C COX-2 polymorphism was associated with lower COX-2 expression, and reduced subclinical atherosclerosis and systemic inflammation compared with GG homozygous, thus conferring atherosclerosis protection in this cardiovascular risk population

Preliminary characterisation of the promoter of the human p22phox gene: identification of a new polymorphism associated with hypertension

The p22(phox) subunit is an essential protein in the activation of NAD(P)H oxidase. Here we report the preliminary characterisation of the human p22(phox) gene promoter. The p22(phox) promoter contains TATA and CCAC boxes and Sp1, gamma-interferon and nuclear factor kappaB sites. We screened for mutations in the p22(phox) promoter and identified a new polymorphism, localised at position -930 from the ATG codon, which was associated with hypertension. Mutagenesis experiments showed that the G allele had higher promoter activity than the A allele. These results suggest that the -930(A/G) polymorphism in the p22(phox) promoter may be a novel genetic marker associated with hypertension

The CD163-expressing macrophages recognize and internalize TWEAK. Potential consequences in atherosclerosis

Background: CD163 is a new potential scavenger receptor of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) which elicits diverse biologic actions involved in atherosclerosis. We have analyzed the importance of TWEAK-CD163 interaction in atherosclerosis. Methods: TWEAK and CD163 expression was studied in cultured human macrophages. Moreover, TWEAK and CD163 expression was analyzed in carotid atherosclerotic plaques (immunohystochemistry) and plasma (ELISA). We have also assessed their potential association with intima/media thickness (IMT) in asymptomatic subjects. Results: In vitro studies revealed that CD163-expressing macrophages can bind and internalize TWEAK protein exogenously added from supernatants. Accordingly, we observed an inverse correlation between the expression of CD163 and TWEAK (r=-0.51; p=0.008) in the shoulder region of atherosclerotic plaques obtained from twenty-five patients undergoing carotid endarterectomy. The same trend was observed when we analyzed the plasma concentration of both proteins in 90 subjects free from clinical cardiovascular disease (r=-0.25; p=0.016) in which carotid ultrasonography was performed to determine IMT. In these subjects, we found a positive correlation between sCD163 and IMT (r=0.36; p<0.001) and between sCD163/sTWEAK ratio and IMT (r=0.51; p<0.001). This association remained significant after adjusting for traditional cardiovascular risk factors and inflammatory markers explaining 39% (sCD163) or 48% (sCD163/sTWEAK ratio) of IMT variance. Conclusions: Our results suggest that TWEAK-CD163 interaction take place in vivo, probably decreasing TWEAK plasma concentration. Furthermore, we have observed that CD163/TWEAK plasma ratio is a potential biomarker of clinical and subclinical atherosclerosis

Vitamins C and E attenuate plasminogen activator inhibitor-1 (PAI-1) expression in a hypercholesterolemic porcine model of angioplasty

BACKGROUND: The plasminogen activator inhibitor-1 (PAI-1), which modulates fibrinolysis and cell migration, may influence proteolysis and neointimal formation in the arterial wall contributing to restenosis after vascular injury. Antioxidants have been proposed as inhibiting multiple proatherogenic events. We explore the effect of vitamins C and E on PAI-1 expression in an experimental model of angioplasty in hypercholesterolemic pigs. METHODS AND RESULTS: A total of 44 Yucatan minipigs were divided into three diet groups: a normal-cholesterol (NC), a high-cholesterol (HC), and a high-cholesterol plus vitamins C+E (HCV) group. Balloon injury was induced in the right internal iliac artery 4 weeks after initiation of either dietary regimen, and plasma and tissue samples were taken at different time periods to measure PAI-1 activity and vascular inhibitor expression. The cholesterol-rich diet induced an increased in vascular PAI-1 expression in the intima, media and adventitia which was markedly reduced in the HCV group. After injury, severe structural changes were observed in NC and HC animals associated with increased systemic PAI-1 activity (P<0.001) and local PAI-1 expression being more intense in HC group. Vitamins C and E significantly reduced plasma PAI-1 activity (P=0.018) and attenuated the inhibitor expression as compared with HC. CONCLUSIONS: This experimental study in a porcine model of hypercholesterolemia demonstrates that vitamins C and E reduce local and systemic PAI-1 induced after angioplasty as well as the hypercholesterolemia-induced vascular PAI-1