Josune Orbe

Identification of Soluble Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (sTWEAK) as a Possible Biomarker of Subclinical Atherosclerosis

Objectives—Assessment of vascular risk in asymptomatic patients and the response to medical therapy is a major challenge for prevention of cardiovascular events. Our aim was to identify proteins differentially released by healthy versus atherosclerotic arterial walls, which could be found in plasma and serve as markers of atherosclerosis. Methods and Results—We have analyzed supernatants obtained from cultured human carotid plaques and healthy arteries by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry ProteinChip System. Surface-enhanced laser-desorption/ionization analysis unveiled an 18.4-kDa peak released in lower amount by carotid plaques than normal endarteries. This protein was identified as soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK). To confirm that sTWEAK was the protein of interest, Western blot and enzyme-linked immunosorbent assay were performed. Both techniques confirmed that sTWEAK levels were decreased in carotid plaque supernatants. Subsequent measurement of sTWEAK in plasma showed a reduced concentration in subjects with carotid stenosis (N=30) compared with healthy subjects matched by sex and age (N=28) (P<0.001). Furthermore, in a test population of 106 asymptomatic subjects, we showed that sTWEAK concentrations negatively correlated with the carotid intima-media thickness (r=−0.4; P<0.001), an index of subclinical atherosclerosis. Conclusions—These results suggest that sTWEAK could be a potential biomarker of atherosclerosis.

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 interaction was studied in cultured human macrophages. Moreover, TWEAK and CD163 expression was analyzed in carotid atherosclerotic plaques (immunohistochemistry) 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 25 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 takes 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.

Different expression of MMPs/TIMP-1 in human atherosclerotic lesions. Relation to plaque features and vascular bed

BACKGROUND Proteolytic imbalance might determine arterial remodeling and plaque destabilization in atherosclerotic vessels. The aim of this study was to examine differences in the patterns of metalloproteinases (MMPs) and MMP inhibitor (TIMP-1) expression in advanced human atheromas, both in relation to the plaque features and the vascular bed involved. METHODS AND RESULTS Immunohistochemistry for MMP-1, -3, -9 and TIMP-1 as well as the collagen content were measured in vascular sections from patients undergoing peripheral revascularization (carotid n=11, femoral n=23) and aorto-coronary bypass surgery (mammary arteries n=20, as controls). Increased expression of all MMPs was detected in atherosclerotic as compared with control sections (P<0.01). Aneurysmal plaques showed a significant increase of MMP-1 and-3 and a reduction in total collagen (P<0.05) in relation to occlusive lesions. Calcification areas in atherosclerotic plaques were consistently associated with increased TIMP-1 expression (P<0.01). Finally, MMP-9 expression was higher in occlusive lesions from carotid than femoral arteries (P<0.01). CONCLUSIONS Aneurysm lesions expressed higher MMP-1 and-3 expression than occlusive plaques, and MMP-9 was mainly detected in carotid as compared with femoral arteries. TIMP-1 was associated with arterial calcification. These differences in the MMPs/TIMP-1 expression might determine the evolution of advanced atherosclerotic plaques and contribute to its vulnerability.

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

The p22phox subunit is an essential protein in the activation of NAD(P)H oxidase. Here we report the preliminary characterisation of the human p22phox gene promoter. The p22phox promoter contains TATA and CCAC boxes and Sp1, γ‐interferon and nuclear factor κB sites. We screened for mutations in the p22phox 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 −930A/G polymorphism in the p22phox promoter may be a novel genetic marker associated with hypertension.

Matrix metalloproteinase-9, -10, and tissue inhibitor of matrix metalloproteinases-1 blood levels as biomarkers of severity and mortality in sepsis

IntroductionMatrix metalloproteinases (MMPs) play a role in infectious diseases through extracellular matrix (ECM) degradation, which favors the migration of immune cells from the bloodstream to sites of inflammation. Although higher levels of MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) have been found in small series of patients with sepsis, MMP-10 levels have not been studied in this setting. The objective of this study was to determine the predictive value of MMP-9, MMP-10, and TIMP-1 on clinical severity and mortality in a large series of patients with severe sepsis.MethodsThis was a multicenter, observational, and prospective study carried out in six Spanish Intensive Care Units. We included 192 (125 surviving and 67 nonsurviving) patients with severe sepsis and 50 age- and sex-matched healthy controls in the study. Serum levels of MMP-9, MMP-10, TIMP-1, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-10 were measured in patients with severe sepsis at the time of diagnosis and in healthy controls.ResultsSepsis patients had higher levels of MMP-10 and TIMP-1, higher MMP-10/TIMP-1 ratios, and lower MMP-9/TIMP-1 ratios than did healthy controls (P < 0.001). An association was found between MMP-9, MMP-10, TIMP-1, and MMP-9/TIMP-1 ratios and parameters of sepsis severity, assessed by the SOFA score, the APACHE-II score, lactic acid, platelet count, and markers of coagulopathy. Nonsurviving sepsis patients had lower levels of MMP-9 (P = 0.037), higher levels of TIMP-1 (P < 0.001), lower MMP-9/TIMP-1 ratio (P = 0.003), higher levels of IL-10 (P < 0.001), and lower TNF-α/IL-10 ratio than did surviving patients. An association was found between MMP-9, MMP-10, and TIMP-1 levels, and TNF-α and IL-10 levels. The risk of death in sepsis patients with TIMP-1 values greater than 531 ng/ml was 80% higher than that in patients with lower values (RR = 1.80; 95% CI = 1.13 to 2.87;P = 0.01; sensitivity = 0.73; specificity = 0.45).ConclusionsThe novel findings of our study on patients with severe sepsis (to our knowledge, the largest series reporting data about MMP levels in sepsis) are that reduced MMP-9/TIMP-1 ratios and increased MMP-10 levels may be of great pathophysiologic significance in terms of severity and mortality, and that TIMP-1 levels may represent a biomarker to predict the clinical outcome of patients with sepsis.

Phagocytic NADPH Oxidase-Dependent Superoxide Production Stimulates Matrix Metalloproteinase-9: Implications for Human Atherosclerosis

Objective—Data suggest that matrix metalloproteinase-9 (MMP-9) has a role in atherosclerosis. The phagocytic NADPH oxidase has been also associated with atherosclerosis. This study aimed to investigate the association between phagocytic NADPH oxidase and MMP-9 in human atherosclerosis. Methods and Results—In vitro experiments performed in human monocytes showed that NADPH oxidase activation enhanced MMP-9 secretion and activity, determined by enzyme-linked immunosorbent assay and zymography, respectively. Immunohistochemical study showed that phagocytic NADPH oxidase localized with MMP-9 in endarterectomies from patients with carotid stenosis. In addition, a positive relationship (P<0.001) was found between phagocytic NADPH oxidase-dependent superoxide production determined with lucigenin and plasma MMP-9 levels in 188 asymptomatic subjects free of overt clinical atherosclerosis. In multivariate analysis, this association remained significant after adjustment for cardiovascular risk factors. Interestingly, subjects in the upper quartile of superoxide production exhibited the highest values of MMP-9, oxidized low-density lipoprotein, nitrotyrosine, carotid intima media thickness, and an increased presence of carotid plaques. Conclusions—Enhanced NADPH oxidase-dependent ·O2− production stimulates MMP-9 in monocytes and this relationship may be relevant in the atherosclerotic process. Moreover, MMP-9 emerges as an important mediator of the phagocytic NADPH oxidase-dependent oxidative stress in atherosclerosis.

Involvement of leptin in the association between percentage of body fat and cardiovascular risk factors.

OBJECTIVES Recent epidemiologic studies have shown that obesity is associated with elevated blood concentrations of prothrombotic-proinflammatory factors and markers of endothelial dysfunction such as fibrinogen, C-reactive protein (CRP), von Willebrand factor (vWF), and homocysteine. We have assessed whether these markers are associated with percentage of body fat (BF), insulin sensitivity as well as with leptin concentrations. DESIGN AND METHODS Twenty-five men aged 49.6 +/- 12.7 yr (mean +/- SD) underwent whole-body air displacement plethysmography (Bod-Pod(R)) for estimating BF. Blood analyses for leptin and several other metabolic and cardiovascular markers were carried out. RESULTS Obese subjects had higher levels as compared to controls of BF (37.5 +/- 5.1 vs. 26.0 +/- 6.6, p < 0.01), fibrinogen (3.30 +/- 0.43 vs. 2.67 +/- 0.11, p < 0.01), vWF (136.4 +/- 50.4% vs. 81.6 +/- 12.6%, p < 0.05), and leptin (17.6 +/- 8.7 vs. 6.2 +/- 3.3, p < 0.01), lower concentrations of HDL-cholesterol (1.09 +/- 0.20 vs. 1.51 +/- 0.10, p < 0.001) and lower QUICKI (1/[log(Ins(0)) + log(Glu(0))]) (0.31 +/- 0.03 vs. 0.34 +/- 0.02, p < 0.05). No significant changes were observed in CRP (5.7 +/- 3.4 vs. 3.8 +/- 1.6, p = 0.327) and homocysteine (9.4 +/- 4.2 vs. 8.3 +/- 0.9, p = 0.749). A positive correlation was observed between BF and fibrinogen (r = 0.67, p = 0.0003). Plasma leptin concentrations were correlated with fibrinogen (r = 0.71, p = 0.0001) and CRP (r = 0.43, p = 0.044). After adjustment for BF leptin emerged as a significant predictor of fibrinogen (beta = 0.47, p = 0.023; R(2) = 0.59, p < 0.001). QUICKI was positively correlated with HDL-cholesterol (r = 0.59, p = 0.010) and negatively with fibrinogen (r = -0.53, p = 0.025), CRP (r = -0.52, p = 0.028) and vWF (r = -0.56, p = 0.013). CONCLUSIONS Increased BF and impaired insulin sensitivity are associated with increased concentrations of cardiovascular risk factors. Leptin seems to be involved in this elevation and emerges as a predictor of circulating fibrinogen concentrations.

Prothrombin Fragment 1+2 Is Associated With Carotid Intima-Media Thickness in Subjects Free of Clinical Cardiovascular Disease

Background and Purpose— Thrombin, a central enzyme in the clotting cascade, plays a role not only in thrombosis but also in the progression of atherosclerosis. We studied the relationship between prothrombin fragment 1+2 (F1+2), a specific marker of thrombin generation in vivo, and carotid intima-media thickness (IMT), an index of subclinical atherosclerosis. Methods— We examined 181 asymptomatic middle-aged subjects (mean age 55.6 years, 76.7% men) free of overt clinical atherosclerotic disease. F1+2 was measured by enzyme-linked immunosorbent assay and IMT by duplex ultrasonography of carotid artery. Multiple linear regression analysis was used to assess the relationship between the 2 parameters. Results— Compared with individuals in the lowest tertile of F1+2, those in the upper tertile (>0.55 nmol/L) showed significantly higher IMT (P <0.01). In correlation analysis, a positive relationship was found between plasma F1+2 and carotid IMT. F1+2 also correlated positively with cholesterol (P <0.008) and low-density lipoprotein cholesterol (P <0.005), but not with blood pressure or body mass index. In the multivariate analysis, the association of F1+2 with carotid IMT remained significant (P <0.001) after adjustment for age, sex, body mass index, systolic blood pressure, cholesterol, diabetes, and smoking. Conclusions— In a population sample of adults without clinically overt atherosclerotic disease, the plasma levels of F1+2 were significantly associated with carotid IMT, suggesting a relationship between thrombin generation and the development atherosclerosis.

Independent association of matrix metalloproteinase‐10, cardiovascular risk factors and subclinical atherosclerosis

Summary.  Objectives: Circulating levels of matrix metalloproteinase (MMP)‐10 are related to inflammation in asymptomatic subjects with cardiovascular risk factors. Whether MMP‐10 is associated with the severity of atherosclerosis remains to be determined. This study examines the relationship of systemic MMP‐10 levels with atherosclerotic risk factors and subclinical atherosclerosis. Methods and results: Circulating levels of MMP‐1, ‐9 and ‐10, and markers of inflammation [fibrinogen, interleukin‐6, von Willebrand factor, and high‐sensitivity C‐reactive protein (hs‐CRP)] were measured in 400 subjects (mean age 54.3 years, 77.7% men) with cardiovascular risk factors but free from clinical cardiovascular disease. Subclinical atherosclerosis was evaluated by both the mean carotid intima‐media thickness (IMT) and the presence of atherosclerotic plaques with the use of B‐mode ultrasound in all subjects. MMP‐10 levels were positively correlated with fibrinogen (r = 0.24, P < 0.001), hs‐CRP (r = 0.14, P < 0.01) and carotid IMT (r = 0.17, P < 0.01). The association between MMP‐10 and IMT remained significant in multiple regression analysis (P < 0.02) when controlling for traditional atherosclerotic risk factors and inflammatory markers. Such an association was not observed for MMP‐1 and ‐9. Subjects in the highest MMP‐10 tertile had significantly higher carotid IMT (adjusted odds ratio 6.3, 95% confidence interval 1.3–31.4, P = 0.024). In addition, MMP‐10 levels were significantly higher in patients with carotid plaques (n = 78) than in those with no plaques after adjusting for age and sex (P < 0.01). Conclusion: Higher serum MMP‐10 levels were associated with inflammatory markers, increased carotid IMT and atherosclerotic plaques in asymptomatic subjects. Circulating MMP‐10 may be useful to identify subclinical atherosclerosis in subjects free from cardiovascular disease.

Antioxidant vitamins increase the collagen content and reduce MMP-1 in a porcine model of atherosclerosis: implications for plaque stabilization

Degradation of extracellular matrix, particularly interstitial collagen, promotes plaque instability and contributes to restenosis after vascular injury. We have explored the effects of vitamins C and E on the collagen content and metalloproteinase-1 (MMP-1) expression after angioplasty in hypercholesterolemic pigs. Iliac angioplasty was performed on 18 minipigs divided into three diet groups: a normal-cholesterol (NC), a high-cholesterol (HC) and a high-cholesterol plus vitamins C+E (HCV). Four weeks later, after sacrifice, the vascular collagen content and MMP-1 protein expression, along with the plasma caseinolytic activity and lipid peroxidation, were measured. MMP-1 was also determined in arterial rings stimulated with native low-density lipoproteins (LDL) isolated from experimental groups. Cholesterol-rich diet augmented plasma lipid peroxidation (P<0.05), reduced the collagen content and increased vascular MMP-1 expression after injury (P<0.05). Enhanced caseinolytic activity (identified as MMP-1) was also observed in HC plasma samples and in supernatants from arterial rings incubated with HC-LDL. Vitamins C and E markedly increased neointimal collagen content (P<0.01), reduced the hypercholesterolemia-induced changes in vascular MMP-1 (P<0.05) and diminished plasma and ex vivo caseinolytic activity. Vitamins C and E may help stabilize atherosclerotic plaque after angioplasty and favor vascular remodeling by increasing collagen content and reducing vascular MMP-1 expression in porcine hypercholesterolemia.