Low cytochrome oxidase 4I1 links mitochondrial dysfunction to obesity and type 2 diabetes in humans and mice.

OBJECTIVES: Cytochrome oxidase (COX) dysfunction is associated with mitochondrial oxidative stress. We determined the association between COX expression, obesity and type 2 diabetes. SUBJECTS/METHODS: COX4I1 and COX10 genes were measured in monocytes of 24 lean controls, 31 glucose-tolerant and 67 diabetic obese patients, and 17 morbidly obese patients before and after bariatric surgery. We investigated the effect of caloric restriction and peroxisome proliferator-activated receptor (PPAR) agonist treatment on Cox in obese diabetic mice, and that of diet-induced insulin resistance in Streptozotocin-treated mice. RESULTS: Low COX4I1 was associated with type 2 diabetes in obese patients, adjusting for age, gender, smoking, interleukin-6 and high-sensitivity C-reactive protein, all related to metabolic syndrome (MetS; odds ratio: 6.1, 95% confidence interval: 2.3-16). In contrast, COX10 was low in glucose-tolerant and diabetic obese patients. In morbidly obese patients, COX4I1 was lower in visceral adipose tissue collected at bariatric surgery. In their monocytes, COX4I1 decreased after bariatric surgery, and low COX4I1 at 4 months was associated with MetS at 7 years. In leptin-deficient obese diabetic mice, Cox4i1 was low in white visceral adipose tissue (n=13; P<0.001) compared with age-matched lean mice (n=10). PPARγ-agonist treatment (n=13), but not caloric restriction (n=11), increased Cox4i1 (P<0.001). Increase in Cox4i1 depended on the increase in glucose transporter 4 (Glut4) expression and insulin sensitivity, independent of the increase in blood adiponectin. In streptozotocin-treated mice (three groups of seven mice, diet-induced insulin resistance decreased Cox4i1 and Glut4 (P<0.001 for both). CONCLUSION: COX4I1 depression is related to insulin resistance and type 2 diabetes in obesity. In peripheral blood monocytes, it may be a diagnostically useful biomarker.

Dietary antioxidants and forced expiratory volume in 1 s decline: the Health, Aging and Body Composition study.

Increased antioxidant defences are hypothesised to decrease age- and smoking-related decline in lung function. The relationship between dietary antioxidants, smoking and forced expiratory volume in 1 s (FEV(1)) was investigated in community-dwelling older adults in the Health, Aging and Body Composition study. 1,443 participants completed a food frequency questionnaire, self-reported smoking history and had measurements taken of FEV(1) at both baseline and after 4 yrs of follow-up. The association of dietary intake of nutrients and foods with antioxidant properties and rate of FEV(1) decline was investigated using hierarchical linear regression models. In continuing smokers (current smokers at both time-points), higher vitamin C intake and higher intake of fruit and vegetables were associated with an 18 and 24 mL · yr(-1) slower rate of FEV(1) decline compared with a lower intake (p < 0.0001 and p = 0.003, respectively). In quitters (a current smoker at study baseline who had quit during follow-up), higher intake was associated with an attenuated rate of decline for each nutrient studied (p ≤ 0.003 for all models). In nonsmoking participants, there was little or no association of diet and rate of decline in FEV(1). The intake of nutrients with antioxidant properties may modulate lung function decline in older adults exposed to cigarette smoke.

Association of endothelial and oxidative stress with metabolic syndrome and subclinical atherosclerosis: multi-ethnic study of atherosclerosis.

BACKGROUND/OBJECTIVES: A cluster of metabolic abnormalities termed metabolic syndrome (MetS) is associated with vascular endothelial dysfunction and oxidative internal milieu. We examined whether the association of MetS with subclinical atherosclerosis is explained by biomarkers of endothelial damage and oxidative stress. SUBJECTS/METHODS: Multi-Ethnic Study of Atherosclerosis (MESA) is a population-based study of 45- to 84-year-old individuals of four US ethnicities without clinical cardiovascular disease. A random sample of 997 MESA participants had data on the following biomarkers: von Willebrand factor, soluble intercellular adhesion molecule-1 (sICAM-1), CD40 ligand (CD40L), soluble thrombomodulin, E-selectin and oxidized LDL (oxLDL). We examined whether the associations of MetS with B-mode ultrasound-defined common and internal carotid intimal-medial thickness (IMT) and coronary artery calcium (CAC) measured using computerized tomography were explained by the biomarkers using multiple regression methods. RESULTS: MetS was associated with higher levels of each of the biomarkers (P<0.001, CD40L-suggestive association P=0.004), with greater IMT (P<0.001), and with greater extent of CAC in those in whom CAC was detectable (P=0.01). The association of MetS with measures of subclinical atherosclerosis remained unchanged after adjustment for the biomarkers. After adjusting for MetS, oxLDL was suggestively associated with greater prevalence of detectable CAC (P=0.005) and thicker internal carotid IMT (P=0.002), whereas sICAM-1 was significantly associated with greater prevalence of detectable CAC (P=0.001). CONCLUSIONS: The association of MetS with subclinical atherosclerosis was independent of its association with biomarkers of endothelial damage and oxidative stress, suggesting that metabolic abnormalities and oxidative endothelial damage may lead to atherosclerotic disease through distinct mechanisms.

Stevioside inhibits atherosclerosis by improving insulin signaling and antioxidant defense in obese insulin-resistant mice.

OBJECTIVE: Stevioside is a non-caloric natural sweetener that does not induce a glycemic response, making it attractive as sweetener to diabetics and others on carbohydrate-controlled diets. Obesity is frequently associated with insulin resistance and increased inflammation and oxidative stress. Therefore, we investigated its effects on insulin resistance, inflammation and oxidative stress related to atherosclerosis in obese insulin-resistant mice. RESEARCH DESIGN: Twelve-week-old mice were treated with stevioside (10 mg kg(-1), n=14) or placebo (n=20) for 12 weeks. RESULTS: Stevioside had no effect on weight and triglycerides, but lowered glucose and insulin. Stevioside treatment improved adipose tissue maturation, and increased glucose transport, insulin signaling and antioxidant defense in white visceral adipose tissues. Together, these increases were associated with a twofold increase of adiponectin. In addition, stevioside reduced plaque volume in the aortic arch by decreasing the macrophage, lipid and oxidized low-density lipoprotein (ox-LDL) content of the plaque. The higher smooth muscle cell-to-macrophage ratio was indicative for a more stable plaque phenotype. The decrease in ox-LDL in the plaque was likely due to an increase in the antioxidant defense in the vascular wall, as evidenced by increased Sod1, Sod2 and Sod3. Circulating adiponectin was associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the aorta of stevioside-treated mice. CONCLUSION: Stevioside treatment was associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the vascular wall, leading to inhibition of atherosclerotic plaque development and inducing plaque stabilization.

Relations between metabolic syndrome, oxidative stress and inflammation and cardiovascular disease.

The metabolic syndrome is a common and complex disorder combining obesity, dyslipidemia, hypertension, and insulin resistance. It is a primary risk factor for diabetes and cardiovascular disease. We showed for the first time that the metabolic syndrome is associated with a higher fraction of oxidized LDL and thus with higher levels of circulating oxidized LDL. Hyperinsulinemia and impaired glycaemic control, independent of lipid levels, were associated with increased in vivo LDL oxidation, as reflected by the higher prevalence of high oxidized LDL. High levels of oxidized LDL were associated with increased risk of future myocardial infarction, even after adjustment for LDL-cholesterol and other established cardiovascular risk factors. This association is in agreement with the finding that accumulation of oxidized LDL, which activates/induces subsets of smooth muscle cells and macrophages to gelatinase production, was associated with upstream localization of a vulnerable plaque phenotype. Dyslipidemia and insulin resistance in obese LDL receptor-deficient mice were associated with increased oxidative stress and impaired HDL-associated antioxidant defence associated with accelerated atherosclerosis due to increased macrophage infiltration and accumulation of oxidized LDL in the aorta. The accumulation of oxidized LDL was partly due to an impaired HDL-associated antioxidant defence due to a decrease in PON. Our data in this experimental model are thus the more relevant because a decrease in PON activity was found to be associated with a defective metabolism of oxidized phospholipids by HDL from patients with type 2 diabetes. Weight loss in leptin-deficient, obese, and insulin-resistant mice was associated with expressional changes of key genes regulating adipocyte differentiation, glucose transport and insulin sensitivity, lipid metabolism, oxidative stress and inflammation, most of which are under the transcriptional control of PPARs. We established an important relationship between PPAR-gamma and SOD1 for the prevention of the oxidation of LDL in the arterial wall. For example we showed that rosuvastatin decreased the oxidized LDL accumulation by increasing the expression of PPAR-gamma and SOD1. In addition, we established a relation between increased PPAR-alpha expression in the adipose tissue and a change in the gene expression pattern, which explains the decrease of free fatty acids, triglycerides and the increase in insulin sensitivity. We demonstrated that plaque oxidized LDL correlated with coronary plaque complexity in a swine atherosclerosis model. Oxidized LDL correlated positively with the expression of IRF1 and TLR2 suggesting a relation between oxidative stress and inflammation in coronary atherosclerotic plaques. Oxidized LDL induced further the expression of TLR2 and IRF1 in macrophages in vitro suggesting a causative link. As in the mouse model described above, plaque oxidized LDL correlated negatively with SOD1 expression and ox-LDL inhibited the expression of SOD1 in macrophages in vitro. We showed that TLR2, CXCR4 and MYC are overexpressed in monocytes of obese women at high cardiovascular risk and that weight loss was associated with a concomitant decrease of their expression. This suggests that the transcription factor cMYC has an atherogenic effect by inducing pro-inflammatory genes. The increased expression of TLR2 and CXCR4 were observed in the absence of an increase in ox-LDL but in the presence of an increase in SOD1. Interestingly, the expression of SOD1 correlated also with that of MYC, suggesting that it has an atherogenic effect by inducing the expression of an anti-oxidant enzyme. How ox-LDL prevents this increase remains to be determined. How we plan to do this is explained in the next part. In aggregate, our studies contributed to a better understanding of the relationships between metabolic syndrome, insulin signalling, oxidative stress and inflammation and atherosclerosis. We identified paraoxonase, interferon regulatory factor-1, toll-like receptors, CXCR4 and SOD1 as possible targets for intervention.

Paraoxonase 1 gene transfer lowers vascular oxidative stress and improves vasomotor function in apolipoprotein E-deficient mice with pre-existing atherosclerosis.

BACKGROUND AND PURPOSE: Transgenesis of human paraoxonase 1 (PON1), a HDL-associated enzyme that destroys lipid peroxides, has been reported to reduce early atherogenesis in mice. The present study explored the therapeutic potential of human PON1 gene transfer in old apolipoprotein E-deficient (apoE(-/-)) mice with advanced atherosclerosis. EXPERIMENTAL APPROACH: ApoE(-/-) mice (18 months, regular chow) were transfected with PON1 adenovirus (AdPON1, n=10) or control adenovirus (AdRR5, n=10). Non-transfected apoE(-/-) (n=9) and C57Bl/6J (WT, n=6) mice served as controls. Three weeks later, plaque size and composition, and endothelial cell (EC) and smooth muscle cell (SMC) function were assessed in the aorta. KEY RESULTS: PON1 gene transfer raised total PON1 serum activity 13-15 fold during the 3-week study period, without affecting hypercholesterolaemia or lesion size. However, PON1 decreased the oxLDL content of the plaque. Plaque-free thoracic aorta rings from apoE(-/-) mice displayed, like rings from WT mice, complete relaxation to acetylcholine (ACh, 86+/-2%), ATP (90+/-2%) or UTP (83+/-3%). In contrast, in plaque-bearing segments amplitude (55+/-7%, 68+/-8%, 52+/-8% respectively) and sensitivity were decreased. EC function was completely (ATP, UTP) or largely (ACh) restored by AdPON1. Furthermore, apoE(-/-) SMCs released less intracellular calcium than WT upon sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibition by cyclopiazonic acid. This defect was also restored by AdPON1 transfection. CONCLUSIONS AND IMPLICATIONS: These data indicate that AdPON1 gene transfer improved vascular wall oxidative stress, EC function, and SMC Ca(2+) homeostasis in segments with pre-existing atherosclerosis, independently of an effect on plaque size.

Oxidized low-density lipoprotein-induced expression of ABCA1 in blood monocytes precedes coronary atherosclerosis and is associated with plaque complexity in hypercholesterolemic pigs.

BACKGROUND: Elevated oxidized low-density lipoprotein (oxLDL) is associated with atherosclerosis and high cardiovascular risk. Previously, we identified 18 genes in coronary plaque macrophages of hypercholesterolemic pigs that correlated with plaque oxLDL. OBJECTIVE: To determine which of these genes were differentially expressed in blood monocytes and correlated with blood and plaque oxLDL and with plaque complexity. METHODS: RNA expression in monocytes of 27 hypercholesterolemic and 12 control pigs was analyzed with quantitative real-time polymerase chain reaction. RESULTS: Five of 12 genes with detectable expression in monocytes were overexpressed (at P < 0.01 level) in blood monocytes of hypercholesterolemic pigs: ABCA1, SCD, IRF1, SDC2, and TLR2. ABCA1 RNA expression in blood monocytes correlated with blood oxLDL, and its RNA and protein expression was increased prior to atherosclerotic plaque formation. Higher expression of ABCA1 in monocytes was associated with higher plaque complexity and higher plaque oxLDL. Immunostaining of coronary plaques showed the association of ABCA1 with macrophages, lipids, and oxLDL; ABCA1 protein correlated with plaque oxLDL (R(2) = 0.66; P < 0.0001). In THP-1 monocytes, oxLDL induced ABCA1 expression. OxLDL-induced foam cell generation in THP-1 and human monocyte-derived macrophages was associated with a further increase of ABCA1 expression. CONCLUSIONS: The increase of ABCA1 in monocytes in association with blood oxLDL prior to atherosclerotic lesion formation and the association of higher ABCA1 with higher plaque complexity suggests that ABCA1 is an early biomarker of atherosclerosis. Studies in humans are warranted.

Rosuvastatin restores superoxide dismutase expression and inhibits accumulation of oxidized LDL in the aortic arch of obese dyslipidemic mice.

BACKGROUND AND PURPOSE: Our goal was to elucidate mechanisms of the inhibitory effect of rosuvastatin on the accumulation of plaque oxidized low density lipoproteins (oxLDL) and on plaque volume, without lowering cholesterol, in mice with combined leptin and LDL-receptor deficiency (DKO). EXPERIMENTAL APPROACH: Twelve-week old DKO mice were treated with rosuvastatin (10 mg kg(-1) day(-1), s.c.) or placebo or no treatment for 12 weeks. The effect on blood variables, aortic plaque volume and composition and gene expression in the aorta and in THP-1 cells was assessed. KEY RESULTS: Rosuvastatin lowered free fatty acids (FFA), triglycerides, and increased insulin sensitivity, without affecting cholesterol. Rosuvastatin lowered the plaque volume, inhibited macrophage, lipid and oxLDL accumulation, and decreased the oxLDL-to-LDL ratio of plaques in the aortic arch. It increased superoxide dismutase 1 (SOD1), CD36, LXR-alpha, ABCA-1 and PPAR-gamma RNA expression in aortic extracts. SOD1 was the strongest inverse correlate of oxLDL. In THP-1 macrophages and foam cells, expression of SOD1 was lower than in THP-1 monocytes. Rosuvastatin restored expression of SOD1 in THP-1 macrophages and foam cells. CONCLUSIONS AND IMPLICATIONS: Rosuvastatin restored SOD1 expression in THP-1 macrophages and foam cells in vitro and in the aorta of DKO mice. The latter was associated with less oxLDL accumulation within atherosclerotic plaques and inhibition of plaque progression. This effect was obtained at a dose not affecting cholesterol levels but improving insulin sensitivity. SOD1 is a potentially important mediator of the prevention of oxLDL accumulation within atherosclerotic plaques.

Molecular and functional characterization of ovine cardiac valve-derived interstitial cells in primary isolates and cultures.

At present the involvement of cardiac valve interstitial cells (VICs) in growth, repair, and tissue engineering is understudied. Therefore, this study aims at characterizing ovine VICs in order to provide a solid base for tissue engineering of heart valves. Ovine ICs of the four heart valves were isolated by the explant outgrowth method and expanded in vitro up to passage 5. Vimentin and collagen I gene expression from freshly isolated or cultured ICs was measured by reverse transcriptase-polymerase chain reaction. Immunocytochemical stainings of vimentin, alpha-smooth muscle actin (ASMA), smooth muscle myosin, and procollagen I were performed on aortic VICs. In addition, migration and extracellular matrix deposition were studied in vitro in aortic VICs. ICs show stable vimentin and collagen I expression in culture. Expression is approximately doubled in cultured ICs compared with fresh isolates. More than 95% of ICs in each passage stain for vimentin and procollagen I. Freshly isolated ICs are ASMA and myosin negative, but ICs in culture partially stain for these contractile markers. ICs have stable matrix production for up to five passages, associated with stable migration of the cells. We conclude that ovine valve interstitial cells undergo phenotypic modulation to activated myofibroblasts under culture conditions but retain stable matrix production.

Effects of homocysteine on the cellular responses of murine endothelial cells cultured in vitro.

Homocysteine, derived from the metabolism of methionine, is claimed as a proatherogenic factor that leads to vascular dysfunction. To gain better insight into the molecular mechanisms involved, homocysteine was tested on a model of murine endothelial cells cultured in vitro, using a prototype DNA chip. The DNA chip was designed to follow the expression at the mRNA level of some major proinflammatory genes; TNF-alpha was used as a positive control.

Raman scattering determination of the depth of cure of light-activated composites: influence of different clinically relevant parameters.

The purpose of this research was to determine the depth of cure of light-activated composites in relation with different clinically relevant parameters. A Raman spectroscopic method has been used. The measurement of cure is made on a relative basis by comparing the vibration band of the residual unpolymerized methacrylate C=C bond at 1640 cm-1 against the aromatic C=C stretching band at 1610 cm-1 used as an internal standard. The information gained draw attention to the importance of light transmission during the exposure. The influence of sample's thickness on the depth of cure is illustrated by a second order polynomial regression. The shade and translucency of the resin composite also modify the light transmission and thus have a significant influence on the degree of conversion. Moreover the light-source intensity and the distance from the curing tip are important parameters of influence. A significant reduction of the depth of cure is observed for all sample thickness of resin composite tested when using a light device with an intensity of 300 mW cm-2 as well as using a distance from the curing tip higher than 20 mm.

Antineutrophil cytoplasmatic antibodies in patients with premature atherosclerosis: prevalence and association with risk factors.

OBJECTIVES: Autoimmunity is suggested to play a role in premature atherosclerosis. Antineutrophil cytoplasmatic antibodies (ANCA) are a group of autoantibodies found in several inflammatory disorders in which they supposedly amplify the inflammatory process. In this study the hypothesis is tested that ANCA play a role in premature atherosclerosis. DESIGN: Cross-sectional study followed by nested case-control study. In a total of 286 consecutive patients with premature atherosclerosis (age < 55 years) ANCA were tested. Within the same cohort, a nested case-control study in 16 ANCA-positive patients and 32 ANCA-negative controls matched for sex, and site of atherosclerosis, was executed. SETTING: University hospital outpatient clinic for lipids and premature atherosclerosis. SUBJECTS: A total of 286 consecutive patients with premature atherosclerosis (age < 55 years). RESULTS: Prevalence of ANCA was 5.6% (16 of 286). All cases had perinuclear ANCA (pANCA); no cytoplasmatic ANCA was found. Mean age was 42 +/- 7 in the ANCA-positive vs. 42 +/- 9 years in the ANCA-negative group (P=ns). More female parents were ANCA-positive (8M/8F vs. 200M/70F, P=0.03). Patients with ANCA had more often peripheral vascular disease (37.5 vs. 15.2%, P=0.03). In the case-control study levels of Lp(a) were higher (43.8 vs. 15.6% >300 mg x L(-1), P=0.05), whereas levels of HDL-c were lower in ANCA-positive patients (0.84 +/- 0.26 vs. 1.06 +/- 0.27 mmol x L(-1), P=0.01). Markers of inflammation, C-reactive protein (CRP) and serum amyloid A (SAA), did not differ, nor did antibodies against oxidized-LDL and malondialdehyde (MDA)-LDL, markers for the extent of atherosclerosis. CONCLUSIONS: Our results suggest that ANCA do not appear to play a major role in premature atherosclerosis as there was no increased prevalence of the autoantibody. Moreover, no differences in the incidence of classical cardiovascular risk factors nor in serum levels of markers of inflammation were found between the ANCA-positive group as compared with the ANCA-negative group.

Restenosis and gene therapy.

Atherosclerosis is one of the main causes of mortality and morbidity in westernised countries. Treatment of symptomatic atherosclerosis by angioplasty involves major vascular responses such as neointima formation and constrictive vascular remodelling leading to restenosis. Stent placement prevents vasoconstriction but is associated with in-stent neointima formation. Therefore, stent placement requires adjunctive therapy. In this review we discuss the potential of local gene therapy for restenosis. More particularly, we focus on strategies to inhibit smooth muscle cell (SMC) proliferation and migration, prevent thrombosis, decrease oxidative stress in the arterial wall and enhance re-endothelialisation associated with adaptive remodelling. The potential of different vector systems and devices for local gene transfer in the arterial wall is discussed.

Arg123-Tyr166 domain of human ApoA-I is critical for HDL-mediated inhibition of macrophage homing and early atherosclerosis in mice.

Atherosclerosis was studied in apolipoprotein E (apoE) knockout mice expressing human apolipoprotein A-I (apoA-I) or an apoA-I/apolipoprotein A-II (apoA-II) chimera in which the Arg123-Tyr166 central domain of apoA-I was substituted with the Ser12-Ala75 segment of apoA-II. High density lipoprotein (HDL) cholesterol levels were identical in apoA-I and apoA-I/apoA-II mice, but at 4 months, plaques were 2.7-fold larger in the aortic root of the apoA-I/apoA-II mice (P<0.01). The macrophage-to-smooth muscle cell ratio of lesions was 2.1-fold higher in apo-I/apoA-II mice than in apoA-I mice (P<0.01). This was due to a 2.7-fold higher (P<0.001) in vivo macrophage homing in the aortic root of apoA-I/apoA-II mice. Plasma platelet-activating factor acetyl hydrolase activity was lower (P<0.01) in apoA-I/apoA-II mice, resulting in increased oxidative stress, as evidenced by the higher titer of antibodies against oxidized low density lipoprotein (P<0.01). Increased oxidative stress resulted in increased stimulation of ex vivo macrophage adhesion by apoA-I/apoA-II beta-very low density lipoprotein and decreased inhibition of beta-very low density lipoprotein-induced adhesion by HDL from apoA-I/apoA-II mice. The cellular cholesterol efflux capacity of HDL from apoA-I/apoA-II mice was very similar to that of apoA-I mice. Thus, the Arg123-Tyr166 central domain of apoA-I is critical for reducing oxidative stress, macrophage homing, and early atherosclerosis in apoE knockout mice independent of its role in HDL production and cholesterol efflux.

Oxidized LDL and HDL: antagonists in atherothrombosis.

Increased LDL oxidation is associated with coronary artery disease. The predictive value of circulating oxidized LDL is additive to the Global Risk Assessment Score for cardiovascular risk prediction based on age, gender, total and HDL cholesterol, diabetes, hypertension, and smoking. Circulating oxidized LDL does not originate from extensive metal ion-induced oxidation in the blood but from mild oxidation in the arterial wall by cell-associated lipoxygenase and/or myeloperoxidase. Oxidized LDL induces atherosclerosis by stimulating monocyte infiltration and smooth muscle cell migration and proliferation. It contributes to atherothrombosis by inducing endothelial cell apoptosis, and thus plaque erosion, by impairing the anticoagulant balance in endothelium, stimulating tissue factor production by smooth muscle cells, and inducing apoptosis in macrophages. HDL cholesterol levels are inversely related to risk of coronary artery disease. HDL prevents atherosclerosis by reverting the stimulatory effect of oxidized LDL on monocyte infiltration. The HDL-associated enzyme paraoxonase inhibits the oxidation of LDL. PAF-acetyl hydrolase, which circulates in association with HDL and is produced in the arterial wall by macrophages, degrades bioactive oxidized phospholipids. Both enzymes actively protect hypercholesterolemic mice against atherosclerosis. Oxidized LDL inhibits these enzymes. Thus, oxidized LDL and HDL are indeed antagonists in the development of cardiovascular disease.

S-nitrosothiols do not induce oxidative stress, contrary to other nitric oxide donors, in cultures of vascular endothelial or smooth muscle cells.

Nitric oxide (NO) has been described to exert various anti-atherogenic actions. However, NO, in some cases, has been shown to stimulate the oxidation of low-density lipoprotein (LDL), which constitute an important triggering event in atherosclerosis. Thus, some NO donors, despite their advantages, might also induce oxidative stress. Therefore, the purpose of this study is to examine the effect of three different NO donors on LDL oxidation, in acellular system as well as in cultures of normal endothelial cells or smooth muscle cells, which constitute the two major cellular components of the arterial wall. Sodium nitroprusside oxidized strongly LDL in medium alone as well as in endothelial or smooth muscle cell cultures. Sydnonimine-1 (SIN-1) oxidized LDL already in the absence of cells and enhanced clearly the LDL oxidation in the cultures. S-nitroso-N-acetylpenicillamine was unable to oxidize LDL in synthetic medium alone as well as in the presence of cells, showing that the amount of superoxide and other reactive oxygen species released by these cells did not suffice, contrary to those liberated by macrophages, to combine to NO providing oxidant activity.

Smooth muscle cells influence monocyte response to LDL as well as their adhesion and transmigration in a coculture model of the arterial wall.

We investigated the possible interference of smooth muscle cells with monocyte response to LDL as well as with their adhesion and transmigration in a coculture of porcine endothelial and smooth muscle cells. Lysophosphatidylcholine (LPC), a component of oxidized LDL (oxLDL), stimulated the adhesion of THP-1 cells to endothelial cells both in mono- and in coculture with smooth muscle cells. When THP-1 cells were incubated with endothelial cells in the presence of copper oxLDL, their adhesion was increased, but only in coculture. The addition of sodium nitroprusside (SNP) together with oxLDL markedly increased the adhesion of THP-1 cells in coculture. Close proximity between endothelial and smooth muscle cells was necessary to observe that effect. Furthermore, this increase in adhesion of THP-1 cells can, at least in part, be attributed to the augmented production of monocyte chemoattractant protein-1 (MCP-1) observed in coculture under the influence of oxLDL and SNP. The passage of THP-1 cells through the coculture was stimulated by MCP-1 and LPC. These results show that physical contacts or close proximity between endothelial and smooth muscle cells play a key role in the adhesion of monocytes and their infiltration into the intima in response to oxLDL.

Adipose tissue expression of gelatinases in mouse models of obesity.

Following the observation by Brown et al. (Am J Physiol 1997; 272: C937-49) that primary rat adipocytes in culture secrete gelatinase A (MMP-2), we have evaluated gelatinase expression in adipose tissue with the use of mouse models of obesity. Wild-type mice were kept on a standard fat diet (SFD) or on a high fat diet (42% fat, HFD) and- genetically obese db/db mice were kept on SFD; gonadal and subcutaneous fat pads were removed and analysed ex vivo. These studies revealed that: 1) the HFD induced adipocyte hypertrophy; 2) after 32 weeks, significantly higher levels of 70 kDa (p <0.05) and 65 kDa proMMP-2 (p <0.01) were observed in extracts of gonadal fat pads of mice on HFD; 3) the contribution of active MMP-2 to the total level was comparable in SFD and HFD groups (20 to 30%); and 4) gelatinase B (MMP-9) was not consistently detected. These findings were confirmed by gelatin zymography and by mRNA determination using competitive RT-PCR. The presence of MMP-2 in the adipose tissue was confirmed immunologically and its localization in adipocytes revealed by immunogold electron microscopy. The potential functional role of MMP-2 in adipose tissue remains to be determined.

Oxidative modification of low-density lipoproteins and the outcome of renal allografts at 1 1/2 years.

BACKGROUND: Previous studies reported a significant association between hyperlipidemia of the recipient and chronic allograft nephropathy (CAN). However, the nature and the pathogenic mechanism of circulating lipid abnormalities in CAN remain unclear. METHODS: In a prospective study of 50 consecutive adult recipients of a cadaveric renal allograft, we investigated the impact of lipid abnormalities on the outcome of the graft at 1 1/2 years. Besides morphometric analysis of implantation and protocol biopsies, clinical and biochemical variables were studied at three-month intervals. Plasma concentrations of oxidized low-density lipoprotein (OxLDL) were determined by means of enzyme-linked immunosorbent assay. Immunohistochemical staining for OxLDL and macrophages was performed on paired renal biopsies. Study end points were the fractional interstitial volume and the 24-hour creatinine clearance at 11/2 years. RESULTS: High-density lipoprotein (HDL) cholesterol of the recipient < or =47 mg/dL was a risk factor for the functional (RR = 1.56; 95% CI, 0.978 to 2.497) and the morphological (RR = 2.75; 95% CI, 1.075 to 7.037) outcome of the graft, mainly in patients without acute rejection (RR = 2.03; 95% CI, 1.13 to 3.65, and RR = 4.67; 95% CI, 1.172 to 18.582, respectively). Interstitial accumulation of OxLDL was inversely associated with HDL cholesterol (R = -0.476, P = 0.019), and was associated with a higher density of tubulointerstitial macrophages (R = 0.656, P = 0.001) and a higher fractional interstitial volume at 11/2 years (P = 0.049). CONCLUSION: Decreased HDL cholesterol levels of the recipient adversely affect the outcome of renal allografts through the accumulation of OxLDL in the renal interstitium of the graft. Interstitial accumulation of OxLDL was associated with the presence of macrophages and the development of interstitial fibrosis.

Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E-deficient mice. METHODS AND RESULTS: Intravenous administration of an adenovirus (5 x 10(8) plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P<0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P<0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P<0.001), macrophages by 69% (P=0.006), and smooth muscle cells by 84% (P=0.002) in the arterial wall. This resulted in a 77% reduction (P<0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P=0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P=0.01 and P=0.04, respectively). CONCLUSIONS: Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E-deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.