L. Masana

Elevated levels of small, low-density lipoprotein with high affinity for arterial matrix components in patients with rheumatoid arthritis: possible contribution of phospholipase A2 to this atherogenic profile.

OBJECTIVE This work studied the presence of inflammatory and atherogenic lipoprotein markers that could explain the high incidence of cardiovascular disease (CVD) reported in rheumatoid arthritis (RA) patients. METHODS Inflammatory markers were 1) soluble adhesion molecules (intercellular adhesion molecule [ICAM] and vascular cell adhesion molecule [VCAM]), 2) C-reactive protein (CRP), 3) fibrinogen (Fb), 4) cytokines (interferon-gamma [IFNgamma], tumor necrosis factor alpha [TNFalpha]), and 5) secretory group IIA phospholipase A2 (sPLA2-IIA). Atherogenic lipoprotein markers were 1) the size distribution of plasma lipoprotein subclasses, and 2) the binding affinity of low-density lipoprotein (LDL) to chondroitin 6-sulfate glycosaminoglycan (GAG). RESULTS RA patients (n = 31) and matched controls (n = 28) had similar plasma concentrations of total cholesterol, triglycerides, Apo B, Apo A-I, very low-density lipoprotein, intermediate-density lipoprotein, and high-density lipoprotein (HDL). RA patients had significantly higher plasma levels of sPLA2-IIA, ICAM, CRP, Fb, TNFalpha, and IFNgamma compared with controls. RA patients also had significantly higher levels of small, dense LDL-1 (P < 0.05) and lower levels of small HDL-2 particles (P < 0.001) compared with controls. In addition, LDL from RA patients had a significantly higher binding affinity (Kd) to GAG (mean +/- SD Kd 204+/-22.4 nM Apo B) than did LDL from control subjects (Kd 312+/-36 nM Apo B) (P < 0.05). This Kd value showed a significant negative correlation with the plasma levels of LDL-1 (r = -0.566, P < or = 0.004). In RA patients, a significant positive correlation was obtained between sPLA2-IIA and CRP, ICAM, and LDL-1. HDL-2 showed a negative correlation with sPLA2-IIA. CONCLUSION These atherogenic lipoprotein factors combined with the presence of chronic inflammation may contribute to the high CVD-related mortality in RA patients.

Simvastatin decreases aldehyde production derived from lipoprotein oxidation.

Treatment with statins are known to lower plasma and low-density lipoprotein (LDL) cholesterol levels with resultant prevention and regression of atherosclerosis. It has been recently suggested that the action of the statins may also have a direct effect on other mechanisms involved in the atherosclerotic plaque formation. Thus, we investigated whether simvastatin could have an antioxidant effect on plasma lipoproteins. The rate of oxidation of LDL and high-density lipoproteins (HDL) was measured by conjugated diene formation with and without the addition of increasing concentrations of simvastatin (in vitro) and in patients with and without treatment with simvastatin (in vivo). A strong correlation was observed between increasing simvastatin concentration and the lag phase, a negative correlation was observed for maximal rate and maximum diene production in LDL samples (r2 = +0.97, p <0.0001; r2 = -0.92, p <0.0001; r2 = -0.98, p <0.0001, respectively). For HDL no clear correlation could be established with the lag phase, but a strong negative correlation was also observed between simvastatin concentration and maximal rate and maximum diene production (r2 = -0.69, p <0.01; r2 = -0.98, p <0.0001, respectively). After 6 hours of oxidation the production of aldehydes in LDL and HDL was lower (30% and 5%, respectively) in samples obtained during simvastatin therapy with respect to those obtained without treatment. The 2,4-decadienal showed a decrease of 37% and 64% (p <0.05) in both oxidized-LDL and oxidized-HDL particles, respectively, with simvastatin treatment. Our findings demonstrate that simvastatin acts as an antioxidant in lipoprotein particles and, together with its lipid-lowering properties, could play an important role in preventing atherosclerosis.

Reversal of atherogenic lipoprotein profile in HIV-1 infected patients with lipodystrophy after replacing protease inhibitors by nevirapine

BackgroundThe widespread use of protease inhibitors (PI) has been associated with abnormalities in the lipid profile of HIV-1-infected patients. Treatment simplification approaches in which PI are replaced by nevirapine (NVP) have been shown to improve PI-related toxicity. ObjectiveTo assess the impact on plasma lipids of replacing the PI by NVP in HIV-1 infected patients with lipodystrophy. MethodsWe studied 34 patients with lipodystrophy who had been the first to be enrolled in a prospective, randomized trial of continuing current treatment, or replacing PI with NVP. Sixteen patients replaced their PI with NVP and 18 continued their current PI-containing treatment. Total, low density lipoprotein (LDL), very low density lipoprotein (VLDL), intermediate density lipoprotein and high density lipoprotein (HDL) cholesterol and triglyceride levels, the size and particle number of LDL were determined at baseline and after 24 weeks, by nucleic magnetic resonance spectroscopy. FindingsAfter 24 weeks of replacing the PI with NVP, we observed a reduction of total cholesterol (P = 0.028), LDL-cholesterol (P = 0.001), the number of circulating LDL particles (P = 0.003) and the VLDL-1 triglyceride level (P = 0.032). A concomitant significant increase was observed in both HDL-cholesterol level (P = 0.002) and HDL particle size (P < 0.001). No significant changes were observed in the group that continued taking the PI. ConclusionsThe replacement of PI by NVP improved the lipid profile both by reducing the number and lipid content of atherogenic LDL particles, and increasing the protective HDL fraction. Although total triglyceride levels remained unchanged, a reduction in the VLDL-1 fraction contributes to the reduction of LDL particles. These changes are expected to reduce the risk of cardiovascular disease in HIV-1-infected patients on highly active antiretroviral therapy.

Atherosclerosis in Patients Infected With HIV Is Influenced by a Mutant Monocyte Chemoattractant Protein-1 Allele

Background—Patients infected with HIV present with premature atherosclerosis, and the 2 diseases share common pathogenic pathways. We investigated mutations in the monocyte chemoattractant protein-1 (MCP-1) and CCR-2 genes, which are known to control aspects of these pathways, to ascertain whether they are involved in atherogenesis in these patients. Methods and Results—We performed carotid and femoral artery ultrasonography to detect subclinical atherosclerosis in patients infected with HIV (n=183). MCP-1–2518G and CCR-2 64I polymorphisms were determined in the HIV group and in a population-based control group (n=348). We also determined MCP-1 circulating levels in the HIV group. The presence of MCP-1–2518G in the group of patients with subclinical atherosclerosis was significantly higher than in patients without atherosclerotic lesions (47.5% versus 18.2%, respectively; P<0.001). Furthermore, the patients with atherosclerotic lesions had higher MCP-1 plasma concentrations than did patients without lesions (74.15 [4.03] versus 57.81 [3.67] pg/mL, respectively; P=0.03). When adjusted for known cardiovascular risk factors, the MCP-1–2518G allele was associated with subclinical atherosclerosis (OR 5.72, 95% CI 1.74 to 18.80, P=0.004). Compared with measurements conducted ≈2.5 years earlier in a subset of 40 patients, intima-media thickness (IMT) in the carotid artery progressed at a mean rate of 0.06 mm/y more rapidly in patients bearing the MCP-1–mutated allele (P=0.08). Conclusions—HIV-infected patients with the MCP-1–2518G allele have a 5-fold increased risk for atherosclerosis, as assessed by ultrasonography.

Retinol-binding protein 4 as a plasma biomarker of renal dysfunction and cardiovascular disease in type 2 diabetes

Objective.  The retinol‐binding protein 4 (RBP4) has been linked to the insulin resistance state in obesity and type 2 diabetes in animal studies. Data in humans are controversial and their relationship with organ damage in diabetic patients is lacking. We studied the association of plasma RBP4 with organ complications in type 2 diabetic patients.

The Role of Immunity and Inflammation in the Progression of Atherosclerosis in Patients With HIV Infection

Background and Purpose— The initial steps of atherosclerosis and the entry of HIV into the cell share similar biological mechanisms. Therefore, our hypothesis is that the progression of atherosclerosis in patients with HIV infection can be influenced by variations in genes implicated in both processes. Methods and Results— The progression of atherosclerosis over a 2-year follow-up period was measured as the combined carotid and femoral intima media thickness (IMT) in 141 patients with HIV infection. The &Dgr;IMT (IMTfollow-up−IMTbaseline) values were used to segregate patients as minimal progressors or regressors (lowest &Dgr;IMT tertile), slow progressors (mid &Dgr;IMT tertile), and rapid progressors (highest &Dgr;IMT tertile). Mutations CCR-5&Dgr;32, CCR-2 64I, MCP-1-2518G, SDF1-3′A, and CX3CR-1 (T280 mol/L and V249I) in the host DNA were determined. Mean age of the patients was 38.96 (SEM: 0.61) and 68.8% were male. The mean &Dgr;IMT was 0.045 mm (0.01) per year, which represented a significant progression (P<0.001) with respect to baseline values. Patients with minimal progression or regression had a significantly (P=0.01) higher CD4 cell count than slow progressors and rapid progressors. Multivariate analyses indicated that age and total cholesterol were positively associated with IMT progression. In contrast, the CD4 cell count, the SDF1-3′A, and the CX3CR-1 249 I mutated alleles were associated with lesser IMT progression. Conclusion— The course of atherosclerosis in patients with HIV infection is influenced by polymorphisms in the SDF1 and CX3CR1 genes by metabolic variables and by the CD4 cell count. These data would be of help in assessing therapeutic needs of these patients.

A proposal to redefine familial combined hyperlipidaemia -- third workshop on FCHL held in Barcelona from 3 to 5 May 2001, during the scientific sessions of the European Society for Clinical Investigation.

Familial combined hyperlipidaemia (FCHL) was described in 1973 by three separate groups as a common familial disorder characterized by multiple lipoprotein phenotypes and an increased risk of premature coronary artery disease [1– 3]. No metabolic explanation was offered for the variable lipid phenotypes and opinion differed as to whether this was likely to be a monogenic or polygenic disorder. In 1986, the first FCHL workshop was held in Seattle and at that meeting an elevated plasma apolipoprotein B (apoB) was added to the list of characteristics. However, it was not made an essential feature, nor was any change to the fundamental approach to phenotypic classification suggested, notwithstanding that complexity of diagnosis severely limits clinical application and comparison of research results [4]. In 1998, investigators met in Helsinki for the second FCHL workshop and heard of the newest efforts to identify the genetic and metabolic bases for FCHL. All of the analyses were presented within the context of the original diagnostic approach. At the most recent meeting of the European Society for Clinical Investigation in Barcelona, the third workshop on FCHL was organized by Dr J. Ribalta (Reus, Spain) and Dr M. Castro Cabezas (Utrecht, the Netherlands) to reconsider this most common, but least well characterized, familial atherogenic dyslipoproteinaemia. Our objective became to search for the most important pathophysiological features. From the outset, as outlined by Professor M-R. Taskinen (Helsinki, Finland) , the two most well-documented features are increased very low-density lipoprotein (VLDL) secretion and impaired clearance of postprandial lipoproteins [5,6]. The increased VLDL2 secretion results in hypertriglyceridaemia and an elevated plasma apoB. Long residence time of VLDL1 particles favour the formation of small dense low-density lipoprotein (LDL). Based on this, we considered the hypothesis that the phenotype of FCHL might not be multiple but unitary – namely, hypertriglyceridaemic (hyperTg) hyperapoB. If so, FCHL phenotype could be defined more simply and consistently as follows. The phenotype of hyperTg hyperapoB would have to be present in more than one family member and at least one individual in the family must have premature symptomatic coronary artery disease. Other genetic disorders and secondary causes of dyslipidaemia, including type 1 and type 2 diabetes would, of course, have to be excluded [4,5]. It was emphasized that such a change only represents an evolution in diagnosis based on the advances in knowledge and technology that have occurred since the disorder was Mike Rosenbloom Laboratory for Cardiovascular Research, McGill University Health Centre, McGill University, Montreal, Quebec, Canada (A. D. Sniderman); Departments of Internal Medicine and Endocrinology, University Medical Centre, Utrecht, the Netherlands (M. Castro Cabezas, D. W. Erkelens); Unitat de Recerca de Lípids i Arteriosclerosi, Facultat de Medicina, Hospital Universitari de Sant Joan, Universitat Rovira i Virgili, Reus, Spain (J. Ribalta, L. Masana); Servicio de Endocrinología, Departamento de Medicina, Universidad de Valencia, Valencia, Spain (R. Carmena, J. T. Real); Departments of Medicine and Endocrinology, Academic Hospital, 6202 AZ Maastricht, the Netherlands (T. W. A. de Bruin); Department General Internal Medicine 541, UMC Nijmegen, the Netherlands (J. de Graaf); Division of Cardiovascular Genetics, Department of Medicine, Royal Free and University College Medical School, London WC1E 6JJ, UK (S. E. Humphries, P. J. Talmud); Department of Medicine, University of Helsinki, Helsinki, Finland (M. R. Taskinen).

Oleic Acid Rich Diet Protects Against the Oxidative Modification of High Density Lipoprotein

Oxidative modifications of lipoproteins could contribute to the development of atherosclerosis, but the influence of dietary fats on high density lipoprotein (HDL) oxidative modification is unknown. This study was designed to determine whether a diet rich in oleic acid could modulate the oxidative modification of HDL3. Twenty two healthy men were randomly placed on a 32-wk crossover study of an oleic acid rich diet supplied by a variant of sunflower oil vs a linoleic acid rich diet provided by conventional sunflower oil. Plasma HDL3 obtained after the diet rich in oleic acid showed a significantly higher oleic acid content in the phospholipid than lipoprotein isolated after the linoleic acid rich diet. HDL3 isolated after the oleic acid rich diet had lower values of thiobarbituric acid reactive substances (TBARS) than HDL3 obtained after the linoleic acid rich diet both for native (mean +/- SE; 0.24 +/- 0.02 vs 0.42 +/- 0.08 nmol MDA/mg protein; p < 0.01) and copper oxidized HDL3 (0.75 +/- 0.06 vs 0.95 +/- 0.07 nmol MDA/mg protein; p < 0.01). Indeed, TBARS for native HDL3 were negatively correlated with the oleic acid to linoleic acid ratio and positively with the percentage of linoleic acid in their phospholipids. Interestingly, HDL3 after both diets had similar antioxidant vitamins A and E content. HDL3 overall composition and fluidity were similar after the two diets. Moreover, HDL3 obtained after both diets produced identical [3H] free cholesterol efflux from human monocyte-derived macrophages (29%) and fibroblasts (26%). In conclusion, HDL3 rich in oleic acid was less easily oxidized regardless of the content of antioxidants such as vitamins A and E. Therefore, dietary monounsaturated fatty acid prevent the oxidative modification of lipoproteins.

Unsaturated fatty acids and their oxidation products stimulate CD36 gene expression in human macrophages

Fatty acids (FA) have been implicated in the control of expression of several atherosclerosis-related genes. Similarly, the CD36 receptor has recently been shown to play an important role in atherosclerosis and other pathologies. The aim of the present study was to evaluate the direct effect of FA and their oxidation products (aldehydes), on the expression of CD36 in both THP-1 macrophages and human monocyte-derived macrophages (HMDM). The FA tested included the saturated FA (SFA) lauric, myristic, palmitic and stearic acid; the monounsaturated FA oleic acid; and the unsaturated FA (UFA) linoleic, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Aldehydes used were malondialdehyde (MDA), hexanal, 2,4-decadienal (DDE) and 4-hydroxynonenal (HNE). CD36 expression was measured by RT-PCR, Western blot and immunofluorescence. Incubation of THP-1 macrophages for 24 h with non-cytotoxic concentrations of UFA significantly increased CD36 mRNA expression. By contrast, exposure of THP-1 macrophages to SFA did not affect the levels of CD36 mRNA. Among all UFAs tested, EPA and DHA were the strongest inducers of CD36 mRNA levels, followed by oleic and linoleic acid. Incubation of HMDM with either oleic or linoleic acid significantly increased steady-state CD36 mRNA in a dose-dependent manner. Consistent with the increase of CD36 mRNA expression, incubation of THP-1 macrophages with oleic and linoleic acid for 24 h markedly increased CD36 protein expression. Treatment of THP-1 macrophages with MDA or hexanal for 24 h significantly increased CD36 mRNA expression in a dose dependent manner. In contrast, DDE and HNE significantly decreased this parameter. The data provide evidence for a direct regulatory effect of UFA on CD36 gene expression and support a role for aldehydes in the regulation of CD36 expression by FA.

Gene expression analysis of a human enterocyte cell line reveals downregulation of cholesterol biosynthesis in response to short-chain fatty acids

It has been suggested that the short‐chain fatty acids (SCFAs) produced by anaerobic bacterial intestinal fermentation of soluble fiber may regulate lipid metabolism in intestine, thus reducing plasma cholesterol levels. However, the exact mechanism of action of SCFAs in lowering cholesterol levels is not fully understood. The aims of this study were to test the effects of SCFAs on gene expression in a human enterocyte cell line Caco‐2/TC‐7 and to validate microarray data by real‐time PCR. Human Caco‐2/TC‐7 enterocytes were cultured on transwell filter inserts and incubated with the SCFAs acetate (Ac), propionate (Pr), and butyrate (Bu). Total RNA was then isolated for microarrays and quantitative real‐time PCR analysis. Treatment of human enterocytes with Pr and Bu affects a wide variety of genes. These genes were classified according to the PANTHER classification system, and the results showed that different biological processes and metabolic pathways were modified by Pr and Bu treatment, including the intestinal cholesterol biosynthesis pathway. Differential array expression analysis showed that nine genes were downregulated in this pathway, and these results were validated by real‐time PCR. This in vitro study allowed us to identify a wide variety of biological processes and metabolic pathways affected by the SCFAs tested. Importantly, our results show that the global effect of Pr and Bu is to downregulate the expression of nine key genes involved in intestinal cholesterol biosynthesis, thus possibly inhibiting this pathway.