Graciela Castro

Increased ABCA1 activity protects against atherosclerosis.

The ABC transporter ABCA1 plays a key role in the first steps of the reverse cholesterol transport pathway by mediating lipid efflux from macrophages. Previously, it was demonstrated that human ABCA1 overexpression in vivo in transgenic mice results in a mild elevation of plasma HDL levels and increased efflux of cholesterol from macrophages. In this study, we determined the effect of overexpression of ABCA1 on atherosclerosis development. Human ABCA1 transgenic mice (BAC(+)) were crossed with ApoE(-/-) mice, a strain that spontaneously develop atherosclerotic lesions. BAC(+)ApoE(-/-) mice developed dramatically smaller, less-complex lesions as compared with their ApoE(-/-) counterparts. In addition, there was increased efflux of cholesterol from macrophages isolated from the BAC(+)ApoE(-/-) mice. Although the increase in plasma HDL cholesterol levels was small, HDL particles from BAC(+)ApoE(-/-) mice were significantly better acceptors of cholesterol. Lipid analysis of HDL particles from BAC(+)ApoE(-/-) mice revealed an increase in phospholipid levels, which was correlated significantly with their ability to enhance cholesterol efflux.

Protection Against Atherogenesis in Mice Mediated by Human Apolipoprotein A-IV

Apolipoproteins are protein constituents of plasma lipid transport particles. Human apolipoprotein A-IV (apoA-IV) was expressed in the liver of C57BL/6 mice and mice deficient in apoE, both of which are prone to atherosclerosis, to investigate whether apoA-IV protects against this disease. In transgenic C57BL/6 mice on an atherogenic diet, the serum concentration of high density lipoprotein (HDL) cholesterol increased by 35 percent, whereas the concentration of endogenous apoA-I decreased by 29 percent, relative to those in transgenic mice on a normal diet. Expression of human apoA-IV in apoE-deficient mice on a normal diet resulted in an even more severe atherogenic lipoprotein profile, without affecting the concentration of HDL cholesterol, than that in nontransgenic apoE-deficient mice. However, transgenic mice of both backgrounds showed a substantial reduction in the size of atherosclerotic lesions. Thus, apoA-IV appears to protect against atherosclerosis by a mechanism that does not involve an increase in HDL cholesterol concentration.

Inhibition of Atherosclerosis Development in Cholesterol-Fed Human Apolipoprotein A-I–Transgenic Rabbits

BACKGROUND Prospective epidemiological studies support the hypothesis that high levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I limit atherosclerosis development. However, more data from studies with animal models of atherosclerosis that resemble the human disease are required to demonstrate the effect of apo A-I in the inhibition of atherogenesis. The rabbit is a good animal model for human atherosclerosis. METHODS AND RESULTS Human apo A-I-transgenic rabbits have been produced, and we have evaluated the effect of apo A-I on the development of atherosclerosis in transgenic rabbits fed a cholesterol-rich diet for 14 weeks. Plasma cholesterol levels of atherogenic apo B-containing lipoproteins were similar for transgenic and control rabbits (> 1000 mg/dL), while plasma levels of HDL cholesterol in the transgenic group were always about twice that of the control group (68 +/- 11 versus 37 +/- 3 mg/dL at 14 weeks; P < .001). At the end of the experiment, the amount of aortic surface area covered by lesions as well as the amount of lipid accumulation in the aorta were significantly less in transgenic rabbits compared with the control group (15 +/- 12% versus 30 +/- 8%, P < .0027 for the surface area of the thoracic aorta; 116 +/- 31 versus 247 +/- 39 mumol/g aorta, P < .0068 for cholesterol content in total aorta). CONCLUSIONS Overexpression of human apo A-I in rabbits inhibits the development of atherosclerosis in this animal model that resembles, in many respects, human atherosclerosis.

Adipophilin Enhances Lipid Accumulation and Prevents Lipid Efflux From THP-1 Macrophages: Potential Role in Atherogenesis

Objective—Uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors results in lipid droplets accumulation and foam cell formation. Excess lipid deposition in macrophages has been reported to modulate expression of several genes including adipophilin. In this study, we investigated the function of adipophilin in lipid accumulation and cholesterol efflux in THP-1 macrophages. Methods and Results—Adipophilin mRNA expression was 3.5-fold higher in human atherosclerotic plaques compared with healthy areas of the same arteries. Moreover, in the presence of acetylated LDL (AcLDL), triglycerides and cholesteryl esters were increased in macrophages overexpressing adipophilin by 40% and 67%, respectively, whereas their accumulation was reduced when endogenous cellular adipophilin was depleted using siRNA approach. In addition, neither overexpression nor downregulation of adipophilin altered expression of genes involved in lipid efflux. However, the affinity and the number of AcLDL receptors were not affected. After 24-hour incubation of lipid-loaded macrophages with apolipoprotein A-I, cholesterol efflux was reduced by 47% in adipophilin transfected cells versus control cells. Conclusion—Our results showed that stimulation of adipophilin expression in macrophages by modified LDL promotes triglycerides and cholesterol storage and reduces cholesterol efflux. Therefore, adipophilin might contribute, in vivo, to lipid accumulation in the intima of the arterial wall.

The National Niemann–Pick C1 disease database: Report of clinical features and health problems

Niemann–Pick type C1 (NPC1) disease is an autosomal recessive disorder characterized clinically by neonatal jaundice, hepatosplenomegaly, vertical gaze palsy, ataxia, dystonia, and progressive neurodegeneration. The present study provides basic clinical and health information from the National Niemann–Pick C1 disease database that was obtained using a clinical questionnaire of 83 questions mailed to families affected by NPC1 disease living in the United States. The study was conducted over a 1‐year period, during which time parents/caregivers and physicians completed the clinical questionnaire. Sixty‐four percent (87/136) of the questionnaires were returned, with 53% and 47% representing male and female NPC1 patients, respectively. The average age of diagnosis for NPC1 disease was 10.4 years, with one‐half of patients being diagnosed before the age of 6.9 years. The average age of death for NPC1 disease was 16.2 years, with one‐half of patients dying before the age of 12.5 years. A common clinical symptom reported at birth was neonatal jaundice (52%), followed by enlargement of the spleen (36%) and liver (31%); ascites (19%) and neonatal hypotonia (6%) were much less frequent. With respect to developmental difficulties, the most common findings included clumsiness (87%), learning difficulties (87%), ataxia (83%), dysphagia (80%), and vertical gaze palsy (81%). Together, these findings confirm and extend previous reports investigating the clinical features associated with NPC1 disease.

Cholesterol efflux from Fu5AH hepatoma cells induced by plasma of subjects with or without coronary artery disease and non-insulin-dependent diabetes: importance of LpA-I:A-II particles and phospholipid transfer protein

We measured the capacity of human plasma to induce cholesterol efflux from Fu5AH rat hepatoma cells in four groups of men with or without non-insulin-dependent diabetes mellitus (NIDDM) and coronary artery disease (CAD). Plasma from men with both NIDDM and CAD (n = 47) had the lowest efflux capacity (17.3 +/- 3.6%) whereas healthy control subjects with neither diabetes nor CAD (n = 25) had the highest capacity (19.8 +/- 3.4%). The groups with CAD but no diabetes (n = 44) and with NIDDM but no CAD (n = 35) had intermediate efflux values (18.5 +/- 3.8 and 18.5 +/- 3.9%, respectively). In a 2 x 2 factorial ANOVA, the differences were significant with respect to the presence of CAD (P = 0.038) and NIDDM (P = 0.041), with no interaction between the factors. The concentration of HDL particles containing apolipoprotein (apo) A-I but no apo A-II (LpA-I) was not related to efflux capacity in univariate or multivariate analyses. A multivariate regression analysis showed that when controlled for the presence of NIDDM and CAD, the concentration of particles containing both apo A-I and apo A-II (LpA-I:A-II) and plasma phospholipid transfer protein activity were both positively, independently, and significantly (P < 0.001) related to cholesterol efflux capacity.

Human Apolipoprotein A-IV Reduces Secretion of Proinflammatory Cytokines and Atherosclerotic Effects of a Chronic Infection Mimicked by Lipopolysaccharide

Objective—Expression of human apolipoprotein (h-apo) A-IV in apoE-deficient (apoE0) mice (h-apoA-IV/E0) reduces susceptibility to atherosclerosis. Chronic infection mimicked by exposure to lipopolysaccharide (LPS) increases the size of atherosclerosis lesions in apoE0 mice. Thus, we used h-apoA-IV/E0 mice to determine whether h-apoA-IV plays a protective role after LPS administration. Methods and Results—We injected apoE0, h-apoA-IV/E0, and C57Bl/6 (wild-type) mice intraperitoneally with either LPS or phosphate-buffered saline (PBS) every week for 10 weeks. Atherosclerotic lesions were significantly smaller in h-apoA-IV/E0 mice treated with LPS than in their apoE0 counterparts. The titers of IgG2a and IgG2b autoantibodies to oxidized low-density lipoprotein (LDL) were higher in the LPS-group of h-apoA-IV/E0 mice than in apoE0 mice, suggesting that the Th1 response is stronger in the presence of h-apoA-IV. Lymphocytes from the blood, liver, spleen, and thymus of h-apoA-IV/E0 mice treated with LPS produced less IL-4, INF-&ggr;, and TNF-&agr; proinflammatory cytokines than their apoE0 counterparts. Furthermore, we demonstrated that recombinant h-apoA-IV blocks the LPS-induced stimulation of monocytes. Conclusions—The expression of h-apoA-IV in apoE0 mice reduces the susceptibility to atherogenesis and decreases the secretion of proinflammatory cytokines after LPS administration.

Alterations in the main steps of reverse cholesterol transport in male patients with primary hypertriglyceridemia and low HDL-cholesterol levels.

Hypertriglyceridemia is a complex pathological entity strongly connected to low HDL-C levels but controversially related to the risk of coronary artery disease. In this study, we evaluated the main steps of the antiatherogenic pathway called reverse cholesterol transport in a group of patients with primary hypertriglyceridemia and low HDL-C levels in comparison to normotriglyceridemic subjects with or without hypoalphalipoproteinemia. In patients with primary hypertriglyceridemia, low HDL-C levels were accompanied by decreased apo A-I and apo A-II concentrations. These reductions were manifested by a selective reduction in LpA-I:A-II particles. In addition, apo C-III Lp non B was found to be elevated and HDL lipid percentage composition showed a triglyceride enrichment and cholesterol depletion. The capacity of serum samples from hypertriglyceridemic patients to promote cellular cholesterol efflux was reduced, as evidenced by using two different cellular models, Fu5AH and J774 cells. This impaired cholesterol efflux promotion was also corroborated by incubations of isolated HDL fractions with Fu5AH cells. Lecithin:cholesterol acyltransferase (LCAT) activity, the driving force of reverse cholesterol transport, showed a tendency towards lower values in hypertriglyceridemic patients, but this difference was not statistically significant. Additionally, cholesteryl ester transfer protein (CETP) activity was increased in this group of patients. Therefore, hypertriglyceridemia was found to induce quantitative and qualitative alterations in HDL and its subclasses and, consequently, in some steps of reverse cholesterol transport. The abnormalities found in this antiatherogenic pathway and its promoters could constitute a possible connection between hypertriglyceridemia and atherosclerosis.

Complete Atherosclerosis Regression After Human ApoE Gene Transfer in ApoE-Deficient/Nude Mice

The apolipoprotein E (apoE)-deficient mouse is a relevant animal model of human atherosclerosis. Although the prevention of atherosclerosis development has been documented after somatic gene transfer into animal models, regression of lesions remains to be demonstrated. Thus, we used this genetically defined mouse model nn the nude background to show atherosclerosis regression. ApoE-deficient nude mice were infected with 5 x 10(8) or 10(9) plaque-forming units of a first-generation adenovirus encoding human apoE cDNA. The secretion of human apoE resulted in a rapid decrease of total cholesterol, which normalized the hypercholesterolemic phenotype within 14 days (from 600+/-100 to <100 microg/mL). Transgene expression was observed during a period of >4 months, with a normalization of cholesterol and triglyceride levels during 5 months. At that time, we successfully reinjected the recombinant adenovirus and observed the appearance of the human protein as well as the correction of lipoprotein phenotype. In mice killed 6 months-after the first infection, we observed a dose-dependent regression of fatty streak lesions in the aorta. We showed sustained expression of a transgene with a first-generation adenoviral vector and a correction of dyslipoproteinemia phenotype leading to lesion regression. These data demonstrate that somatic gene transfer can induce plaque regression.

Abnormal Reverse Cholesterol Transport in Controlled Type II Diabetic Patients: Studies on Fasting and Postprandial LpA-I Particles

The high incidence and prevalence of coronary heart disease in diabetes mellitus is clearly established. The usual lipid pattern found in type II diabetic patients is a moderate increase in fasting triglyceride levels associated with low HDL cholesterol levels. These abnormalities are further amplified in the postprandial state. To study the effect of these alterations on reverse cholesterol transport, we isolated lipoprotein containing apoA-I but not apoA-II (LpA-I) particles by immunoaffinity chromatography from the plasma of well-controlled type II diabetic patients and nondiabetic matched control subjects. Different parameters involved in this antiatherogenic pathway were measured in both fasting and postprandial states. Diabetic patients had reduced levels of LpA-I particles that were protein enriched and phospholipid depleted. Gradient gel electrophoresis showed that control LpA-I particles had five distinct populations, whereas diabetic particles lacked the largest one. LpA-I isolated from diabetic plasma exhibited a decreased capacity to induce cholesterol efflux from Ob 1771 adipose cells both in fasting (15.1 +/- 10.0% versus 7.5 +/- 2.7%, P < .05) and postprandial (17.7 +/- 11.2% versus 7.7 +/- 3.9%, P < .05) states, whereas only control particles showed significantly higher ability to promote cholesterol efflux after the test meal (P = .02). Lecithin:cholesterol acyltransferase activity measured with an exogenous substrate showed a 54% increase and an 18% decrease postprandially for control subjects and patients, respectively. Thus, the different abnormalities found in the fasting state were further amplified in the postprandial situation. This resulted in LpA-I particles with aberrant size and composition and decreased ability to accomplish their antiatherogenic role in type II diabetic patients.