David Usher

Regression of Atherosclerosis Induced by Liver-Directed Gene Transfer of Apolipoprotein A-I in Mice

BACKGROUND The ability of apolipoprotein (apo)A-I to induce regression of preexisting atherosclerotic lesions has not been determined, and a mouse model of atherosclerosis regression has not yet been reported. METHODS AND RESULTS LDL receptor-deficient mice were fed a western-type diet for 5 weeks to induce atherosclerotic lesions. A second-generation recombinant adenovirus encoding human apoA-I or a control adenovirus were injected intravenously in order to express apoA-I in the liver. Three days after injection, total apoA-I levels in mice injected with the apoA-I-expressing adenovirus were 216+/-16.0 mg/dL, compared with 68.0+/-3.0 mg/dL in control virus-injected mice (P<0.001). HDL cholesterol levels in mice injected with the AdhapoA-I vector 7 days after injection were 189+/-21.0 mg/dL, compared with 123+/-8.0 mg/dL in control virus-injected mice (P<0.02). Total and non-HDL cholesterol levels did not differ between the 2 groups. Atherosclerotic lesion area was quantified by en face analysis of the aorta and cross-sectional analysis of the aortic root. Compared with baseline mice, atherosclerosis progressed in mice injected with the control adenovirus. In contrast, in mice expressing apoA-I compared with baseline mice, total en face aortic lesion area was reduced by 70% and aortic root lesion was reduced by 46%. Expression of apoA-I was associated with a significant reduction in the fraction of lesions occupied by macrophages and macrophage-derived foam cells. CONCLUSIONS Liver-directed gene transfer of human apoA-I resulted in significant regression of preexisting atherosclerotic lesions in LDL receptor-deficient mice as assessed by 2 independent methods.

The inverse association of plasma lipoprotein(a) concentrations with apolipoprotein(a) isoform size is not due to differences in Lp(a) catabolism but to differences in production rate.

Lipoprotein(a) (Lp[a]) is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL) but contains an additional protein called apolipoprotein(a) (apo[a]). Apo(a) is highly polymorphic in size, and there is a strong inverse association between the size of the apo(a) isoform and the plasma concentration of Lp(a). We directly compared the in vivo catabolism of Lp(a) particles containing different size apo(a) isoforms to establish whether there is an effect of apo(a) isoform size on the catabolic rate of Lp(a). In the first series of studies, four normal subjects were injected with radio-labeled S1-Lp(a) and S2-Lp(a) and another four subjects were injected with radiolabeled S2-Lp(a) and S4-Lp(a). No significant differences in fractional catabolic rate were found between Lp(a) particles containing different apo(a) isoforms. To confirm that apo(a) isoform size does not influence the rate of Lp(a) catabolism, three subjects heterozygous for apo(a) were selected for preparative isolation of both Lp(a) particles. The first was a B/S3-apo(a) subject, the second a S4/S6-apo(a) subject, and the third an F/S3-apo(a) subject. From each subject, both Lp(a) particles were preparatively isolated, radiolabeled, and injected into donor subjects and normal volunteers. In all cases, the catabolic rates of the two forms of Lp(a) were not significantly different. In contrast, the allele-specific apo(a) production rates were more than twice as great for the smaller apo(a) isoforms than for the larger apo(a) isoforms. In a total of 17 studies directly comparing Lp(a) particles of different apo(a) isoform size, the mean fractional catabolic rate of the Lp(a) with smaller size apo(a) was 0.329 +/- 0.090 day-1 and of the Lp(a) with the larger size apo(a) 0.306 +/- 0.079 day-1, not significantly different. In summary, the inverse association of plasma Lp(a) concentrations with apo(a) isoform size is not due to differences in the catabolic rates of Lp(a) but rather to differences in Lp(a) production rates.

Variation in lipoprotein(a) concentrations among individuals with the same apolipoprotein (a) isoform is determined by the rate of lipoprotein(a) production.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to, but metabolically distinct from, LDL. Factors regulating plasma concentrations of Lp(a) are poorly understood. Apo(a), the protein that distinguishes Lp(a) from LDL, is highly polymorphic, and apo(a) size is inversely correlated with plasma Lp(a) level. Even within the same apo(a) isoform class, however, plasma Lp(a) concentrations vary widely. A series of in vivo kinetic studies were performed using purified radiolabeled Lp(a) in individuals with the same apo(a) isoform but different Lp(a) levels. In a group of seven subjects with a single S4-apo(a) isoform and Lp(a) levels ranging from 1 to 13.2 mg/dl, the fractional catabolic rate (FCR) of 131I-labeled S2-Lp(a) (mean 0.328 day-1) was not correlated with the plasma Lp(a) level (r = -0.346, P = 0.45). In two S4-apo(a) subjects with a 10-fold difference in Lp(a) level, the FCRs of 125I-labeled S4-Lp(a) were very similar in both subjects and not substantially different from the FCRs of 131I-S2-Lp(a) in the same subjects. In four subjects with a single S2-apo(a) isoform and Lp(a) levels ranging from 9.4 to 91 mg/dl, Lp(a) concentration was highly correlated with Lp(a) production rate (r = 0.993, P = 0.007), but poorly correlated with Lp(a) FCR (mean 0.304 day-1). Analysis of Lp(a) kinetic parameters in all 11 subjects revealed no significant correlation of Lp(a) level with Lp(a) FCR (r = -0.53, P = 0.09) and a strong correlation with Lp(a) production rate (r = 0.99, P < 0.0001). We conclude that the substantial variation in Lp(a) levels among individuals with the same apo(a) phenotype is caused primarily by differences in Lp(a) production rate.

The low density lipoprotein receptor is not required for normal catabolism of Lp(a) in humans.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL). The role of the LDL receptor in the catabolism of Lp(a) has been controversial. We therefore investigated the in vivo catabolism of Lp(a) and LDL in five unrelated patients with homozygous familial hypercholesterolemia (FH) who have little or no LDL receptor activity. Purified 125I-Lp(a) and 131I-LDL were simultaneously injected into the homozygous FH patients, their heterozygous FH parents when available, and control subjects. The disappearance of plasma radioactivity was followed over time. As expected, the fractional catabolic rates (FCR) of 131I-LDL were markedly decreased in the homozygous FH patients (mean LDL FCR 0.190 d-1) and somewhat decreased in the heterozygous FH parents (mean LDL FCR 0.294 d-1) compared with controls (mean LDL FCR 0.401 d-1). In contrast, the catabolism of 125I-Lp(a) was not significantly different in the homozygous FH patients (mean FCR 0.251 d-1), heterozygous FH parents (mean FCR 0.254 d-1), and control subjects (mean FCR 0.287 d-1). In summary, absence of a functional LDL receptor does not result in delayed catabolism of Lp(a), indicating that the LDL receptor is not a physiologically important route of Lp(a) catabolism in humans.

Reduction of Isoprostanes and Regression of Advanced Atherosclerosis by Apolipoprotein E

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR− /−) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivooxidant stress, we quantitated the isoprostane iPF2α-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF2α-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.

Effect of transdermal testosterone treatment on serum lipid and apolipoprotein levels in men more than 65 years of age

PURPOSE Because the effects of androgen replacement on lipoprotein levels are uncertain, we sought to determine the effect of transdermal testosterone treatment on serum lipid and apolipoprotein levels in elderly men. SUBJECTS AND METHODS One hundred and eight healthy men more than 65 years of age who had serum testosterone concentrations >1 SD below the mean for young men were randomly assigned to receive either testosterone (54 men; 6 mg/day) or placebo (54 men) transdermally in a double-blind fashion for 36 months. Serum concentrations of lipids and apolipoproteins were measured, and cardiovascular events recorded. RESULTS Serum total cholesterol concentrations decreased in both the testosterone-treated men and placebo-treated men, but the 3-year mean (+/- SD) decreases in the two groups (testosterone treated, -17 +/- 29 mg/dL; placebo treated, -12 +/- 38 mg/dL) were not significantly different from each other (P = 0.4). Similarly, serum low-density lipoprotein (LDL) cholesterol levels decreased in both treatment groups, but the decreases in the two groups (testosterone treated, -16 +/- 24 mg/dL; placebo treated, -16 +/- 33 mg/dL) were similar (P = 1.0). Levels of high-density lipoprotein (HDL) cholesterol, triglycerides, and apolipoproteins A-I and B did not change. Lipoprotein(a) levels increased in both groups by similar amounts (testosterone treated, 3 +/- 9 mg/dL; placebo treated, 4 +/- 6 mg/dL; P = 1.0). The number of cardiovascular events was small and did not differ significantly between the testosterone-treated men (9 events) and the placebo-treated men (5 events) during the 3-year study (relative risk = 1.8; 95% confidence interval: 0.7 to 5.0). CONCLUSIONS As compared with placebo, transdermal testosterone treatment of healthy elderly men for 3 years did not affect any of the lipid or apolipoprotein parameters that we measured. The effect of testosterone treatment on cardiovascular events was unclear, because the number of events was small.

A PstI Polymorphism Detects the Mutation of Serine128 to Arginine in CD 62E Gene – a Risk Factor for Coronary Artery Disease

The mutation of serine128 to arginine in the CD 62E gene is a risk factor for coronary artery disease (CAD). We designed a new method to detect this mutation based on the observation that it is due to a transversion of nucleotide A561 to C, which abolishes a PstI recognition site. Two alleles, A and C, are easily typed when genomic DNA is amplified by PCR, digested with PstI, and separated on agarose gels. Among 153 people who underwent an elective, diagnostic arteriography in Johns Hopkins Hospital, we found that the C allele accounts for 19.5% in angiographically documented CAD patients (n = 82). It is significantly higher than the 10.6% frequency observed in normal controls (n = 71, p < 0.05). It indicates that the C allele is associated with early-onset CAD. This new method should facilitate the screening of this mutant allele in large populations and contribute to the understanding of the molecular mechanism underlying the association of this mutation with CAD.

Gene expression profiling of human diseases by serial analysis of gene expression

Until recently, the approach to understanding the molecular basis of complex syndromes such as cancer, coronary artery disease, and diabetes was to study the behavior of individual genes. However, it is generally recognized that expression of a number of genes is coordinated both spatially and temporally and that this coordination changes during the development and progression of diseases. Newly developed functional genomic approaches, such as serial analysis of gene expression (SAGE) and DNA microarrays have enabled researchers to determine the expression pattern of thousands of genes simultaneously. One attractive feature of SAGE compared to microarrays is its ability to quantify gene expression without prior sequence information or information about genes that are thought to be expressed. SAGE has been successfully applied to the gene expression profiling of a number of human diseases. In this review, we will first discuss SAGE technique and contrast it to microarray. We will then highlight new biological insights that have emerged from its application to the study of human diseases.

Microarray, SAGE and their applications to cardiovascular diseases.

ABSTRACTThe wealth of DNA data generated by the human genome project coupling with recently invented high-throughput gene expression profiling techniques has dramatically sped up the process for biomedical researchers on elucidating the role of genes in human diseases. One powerful method to reveal insight into gene functions is the systematic analysis of gene expression. Two popular high-throughput gene expression technologies, microarray and Serial Analysis of Gene Expression (SAGE) are capable of producing large amounts of gene expression data with the potential of providing novel insights into fundamental disease processes, especially complex syndromes such as cardiovascular disease, whose etiologies are due to multiple genetic factors and their interplay with the environment. Microarray and SAGE have already been used to examine gene expression patterns of cell-culture, animal and human tissues models of cardiovascular diseases. In this review, we will first give a brief introduction of microarray and SAGE technologies and point out their limitations. We will then discuss the major discoveries and the new biological insights that have emerged from their applications to cardiovascular diseases. Finally we will touch upon potential challenges and future developments in this area.

Anti-atherogenic effects of the acyl-CoA:cholesterol acyltransferase inhibitor, avasimibe (CI-1011), in cultured primary human macrophages.

Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been shown to reduce atherosclerotic lesions in animals; however, the mechanism(s) for this effect remains unclear. Therefore, we used cultured primary human monocyte-derived macrophages (HMMs) to examine the effect of the ACAT inhibitor, avasimibe (CI-1011), during foam cell formation and during cholesterol efflux from established foam cells. To examine the effect of CI-1011 on foam cell development, HMMs were incubated with aggregated acetylated LDL (ag-acLDL)+/-CI-1011 for 48 h. Total cholesterol (TC) was 29% lower in HMMs incubated with ag-acLDL and CI-1011 compared with ag-acLDL (P<0.05). To determine if TC reduction was due to reduced ag-acLDL uptake by CI-1011, 125I-acLDL binding at 4 degrees C for 4 h to HMMs preincubated with acLDL or ag-acLDL, CI-1011, acLDL+CI-1011, or ag-acLDL+CI-1011 for 48 h was measured. Specific binding was 40% lower in cells preincubated with acLDL+CI-1011, 52% lower in cells preincubated with ag-acLDL+CI-1011 and 49% lower in cells preincubated with CI-1011 compared with cells preincubated with acLDL (P<0.0003). Because CI-1011 appeared to directly affect acLDL binding, 125I-acLDL (3-80 microg protein/ml) binding was done in HMMs preincubated with CI-1011 (0-10 microg/ml) for 48 h. The calculated B(max) decreased in HMMs exposed to increasing concentrations of CI-1011, suggesting that CI-1011 altered scavenger receptor function and/or number. To examine the effects of CI-1011 on cholesterol efflux from established foam cells, we first examined whether CI-1011 was cytotoxic. HMMs were preincubated with ag-acLDL for 24 h, and then radiolabeled with [14C]adenine for 2 h (time zero). The radiolabeled cells were exposed to control RPMI medium or the same medium+HDL, CI-1011, or HDL+CI-1011 for 24 h. The release of [14C]adenine into the medium was not significantly different between cells exposed to RPMI, HDL, CI-1011, or HDL+CI-1011, suggesting that CI-1011 was not cytotoxic. Foam cells exposed to RPMI and CI-1011 (1-10 microg/ml) for 48 h showed time dependent reduction in cellular TC mass, with a corresponding increase in radiolabeled unesterified cholesterol into the medium. We then asked whether CI-1011 enhanced apoE mediated cholesterol efflux. Although cellular apoE increased between 2- and 7-fold in foam cells compared to control macrophages, apoE secreted into the medium was not significantly different between cells exposed to RPMI or CI-1011. Thus, CI-1011 exerted anti-atherogenic effects by reducing TC accumulation, inhibiting acLDL binding, and by limiting lipid storage in HMMs.