Apo Expression Research Articles

Targeting Lipoprotein(a): Can RNA Therapeutics Provide the Next Step in the Prevention of Cardiovascular Disease?

Numerous genetic and epidemiologic studies have demonstrated an association between elevated levels of lipoprotein(a) (Lp[a]) and cardiovascular disease. As a result, lowering Lp(a) levels is widely recognized as a promising strategy for reducing the risk of new-onset coronary heart disease, stroke, and heart failure. Lp(a) consists of a low-density lipoprotein-like particle with covalently linked apolipoprotein A(apo[a]) and apolipoprotein B-100, which explains its pro-thrombotic, pro-inflammatory, and pro-atherogenic properties. Lp(a) serum concentrations are genetically determined by the apo(a) isoform, with shorter isoforms having a higher rate of particle synthesis. To date, there are no approved pharmacological therapies that effectively reduce Lp(a) levels. Promising treatment approaches targeting apo(a) expression include RNA-based drugs such as pelacarsen, olpasiran, SLN360, and lepodisiran, which are currently in clinical trials. In this comprehensive review, we provide a detailed overview of RNA-based therapeutic approaches and discuss the recent advances and challenges of RNA therapeutics specifically designed to reduce Lp(a) levels and thus the risk of cardiovascular disease.

Methionine-Mediated Regulation of Intestinal Structure and Lipid Transport in the Rice Field Eel (Monopterus albus)

An eight-week feeding trial discusses how methionine affects intestinal barrier and lipid transport on rice field eel (Monopterus albus). Six isoenergetic and isonitrogenous feeds contained different levels of methionine (0, 2 g/kg, 4 g/kg, 6 g/kg, 8 g/kg, or 10 g/kg). Compared with M0 (0 g/kg), gastric amylase, lipase, and trypsin were remarkably increased as dietary methionine ( P < 0.05 ); intestinal amylase, lipase, and trypsin were remarkably increased in M8 (8 g/kg) ( P < 0.05 ). Compared with M0, gastric fovea was remarkably increased ( P < 0.001 ), gastric epithelium is neater in M8 than that in M0, intestinal villus height and muscular thickness are remarkably increased in M8 ( P < 0.001 ), and amounts of goblet cells per root in M8 were increased ( P > 0.05 ), while intestinal crypt depth was markedly decreased ( P < 0.001 ). Lipid droplets in the intestinal villus and mucosal layer in the M8 (8 g/kg) group were more than that in M0 (0 g/kg). Compared with M0 (0 g/kg), the intestinal gcn2 and eif2α were downregulated in M8 (8 g/kg) ( P < 0.01 and P < 0.05 , respectively), while occ, cl12, cl15, zo-1, zo-2, hdlbp, ldlrap, npc1l1, cd36, fatp1, fatp2, fatp6, apo, apoa, apob, apoc, apoe, mct1, mct2, mct8, lpl, mttp, moat2, and dgat2 were upregulated markedly in M8 (8 g/kg). Intestinal eif2α expression was positively correlated with gcn2, and intestinal zo-1, cl15, fatp6, ldlrap, mct2, mct8, apo, apob, mct1, apoc, fatp1, mttp, cd36, occ, npc1l1, hdlbp, fatp2, apoe, lpl, and moat2 gene expression was negatively correlated with gcn2. In conclusion, methionine deficiency affected the gastric and intestinal structures, damaged the intestinal barrier, and decreased lipid and fatty acid transport. Besides, gcn2 could be activated when M. albus was fed methionine-deficient feed.

Diallyl disulphide inhibits apolipoprotein(a) expression in HepG2 cells through the MEK1-ERK1/2-ELK-1 pathway

BackgroundLipoprotein(a) [LP(a)] is implicated as a common and independent risk factor for cardiovascular diseases. The therapeutic options currently available for reducing plasma LP(a) concentrations are limited. Diallyl disulphide (DADS), the main component of garlic, regulates lipid metabolism in hepatocytes and adipocytes through ERK1/2 signalling. This study aimed to assess the effect of DADS on apolipoprotein(a) [apo(a)] in HepG2 cells. We also determined the effects of DADS on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms.MethodsWe examined the role of DADS on apo(a) expression in HepG2 cells by treating cell with different concentrations of DADS (10, 20, 40 and 80 μg/mL) for 24 h or treating cells with 40 μg/mL DADS for 0, 6, 12, 24 and 48 h. Then we used quantitative real-time PCR to analysis apo(a) mRNA levels, used Western blot to analysis apo(a) protein levels and used enzyme-linked immunosorbent assay to test apo(a) secreted levels. To farther determined the role of DADS, we applied Transfection of small interfering RNA to knockdown ELK-1levels and applied PD98059, a specific inhibitor of ERK1/2, to block ERK1/2 signal.ResultsThe results show DADS inhibited apo(a) at both the mRNA and protein levels in HepG2 cells in a dose-dependent manner. DADS-mediated inhibition of apoa(a) expression in HepG2 cells was attenuated when the cells were cultured in medium containing PD98059 (ERK1/2 inhibitor) or were transfected with siRNAs against MEK1 or ELK-1. Overexpression of apo(a) yielded similar results.ConclusionsThis study reveals that DADS can downregulate apo(a) expression in a dose-dependent manner via the MEK-ERK12-ELK-1 pathway.

MiR-23b-3p and miR-125b-5p downregulate apo(a) expression by targeting Ets1 in HepG2 cells.

High concentrations of plasma lipoprotein(a) [Lp(a)] have been inferred to be an independent risk factor for cardiovascular and cerebrovascular diseases, such as coronary artery diseases, restenosis, and stroke. Apolipoprotein(a) [apo(a)] is one of the most important components of Lp(a) and contributes greatly to the increased concentration of plasma Lp(a). As a critical positive transacting factor of apo(a) gene, Ets1 has been proven as a target gene of several miRNAs, such as miR-193b, miR-125b-5p, miR-200b, miR-1, and miR-499. In this study, a series of experiments on miRNAs and relative miRNAs inhibitor delivered HepG2 cells were conducted, and two miRNAs that downregulate the apo(a) by targeting the 3'-UTR of Ets1 were identified. Results showed that apo(a) and Ets1 were differentially expressed in SMMC7721 and HepG2 cell lines. Meanwhile, apo(a) and Ets1 were inversely correlated with several hepatic endogenous miRNAs, such as miR-125b-5p, miR-23b-3p, miR-26a-5p, and miR-423-5p, which were predicted to bind to Ets1. Results show that miR-125b-5p and miR-23b-3p mimics could inhibit the synthesis of apo(a) by directly targeting Ets1 in HepG2, thereby reducing the plasma Lp (a) concentration.

Vitamin C down-regulate apo(a) expression via Tet2-dependent DNA demethylation in HepG2 cells

Lipoprotein(a)[Lp(a)] is a risk factor for coronary heart diseases. However, the metabolism of this protein remains poorly understood. Efficient and specific drugs that can decrease high plasma levels of Lp(a) have not been developed yet. Vitamin C is responsible for maintaining the catalytic activity of a group of iron and 2-oxoglutarate (2OG)-dependent dioxygenases and induces the generation of 5-hydroxymethylcytosine (5hmC) via Ten-eleven translocation (Tet) dioxygenases. In addition, It has been reported vitamin C deficiency induces atherosclerosis and increases Lp(a) and apo(a) plasma levels in Lp(a)+ mice. However, the mechanism is still unclear. In this study, we investigated the effects of vitamin C on apo(a) expression and the possible molecular mechanism of vitamin C that influences apolipoprotein(a) [apo(a)] biosynthesis in HepG2 cells. Results showed that vitamin C significantly inhibited the expression and secretion levels of apo(a). Vitamin C can also increase ELK1 expression and hydroxymethylation of ELK1 promoter and the globle DNA in HepG2 cells. In addition, the effects of vitamin C inhibiting the apo(a) expression were attenuated by ELK1siRNA and Tet2siRNA. These results suggested vitamin C down-regulate apo(a) expression via Tet2-dependent DNA demethylation in HepG2 cells.

H2 S inhibits apo(a) expression and secretion through PKCα/FXR and Akt/HNF4α pathways in HepG2 cells.

Lipoprotein(a) [Lp(a)] is a strong genetic risk factor for coronary heart diseases. However, the metabolism of this protein remains poorly understood. Efficient and specific drugs that can decrease high plasma levels of Lp(a) have not been developed yet. Hydrogen sulfide (H2 S), a member of the gas transmitter family, performs important biological actions, including protection against cardiovascular diseases and maintenance of the lipid metabolism equilibrium in hepatocytes and adipocytes. In this study, we investigated the possible molecular mechanism of H2 S that influences apolipoprotein(a) [apo(a)] biosynthesis. We also determined the effects of H2 S on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms. Results showed that H2 S significantly inhibited the expression and secretion levels of apo(a). These effects were attenuated by the PKCα inhibitor and FXR siRNA. H2 S also reduced HNF4α expression and enhanced FXR expression. The Akt inhibitor partially reversed H2 S-induced inhibition of apo(a) and HNF4α expression and apo(a) secretion. This study reveals that H2 S suppressed apo(a) expression and secretion via the PKCα-FXR and PI3K/Akt-HNF4α pathways.

Abstract 428: Lipoprotein(a) Targeting With RNAi Delivery Platforms in Transgenic Mice and Cynomolgus Monkeys

Background: Meta-analyses of clinical studies have demonstrated that high levels of lipoprotein(a), known as Lp(a), correlate with residual risk of cardiovascular disease (CVD) while Mendelian genetic studies indicate that Lp(a) is an independent cause of CVD. Lp(a) is a complex lipoprotein particle similar in structure to LDL (consisting of cholesterol, triglycerides, phospholipids and apoB-100), but contains the genetically variable liver-expressed protein apo(a). Lp(a) may contribute to atherosclerosis by targeting oxidized phospholipids to vascular injury sites, promoting both inflammation and accumulation of lipids. Methods: RNA interference (RNAi) based approaches for reducing liver apo(a) expression were developed using both a targeted intravenous platform called Dynamic Polyconjugates (DPC) TM and a subcutaneous (SQ) format. The DPC platform comprises an RNAi trigger conjugated to cholesterol, and a hepatocyte-targeted, membrane active peptide to promote endosomal release of the RNAi trigger. The SQ platform is an RNAi trigger targeted to liver by N-acetyl galactosamine in a proprietary format. RNAi activity was assessed in two different humanized transgenic mice and cynomolgus monkeys. Results: In Lp(a) transgenic mice expressing human apo(a) and apoB-100 genes, treatment with a single DPC dose reduced serum Lp(a) by >98% at nadir and >90% for >5 weeks. A single DPC injection in cynomolgus monkeys resulted in >95% reduction in endogenous Lp(a) particles at 5 weeks post injection, without significant changes in toxicity biomarkers. Treatment with a SQ RNAi trigger reduced serum apo(a) in transgenic mice by >90% at nadir, while that in monkeys reached 64% reduction of Lp(a). Additional modifications to the RNAi trigger improved both depth and duration of knockdown in transgenic mice. Conclusion: Long-duration reductions in circulating apo(a) and Lp(a) have been shown with hepatocyte-directed delivery of apo(a)-specific RNAi triggers in mice and monkeys. This represents an intriguing mechanism to evaluate in humans for reducing residual risk of CVD in LDL-controlled individuals with elevated Lp(a).

FGF21 inhibits apolipoprotein(a) expression in HepG2 cells via the FGFR1-ERK1/2-Elk-1 pathway

Lipoprotein(a) [Lp(a)] is a highly atherogenic lipoprotein, whose metabolism is poorly understood. Efficient and secure drugs that can lower elevated plasma Lp(a) concentrations are currently lacking. Fibroblast growth factor-21 (FGF-21), a member of the FGFS super family, regulates glucose and lipid metabolism in hepatocytes and adipocytes via FGFR-ERK1/2 signaling. In this study, we investigated the molecular mechanisms that influence apolipoprotein(a) [apo(a)] biosynthesis. We also determined the effects of FGF21 on HepG2 cell apo(a) expression and secretion, as well as the mechanism of FGF21 in these effects. Results showed that FGF21 inhibited apo(a) expression at both mRNA and protein levels in a dose- and time--dependent manner and then suppressed the secretion of apo(a). These effects were attenuated by PD98059 (ERK1/2 inhibitor) and Elk-1 siRNA. PD166866 (FGFR1 inhibitor) also attenuated the FGF21-mediated inhibition of apo(a) expression and inhibited ERK1/2 and Elk-1 activation. These results demonstrate that FGF21 suppresses apo(a) expression via the FGFR1-ERK1/2-Elk-1 pathway.

When should we measure lipoprotein (a)?

Recently published epidemiological and genetic studies strongly suggest a causal relationship of elevated concentrations of lipoprotein (a) [Lp(a)] with cardiovascular disease (CVD), independent of low-density lipoproteins (LDLs), reduced high density lipoproteins (HDL), and other traditional CVD risk factors. The atherogenicity of Lp(a) at a molecular and cellular level is caused by interference with the fibrinolytic system, the affinity to secretory phospholipase A2, the interaction with extracellular matrix glycoproteins, and the binding to scavenger receptors on macrophages. Lipoprotein (a) plasma concentrations correlate significantly with the synthetic rate of apo(a) and recent studies demonstrate that apo(a) expression is inhibited by ligands for farnesoid X receptor. Numerous gaps in our knowledge on Lp(a) function, biosynthesis, and the site of catabolism still exist. Nevertheless, new classes of therapeutic agents that have a significant Lp(a)-lowering effect such as apoB antisense oligonucleotides, microsomal triglyceride transfer protein inhibitors, cholesterol ester transfer protein inhibitors, and PCSK-9 inhibitors are currently in trials. Consensus reports of scientific societies are still prudent in recommending the measurement of Lp(a) routinely for assessing CVD risk. This is mainly caused by the lack of definite intervention studies demonstrating that lowering Lp(a) reduces hard CVD endpoints, a lack of effective medications for lowering Lp(a), the highly variable Lp(a) concentrations among different ethnic groups and the challenges associated with Lp(a) measurement. Here, we present our view on when to measure Lp(a) and how to deal with elevated Lp(a) levels in moderate and high-risk individuals.

Gene expression in diplosporous and sexual Eragrostis curvula genotypes with differing ploidy levels

The molecular nature of gene expression during the initiation and progress of diplosporous apomixis is still unknown. Moreover, the basis of the close correlation between diplospory and polyploidy is not clarified yet. A comparative expression analysis was performed based on expressed sequence tags (ESTs) sequencing and differential display in an Eragrostis curvula diplosporous tetraploid genotype (T, 4x apo), a sexual diploid derivative obtained from tissue culture (D, 2x sex) and an artificial sexual tetraploid obtained from the diploid seeds after colchicine treatment (C, 4x sex). From a total of 8,884 unigenes sequenced from inflorescence-derived libraries, 112 (1.26%) showed significant differential expression in individuals with different ploidy level and/or variable reproductive mode. Independent comparisons between plants with different reproductive mode (same ploidy) or different ploidy level (same reproductive mode) allowed the identification of genes modulated in response to diplosporous development or polyploidization, respectively. Surprisingly, a group of genes (Group 3) were differentially expressed or silenced only in the 4x sex plant, presenting similar levels of expression in the 4x apo and the 2x sex genotypes. A group of randomly selected differential genes was validated by QR-PCR. Differential display analysis showed that in general the 4x apo and 4x sex expression profiles were more related and different from the 2x sex one, but confirmed the existence of Group 3-type genes, in both inflorescences and leaves. The possible biological significance for the occurrence of this particular group of genes is discussed. In silico mapping onto the rice genome was used to identify candidates mapping to the region syntenic to the diplospory locus.

Cyclosporin A inhibits apolipoprotein AI gene expression

Cyclosporin A (CsA), a calcineurin inhibitor, has been widely used as an immunosuppressant, and is known to induce hyperlipidemia and dyslipoproteinemia with low levels of high-density lipoprotein (HDL). Since apolipoprotein AI (apo AI) is a major protein component of HDL particles and reduction of apo AI results in low levels of HDL, we hypothesized that CsA inhibits apo AI gene expression contributing to its lipid effects. Therefore, we first measured the serum apo AI protein levels in rats with or without CsA treatment, and found that both serum apo AI protein and liver apo AI mRNA levels were significantly reduced in response to CsA treatment. In stably transfected Hep G2 cells harboring an apo AI-474-CAT reporter gene, we found that intracellular calcium mobilization by A23187 a calcium ionophore stimulated apo AI gene expression and the calcineurin inhibitors, CsA and FK605, selectively inhibited this stimulation. Therefore, we conclude that activation of the calcineurin pathway by intracellular calcium mobilization stimulates apo AI gene expression and calcineurin inhibition by CsA results in reduced apo AI gene expression.

Regulation of the expression of the apolipoprotein(a) gene: evidence for a regulatory role of the 5' distal apolipoprotein(a) transcription control region enhancer in yeast artificial chromosome transgenic mice.

The apolipoprotein(a) [apo(a)] gene locus is the major determinant of the circulating concentration of the atherothrombogenic lipoprotein Lp(a). In vitro analysis of the intergenic region between the apo(a) and plasminogen genes revealed the presence of a putative apo(a) transcription control region (ACR) approximately 20 kb upstream of the apo(a) gene that significantly increases the minimal promoter activity of the human apo(a) gene. To examine the function of the ACR in its natural genomic context, we used the Cre-loxP recombination system to generate 2 nearly identical apo(a)-yeast artificial chromosome transgenic mouse lines that possess a single integration site for the human apo(a) transgene in the mouse genome but differ by the presence or absence of the ACR enhancer. Analysis of the 2 groups of animals revealed that the deletion of the ACR was associated with 30% reduction in plasma and mRNA apo(a) levels. Apo(a)-yeast artificial chromosome transgenic mice with and without the ACR sequence were similar in all other aspects of apo(a) regulation, including liver-specific apo(a) expression and alteration in expression levels in response to sexual maturation and a high-fat diet. This study provides the first experimental in vivo evidence for a functional role of the ACR enhancer in determining levels of apo(a) expression.

4P-1008 Effect of cholesterol feeding on apo(a), CYP7A1 and LXRα expression in LPA transgenic mice

4P-1008 Effect of cholesterol feeding on apo(a), CYP7A1 and LXRα expression in LPA transgenic mice

Metabolism of triglyceride-rich lipoproteins and their role in atherosclerosis.

Recent large-scale clinical trials indicate that hypertriglyceridemia is a risk factor in coronary artery disease; however, the mechanism has not yet been completely clarified. We are currently studying the metabolism of triglyceride-rich lipoproteins and their role in atherosclerosis. Remnants, one of atherogenic lipoproteins, showed a marked increase and remained high even 8 hours after fat loading, especially in patients with coronary artery disease or diabetes mellitus. This shows that the postprandial state persists almost the whole day in these patients. Accordingly, it may be important to assess post-prandial remnant concentrations when evaluating risk factors for atherosclerosis. We identified apo B100 expression in the epithelial cells of the small intestine by immunoblotting with anti-apo B100 monoclonal antibody and dot-blotting of PCR-amplified cDNA. This indicates that not only apo B48, but also apo B100 is expressed in human small intestinal epithelium. The expression of apo B100 suggests that dietary VLDL may be synthesized in human small intestinal epithelium and converted into LDL, which may play an important role in atherosclerosis. A new receptor, apo B48, which binds and internalizes triglyceride-rich lipoproteins via a domain in apo B48, was identified in human monocyte-macrophages. The receptor differs from the scavenger receptor family and LDL receptor family because it does not bind acetyl LDL and it does bind VLDL devoid of apo E. Immunohistochemical studies indicate colocalization of anti-apo B48 receptor antibody in human atherosclerotic lesion foam cells, suggesting that apo B48 receptor may contribute to foam cell formation and atherosclerosis.

SstI polymorphism of the apo C3 gene and expression of dysbetalipoproteinemia (type III) in individuals heterozygous for familial LPL deficiency

SstI polymorphism of the apo C3 gene and expression of dysbetalipoproteinemia (type III) in individuals heterozygous for familial LPL deficiency

Human growth hormone increases apo(a) expression in transgenic mice.

Levels of Lp(a), an atherogenic lipoprotein that circulates in human plasma, are increased by the administration of growth hormone (GH). Many of the physiological effects of GH are mediated through insulin-like growth factor-1 (IGF-1), but ironically, IGF-1 treatment of humans is associated with a fall in plasma Lp(a) levels. To glean insight into the mechanism responsible for the GH-associated increase in plasma levels of Lp(a), we administered recombinant human GH (rhGH) to mice expressing a 370-kb human genomic fragment containing the apo(a) gene, 40 kb of 5'-, and 200 kb of 3'-flanking sequence [YAC-apo(a) transgenic mice]. The plasma levels of apo(a) and hepatic levels of apo(a) mRNA rose dramatically in the post-pubertal male mice in response to rhGH treatment. To determine whether the increase in plasma apo(a) was mediated by IGF-1, we treated castrated and noncastrated YAC-apo(a) transgenic mice with a continuous infusion of IGF-1 (100 microg/d) for 2 weeks, and plasma levels of apo(a) fell by approximately 50%. Thus the effects of rhGH and IGF-1 administration on plasma levels of apo(a) in the YAC-apo(a) transgenic mice simulate those seen in humans. The coordinate changes in apo(a) mRNA and plasma levels of apo(a) in response to rhGH and IGF-1 strongly suggest that these 2 hormones have independent effects on the transcription of the apo(a) gene.

Estrogen Modulation of Apolipoprotein(a) Expression: IDENTIFICATION OF A REGULATORY ELEMENT

Elevated plasma levels of the lipoprotein particle Lp(a) are a major risk factor for cardiovascular disease. Lp(a) plasma levels are determined by the level of expression of its characteristic protein component, apo(a). Apo(a) expression is modulated by several hormones, of which estrogens are the best known. The chromosomal region responsible for estrogen response was identified within an apo(a) enhancer located at approximately 26 kilobases from the apo(a) promoter. Although the estrogen-responsive unit contains a potential estrogen response element, binding of estrogen receptor-alpha to DNA was not necessary. The receptor, activated by bound estradiol, interacts through its transactivation domains with a transcription factor necessary for the function of the enhancer, preventing its binding to DNA.

Cell Culture Conditions Determine Apolipoprotein CIII Secretion and Regulation by Fibrates in Human Hepatoma HepG2 Cells

Fibrates are widely used drugs which lower triglycerides and increase HDL concentrations in serum. Recent findings from our laboratory have shown that fibrates repress apolipoprotein (apo) CIII gene expression, an effect that explains partially the triglyceride-lowering activity of these drugs. The goal of the present study was to compare the effect of various fibrates on apo CIII gene expression in the human hepatoblastoma cell line HepG2. First, we demonstrate that the level of apo CIII secretion by HepG2 cells is controlled by serum factors whereas apo CIII mRNA levels are not and even increase under conditions when apo CIII secretion dramatically decreases. Twelve different fetal calf serum batches were tested during this study and apo CIII secretion in cell medium could only be detected with three of them. The effect of serum on apolipoprotein secretion was more pronounced for apo CIII whereas other apolipoproteins (apo E, apo B, apo AII and apo AI) were affected to a lesser extent. Under serum conditions allowing apo CIII secretion, treatment with the peroxisome-proliferator activated receptor (PPAR)α activators fenofibrate, gemfibrozil and Wy-14643 result in a marked lowering of apo CIII secretion and gene expression, this effect being most pronounced with Wy-14643. Comparison of the activity of a PPARγ-specific ligand, the antidiabetic thiazolidinedione, BRL-49653 and a PPARα ligand Wy-14643 showed a marked decrease of apo CIII secretion and gene expression after activation of PPARα but not PPARγ. In conclusion, fibrates down-regulate apo CIII gene expression in human HepG2 cells, most likely via PPARα but not via PPARγ. However, these effects are only observed in HepG2 cells cultured under appropriate conditions.

Novel therapeutic strategy for atherosclerosis: ribozyme oligonucleotides against apolipoprotein(a) selectively inhibit apolipoprotein(a) but not plasminogen gene expression.

Because mechanisms of atherosclerosis by lipoprotein(a) [Lp(a)] have been postulated in the decrease in active transforming growth factor-beta conversion by decreased plasmin, selective decrease in apolipoprotein(a) [apo(a)] independent of plasminogen may have therapeutic values. Although antisense can decrease apo(a), its application may be difficult because of very high homology of apo(a) gene to plasminogen. Thus we used ribozyme strategy that actively cleaves targeted genes to selectively inhibit apo(a) expression. We constructed ribozyme oligonucleotides containing phosphorothioate DNA- and RNA-targeted kringle 4 of the apo(a) gene that showed 80% homology to plasminogen. Transfection of human apo(a) gene produced Lp(a) in medium of HepG2 cells, whereas Lp(a) could not be detected in control cells. Cotransfection of ribozyme and apo(a) gene resulted in the decrease in mRNA of apo(a) but not plasminogen. Moreover, marked decrease in Lp(a) was also observed in the medium transfected with ribozyme and apo(a) gene compared with apo(a) gene alone (P<0.01), whereas there was no significant change in plasminogen level between ribozyme-transfected and control cells. Incubation of human vascular smooth muscle cells (VSMC) with conditioned medium from apo(a)-transfected HepG2 cells resulted in a significant increase in VSMC number, whereas addition of conditioned medium from cells cotransfected with ribozyme oligonucleotides and apo(a) gene resulted in no VSMC growth (P<0.01). DNA-based control oligonucleotides and mismatched ribozyme oligonucleotides did not have an inhibitory effect on Lp(a) production. Overall, our data revealed that transfection of ribozyme against the apo(a) gene resulted in the selective inhibition of the apo(a) but not the plasminogen gene, providing novel therapeutic strategy for treatment of high Lp(a), a risk factor for atherosclerosis.

Differential expression of double-band apolipoprotein(a) phenotypes in healthy Spanish subjects detected by SDS–agarose immunoblotting

A sodium dodecyl sulphate-agarose apolipoprotein(a) [apo(a)] phenotyping method was set up to attain accurate scanning densitometry of proteins. Serum samples from 99 healthy Spanish men were analysed and twenty-five different apo(a) isoforms (12 to 37 kringle 4 repeats) were detected. Double-band phenotypes accounted for 39.4% ( n=39) and three different patterns of protein expression were identified: pattern A (20.5% of double-band phenotyped samples) predominantly expressed the highest molecular weight isoform; pattern B (53.9%) mainly the lowest molecular weight isoform, and pattern AB (25.6%), expressed both isoforms equally. A significant linear association between expression pattern and lipoprotein(a) [Lp(a)] concentration ≥0.30 g/l was observed. Single-band phenotyped samples ( n=60) were stratified according to apo(a) kringle 4 repeat categories and showed that 90% of isoforms <20 K4 repeats had high Lp(a) concentrations (≥0.30 g/l), whereas isoforms with 20 to 24 or more than 24 kringle 4 repeats had Lp(a) concentrations ≥0.30 g/l in 47% and 14%, respectively. A logistic regression model was fitted to test the association between apo(a) size, expression pattern and Lp(a) concentration. In this model, apo(a) isoform <25 kringle 4 repeats was significantly associated with serum Lp(a) concentration ≥0.30 g/l in both single and double-band phenotyped samples (odds ratio=8.9, p<0.001). In the latter, a differential expression pattern with respect to smaller size isoforms (pattern AB vs A) was significantly associated with Lp(a) concentration ≥0.30 g/l (odds ratio=17.97, P=0.045). Heterogeneity in protein apo(a) size expressed according to kringle 4 repeat number could be categorized in heterozygous phenotypes as three patterns. When small-sized isoform was expressed (pattern B) or both isoforms were equally expressed (pattern AB), the probability of having Lp(a)≥0.30 g/l is higher.