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Oxidized phospholipids (OxPL) are key mediators of the pro-atherosclerotic effects of oxidized lipoproteins. They are particularly important for the pathogenicity of lipoprotein(a) (Lp(a)), which is the preferred lipoprotein carrier of phosphocholine-containing OxPL in plasma. Indeed, elevated levels of OxPL-apoB, a parameter that almost entirely reflects the OxPL on Lp(a), are a potent risk factor for atherothrombotic diseases as well as calcific aortic valve stenosis. A substantial fraction of the OxPL on Lp(a) are covalently bound to the KIV

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a causal risk factor for the development of atherothrombotic disorders including coronary heart disease. However, the pathological mechanisms underlying this causal relationship remain incompletely defined. Lp(a) consists of a lipoprotein particle in which apolipoproteinB100 is covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). The remarkable homology between apo(a) and the fibrinolytic proenzyme plasminogen strongly suggests an antifibrinolytic role: apo(a) contains a strong lysine binding site and can block the sites on fibrin and cellular receptors required for plasminogen activation, but itself lacks proteolytic activity. While numerous in vitro and animal model studies indicate that apo(a) can inhibit plasminogen activation and fibrinolysis, this activity may not be preserved in Lp(a). Moreover, elevated Lp(a) does not reduce the efficacy of thrombolytic therapy and is not a risk factor for some non-atherosclerotic thrombotic disorders such as venous thromboembolism. Accordingly, different prothrombotic mechanisms for Lp(a) must be contemplated. Evidence exists that Lp(a) binds to and inactivates tissue factor pathway inhibitor and stimulates expression of tissue factor by monocytes. Moreover, some studies have shown that Lp(a) promotes platelet activation and aggregation, at least in response to some agonists. Lp(a) alters the structure of the fibrin network to make it less permeable and more resistant to lysis. Finally, Lp(a) may promote the development of a vulnerable plaque phenotype that is more prone to rupture and hence the precipitation of atherothrombotic events. Further study, especially in animal models of thrombosis, is required to clarify the prothrombotic effects of Lp(a).

Lipoprotein(a) [Lp(a)] has been established as an independent and causal risk factor for cardiovascular disease. Individuals with elevated levels of Lp(a) (>125 nmol/L; >50 mg/dl) display increased arterial wall inflammation characterized by activation of the endothelium by Lp(a)-carried oxidized phospholipids and recruitment of circulating monocytes. This results in increased secretion of chemoattractants and cytokines, upregulation of adhesion molecules and increased migration of leukocytes through the vessel wall. In addition, Lp(a) is also pivotal in the initiation phase of aortic valve stenosis. The oxidized phospholipids associated, in part, with the apolipoprotein(a) [apo(a)] moiety of Lp(a) stimulate the aortic valve residential cell, the valve interstitial cells (VICs), to either induce osteoblastic differentiation or apoptosis, thereby initiating the process of aortic valve calcification. Lastly, Lp(a) has been linked to systemic inflammation, including the acute phase response. Specifically, the cytokine interleukin 6 (IL-6) has a unique relationship with Lp(a), since the LPA gene contains IL-6 response elements. In this review, we will discuss the pathways and cell types affected by Lp(a) in the context of atherosclerosis, aortic valve stenosis and the acute phase response, highlighting the role of Lp(a) as an inflammatory mastermind. ispartof: Atherosclerosis vol:349 pages:101-109 ispartof: location:Ireland status: published

Epidemiological studies investigating the association between a biomarker and a disease have many limitations. The most prominent among these is that we cannot impute causality purely from a statistical association. If we observe an association, the biomarker might really be causal for the development of the disease, the association might be caused by a confounding variable or by reverse causation. With Mendelian Randomization (MR) methods, we have a potent tool at hand to derive evidence for a direct causal relationship. One of the core assumptions of MR studies is that genetic variants can be identified, which are strongly associated with the biomarker of interest, and can serve as an instrument indicating lifetime exposure. Since Lp(a) is primarily genetically determined by KIV-2 repeats, that in turn determine apo(a) isoform size, and by numerous single nucleotide polymorphisms (SNPs) and SNP-scores, this assumption is definitely fulfilled and it is probably one of the best phenotypes to be studied with Mendelian Randomization methods. The first studies evaluating the causal role of Lp(a) for cardiovascular diseases were performed in the early 1990s and more recently gained interest after several Lp(a)-increasing SNPs were identified in genome wide association studies. In this review, the principles behind MR methods are explained, together with their important role for Lp(a) research, particularly reconsidered in their historic context. MR methods have also been used to estimate the extent of Lp(a) reduction that would be required to yield a clinically meaningful reduction in outcomes in clinical intervention trials.

Lipoprotein(a) [Lp(a)] became besides LDL cholesterol one of the most attractive targets for intervention in cardiovascular disease. Strong genetic evidence supports the causal association between high Lp(a) concentrations and cardiovascular outcomes. Since specific Lp(a)-lowering therapies are under clinical investigation, the interest in measuring Lp(a) has markedly increased. However, the special structure of the lead protein component of Lp(a), named apolipoprotein(a), creates difficulties for an accurate measurement of Lp(a). A highly homologous repetitive structure, called kringle IV repeat with up to more the 40 repeats, causes a highly polymorphic protein. Antibodies raised against apolipoprotein(a) are mostly directed against the repetitive structure of this protein, which complicates the measurement of Lp(a) in molar terms. Both measurements in mass (mg/dL) and molar terms (nmol/L) are described and a conversion from one into the another unit is only approximately possible. Working groups for standardization of Lp(a) measurements are going to prepare widely available and improved reference materials, which will be a major step for the measurement of Lp(a). This review discusses many aspects of the difficulties in measuring Lp(a). It tries to distinguish between academic and practical concerns and warns to make a mountain out of a molehill, which does no longer allow to see the patient behind that mountain by simply staring at the laboratory issues. On the other hand, the calibration of some assays raises major concerns, which are anything else but a molehill. This should be kept in mind and we should start measuring Lp(a) with the aim of a better risk stratification for the patient and to identify those patients who might be in urgent need for a specific Lp(a)-lowering therapy as soon as it becomes available.

Lipoprotein(a) (Lp(a)) is an independent risk factor in the development of atherosclerotic cardiovascular diseases (ASCVD) and calcific aortic valve disease (CAVD). Lp(a) is an LDL-like particle to which apolipoprotein (a) (apo(a)) is covalently bound. Apo(a) contains a variable number of kringle IV repeats, a kringle V and a protease domain. Serum/plasma Lp(a) concentrations are traditionally expressed as total particle mass in mg/L. Concern has arisen lately as flawed Lp(a) mass tests have masked its clinical utility. The determinants of variability in Lp(a) composition were investigated, including the apo(a) size polymorphism, post-translational modifications -N- and O-glycosylation- and the lipid:protein ratio. Depending on the number of kringle IV-2 repeats, the theoretical protein content of the Lp(a) particle varies between 30 and 46 (w/w) %, which inescapably confounds Lp(a) mass measurements. The authors advocate that reporting of Lp(a) particle concentrations in mass units is metrologically inappropriate and should be abandoned, as it results in systematically biased Lp(a) results. Enabling technology, such as mass spectrometry, allows unequivocal molecular characterization of the apo(a) measurand(s) and accurate quantitation of apo(a) in molar units, unaffected by apo(a) size polymorphism. To guarantee that Lp(a)/apo(a) tests are fit-for-clinical-purpose, basic metrology principles should be implemented upfront during test development.

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, ε-aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it “pseudo-LBS”. Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.

Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV(2) repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV(2). We generated hybridomas, screened them, and successfully produced a KIV(2)-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV(9) without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence (4076)LETPTVV(4082) on KIV(9). In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements.

The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV2 and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV9. Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27-1,402nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, 4076LETPTVV4082, on KIV9. In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.

We previously demonstrated an association between lipoprotein (a) [Lp(a)] levels and atherosclerosis in human immunodeficiency virus (HIV)-seropositive women. The effects of antiretroviral therapy (ART) on Lp(a) levels in relation to apo(a) size polymorphism remain unclear. ART effects on allele-specific apo(a) level (ASL), an Lp(a) level associated with individual apo(a) alleles within each allele-pair, were determined in 126 HIV-seropositive women. ART effects were tested by a mixed-effects model across pre-ART and post-ART first and third visits. Data from 120 HIV-seronegative women were used. The mean age was 38 years; most were African-American (∼70%). Pre-ART ASLs associated with the larger (4.6 mg/dl vs. 8.0 mg/dl, P = 0.024) or smaller (13 mg/dl vs. 19 mg/dl, P = 0.041) apo(a) sizes were lower in the HIV-seropositive versus HIV-seronegative group, as was the prevalence of a high Lp(a) level (P = 0.013). Post-ART ASL and prevalence of high Lp(a) or apo(a) sizes and frequency of small size apo(a) (22 kringles) did not differ between the two groups. ART increased Lp(a) level (from 18 to 24 mg/dl, P < 0.0001) and both ASLs (P < 0.001). In conclusion, regardless of genetic control, Lp(a) can be modulated by HIV and its treatment. ART initiation abrogates HIV-induced suppression of Lp(a) levels and ASLs, contributing to promote CVD risk in HIV-seropositive individuals.

Background Lipoprotein(a) [Lp(a)] is reported as Lp(a) particle mass (mg/dL) or molar concentration of apolipoprotein(a) [apo(a)] (nmol/L), which is considered the gold standard. Values are often converted from one measurement to the other but the validity of this is unknown. Objectives To quantify the relationship between Lp(a) molar concentration and Lp(a) mass in the context of various Lp(a) level thresholds and apo(a) isoform size. Methods In all samples, Lp(a) levels in molar concentration and apo(a) isoform size were determined at the Northwest Lipid Metabolism and Diabetes Research Laboratories (NLMDRL). Lp(a) mass levels were determined at the University of California, San Diego (UCSD) (1635 samples), by 5 commercially available assays: Denka 1 and Denka 2 (each 80 samples), 2 turbidimetric assays (2545 and 2673 samples, respectively), and an enzyme-linked immunosorbent assay (2605 samples). The ratios between Lp(a) molar concentration and mass (eg, nmol/L/mg/dL) were calculated and related to apo(a) isoform size. Results The mean (SD) ratios for NLMDRL/UCSD, NLMDRL/Denka1, and NLMDRL/Denka2 were 2.42 (1.25), 1.64 (0.18), and 2.02 (0.22), respectively. The ratios for NLMDRL/UCSD, NLMDRL/Denka1, and NLMDRL/Denka2 increased by Lp(a) cutoffs, with ratios of 1.82, 1.52, and 1.87, respectively, for Lp(a) 125 nmol/L. For the commercial turbidimetric assays and enzyme-linked immunosorbent assay, the ratios ranged from 5. Conclusions Lp(a) molar/mass ratios are threshold, method, and isoform dependent. A single conversion factor between assays is not appropriate. These data support the transition of Lp(a) mass assays to molar concentration to improve diagnostic and clinical interpretation of Lp(a)-mediated risk.

Summary Background Elevated lipoprotein(a) (Lp[a]) is a highly prevalent (around 20% of people) genetic risk factor for cardiovascular disease and calcific aortic valve stenosis, but no approved specific therapy exists to substantially lower Lp(a) concentrations. We aimed to assess the efficacy, safety, and tolerability of two unique antisense oligonucleotides designed to lower Lp(a) concentrations. Methods We did two randomised, double-blind, placebo-controlled trials. In a phase 2 trial (done in 13 study centres in Canada, the Netherlands, Germany, Denmark, and the UK), we assessed the effect of IONIS-APO(a) Rx , an oligonucleotide targeting apolipoprotein(a). Participants with elevated Lp(a) concentrations (125–437 nmol/L in cohort A; ≥438 nmol/L in cohort B) were randomly assigned (in a 1:1 ratio in cohort A and in a 4:1 ratio in cohort B) with an interactive response system to escalating-dose subcutaneous IONIS-APO(a) Rx (100 mg, 200 mg, and then 300 mg, once a week for 4 weeks each) or injections of saline placebo, once a week, for 12 weeks. Primary endpoints were mean percentage change in fasting plasma Lp(a) concentration at day 85 or 99 in the per-protocol population (participants who received more than six doses of study drug) and safety and tolerability in the safety population. In a phase 1/2a first-in-man trial, we assessed the effect of IONIS-APO(a)-L Rx , a ligand-conjugated antisense oligonucleotide designed to be highly and selectively taken up by hepatocytes, at the BioPharma Services phase 1 unit (Toronto, ON, Canada). Healthy volunteers (Lp[a] ≥75 nmol/L) were randomly assigned to receive a single dose of 10–120 mg IONIS-APO(a)L Rx subcutaneously in an ascending-dose design or placebo (in a 3:1 ratio; single-ascending-dose phase), or multiple doses of 10 mg, 20 mg, or 40 mg IONIS-APO(a)L Rx subcutaneously in an ascending-dose design or placebo (in an 8:2 ratio) at day 1, 3, 5, 8, 15, and 22 (multiple-ascending-dose phase). Primary endpoints were mean percentage change in fasting plasma Lp(a) concentration, safety, and tolerability at day 30 in the single-ascending-dose phase and day 36 in the multiple-ascending-dose phase in participants who were randomised and received at least one dose of study drug. In both trials, the randomised allocation sequence was generated by Ionis Biometrics or external vendor with a permuted-block randomisation method. Participants, investigators, sponsor personnel, and clinical research organisation staff who analysed the data were all masked to the treatment assignments. Both trials are registered with ClinicalTrials.gov, numbers NCT02160899 and NCT02414594. Findings From June 25, 2014, to Nov 18, 2015, we enrolled 64 participants to the phase 2 trial (51 in cohort A and 13 in cohort B). 35 were randomly assigned to IONIS-APO(a) Rx and 29 to placebo. At day 85/99, participants assigned to IONIS-APO(a) Rx had mean Lp(a) reductions of 66·8% (SD 20·6) in cohort A and 71·6% (13·0) in cohort B (both p vs pooled placebo). From April 15, 2015, to Jan 11, 2016, we enrolled 58 healthy volunteers to the phase 1/2a trial of IONIS-APO(a)-L Rx . Of 28 participants in the single-ascending-dose phase, three were randomly assigned to 10 mg, three to 20 mg, three to 40 mg, six to 80 mg, six to 120 mg, and seven to placebo. Of 30 participants in the multiple-ascending-dose phase, eight were randomly assigned to 10 mg, eight to 20 mg, eight to 40 mg, and six to placebo. Significant dose-dependent reductions in mean Lp(a) concentrations were noted in all single-dose IONIS-APO(a)-L Rx groups at day 30. In the multidose groups, IONIS-APO(a)-L Rx resulted in mean reductions in Lp(a) of 66% (SD 21·8) in the 10 mg group, 80% (SD 13·7%) in the 20 mg group, and 92% (6·5) in the 40 mg group (p=0·0007 for all vs placebo) at day 36. Both antisense oligonucleotides were safe. There were two serious adverse events (myocardial infarctions) in the IONIS-APO(a) Rx phase 2 trial, one in the IONIS-APO(a) Rx and one in the placebo group, but neither were thought to be treatment related. 12% of injections with IONIS-APO(a) Rx were associated with injection-site reactions. IONIS-APO(a)-L Rx was associated with no injection-site reactions. Interpretation IONIS-APO(a)-L Rx is a novel, tolerable, potent therapy to reduce Lp(a) concentrations. IONIS-APO(a)-L Rx might mitigate Lp(a)-mediated cardiovascular risk and is being developed for patients with elevated Lp(a) concentrations with existing cardiovascular disease or calcific aortic valve stenosis. Funding Ionis Pharmaceuticals.

Background and aims Circulating lipoprotein (a) [Lp(a)] level relates inversely to apolipoprotein (a) [apo(a)] size. Both smaller apo(a) isoforms and higher Lp(a) levels have been linked to coronary heart disease and stroke, but their independent contributions are less well defined. We examined the role of Lp(a) in younger adults with cryptogenic stroke. Methods Lp(a) and apo(a) isoforms were evaluated in a prospectively designed case-control study of patients with unexplained ischemic stroke and stroke-free controls, ages 18 to 64. Serum Lp(a) was measured among 255 cases and 390 controls with both apo(a)-size independent and dependent assays. Apo(a) size was determined by agarose gel electrophoresis. Results Cases and controls were similar in socio-demographic characteristics, but cases had more hypertension, diabetes, smoking, and migraine with aura. In race-specific analyses, Lp(a) levels showed positive associations with cryptogenic stroke in whites, but not in the smaller subgroups of blacks and Hispanics. After full adjustment, comparison of the highest versus lowest quartile in whites was significant for apo(a)-size-independent (OR = 2.10 [95% CI = 1.04, 4.27], p = 0.040), and near-significant for apo(a)-size-dependent Lp(a) (OR = 1.81 [95% CI = 0.95, 3.47], p = 0.073). Apo(a) size was not associated with cryptogenic stroke in any race-ethnic subgroup. Conclusions This study underscores the importance of Lp(a) level, but not apo(a) size, as an independent risk factor for unexplained ischemic stroke in young and middle-aged white adults. Given the emergence of effective Lp(a)-lowering therapies, these findings support routine testing for Lp(a) in this setting, along with further research to assess the extent to which such therapies improve outcomes in this population.

Background Coronary artery disease (CAD) risk is greater with higher plasma lipoprotein(a)[Lp(a)] concentrations or smaller apoisoform size and putatively with increased cellular cholesterol loading capacity (CLC). The relationship between Lp(a) and CLC is not known. Information on Lp(a) polymorphisms in Italian patients is lacking. Objective The objective of this study was to determine relationships between Lp(a) and CLC, the impact of lipoprotein apheresis (LA), and describe the genetic profile of Lp(a). Methods We conducted a multicenter, observational study in Italian patients with hyperLp(a) and premature CAD with (n = 18)/without (n = 16) LA in which blood samples were analyzed for Lp(a) parameter and CLC. Genetic profiling of LPA was conducted in patient receiving LA. Results Mean macrophage CLC of the pre-LA serum was significantly higher than that of normolipidemic controls (19.7 ± 0.9 μg/mg vs 16.01 ± 0.98 μg/mg of protein, respectively). After LA, serum macrophage CLC was markedly lower relative to preapheresis (16.1 ± 0.8 μg/mg protein; P = .003) and comparable with CLC of the normolipidemic serum. LA did not significantly affect average apo(a) isoform size distribution. No anthropometric or lipid parameters studied were related to serum CLC, but there was a relationship between CLC and the Lp(a) plasma concentration (P = .035). DNA analysis revealed a range of common genetic variants. Two rare, new variants were identified: LPA exon 21, c.3269C>G, p.Pro1090Arg, and rs41259144 p.Arg990Gln, c.2969G>A Conclusions LA reduces serum Lp(a) and also reduces macrophage CLC. Novel genetic variants of the LPA gene were identified, and geographic variations were noted. The complexity of these polymorphisms means that genetic assessment is not a predictor of CAD risk in hyperLp(a).

Summary Background Lipoprotein(a) (Lp[a]) is a risk factor for cardiovascular disease and calcific aortic valve stenosis. No effective therapies to lower plasma Lp(a) concentrations exist. We have assessed the safety, pharmacokinetics, and pharmacodynamics of ISIS-APO(a) Rx , a second-generation antisense drug designed to reduce the synthesis of apolipoprotein(a) (apo[a]) in the liver. Methods In this randomised, double-blind, placebo-controlled, phase 1 study at the PAREXEL Clinical Pharmacology Research Unit (Harrow, Middlesex, UK), we screened for healthy adults aged 18–65 years, with a body-mass index less than 32·0 kg/m 2 , and Lp(a) concentration of 25 nmol/L (100 mg/L) or more. Via a randomisation technique, we randomly assigned participants to receive a single subcutaneous injection of ISIS-APO(a) Rx (50 mg, 100 mg, 200 mg, or 400 mg) or placebo (3:1) in the single-dose part of the study or to receive six subcutaneous injections of ISIS-APO(a) Rx (100 mg, 200 mg, or 300 mg, for a total dose exposure of 600 mg, 1200 mg, or 1800 mg) or placebo (4:1) during a 4 week period in the multi-dose part of the study. Participants, investigators, and study staff were masked to the treatment assignment, except for the pharmacist who prepared the ISIS-APO(a) Rx or placebo. The primary efficacy endpoint was the percentage change from baseline in Lp(a) concentration at 30 days in the single-dose cohorts and at 36 days for the multi-dose cohorts. Safety and tolerability was assessed 1 week after last dose and included determination of the incidence, severity, and dose relation of adverse events and changes in laboratory variables, including lipid panel, routine haematology, blood chemistry, urinalysis, coagulation, and complement variables. Other assessments included vital signs, a physical examination, and 12-lead electrocardiograph. This trial is registered with European Clinical Trials Database, number 2012-004909-27. Findings Between Feb 27, 2013, and July 15, 2013, 47 (23%) of 206 screened volunteers were randomly assigned to receive ISIS-APO(a) Rx as a single-dose or multi-dose of ascending concentrations or placebo. In the single-dose study, we assigned three participants to receive 50 mg ISIS-APO(a) Rx , three participants to receive 100 mg ISIS-APO(a) Rx , three participants to receive 200 mg ISIS-APO(a) Rx , three participants to receive 400 mg ISIS-APO(a) Rx , and four participants to receive placebo. All 16 participants completed treatment and follow-up and were included in the pharmacodynamics, pharmacokinetics, and safety analyses. For the multi-dose study, we assigned eight participants to receive six doses of 100 mg ISIS-APO(a) Rx , nine participants to receive six doses of 200 mg ISIS-APO(a) Rx , eight participants to receive six doses of 300 mg ISIS-APO(a) Rx , and six participants to receive six doses of placebo. Whereas single doses of ISIS-APO(a) Rx (50–400 mg) did not decrease Lp(a) concentrations at day 30, six doses of ISIS-APO(a) Rx (100–300 mg) resulted in dose-dependent, mean percentage decreases in plasma Lp(a) concentration of 39·6% from baseline in the 100 mg group (p=0·005), 59·0% in the 200 mg group (p=0·001), and 77·8% in the 300 mg group (p=0·001). Similar reductions were observed in the amount of oxidized phospholipids associated with apolipoprotein B-100 and apolipoprotein(a). Mild injection site reactions were the most common adverse events. Interpretation ISIS-APO(a) Rx results in potent, dose-dependent, selective reductions of plasma Lp(a). The safety and tolerability support continued clinical development of ISIS-APO(a) Rx as a potential therapeutic drug to reduce the risk of cardiovascular disease and calcific aortic valve stenosis in patients with elevated Lp(a) concentration. Funding Isis Pharmaceuticals.

Lipoprotein (a) [Lp(a)] is a highly atherogenic lipid particle. Although earlier reports suggested that Lp(a) levels are mostly determined by genetic factors, several recent studies have revealed that Lp(a) induction is also caused by chronic inflammation. Therefore, we aimed to examine whether cytokine blockade by monoclonal antibodies may inhibit Lp(a) metabolism. We found that interleukin 6 (IL-6) blockade by tocilizumab (TCZ) reduced Lp(a) while TNF-α-inhibition by adalimumab in humans had no effect. The specificity of IL-6 in regulating Lp(a) was further demonstrated by serological measurements of human subjects (n = 1,153) revealing that Lp(a) levels are increased in individuals with elevated serum IL-6. Transcriptomic analysis of human liver biopsies (n = 57) revealed typical IL-6 response genes being correlated with the LPA gene expression in vivo. On a molecular level, we found that TCZ inhibited IL-6-induced LPA mRNA and protein expression in human hepatocytes. Furthermore, examination of IL-6-responsive signal transducer and activator of transcription 3 binding sites within the LPA promoter by reporter gene assays, promoter deletion experiments, and electrophoretic mobility shift assay analysis showed that the Lp(a)-lowering effect of TCZ is specifically mediated via a responsive element at −46 to −40. Therefore, IL-6 blockade might be a potential therapeutic option to treat elevated Lp(a) serum concentrations in humans and might be a noninvasive alternative to lipid apheresis in the future.

Background The lipoprotein(a) pathway is a causal factor in coronary heart disease. We used a genetic approach to distinguish the relevance of two distinct components of this pathway, apolipoprotein(a) isoform size and circulating lipoprotein(a) concentration, to coronary heart disease. Methods In this mendelian randomisation study, we measured lipoprotein(a) concentration and determined apolipoprotein(a) isoform size with a genetic method (kringle IV type 2 [KIV2] repeats in the LPA gene) and a serum-based electrophoretic assay in patients and controls (frequency matched for age and sex) from the Pakistan Risk of Myocardial Infarction Study (PROMIS). We calculated odds ratios (ORs) for myocardial infarction per 1-SD difference in either LPA KIV2 repeats or lipoprotein(a) concentration. In a genome-wide analysis of up to 17 503 participants in PROMIS, we identified genetic variants associated with either apolipoprotein(a) isoform size or lipoprotein(a) concentration. Using a mendelian randomisation study design and genetic data on 60 801 patients with coronary heart disease and 123 504 controls from the CARDIoGRAMplusC4D consortium, we calculated ORs for myocardial infarction with variants that produced similar differences in either apolipoprotein(a) isoform size in serum or lipoprotein(a) concentration. Finally, we compared phenotypic versus genotypic ORs to estimate whether apolipoprotein(a) isoform size, lipoprotein(a) concentration, or both were causally associated with coronary heart disease. Findings The PROMIS cohort included 9015 patients with acute myocardial infarction and 8629 matched controls. In participants for whom KIV2 repeat and lipoprotein(a) data were available, the OR for myocardial infarction was 0·93 (95% CI 0·90–0·97; p

To evaluate the relationships of lipoprotein(a) (Lp(a)) concentration and apolipoprotein(a) (apo(a)) phenotype to major adverse cardiovascular events after coronary artery bypass grafting (CABG) in long-term follow-up.This single-center study included 356 patients with stable coronary heart disease (CHD) who underwent successful CABG. At baseline, we assessed the patient's risk factor profile for atherosclerosis, Lp(a) concentration and apo(a) phenotype. The primary endpoint was the composite of cardiovascular death and non-fatal myocardial infarction (MI). The secondary endpoint also included hospitalization for recurrent or unstable angina and repeat revascularization.Over a mean of 8.5 ± 3.5 years (range 0.9-15.0 years), the primary and secondary endpoints were registered in 46 (13%) and 107 (30%) patients, respectively. Patients with Lp(a) ≥30 mg/dL were at significantly greater risk for the primary endpoint (hazard ratio (HR) 2.98, 95% confidence interval (CI) 1.76-5.03, p 0.001) and secondary endpoint (HR 3.47, 95% CI 2.48-4.85, p 0.001) than patients with Lp(a) values30 mg/dL. The low molecular-weight apo(a) phenotype was also associated with higher risk of both primary and secondary endpoints (3.57 (1.87-6.82) and 3.05 (2.00-4.62), respectively; p 0.001 for both), regardless of conventional risk factors and statins use.In stable CHD patients Lp(a) concentration and low molecular-weight apo(a) phenotype are independently associated with three-fold increase in risk of major adverse cardiovascular events within 15 years after CABG. Lp(a) levels may provide an additional information for postoperative cardiovascular risk assessment.

Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular diseases, but the mechanism is unclear. The pathogenic risk of Lp(a) is associated with elevated plasma concentration, small isoforms of apolipoprotein [apo(a)], the unique apolipoprotein of Lp(a), and a mimic of plasminogen. Inflammation is associated with both the initiation and recovery of cardiovascular diseases, and plasminogen plays an important role in leukocyte recruitment. Because Lp(a)/apo(a) is expressed only in primates, transgenic mice were generated, apo(a)tg and Lp(a)tg mice, to determine whether Lp(a)/apo(a) modifies plasminogen-dependent leukocyte recruitment or whether apo(a) has an independent role in vivo . Plasminogen activation was markedly reduced in apo(a)tg and Lp(a)tg mice in both peritonitis and vascular injury inflammatory models, and was sufficient to reduce matrix metalloproteinase-9 activation and macrophage recruitment. Furthermore, neutrophil recruitment and the neutrophil cytokines, CXCL1/CXCL2, were suppressed in apo(a)tg mice in the abdominal aortic aneurysm model. Reconstitution of CXCL1 or CXCL2 restored neutrophil recruitment in apo(a)tg mice. Apo(a) in the plasminogen-deficient background and Lp(a)tg mice were resistant to inhibition of macrophage recruitment that was associated with an increased accumulation of apo(a) in the intimal layer of the vessel wall. These data indicate that, in inflammation, Lp(a)/apo(a) suppresses neutrophil recruitment by plasminogen-independent cytokine inhibition, and Lp(a)/apo(a) inhibits plasminogen activation and regulates matrix metalloproteinase-9 activation and macrophage recruitment.

Graphical abstract, Highlights • Acute application of apo(a) to smooth muscle cells induced chemorepulsion. • Chronic application (>24 h) induced stress fibre formation and cell spreading. • Effects of apo(a) were mediated by integrin αVβ3, tyrosine kinases and RhoA/ROCK. • Apo(a) impaired SMC motility which potentially contributes to vascular dysfunction., Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVβ3-dependent, but transforming growth factor β-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVβ3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.

Objectives The purpose of this study was to examine the relationship between apolipoprotein (a) isoforms and early atherosclerosis, assessed by carotid artery intima-media thickness in a sample of adults of the general population of Burgos, a city in the north of Spain. Design and methods Lipids, lipoprotein (a), number of carotid atherosclerotic plaques, if any, and the intima-media thickness in the far wall of both common carotid arteries by B-mode ultrasound were determined in a group of 171 adults from the general population of Burgos, Spain. Apolipoprotein (a) isoforms were determined in a random subset of 119 subjects. Results Increasing age, male sex, and past personal cardiovascular history were significantly associated with increased left, right, or average intima-media thickness of both carotid arteries in multivariate analysis. No statistically significant association was found between apolipoprotein (a) isoforms and mean carotid intima-media thickness by bivariate or multivariate regression analysis. Conclusions In this sample of the general Spanish population, no association was found between apolipoprotein (a) isoforms and carotid artery intima-media thickness.

High lipoprotein (a) [Lp(a)] concentrations are an independent risk factor for cardiovascular outcomes. Concentrations are strongly influenced by apo(a) kringle IV repeat isoforms. We aimed to identify genetic loci associated with Lp(a) concentrations using data from five genome-wide association studies (n = 13,781). We identified 48 independent SNPs in the LPA and 1 SNP in the APOE gene region to be significantly associated with Lp(a) concentrations. We also adjusted for apo(a) isoforms to identify loci affecting Lp(a) levels independently from them, which resulted in 31 SNPs (30 in the LPA, 1 in the APOE gene region). Seven SNPs showed a genome-wide significant association with coronary artery disease (CAD) risk. A rare SNP (rs186696265; MAF ∼1%) showed the highest effect on Lp(a) and was also associated with increased risk of CAD (odds ratio = 1.73, P = 3.35 × 10−30). Median Lp(a) values increased from 2.1 to 91.1 mg/dl with increasing number of Lp(a)-increasing alleles. We found the APOE2-determining allele of rs7412 to be significantly associated with Lp(a) concentrations (P = 3.47 × 10−10). Each APOE2 allele decreased Lp(a) by 3.34 mg/dl corresponding to ∼15% of the population’s mean values. Performing a gene-based test of association, including suspected Lp(a) receptors and regulators, resulted in one significant association of the TLR2 gene with Lp(a) (P = 3.4 × 10−4). In summary, we identified a large number of independent SNPs in the LPA gene region, as well as the APOE2 allele, to be significantly associated with Lp(a) concentrations.

ObjectivesThis study sought to assess whether an antisense oligonucleotide (ASO) directed to apolipoprotein (a) [apo(a)] reduces apo(a) and lipoprotein (a) [Lp(a)] levels in transgenic mouse models.BackgroundElevated Lp(a) is a causal, independent, genetic risk factor for cardiovascular disease and myocardial infarction. Effective therapies to specifically lower plasma Lp(a) levels are lacking.MethodsThree transgenic mouse models were utilized: 8K-apo(a) mice expressing 8 kringle IV (KIV) repeats with a single copy of KIV-2; 8K-Lp(a) mice expressing both the 8K apo(a) plus human apolipoprotein B-100; and 12K-apo(a) mice expressing a 12K apo(a) with 3 KIV-2 repeats. The mice were treated intraperitoneally with saline, a control ASO, or ASO 144367 directed to KIV-2 for 4 to 6 weeks. Apo(a), Lp(a), and oxidized phospholipids present on human apoB (OxPL/h-apoB) or apo(a) [OxPL/apo(a)] were measured at baseline and on and off therapy.ResultsASO 144367 significantly reduced Lp(a) by 24.8% in 8K-Lp(a) mice, and reduced apo(a) levels by 19.2% in 8K-Lp(a) mice, 30.0% in 8K-apo(a) mice, and 86% in 12K-apo(a) mice; ASO 144367 also significantly reduced OxPL/apoB 22.4% in 8K-Lp(a) mice, and OxPL/apo(a) levels by 19.9% in 8K-Lp(a) mice, 22.1% in 8K-apo(a) mice, and 92.5% in 12K-apo(a) mice (p < 0.004, or less, for all). No significant changes occurred in Lp(a), apo(a), OxPL/apoB, or OxPL/apo(a) levels with control ASO or saline.ConclusionsThis study documents the first specific therapy, to our knowledge, for lowering apo(a)/Lp(a) levels and their associated OxPL. A more potent effect was documented in mice expressing apo(a) with multiple KIV-2 repeats. Targeting liver expression of apo(a) with ASOs directed to KIV-2 repeats may provide an effective approach to lower elevated Lp(a) levels in humans.

Human apolipoprotein(a) (apo(a)), synthesized in the liver, contains oxidized phosphatidylcholine (oxPtdPC) adducts probably generated at the hepatic site. Since plasminogen (Plg), also synthesized in the liver, is genetically related and structurally homologous to apo(a), we wanted to determine whether it contains oxPtdPCs and their location. We used Plg isolated from fresh or frozen normal human plasma and several commercial preparations. Some were freed of non-covalently bound lipids by organic solvent extraction. By immunoblot analyses all products reacted against T15, a natural IgM monoclonal antibody specific for phosphorylcholine -containing oxidized phospholipids (ox-PLs). This immunoreactivity was retained in urokinase type plasminogen activator -generated plasmin and was abrogated in Plg previously digested with lipoprotein-associated phospholipase A2 (Lp-PLA2), a reaction that generated that predominantly C16:0 lysophosphatidylcholine species as determined by mass spectrometry. Lysoderivatives were also generated upon the cleavage by Lp-PLA2 of a model ox-PL chemically linked to a lysine-containing pentapeptide. From inorganic phosphorous analyses, we found 2 moles of oxPtdPC/mole of Plg distributed between the kringles 1-4 and mini-Plg domain. OxPtdPCs were also present in the Plg isolated from the serum-free medium of cultured human HepG2 cells. In conclusion, our results provide strong evidence that naturally occurring Plg contains oxPtdPC probably linked by a Schiff base and also suggest that the linkage occurs at the hepatic site. Given the emerging evidence for the cardiovascular pathogenicity of oxPtdPCs we speculate that they may impart athero-thrombogenic properties to Plg under inflammatory conditions.

Lipoprotein(a) (Lp(a)) is an independent, genetically regulated cardiovascular risk factor. Lp(a) plasma levels are largely determined by the apolipoprotein(a) (apo(a)) component, and differ across ethnicity. Although a number of polymorphisms in the apo(a) gene have been identified, apo(a) genetic regulation is not fully understood. To study the relation between apo(a) gene variants, we constructed haplotypes and assessed linkage equilibrium in African Americans and Caucasians for three widely studied apo(a) gene polymorphisms (apo(a) size, +93 C/T and pentanucleotide repeat region (PNR)). Apo(a) size allele frequency distributions were different across ethnicity (p0.01). For African Americans, PNR frequencies were similar across apo(a) sizes, suggesting linkage equilibrium. For Caucasians, the PNR and the PNR-C/T haplotype frequencies differed for large and small apo(a), with the T and PNR 9 alleles associated with large apo(a) size (p0.0002); also, the PNR 9 allele was more common on a T allele, while PNR 8 was more common on a C allele. On a C allele background, small PNR alleles were more common and the PNR 10 allele less common among African Americans than Caucasians (p0.001). The ethnic difference in apo(a) size distribution remained controlling for C/T and PNR alleles (p=0.023). In conclusion, allele and haplotype frequencies and the nature of the linkage disequilibrium differed between African Americans and Caucasians at three apo(a) gene polymorphisms.

Introduction: Cardio- and/or cerebro-vascular risk are associated with high lipoprotein (a) [Lp(a)] levels and low-molecular-weight (LMW) apo(a) isoforms. Aims of this study were to evaluate the deposition of apo(a) isoforms and apoprotein B (apo B) in atherosclerotic plaque from patients (males and females) who had carotid endarterectomy for severe stenosis, and to identify differences between patients classified by gender and divided according to the stability or instability of their plaques. Materials and methods: We determined lipids, apo B and Lp(a) in serum and plaque extracts from 55 males and 25 females. Apo(a) was phenotyped and isoforms were classified by number of kringle IV (KIV) repeats. Results: Lp(a) levels were higher in female serum and plaque extracts than in male samples, while apo B levels were lower. More Lp(a) than apo B deposition was observed in plaque after normalization for serum levels. Thirty-one different apo(a) isoforms were detected in our patients, with a double band phenotype in 94% of cases. In both sexes, the low/high (L/H) molecular weight apo(a) isoform expression ratio was significantly higher in plaque than in serum. Females with unstable plaques had higher Lp(a) levels in both serum and tissue extracts, and fewer KIV repeats of the principal apo(a) isoform in the serum than the other female group or males. Conclusions: In both sexes, the same apo(a) isoforms are found in serum and atherosclerotic plaque, but in different proportions: in plaque, LMW apo(a) is almost always more strongly accumulated than HMW apo(a), irrespective of any combination of apo(a) isoforms in double band phenotypes or Lp(a) serum levels. Moreover, serum and tissue Lp(a) levels were higher in females than in males, and particularly in the group with unstable plaques.

Lipoprotein(a) [Lp(a)] is a LDL-like particle containing a single copy of apolipoprotein B-100 (apoB-100), covalently attached to apolipoprotein(a) [apo(a)]. Apo(a) is encoded by LPA gene (6q26-27), and it has been hypothesized that LPA +93C>T and +121G>A polymorphisms in the 5' flanking region could influence the apolipoprotein(a) synthesis, so affecting Lp(a) levels. In order to permit a rapid detection of LPA polymorphisms, we performed an analysis protocol for the SNPs detection through Nanogen Technology with the Universal Reporting System, and we compared our results with those obtained with a more conventional method, such as PCR-RFLP assay. Our experiments evidenced that Nanogen Technology may be used as a high-throughput tool in LPA +93C>T and +121G>A polymorphisms analysis, minimizing the hands-on time and the costs for the SNPs detection. In particular, this Technology allows the analysis of polymorphisms at the LPA locus, able to modulate the levels of Lp(a), a relevant marker of atherosclerosis.

Few and conflicting data are available in the literature on the association between Lp(a) levels and the severity of coronary artery disease (CAD) in diabetic patients. In addition, no studies took into account the role of apo(a) polymorphism. The purpose of the present study was to analyse the association of the degree of coronary atherosclerosis with Lp(a) levels and apo(a) polymorphism in a large group of type 2 diabetic patients.The study population consisted of 227 consecutive type 2 diabetic patients undergoing a routine coronary angiography to evaluate chest pain or suspected CAD. The patients were subdivided into four subgroups according to the number of coronary arteries diseased: normal arteries (n=26), mono-vessel disease (n=67), bi-vessel disease (n=54) and multi-vessel disease (n=80).Lp(a) levels (normal arteries: 14.6+/-19.6 mg/dl; mono-vessel disease: 19.0+/-16.4 mg/dl; bi-vessel disease: 19.3+/-15.1 mg/dl; multi-vessel disease: 26.5+/-16.8 mg/dl; p0.001) and the percentages of patients with at least one isoform of low molecular weight (normal arteries: 23.1%; mono-vessel disease: 38.8%; bi-vessel disease: 75.9%; multi-vessel disease: 81.2%; p0.001) were significantly correlated with increasing number of coronary vessels diseased. Multiple logistic regression analysis showed that both Lp(a) levels (OR: 1.31; 95% CI: 1.02-4.11) and apo(a) polymorphism (OR: 3.43; 95% CI: 1.67-7.05) were independent predictors of CAD severity.Our data suggest that Lp(a) levels and apo(a) polymorphism may be reliable predictors of CAD severity in type 2 diabetic patients.

ObjectivesThis study sought to determine the effect of lipoprotein(a), or Lp(a), levels and apolipoprotein(a), or apo(a), sizes on endothelial function and to explore ethnic differences in their effects.BackgroundAlthough high levels of Lp(a) have been shown to confer increased cardiovascular risk in Caucasians, its significance in non-Caucasian populations is uncertain. The pathogenic role of the apo(a) component of Lp(a) is also unclear.MethodsThe relationship of Lp(a) levels and apo(a) sizes to endothelial function was examined in a multiethnic cohort of 89 healthy subjects (age 42 ± 9 years; 50 men, 39 women) free of other cardiac risk factors. Endothelium-dependent, flow-mediated dilation (FMD) and endothelium-independent, nitrate-induced dilation (NTG) were assessed by ultrasound imaging of the brachial artery.ResultsPlasma Lp(a) levels were lowest in Caucasians (18.3 ± 21.1 mg/dl, n = 40); intermediate in Hispanics (30.2 ± 30.5 mg/dl, n = 21); and highest in African Americans (68.8 ± 46.0 mg/dl, n = 28). Lipoprotein(a) levels were found to correlate inversely to FMD (r = −0.33, p < 0.005) but not to NTG (r = 0.06, p = 0.60). This association remained significant after adjusting for gender (p = 0.002). In addition, subjects with small apo(a) size of ≤22 kringle 4 repeats had significantly lower FMD than those with large apo(a) (2.23 ± 2.37% vs. 6.26 ± 4.29%, p < 0.0001), irrespective of Lp(a) levels.ConclusionsThese findings support an independent role of Lp(a) in atherogenesis, an effect that is particularly evident in African Americans. The proatherogenic property of Lp(a) can be attributed in part to its apo(a) component.

Accumulation of lipoprotein(a) (Lp(a)) in atherosclerotic plaques is mediated through interaction of fibrin-(ogen) deposits with the apolipoprotein(a) (apo(a)) moiety of Lp(a). It was suggested that because apo(a) competes with plasminogen for binding to fibrin, causing inhibition of fibrinolysis, it could also promote atherothrombosis. Because the fibrin(ogen) alphaC-domains bind plasminogen and tissue-type plasminogen activator with high affinity in a Lys-dependent manner, we hypothesized that they could also bind apo(a). To test this hypothesis, we studied the interaction between the recombinant apo(a) A10 isoform and the recombinant alphaC-fragment (Aalpha-(221-610)) corresponding to the alphaC-domain by enzyme-linked immunosorbent assay and surface plasmon resonance. Both methods revealed a high affinity interaction (Kd = 19-21 nm) between the immobilized alphaC-fragment and apo(a), indicating that the former contains an apo(a)-binding site. This affinity was comparable to that of apo(a) for fibrin. At the same time, no interaction was observed between soluble fibrinogen and immobilized apo(a), suggesting that, in the former, this and other apo(a)-binding sites are cryptic. Further experiments with truncated recombinant variants of the alphaC-fragment allowed localization of the apo(a)-binding site to the Aalpha-(392-610) region. The presence of epsilon-aminocaproic acid only slightly inhibited binding of apo(a) to the alphaC-fragment, indicating the Lys-independent nature of their interaction. In agreement, the influence of plasminogen or tissue-type plasminogen activator on binding of apo(a) to the alphaC-fragment was minimal. These results indicate that the alphaC-domains contain novel high affinity apo(a)-binding sites that may provide a Lys-independent mechanism for bringing Lp(a) to places of fibrin deposition such as injured vessels or atherosclerotic lesions.

Most studies aiming to detect associations of genetic variation with common complex diseases, e.g. coronary heart disease (CHD) have been performed in populations with a western lifestyle but it is unclear whether associations detected in one geographic group exist also in others. We here have determined lipoprotein(a) levels and apo(a) K-IV-2 repeat genotypes in CHD patients (N=254) and controls (N=480) from two Asian Indian populations (Tamil Nadu and New Delhi). In both populations and also in the pooled dataset median Lp(a) levels were significantly elevated in the patients (27.4 mg/dl) compared with the controls (17.6 mg/dl). Apo(a) K-IV-2 allele frequencies were not different between the CHD patients and controls and thus did not explain the increased Lp(a) levels in CHD patients. Contrary to what has recently been observed in Black and White men short (K-IVor=22) alleles associated with high Lp(a) concentration were not overrepresented in the patients. Rather, short (K-IVor=22), intermediate (K-IV 23-29) and long (K-IVor=30) apo(a) alleles were all associated with higher Lp(a) levels in the patients. Accordingly relative risk (estimated as odds ratio) for CHD rose continuously with increasing Lp(a) but was independent of apo(a) allele length. Together with previous studies our results indicate that the relation between apo(a) genotypes, Lp(a) levels, and CHD may be heterogeneous across ethnic groups and that it depends on the genetic architecture of the Lp(a) trait in a given population whether an association of K-IV-2 repeat length with CHD exists or not.

The sites and mechanisms of the catabolism of atherogenic lipoprotein(a) (Lp(a)) are not well understood. Lp(a) is increased in patients with end-stage renal disease, suggesting a renal catabolism of Lp(a). To gain a better insight into renal handling of Lp(a), we established a heterologous rat model to study the renal catabolism of human Lp(a). Pure human Lp(a) was injected into Wistar rats, and animals were sacrificed at different time points (30 minutes to 24 hours). Intact Lp(a) was cleared from the circulation of injected rats with a half-life time of 14.5 hours. Strong intracellular immunostaining for apolipoprotein(a) (apo(a)) was observed in the cytoplasm of proximal tubular cells after 4, 8, and 24 hours. Apolipoprotein B (apoB) was colocalized with glomerular apo(a) 1 to 8 hours after Lp(a) injection, but renal capillaries and tubules remained negative. No relevant amounts of apo(a) fragments were found in the plasma of rats after injection of Lp(a). During all urine collection periods, apo(a) fragments with molecular weights of 50 to 160 kd were detected in the urine, however. Our results show that human Lp(a) injected into rats accumulates intracellularly in the rat kidney, and apo(a) fragments are excreted in the urine. The kidney apparently plays a major role in fragmentation of Lp(a). Despite the fact that rodents lack endogenous Lp(a), rats injected with human Lp(a) may provide a useful heterologous animal model to study the renal metabolism of Lp(a) further.

Objective: To establish reference estimates of the effects of different hormone replacement therapy (HRT) regimens on lipid and lipoprotein levels. Design: Review and pooled analysis of prospective studies published up until the year 2000. Setting: Clinical trials centers, hospitals, menopause clinics. Patient(s): Healthy postmenopausal women. Intervention(s): Estrogen alone, estrogen plus progestogen, tibolone, or raloxifene in the treatment of menopausal symptoms. Main Outcome Measure(s): Serum high- and low-density lipoprotein (HDL and LDL) cholesterol, total cholesterol, triglycerides, and lipoprotein (a). Result(s): Two-hundred forty-eight studies provided information on the effects of 42 different HRT regimens. All estrogen alone regimens raised HDL cholesterol and lowered LDL and total cholesterol. Oral estrogens raised triglycerides. Transdermal estradiol 17-beta lowered triglycerides. Progestogens had little effect on estrogen-induced reductions in LDL and total cholesterol. Estrogen-induced increases in HDL and triglycerides were opposed according to type of progestogen, in the order from least to greatest effect: dydrogesterone and medrogestone, progesterone, cyproterone acetate, medroxyprogesterone acetate, transdermal norethindrone acetate, norgestrel, and oral norethindrone acetate. Tibolone decreased HDL cholesterol and triglyceride levels. Raloxifene reduced LDL cholesterol levels. In 41 studies of 20 different formulations, HRT generally lowered lipoprotein (a). Conclusion(s): Route of estrogen administration and type of progestogen determined differential effects of HRT on lipid and lipoprotein levels. Future work will focus on the interpretation of the clinical significance of these changes.

The atherogenic lipoprotein Lp(a) consists of an LDL-like core and apo(a), linked to apoB via a thiol bridge. Apo(a) fragments ranging in size from 60 to 220 kDa are excreted into urine and the excretion rate correlates significantly with the plasma levels of Lp(a). In order to study the interrelationship of apo(a) secretion with that of other plasma proteins, urinary apo(a) and protein secretion of five probands were followed for 24 h at different urinary densities. The excretion rate of apo(a) fragments, despite their high molecular weight, was highest, followed by apoD, orosomucoid, albumin and beta(2)-glycoprotein-I (beta2-GI) and plasminogen (1.58, 0.87, 0.095, 0.027, 0.013 and0.001%/day, respectively). There was a highly significant correlation between apo(a), apoD and beta2-GI concentrations but not with albumin and orosomucoid concentrations in urine. The only protein that was fragmented in urine was apo(a) while the other proteins had molecular weights comparable to those in plasma. We conclude that a previously suggested fragmentation of apo(a) by the kidney is not a rate-limiting step in its excretion. Since plasminogen, another kringle-IV-containing plasma compound, and fragments thereof, are undetectable in urine under identical experimental conditions, it is very unlikely that the characteristic kringle structure is responsible for the high excretion rate of apo(a).

In this nested case-control study, lipoprotein (a) [Lp(a)] concentrations and apo(a) isoform size were measured in serum samples obtained from men participating in the prospective Multiple Risk Factor Intervention Trial (MRFIT). Serum from men aged 35 to 57 years and stored for up to 20 years were analyzed for Lp(a) levels (n=736) and isoform size (n=487), respectively. Cases involved nonfatal myocardial infarctions (MI; n=98), documented during the active phase of the study that ended on February 28, 1982 and coronary heart disease (CHD) deaths (n=148) monitored through 1990. Median Lp(a) levels did not differ between cases and controls and mean apo(a) size did not vary between cases and controls in the entire study population. When adjusted for age and Lp(a) concentration, logistic regression analysis indicated that small apo(a) isoforms were associated with CHD deaths among smokers (OR 3.31; 95% CI 1.07-10.28).

The assembly of lipoprotein(a) (Lp(a)) involves an initial noncovalent interaction between apolipoprotein (apo) B100 and apo(a), followed by the formation of a disulfide bond between apoB100 cysteine 4326 and apo(a) cysteine 4057. The structural features of apoB100 that are required for its noncovalent interaction with apo(a) have not been fully defined. To analyze that initial interaction, we tested whether apo(a) could bind noncovalently to two apoB proteins that lack cysteine 4326: mouse apoB100 and human apoB100-C4326G. Our experiments demonstrated that both mouse apoB and the human apoB100-C4326G bind noncovalently to apo(a). We next sought to gain insights into the apoB amino acid sequences required for the interaction between apoB100 and apo(a). Previous studies of truncated human apoB proteins indicated that the carboxyl terminus of human apoB100 (amino acids 4330-4397) is important for Lp(a) assembly. To determine whether the carboxyl terminus of mouse apoB100 can interact with apo(a), transgenic mice were produced with a mutant human apoB gene construct in which human apoB100 amino acids 4279-4536 were replaced with the corresponding mouse apoB100 sequences and tyrosine 4326 was changed to a cysteine. The mutant apoB100 bound to apo(a) and formed bona fide disulfide-linked Lp(a), but Lp(a) assembly was less efficient than with wild-type human apoB100. The fact that Lp(a) assembly was less efficient with the mouse apoB sequences provides additional support for the notion that sequences in the carboxyl terminus of apoB100 are important for Lp(a) assembly.

Lipoprotein(a) and apolipoprotein(a) isoforms and proteinuria in patients with moderate renal failure.BackgroundAtherosclerotic diseases are a major cause of death in patients with renal failure. Increased serum concentrations of lipoprotein(a) [Lp(a)] have been established as a genetically controlled risk factor for these diseases and have been demonstrated in patients with moderate renal failure, suggesting that this lipoprotein contributes to the increased cardiovascular risk seen in these patients. Variable alleles at the apolipoprotein(a) [apo(a)] gene locus are the main determinants of the serum Lp(a) level in the general population. The purpose of this study was to investigate apo(a) isoforms in patients with moderate renal failure and mild proteinuria (less than 1.0 g/day).MethodsIn 250 consecutive subjects recruited at a hypertension clinic, we assessed the renal function by 24-hour creatinine clearance, proteinuria, and microalbuminuria, as well as the prevalence of atherosclerotic disease, and we also measured apo(a) isoforms, serum albumin, and Lp(a) concentrations.ResultsModerate impairment of renal function (creatinine clearance, 30 to 89 ml/min per 1.73 m2 of body surface area) was found in 97 patients. Lp(a) levels were significantly greater in patients with moderate renal failure (21.7 ± 23.9 mg/dl) as compared with patients with normal renal function (15.6 ± 16.4 mg/dl, P < 0.001), and an inverse correlation was observed between log Lp(a) and creatinine clearance (r = -0.181, P < 0.01). However, no difference was found in the frequency of low molecular weight apo(a) isoforms between patients with normal (25.5%) and impaired (27.8%) renal function. Only patients with the smallest size apo(a) isoforms exhibited significantly elevated levels of Lp(a), whereas the large-size isoforms had similar concentrations in patients with normal and impaired renal function. No significant relationship was found between serum Lp(a) and proteinuria. Clinical and laboratory evidence of one or more events attributed to atherosclerosis was found in 9.8% of patients with normal renal function and 25.8% of patients with moderate renal failure (P < 0.001).ConclusionsThese results indicate that renal failure per se or other genes beside the apo(a) gene locus are responsible for the elevation of serum Lp(a) levels in patients with moderate impairment of renal function. The elevation of Lp(a) levels occurs independently of the level of proteinuria and may contribute to the risk for atherosclerotic disease in these patients.

Apolipoprotein (a) (apo(a)) is a component of the atherogenic lipoprotein, Lp(a). The efficiency with which apo(a) escapes the endoplasmic reticulum (ER) and is secreted by the liver is a major determinant of plasma Lp(a) levels. Apo(a) contains a series of domains homologous to plasminogen kringle (K) 4, each of which possesses a potential lysine-binding site. By using primary mouse hepatocytes expressing a 17K4 human apo(a) protein, we found that high concentrations (25–200 mm) of the lysine analog, 6-aminohexanoic acid (6AHA), increased apo(a) secretion 8–14-fold. This was accompanied by a decrease in apo(a) presecretory degradation. 6AHA inhibited accumulation of apo(a) in the ER induced by the proteasome inhibitor, lactacystin. Thus, 6AHA appeared to inhibit degradation by increasing apo(a) export from the ER. Significantly, 6AHA overcame the block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine. 6AHA may therefore circumvent the requirement for calnexin and calreticulin interaction in apo(a) secretion. Sucrose gradients and a gel-based folding assay were unable to detect any influence of 6AHA on apo(a) folding. However, non-covalent or small, disulfide-dependent changes in apo(a) conformation would not be detected in these assays. Proline also increased the efficiency of apo(a) secretion. We propose that 6AHA and proline can act as chemical chaperones for apo(a).

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)] concentrationor = 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 isoforms20 K4 repeats had high Lp(a) concentrations (or = 0.30 g/l), whereas isoforms with 20 to 24 or more than 24 kringle 4 repeats had Lp(a) concentrationsor = 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) isoform25 kringle 4 repeats was significantly associated with serum Lp(a) concentrationor = 0.30 g/l in both single and double-band phenotyped samples (odds ratio = 8.9, p0.001). In the latter, a differential expression pattern with respect to smaller size isoforms (pattern AB vs A) was significantly associated with Lp(a) concentrationor = 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)or = 0.30 g/l is higher.

The human apolipoprotein(a) (apo(a)) gene is a member of a family of related genes including plasminogen, apo(a)rg-B and apo(a)rg-C, which are clustered on chromosome 6q 2,7. Apo(a) contains ten different types of plasminogen-like kringle IV repeats (K-IV 1-10) one of which (K-IV 2) varies in number resulting in a remarkable size polymorphism of the protein. Sequence analysis of human apo(a) alleles and indirect evidence have suggested that K-IV 1 and K-IV 3-10 are each present once in individual alleles and that the 3' apo(a) region encompassing kringles IV 3-10, kringle V and the protease domain is invariable. To directly test this, we have constructed a restriction map of the apo(a) gene region from genomic DNA and from a yeast artificial chromosome (YAC) (K-IV 13) which contains the entire apo(a) gene. The presence of a 63 kb ClaI fragment encompassing kringles IV 3-10, kringle V and the protease domain and a 46 kb SwaI fragment, spanning kringles IV 5-10, kringle V and the protease domain was demonstrated by PFGE/Southern blotting in 30 unrelated subjects, who represented a range of apo(a) size alleles containing from 11 to 49 kringles. Our analysis demonstrates that the number of kringles IV 3-10 is invariable in the human apo(a) gene, suggesting that the 3'domain of Apo(a) is functionally important.

This work reports several aspects concerning dynamic light scattering data of apoprotein A-I in solution. Using the geometrical factor (H) and turbidity (τ) at different pH values ranging from 5 to 10, we have tried to elucidate either if the initial formation of clusters and the trend for aggregation are carried out by nucleation or random mechanisms. Taking into account the solubility of the protein, this behavior depends on the precipitating agent used and the rate of mass transporting capacity carried out by vapor diffusion in small drops. Finally, we demonstrate that dynamic light scattering is a useful tool in order to determine protein precrystallization conditions.

Lipoprotein(a) (Lp(a)), which is generated through the covalent association of apolipoprotein(a) (apo(a)) and apo B-100-LDL, is an independent risk factor for several vascular diseases. Therefore, there is interest in developing therapies for lowering Lp(a). This investigation was carried out to determine the effect of CI-1011, a potent lipid regulator in rodents, on Lp(a) and other lipid parameters in cynomolgus monkeys (Macaca fascicularis). Nine healthy male monkeys on a normal chow diet were orally treated with CI-1011 at 30 mg/kg per day for 3 weeks. Lp(a) and total cholesterol levels were significantly decreased after 1 week and maximally reduced to 68 and 73% of control levels, respectively, after 3 treatment weeks. The decreases in total cholesterol were mainly due to changes in low density lipoprotein (LDL). The LDL:HDL ratio decreased by 30%. Triglycerides were unaffected by treatment. Lp(a) and total cholesterol levels returned to pretreatment values after stopping treatment suggesting a direct effect of the compound on their inhibition. Further studies demonstrated that CI-1011 was effective at a low dose of 3 mg/kg per day after 1 week of administration. CI-1011 also decreased apo B-100 to 80% of control levels, but this change was not sufficient to account for the Lp(a) lowering. There was also no correlation between the changes in Lp(a) and apo B-100 levels. Treatment of cynomolgus monkey primary hepatocyte cultures with CI-1011 resulted in a dose-dependent inhibition of Lp(a) levels suggesting a direct hepatic effect of the compound. Western blot analysis of the samples showed that changes in Lp(a) were associated mainly with decreased apo(a) (47%), but not apo B-100 (17%). These results demonstrate that CI-1011 effectively decreases Lp(a) levels both in vivo and in vitro.

Numerous epidemiological studies have shown that lipoprotein(a) (Lp(a)) is an independent risk factor for the premature development of cardiovascular disease. In spite of such evidence, the structural and functional features of this atherogenic, cholesterol-rich particle are not clearly understood. We have demonstrated the presence of two distinct structural domains in apolipoprotein(a) (apo(a)), which are linked by a flexible and accessible region located between kringles 4-4 and 4-5. We have isolated the Lp(a) particle following removal of the N-terminal domain by proteolytic cleavage; the residual particle, containing the C-terminal domain (comprising the region from Kr 4-5 to the protease domain), is linked to apo B-100 by disulphide linkage, and is termed 'mini-Lp(a)'. Mini-Lp(a) exhibited the same binding affinity to fibrin as the corresponding Lp(a). This finding indicated that the kringles responsible for fibrin binding are restricted to Kr 4-5 to Kr 4-10, an observation consistent with the failure of the N-terminal domain to bind to fibrin. N-terminal fragments of apo(a) have been detected in the urine of normal subjects, thereby indicating that part of the catabolism of Lp(a), which is largely indeterminate, could occur via the renal route.

Apoprotein (a) size polymorphism was evaluated at the genotypic and phenotypic level in 110 individuals. Both methods were well correlated with respect to size (r = 0.971), providing that the protein size was expressed as a number of kringle 4 repeats. Despite the fact that the immunoblotting method used was sensitive enough to detect less than 1 ng of lipoprotein (a), 62 samples had single-band phenotypes and one sample had no detectable band, whereas only seven samples had single-band genotypes. The mean size of the alleles coding for the undetected isoforms was significantly larger (141 kb) than for the detected isoforms (123 kb), corroborating the earlier finding of an inverse relationship between the size and the plasma expression level of apoprotein (a). Furthermore, increasing detectability was achieved by loading the gel with different amounts of plasma for each sample. Our results indicate that genotyping is more resolving and more sensitive, but requires a more specialized technology. Phenotyping was carried out using commercially available reagents.

Plasma levels of lipoprotein(a) (Lp(a)) vary over 1000-fold between individuals and are determined by the gene for its unique apolipoprotein, apo(a), which has greater than 100 alleles. Using primary baboon hepatocyte cultures, we previously demonstrated that differences in the ability of apo(a) allelic variants to escape the endoplasmic reticulum (ER) are a major determinant of Lp(a) production rate. To examine the reason for these differences, the folding of newly synthesized apo(a) was analyzed in pulse-chase experiments. Samples were harvested in the presence of N-ethylmaleimide to preserve disulfide-bonded folding intermediates, and apo(a) was analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Apo(a) required a prolonged period (30-60 min) to reach its fully oxidized form. Multiple folding intermediates were resolved, including a disulfide-linked, apo(a)-containing complex. Unexpectedly, all allelic variants examined showed similar patterns and kinetics of folding. Even "null" apo(a) proteins, which are unable to exit the ER, appeared to fold normally. The ER glucosidase inhibitor, castanospermine, prevented apo(a) secretion, but did not inhibit folding. This suggests that an event which is dependent on trimming of N-linked glucoses, and which occurs after the folding events detectable in our assay, is required for apo(a) secretion. Differences in the ability to undergo this event may explain the variable efficiency with which apo(a) allelic variants exit the ER.

Serum lipoprotein (a) concentrations (Lp(a)) are largely under genetic control, and are strong predictors of coronary heart disease. It has been hypothesised that Lp(a) may contribute to the increased risk of coronary heart disease in familial Type 2 diabetes mellitus. We therefore examined the Lp(a) concentrations and the apolipoprotein (a) (apo(a)) phenotypes in 126 normoglycaemic first degree relatives from families with two or more living Type 2 diabetic patients. These were compared with 147 sex matched normoglycaemic control subjects with no family history of diabetes. Lp(a) concentrations were measured using an enzyme-linked immunosorbent assay (ELISA), and apo(a) isoforms were determined and classified according to the relative mobility of apo(a) on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), relative to that of apolipoprotein B-100. There were no significant differences in Lp(a) concentrations between the relatives (R) and controls (C): 11.2 (R) vs. 11.1 (C) mg/dl (median). The distribution of apo(a) phenotypes was not significantly different between groups 0.65 (R) vs. 0.67 (C). These results show that first degree relatives at risk of developing Type 2 diabetes do not have abnormal Lp(a) concentrations or apo(a) phenotypes.

Five different gel systems were evaluated for their utility in determining the molecular weights of apolipoprotein(a) (apo(a)) polymorphs by SDS polyacrylamide or agarose gel electrophoresis. Three linear polyacrylamide gradient gels (2–16% from Isolab (Akron, OH), 4–15% from Pharmacia (Piscataway, NJ), and 2.5–6% homemade), a 4% polyacrylamide, and a 1.5% agarose gel were examined. Crosslinked phosphorylase B oligomers served as molecular weight standards. Molecular weights of four different apo(a) polymorphs were determined in each gel system and compared to values measured previously by sedimentation equilibrium. The results indicate that molecular weights obtained by gradient polyacrylamide gel electrophoresis were within 10% and often not statistically different from values acquired by sedimentation equilibrium. The use of homogenous 4% polyacrylamide and 1.5% agarose gels led to molecular weights that were overestimated by 20 and 60–70%, respectively. ApoB 100 , which is a commonly used molecular weight marker, was found to have anomalously fast mobility in each of the four polyacrylamide gel systems. Because its use would lead to overestimated apo(a) molecular weights, it was not useful as a molecular weight standard. Our results indicate that SDS–gradient polyacrylamide gel electrophoresis with cross-linked phosphorylase B as standard is a suitable gel system for evaluating apo(a) molecular weights.

Summary We examined the immunohistochemical distribution of tissue factor (TF), apolipoprotein (a) (apo(a)) in atherosclerotic intimas of human thoracic aortas obtained from 51 autopsies in order to analyze the mechanism of fibrinogen-fibrin transition as a part of thrombogenic properties of atherosclerotic intimas. TF was overexpressed mainly by macrophages in both fatty streaks and more advanced lesions, while it was also scatteringly deposited in the matrix of advanced lesions, especially in the atheromatous gruel. TF-positive macrophages were frequently intermingled at the base of fibrin thrombi formed on the eroded intimas. On the other hand, apo(a) was localized in the stroma and within some macrophages, and also in the mural thrombi. Fibrinogen and fibrin were more frequently detected in the matrix of advanced lesions than in that of early lesions. Fibrin was occasionally co-located with cell- and matrix-associated TF and apo(a) deposited in matrix. These findings suggest that the overexpressed TF in the atherosclerotic intima plays a critical role in the initiation of fibrin formation. This could result from either fibrinogen permeating into the intima or from rupture of the fibrous cap overlying atheromas. Apo(a) deposited in the atherosclerotic intima may also participate in the persistent deposition of fibrin.

Lipoprotein (a) is similar to low-density lipoprotein but is unique in having an additional apolipoprotein called apolipoprotein (a) (apo(a)) covalently linked to it. apo(a), which is a member of the plasminogen gene superfamily, has a protease domain which cannot be activated to cause fibrinolysis. Its sequence of kringles is much longer than that of plasminogen and there is remarkable genetic variation in its length. The consequent inherited differences in apo(a) molecular mass are largely responsible for the wide range of serum Lp(a) concentrations in different individuals with low levels predominating in Europid populations. Physiologically Lp(a) may participate in haemocoagulation or in wound-healing. Epidemiological evidence that it is a risk factor for atherosclerosis, particularly in populations with high serum LDL levels, has led to research to uncover its role in atherogenesis and thrombosis. Diseases such as renal disease, and probably atherogenesis and thrombosis. Diseases such as renal disease, and probably atherosclerosis itself, are associated with an increase in Lp(a) above its genetically determined level and it remains a subject of speculation as to whether such increases are as closely involved in atherothrombosis as are spontaneously high levels resulting from low-molecular-mass apo(a) variants.