Apolipoprotein A-IV polymorphisms Q360H and T347S attenuate its endogenous inhibition of thrombosis.

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbß3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

O-glycan structures in apo(a) subunit of human lipoprotein(a) suppresses the pro-angiogenic activity of galectin-1 on human umbilical vein endothelial cells.

Apolipoprotein(a) [apo(a)] is a highly polymorphic O-glycoprotein circulating in human plasma as lipoprotein(a) [Lp(a)]. The O-glycan structures of apo(a) subunit of Lp(a) serve as strong ligands of galectin-1, an O-glycan binding pro-angiogenic lectin abundantly expressed in placental vascular tissues. But the pathophysiological significance of apo(a)-galectin-1 binding is not yet been revealed. Carbohydrate-dependent binding of galectin-1 to another O-glycoprotein, neuropilin-1 (NRP-1) on endothelial cells activates vascular endothelial growth factor receptor 2 (VEGFR2) and mitogen-activated protein kinase (MAPK) signaling. Using apo(a), isolated from human plasma, we demonstrated the potential of the O-glycan structures of apo(a) in Lp(a) to inhibit angiogenic properties such as proliferation, migration, and tube-formation in human umbilical vein endothelial cells (HUVECs) as well as neovascularization in chick chorioallantoic membrane. Further, in vitro protein-protein interaction studies have confirmed apo(a) as a superior ligand to NRP-1 for galectin-1 binding. We also demonstrated that the protein levels of galectin-1, NRP-1, VEGFR2, and downstream proteins in MAPK signaling were reduced in HUVECs in the presence of apo(a) with intact O-glycan structures compared to that of de-O-glycosylated apo(a). In conclusion, our study shows that apo(a)-linked O-glycans prevent the binding of galectin-1 to NRP-1 leading to the inhibition of galectin-1/neuropilin-1/VEGFR2/MAPK-mediated angiogenic signaling pathway in endothelial cells. As higher plasma Lp(a) level in women is an independent risk factor for pre-eclamsia, a pregnancy-associated vascular complication, we propose that apo(a) O-glycans-mediated inhibition of the pro-angiogenic activity of galectin-1 may be one of the underlying molecular mechanism of pathogenesis of Lp(a) in pre-eclampsia.

Relationship of apolipoprotein(a) isoform size with clearance and production of lipoprotein(a) in a diverse cohort.

Lipoprotein(a) [Lp(a)] has two main proteins, apoB100 and apo(a). High levels of Lp(a) confer an increased risk for atherosclerotic cardiovascular disease. Most people have two circulating isoforms of apo(a) differing in their molecular mass, determined by the number of Kringle IV Type 2 repeats. Previous studies report a strong inverse relationship between Lp(a) levels and apo(a) isoform sizes. The roles of Lp(a) production and fractional clearance and how ancestry affects this relationship remain incompletely defined. We therefore examined the relationships of apo(a) size with Lp(a) levels and both apo(a) fractional clearance rates (FCR) and production rates (PR) in 32 individuals not on lipid-lowering treatment. We determined plasma Lp(a) levels and apo(a) isoform sizes, and used the relative expression of the two isoforms to calculate a "weighted isoform size" (wIS). Stable isotope studies were performed, using D3-leucine, to determine the apo(a) FCR and PR. As expected, plasma Lp(a) concentrations were inversely correlated with wIS (R2 = 0.27; P = 0.002). The wIS had a modest positive correlation with apo(a) FCR (R2 = 0.10, P = 0.08), and a negative correlation with apo(a) PR (R2 = 0.11; P = 0.06). The relationship between wIS and PR became significant when we controlled for self-reported race and ethnicity (SRRE) (R2 = 0.24, P = 0.03); controlling for SRRE did not affect the relationship between wIS and FCR. Apo(a) wIS plays a role in both FCR and PR; however, adjusting for SRRE strengthens the correlation between wIS and PR, suggesting an effect of ancestry.

Oxidized phospholipid modification of lipoprotein(a): Epidemiology, biochemistry and pathophysiology.

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 KIV10 domain of apo(a), and the strong lysine binding site (LBS) in this kringle is required for OxPL addition. Using apo(a) species lacking OxPL modification - by mutating the LBS - has allowed direct assessment of the role of apo(a) OxPL in Lp(a)-mediated pathogenesis. The OxPL on apo(a) account for numerous harmful effects of Lp(a) on monocytes, macrophages, endothelial cells, smooth muscle cells, and valve interstitial cells documented both in vitro and in vivo. In addition, the mechanisms underlying these effects have begun to be unraveled by identifying the cellular receptors that respond to OxPL, the intracellular signaling pathways turned on by OxPL, and the changes in gene and protein expression evoked by OxPL. The emerging picture is that the OxPL on Lp(a) are central to its pathobiology. The OxPL modification may explain why Lp(a) is such a potent risk factor for cardiovascular disease despite being present at concentrations an order of magnitude lower than LDL, and they account for the ability of elevated Lp(a) to cause both atherothrombotic disease and calcific aortic valve stenosis.

PCSK9 inhibition with alirocumab decreases plasma lipoprotein(a) concentration by a dual mechanism of action in statin-treated patients with very high apolipoprotein(a) concentration.

BACKGROUND: Inhibition of proprotein convertase subtilisin/kexin type 9 with alirocumab decreases plasma lipoprotein(a) [Lp(a)] levels. The kinetic mechanism for lowering Lp(a) by alirocumab may differ according to pre-treatment apolipoprotein(a) [apo(a)] levels. METHODS: The effect of 12-week alirocumab (150 mg subcutaneously fortnightly) on the kinetics of apo(a) was compared in statin-treated patients with high (n = 10) and very high Lp(a) concentrations (n = 11). RESULTS: In patients with high apo(a) concentrations, alirocumab lowered plasma apo(a) pool size (-17%, p < 0.01) chiefly by increasing the fractional catabolic rate (FCR) of apo(a) (+27%, p < 0.001). By contrast in patients with very high apo(a) concentrations, alirocumab significantly lowered plasma apo(a) pool size (-32%, p < 0.001) by both increasing apo(a) FCR (+30%, p < 0.001) and lowering production rate (-11%, p < 0.05). CONCLUSIONS: In statin-treated patients with very high apo(a) concentrations, alirocumab lowers plasma Lp(a) concentration by a dual mode of action that increases the clearance and decreases the production of Lp(a) particles.

High resolution structure of human apolipoprotein (a) kringle IV type 2: beyond the lysine binding site.

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.

ApoCIII-Lp(a) complexes in conjunction with Lp(a)-OxPL predict rapid progression of aortic stenosis.

OBJECTIVE: This study assessed whether apolipoprotein CIII-lipoprotein(a) complexes (ApoCIII-Lp(a)) associate with progression of calcific aortic valve stenosis (AS). METHODS: Immunostaining for ApoC-III was performed in explanted aortic valve leaflets in 68 patients with leaflet pathological grades of 1-4. Assays measuring circulating levels of ApoCIII-Lp(a) complexes were measured in 218 patients with mild-moderate AS from the AS Progression Observation: Measuring Effects of Rosuvastatin (ASTRONOMER) trial. The progression rate of AS, measured as annualised changes in peak aortic jet velocity (Vpeak), and combined rates of aortic valve replacement (AVR) and cardiac death were determined. For further confirmation of the assay data, a proteomic analysis of purified Lp(a) was performed to confirm the presence of apoC-III on Lp(a). RESULTS: Immunohistochemically detected ApoC-III was prominent in all grades of leaflet lesion severity. Significant interactions were present between ApoCIII-Lp(a) and Lp(a), oxidised phospholipids on apolipoprotein B-100 (OxPL-apoB) or on apolipoprotein (a) (OxPL-apo(a)) with annualised Vpeak (all p<0.05). After multivariable adjustment, patients in the top tertile of both apoCIII-Lp(a) and Lp(a) had significantly higher annualised Vpeak (p<0.001) and risk of AVR/cardiac death (p=0.03). Similar results were noted with OxPL-apoB and OxPL-apo(a). There was no association between autotaxin (ATX) on ApoB and ATX on Lp(a) with faster progression of AS. Proteomic analysis of purified Lp(a) showed that apoC-III was prominently present on Lp(a). CONCLUSION: ApoC-III is present on Lp(a) and in aortic valve leaflets. Elevated levels of ApoCIII-Lp(a) complexes in conjunction with Lp(a), OxPL-apoB or OxPL-apo(a) identify patients with pre-existing mild-moderate AS who display rapid progression of AS and higher rates of AVR/cardiac death. TRIAL REGISTRATION: NCT00800800.

Apolipoprotein(a) Kinetics in Statin-Treated Patients With Elevated Plasma Lipoprotein(a) Concentration.

BACKGROUND: Lipoprotein(a) [Lp(a)] is a low-density lipoprotein‒like particle containing apolipoprotein(a) [apo(a)]. Patients with elevated Lp(a), even when treated with statins, are at increased risk of cardiovascular disease. We investigated the kinetic basis for elevated Lp(a) in these patients. OBJECTIVES: Apo(a) production rate (PR) and fractional catabolic rate (FCR) were compared between statin-treated patients with and without elevated Lp(a). METHODS: The kinetics of apo(a) were investigated in 14 patients with elevated Lp(a) and 15 patients with normal Lp(a) levels matched for age, sex, and body mass index using stable isotope techniques and compartmental modeling. All 29 patients were on background statin treatment. Plasma apo(a) concentration was measured using liquid chromatography-mass spectrometry. RESULTS: The plasma concentration and PR of apo(a) were significantly higher in patients with elevated Lp(a) than in patients with normal Lp(a) concentration (all P < 0.01). The FCR of apo(a) was not significantly different between the groups. In univariate analysis, plasma concentration of apo(a) was significantly associated with apo(a) PR in both patient groups (r = 0.699 and r = 0.949, respectively; all P < 0.01). There was no significant association between plasma apo(a) concentration and FCR in either of the groups (r = 0.160 and r = -0.137, respectively). CONCLUSION: Elevated plasma Lp(a) concentration is a consequence of increased hepatic production of Lp(a) particles in these patients. Our findings provide a kinetic rationale for the use of therapies that target the synthesis of apo(a) and production of Lp(a) particles in patients with elevated Lp(a).

Redox equilibrium of serum apolipoprotein E3: a buffering effect of disulfide-linked complexes against oxidative stress on apolipoprotein E3-containing lipoproteins.

Reversible redox modification of cysteine thiols is crucial for protecting proteins from irreversible detrimental change. However, the physiological significance of the redox modification of apolipoprotein (apo) E is unclear. Here, we hypothesized that the disulfide-linked complexes of apoE3 corresponding to the representative reversible-modified apoE3 play a protective role against oxidative stress. The effects of disulfide bond formation on oxidative stress on apoE3 were evaluated with a band-shift assay. Maleimide-labeled apoE3 and unlabeled apoE3 were defined as the reduced (r)-apoE3 and non-reduced (nr)-apoE3 forms, respectively. Hydrogen peroxide-induced oxidation decreased for reduced-form apoE (r-apoE3) but increased for nr-apoE3. Induction of apoE3-AII complex formation with excess of apoAII markedly suppressed the oxidative stress-induced increase in nr-apoE3 (P<0.001) and enhanced homodimer formation. The apoE3-AII complex was more dominant in high-density lipoprotein (HDL) than in very low-density lipoprotein. Under oxidative stress, HDL showed a significant decrease, rather than an increase, in nr-apoE3 levels with a concomitant significant increase in apoE3-AII levels (P<0.005). This finding suggests that the majority of nr-apoE3 in HDL exists in a reversible oxidized form. The apoE3-AII complex, formed from the reversible oxidized apoE3, is beneficial for maintaining the redox equilibrium of apoE3 by preventing the modification of apoE3 to its irreversible oxidized form. The apoE3-AII complex may be possibly implicated in the pathophysiology of various apoE-related diseases.

Relationship of lipoprotein(a) molar concentrations and mass according to lipoprotein(a) thresholds and apolipoprotein(a) isoform size.

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) < 75 nmol/L and 2.80, 1.89, and 2.24, respectively, for Lp(a) > 125 nmol/L. For the commercial turbidimetric assays and enzyme-linked immunosorbent assay, the ratios ranged from <1 to >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.

Inhibition of pericellular plasminogen activation by apolipoprotein(a): Roles of urokinase plasminogen activator receptor and integrins αMß2 and αVß3.

BACKGROUND AND AIMS: Lipoprotein(a) (Lp(a)) is a causal risk factor for cardiovascular disorders including coronary heart disease and calcific aortic valve stenosis. Apolipoprotein(a) (apo(a)), the unique glycoprotein component of Lp(a), contains sequences homologous to plasminogen. Plasminogen activation is markedly accelerated in the presence of cell surface receptors and can be inhibited in this context by apo(a). METHODS: We evaluated the role of potential receptors in regulating plasminogen activation and the ability of apo(a) to mediate inhibition of plasminogen activation on vascular and monocytic/macrophage cells through knockdown (siRNA or blocking antibodies) or overexpression of various candidate receptors. Binding assays were conducted to determine apo(a) and plasminogen receptor interactions. RESULTS: The urokinase-type plasminogen activator receptor (uPAR) modulates plasminogen activation as well as plasminogen and apo(a) binding on human umbilical vein endothelial cells (HUVECs), human acute monocytic leukemia (THP-1) cells, and THP-1 macrophages as determined through uPAR knockdown and overexpression. Apo(a) variants lacking either the kringle V or the strong lysine binding site in kringle IV type 10 are not able to bind to uPAR to the same extent as wild-type apo(a). Plasminogen activation is also modulated, albeit to a lower extent, through the Mac-1 (αMß2) integrin on HUVECs and THP-1 monocytes. Integrin αVß3 can regulate plasminogen activation on THP-1 monocytes and to a lesser extent on HUVECs. CONCLUSIONS: These results indicate cell type-specific roles for uPAR, αMß2, and αVß3 in promoting plasminogen activation and mediate the inhibitory effects of apo(a) in this process.

Temporal variability in lipoprotein(a) levels in patients enrolled in the placebo arms of IONIS-APO(a)Rx and IONIS-APO(a)-LRx antisense oligonucleotide clinical trials.

BACKGROUND: Lipoprotein(a) [Lp(a)] levels are primarily genetically determined, but their natural variability is not well known. OBJECTIVE: The aim of the study was to evaluate the short-term temporal variability in Lp(a) in 3 placebo groups from the IONIS-APO(a)Rx and IONIS-APO(a)-LRx trials. METHODS: The placebo groups comprised 3 studies: Study 1 with 10 subjects with any Lp(a) concentration; Study 2 with 13 subjects with Lp(a) ≥75 nmol/L (∼30 mg/dL); and Study 3 with 29 patients with Lp(a) ≥125 nmol/L (≥∼50 mg/dL). Lp(a) was measured in serial blood samples (range 7-12 samples up to 190 days of follow-up) and analyzed as absolute change and mean percent change from baseline. Outliers were defined as having a > ±25% difference in Lp(a) from baseline at any future time point. RESULTS: No significant temporal differences in mean absolute Lp(a) levels were present in any group. However, among individuals, the mean change in absolute Lp(a) levels at any time point ranged from -16.2 to +7.0 nmol/L in Study 1, -15.8 to +9.8 nmol/L in Study 2, and -60.2 to +16.6 nmol/L in Study 3. The mean percent change from baseline ranged from -9.4% to +21.6% for Study 1, -13.1% to 2.8% for Study 2, and -12.1% to +4.9% in Study 3. A total of 21 of 52 subjects (40.4%) were outliers, with 13 (62%) >25% up and 8 (38%) >25% down. Significant variability was also noted in other lipid parameters, but no outliers were noted with serum albumin. CONCLUSION: In subjects randomized to placebo in Lp(a) lowering trials, modest intra-individual temporal variability of mean Lp(a) levels was present. Significant number of subjects had > ±25% variation in Lp(a) in at least 1 time point. Although Lp(a) levels are primarily genetically determined, further study is required to define additional factors mediating short-term variability.

The roles of apo(a) size, phenotype, and dominance pattern in PCSK9-inhibition-induced reduction in Lp(a) with alirocumab.

An elevated level of lipoprotein (a) [Lp(a)] is a risk factor for CVD. Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9, is reported to reduce Lp(a) levels. The relationship of Lp(a) reduction with apo(a) size polymorphism, phenotype, and dominance pattern and LDL cholesterol (LDL-C) reduction was evaluated in a pooled analysis of 155 hypercholesterolemic patients (75 with heterozygous familial hypercholesterolemia) from two clinical trials. Alirocumab significantly reduced total Lp(a) (pooled median: -21%, P = 0.0001) and allele-specific apo(a), an Lp(a) level carried by the smaller (median: -18%, P = 0.002) or the larger (median: -37%, P = 0.0005) apo(a) isoform, at week 8 versus baseline. The percent reduction in Lp(a) level with alirocumab was similar across apo(a) phenotypes (single vs. double bands) and carriers and noncarriers of a small size apo(a) (≤22 kringles). The percent reduction in LDL-C correlated significantly with the percent reduction in Lp(a) level (r = 0.407, P < 0.0001) and allele-specific apo(a) level associated with the smaller (r = 0.390, P < 0.0001) or larger (r = 0.270, P = 0.0183) apo(a) sizes. In conclusion, alirocumab-induced Lp(a) reduction was independent of apo(a) phenotypes and the presence or absence of a small size apo(a).

Lipoprotein(a) level and apolipoprotein(a) phenotype as predictors of long-term cardiovascular outcomes after coronary artery bypass grafting.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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) values <30 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. CONCLUSION: 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) metabolism.

PURPOSE OF REVIEW: Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein. The metabolism of this lipoprotein is still not well understood. RECENT FINDINGS: It has long been known that the plasma concentration of Lp(a) is highly heritable, with its genetic determinants located in the apo(a) locus and regulating the rate of hepatic apo(a) production. Recent human intervention trials have convincingly established that, in addition to apo(a) production, hepatic apoB100 production plays an important role in Lp(a) levels. Although the major site and mode of Lp(a) clearance remain unidentified, a recent cell and animal study points to the involvement of the hepatic scavenger receptor class B type I in the uptake of both the lipid and protein constituents of Lp(a) from plasma. SUMMARY: Progress in the understanding of Lp(a) metabolism has the potential to lead to the development of novel and specific treatments for the reduction of Lp(a) levels and the associated risk of cardiovascular disease.

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.

Lp(a)/apo(a) modulate MMP-9 activation and neutrophil cytokines in vivo in inflammation to regulate leukocyte recruitment.

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.

Impact of lipoprotein(a) levels and apolipoprotein(a) isoform size on risk of coronary heart disease.

OBJECTIVES: Observational and genetic studies have shown that lipoprotein(a) [Lp(a)] levels and apolipoprotein(a) [apo(a)] isoform size are both associated with coronary heart disease (CHD) risk, but the relative independence of these risk factors remains unclear. Clarification of this uncertainty is relevant to the potential of future Lp(a)-lowering therapies for the prevention of CHD. METHODS: Plasma Lp(a) levels and apo(a) isoform size, estimated by the number of kringle IV (KIV) repeats, were measured in 995 patients with CHD and 998 control subjects. The associations between CHD risk and fifths of Lp(a) levels were assessed before and after adjustment for KIV repeats and, conversely, the associations between CHD risk and fifths of KIV repeats were assessed before and after adjustment for Lp(a) levels. RESULTS: Individuals in the top fifth of Lp(a) levels had more than a twofold higher risk of CHD compared with those in the bottom fifth, and this association was materially unaltered after adjustment for KIV repeats [odds ratio (OR) 2.05, 95% confidence interval (CI) 1.38-3.04, P < 0.001]. Furthermore, almost all of the excess risk was restricted to the two-fifths of the population with the highest Lp(a) levels. Individuals in the bottom fifth of KIV repeats had about a twofold higher risk of CHD compared with those in the top fifth, but this association was no longer significant after adjustment for Lp(a) levels (OR 1.13, 95% CI 0.77-1.66, P = 0.94). CONCLUSIONS: The effect of KIV repeats on CHD risk is mediated through their impact on Lp(a) levels, suggesting that absolute levels of Lp(a), rather than apo(a) isoform size, are the main determinant of CHD risk.

Apolipoprotein (a) impairs endothelial progenitor cell-mediated angiogenesis.

Improvement of blood flow and promotion of angiogenesis are important therapeutic measures for the treatment of ischemic peripheral vascular diseases. Since apolipoprotein (a) (apo (a)) is a glycoprotein with repetitive kringle domains exhibiting 75% to 98% structural homology with plasminogen (Plg), apo (a) may also have a negative effect on endothelial progenitor cell (EPC)-induced angiogenesis through Plg-like inhibitory effects on EPC proliferation, adhesion, migration, and angiogenesis. To evaluate the effect of apo (a) on EPCs-induced angiogenesis, EPCs were isolated from the bone marrow of apo (a) transgenic mice, wild-type litter mates, and normal mice. These cells were cultured without or with apo (a) before transplantation. Hindlimb ischemia models were surgically induced in mice, which then received an intravenous injection of 3×10(5) EPCs. At 3, 7, and 14 days post EPC transplantation, the adhesion, migration abilities, and capillary density in calf muscles were assessed. Results indicate that apo (a) significantly reduced the adhesion and migration abilities of EPCs. Furthermore, the tubule-like formation of EPCs on Matrigel gels was damaged. In vivo experiments showed the homing of EPCs to ischemic peripheral vascular, and the number of capillary vessels decreased significantly in apo(a) transgenic mice. This study demonstrated that apo (a) could attenuate the adhesion, migration, and homing abilities of EPCs and could impair the angiogenesis ability of EPCs.

[The secretory phospholipase A2 and transport of lipids by lipoproteins in patients with the risk of cardio-vascular pathology (the system "score") of lower and average degree. The diagnostic significance of the test].

The clinical and pathomorphologic data demonstrate that the most frequent cause of cardiac infarction is the formation of "soft" atheromatosis plaques in the intima of arteries. Their rupture results in thrombosis of coronary arteries. The plaques are characterized by higher content of triglycerides. On the basis of the research data, it is possible to validly consider that the detection of secretary phospholipase content A2 conjugated with lipoproteins is the test of systemic inflammatory response. This response is formed under atherosclerosis in vivo as a feedback to the accumulation in the intercellular medium of the endogenic flogogens (initiators of biological reaction of inflammation)--lipoproteins of lower density subclass A. Their utilization in the intima, as a pool of local interstitial tissue, by the resident macrophagocytes transformed from monocytes result in the formation of doth soft and disposed to laceration atheromatosis plaques and the atherothrombosis of coronary arteries and rarer of carotids. Concurrently, the increase of lipoproteins content in blood plasma is supposed to be the test of proliferation of cells in vivo, the smooth muscle cells of medium in particular. The simultaneous detection of content of secretory associated with lipoproteins phospholipase A2 and lipoprotein (a) can be considered as a valid risk factor of atherosclerosis and atherothrombosis--atheromatosis of intima of arteries with the formation of "soft" plaques in the intima, their laceration and thrombosis of coronary arteries and clinical presentation of cardiac infarction. The diagnostic triad of formation of soft plaques in the intima can be composed of the higher level of triglycerides, the content of protein of phospholipase A2 and lipoprotein (a).