Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function.

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT

Localization of apolipoprotein A-I epitopes involved in the activation of lecithin: Cholesterol acyltransferase

Eight murine monoclonal antibodies (Mab) to apolipoprotein A-I were characterized for their epitopes and for their ability to interfere with lecithin:cholesterol acyltransferase (LCAT) activation mediated by apo apoA-I using a synthetic substrate. Using overlapping synthetic peptides we have identified six continuous epitopes that span amino acids 1-10 (Mab A-I-19), 96-101 (Mab A-I-15), 133-141 (Mab A-I-5), 140-145 (Mab A-I-9), 144-148 (Mab A-I-8), and 167-174 (Mab A-I-57). Furthermore, antibodies A-I-11 and A-I-16 recognized discontinuous epitopes, namely amino acids 124-128 and 144-148. When antibodies were tested for their ability to inhibit LCAT activation, an inhibitory effect was observed with those whose epitopes covered the area of apoA-I encompassing amino acids 96-174. From these data we conclude that several areas of apoA-I spanning the middle region of the apolipoprotein act in concert to stimulate LCAT activity, possibly by cooperative interaction with the enzyme

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor

Predictive value of HDL function in patients with coronary artery disease: relationship with coronary plaque characteristics and clinical events

BACKGROUND: HDL is endowed with several metabolic, vascular, and immunoinflammatory protective functions. Among them, a key property is to promote reverse cholesterol transport from cells back to the liver. The aim of this study was to estimate the association of scavenger receptor class B type I (SR-BI)- and ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux (the two major routes for cholesterol efflux to HDL) with the presence, extent, and severity of coronary artery disease (CAD), vascular wall remodelling processes, coronary plaque characteristics, and the incidence of myocardial infarction in the different subgroups of patients from the CAPIRE study. METHODS: Patients (n = 525) from the CAPIRE study were divided into two groups: low-risk factors (RF), with 0–1 RF (n = 263), and multiple-RF, with ≥2 RFs; within each group, subjects were classified as no-CAD or CAD based on the segment involvement score (SIS) evaluated by coronary computed tomography angiography (SIS = 0 and SIS > 5, respectively). SR-BI- and ABCA1-mediated cholesterol efflux were measured using the plasma of all patients. RESULTS: SR-BI-mediated cholesterol efflux was significantly reduced in patients with CAD in both the low-RF and multiple-RF groups, whereas ABCA1-mediated cholesterol efflux was similar among all groups. In CAD patients, multivariable analysis showed that SR-BI-mediated cholesterol efflux <25(th) percentile predicted cardiovascular outcome (odds ratio 4.1; 95% CI: 1.3–13.7; p = .019), whereas ABCA-1-mediated cholesterol efflux and HDL-C levels significantly did not. Despite this finding, reduced SR-BI-mediated cholesterol efflux was not associated with changes in high-risk plaque features or changes in the prevalence of elevated total, non-calcified, and low-attenuation plaque volume. CONCLUSION: KEY MESSAGES: Increased cholesterol efflux capacity, an estimate of HDL function, is associated with a reduced CVD risk, regardless of HDL-C levels. HDL-C levels are significantly lower in patients with CAD. Lower SR-BI-mediated cholesterol efflux capacity is observed in patients with diffuse coronary atherosclerosis and is associated with the worst clinical outcomes in patients with CAD, independently of atherosclerotic plaque features

Dendritic cell marker Clec4a4 deficiency limits atherosclerosis progression

Background and aims: Atherogenesis results from altered lipid metabolism and impaired immune response. Emerging evidence has suggested that dendritic cells (DCs) participate to atherosclerosis-related immune response, but their impact is scarcely characterized. Clec4a4 or DCIR2 (Dendritic cell immunoreceptor 2) is a C-type lectin receptor, mainly expressed by CD8α− DCs, able to modulate T cell immunity. However, whether this DC subset could play a role in the atherogenesis is still poorly understood. Thus, the aim of this study is to investigate whether the absence of Clec4a4 could affect atherosclerosis-related immune response and atherosclerosis itself. Methods: Dcir2−/− Ldlr−/− and Ldlr−/− mice were fed a standard diet or cholesterol-enriched diet for 12 weeks. Subsequently, the profile of circulating and lymph nodes-resident immune cells was investigated together with the analysis of plasma lipid levels and atherosclerotic plaque extension in the aorta. Results: Here, we show that Clec4a4 expression is downregulated under hypercholesterolemia and its deficiency in Ldlr−/− mice results in the reduction of atherosclerotic plaque formation, together with altered lipid metabolism and impaired myeloid immune cell distribution. Conclusions: Our findings suggest a pro-atherosclerotic role of Clec4a4 in experimental atherosclerosis

The Association of Proprotein Convertase Subtilisin/Kexin Type 9 to Plasma Low-Density Lipoproteins: An Evaluation of Different Methods

Background: Proprotein convertase subtilisin/kexin type-9 (PCSK9) is key regulator of low-density lipoprotein (LDL) metabolism. A significant proportion of PCSK9 is believed to be associated with LDL in plasma as it circulates, although this finding is still a matter of debate. The purpose of this study was to establish an experimental method to investigate the presence of such an interaction in the bloodstream. Methods: We compared a number of well-established methods for lipoprotein (LP) isolation to clarify whether PCSK9 associates differently to circulating lipoproteins, such as KBr gradient ultracentrifugation, physical precipitation of ApoB-LPs, fast protein liquid chromatography (FPLC) and iodixanol gradient ultracentrifugation. Results: Our data show heterogeneity in PCSK9 association to lipoproteins according to the method used. Two methods, iodixanol ultracentrifugation and column chromatography, which did not involve precipitation or high salt concentration, consistently showed an interaction of PCSK9 with a subfraction of LDL that appeared to be more buoyant and have a lower size than average LDL. The percent of PCSK9 association ranged from 2 to 30% and did not appear to correlate to plasma or LDL cholesterol levels. Conclusions: The association of PCSK9 to LDL appeared to be sensitive to high salt concentrations. FPLC and iodixanol gradient ultracentrifugation appeared to be the most suitable methods for the study of this association

PCSK9 knock-out mice are protected from neointimal formation in response to perivascular carotid collar placement

Background and aims: Proprotein convertase subtilisin kexin type 9 (PCSK9) induces degradation of the low-density lipoprotein-receptor (LDLR). Smooth muscle cells (SMCs) in human atherosclerotic plaques and cultured SMCs express PCSK9. The present study aimed at defining the role of PCSK9 on vascular response to injury. Methods: Carotid neointimal lesions were induced by positioning a non-occlusive collar in PCSK9 knockout (PCSK9(-/-)) and wild type littermate (PCSK9(+/+)) mice. Results: In PCSK9(-/-) mice, we observed a significantly less intimal thickening (p < 0.05), a lower intimal media ratio (p < 0.02), and a tendency to higher lumen area, compared to PCSK9(+/+) mice. When compared with PCSK9(-/-), lesions of PCSK9(+/+) mice had a higher content of SMCs (p < 0.05) and collagen (p < 0.05), while no difference was observed in the accumulation of macrophages. PCSK9 was detectable in both left and right carotids artery in regions occupied by medial and neointimal SMCs. SMCs freshly isolated from PCSK9(-/-), when compared to PCSK9(+/+) cells, showed higher levels of alpha-smooth muscle actin (alpha-SMA; 2.24 +/- 0.36 fold; p < 0.01) and myosin heavy chain II (MHC-II; 8.65 +/- 1.55 fold; p < 0.01), and lower levels of caldesmon mRNA (-54 +/- 14%; p < 0.01). PCSK9(-/-) cells also showed a slower proliferation rate, and an impaired migratory capacity and G1/S progression of the cell cycle. The reconstitution of PCSK9 expression, by retroviral infection of PCSK9(-/-) SMCs, led to a downregulation of a-SMA (-56 +/- 2%; p < 0.01), MHC-II(-45% +/- 25.5 fold: p = 0.06) and calponin (-25% +/- 0.8 fold: p < 0.05) and induction of caldesmon mRNA (1.46 +/- 0.3 fold; p < 0.05). Proliferation rate of SMCs PCSK9(-/-) was significantly lower compared to PCSK9 reconstituted cells. Conclusions: Taken together, the present results suggest that PCSK9, by sustaining SMC synthetic phenotype, proliferation, and migration, may play a pro-atherogenic role in the arterial wall

The arachidonic acid metabolome serves as a conserved regulator of cholesterol metabolism

© 2014 The Authors. Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in &gt; 100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolome as conserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans. Omega-6 polyunsaturated fatty acids, including arachidonic acid (AA), have beneficial cardiovascular effects. Demetz et al. show that Alox5, a key enzyme of the AA pathway, regulates cholesterol in humans. Modulation of the AA pathways genetically or pharmacologically, with aspirin or bioactive AA-mimetics influences cholesterol metabolism including reverse cholesterol transport