G. M. Anantharamaiah

Antiinflammatory Properties of HDL

There are several well-documented functions of high-density lipoprotein (HDL) that may explain the ability of these lipoproteins to protect against atherosclerosis. The best recognized of these is the ability of HDL to promote the efflux of cholesterol from cells. This process may minimize the accumulation of foam cells in the artery wall. However, HDL has additional properties that may also be antiatherogenic. For example, HDL is an effective antioxidants. The major proteins of HDL, apoA-I and apoA-II, as well as other proteins such as paraoxonase that cotransport with HDL in plasma, are well-known to have antioxidant properties. As a consequence, HDL has the capacity to inhibit the oxidative modification of low-density lipoprotein (LDL) in a process that reduces the atherogenicity of these lipoproteins. HDL also possesses other antiinflammatory properties. By virtue of their ability to inhibit the expression of adhesion molecules in endothelial cells, they reduce the recruitment of blood monocytes into the artery wall. These antioxidant and antiinflammatory properties of HDL may be as important as its cholesterol efflux function in terms of protecting against the development of atherosclerosis.

Oral D-4F Causes Formation of Pre-β High-Density Lipoprotein and Improves High-Density Lipoprotein–Mediated Cholesterol Efflux and Reverse Cholesterol Transport From Macrophages in Apolipoprotein E–Null Mice

Background— These studies were designed to determine the mechanism of action of an oral apolipoprotein (apo) A-I mimetic peptide, D-4F, which previously was shown to dramatically reduce atherosclerosis in mice. Methods and Results— Twenty minutes after 500 μg of D-4F was given orally to apoE-null mice, small cholesterol-containing particles (CCPs) of 7 to 8 nm with pre-β mobility and enriched in apoA-I and paraoxonase activity were found in plasma. Before D-4F, both mature HDL and the fast protein liquid chromatography fractions containing the CCPs were proinflammatory. Twenty minutes after oral D-4F, HDL and CCPs became antiinflammatory, and there was an increase in HDL-mediated cholesterol efflux from macrophages in vitro. Oral D-4F also promoted reverse cholesterol transport from intraperitoneally injected cholesterol-loaded macrophages in vivo. In addition, oral D-4F significantly reduced lipoprotein lipid hydroperoxides (LOOH), except for pre-β HDL fractions, in which LOOH increased. Conclusions— The mechanism of action of oral D-4F in apoE-null mice involves rapid formation of CCPs, with pre-β mobility enriched in apoA-I and paraoxonase activity. As a result, lipoprotein LOOH are reduced, HDL becomes antiinflammatory, and HDL-mediated cholesterol efflux and reverse cholesterol transport from macrophages are stimulated.

The Amphipathic α Helix: A Multifunctional Structural Motif in Plasma Apolipoproteins

Publisher Summary The dominant structural motif of the peripheral apolipoproteins is the amphipathic helix, which is responsible for the reversible association of these proteins with lipids, as well as for many biological functions mediated by these apolipoproteins. This chapter reviews the different classes of amphipathic helices, using a combination of powerful computer programs to develop a comparison database and to analyze these structures. It also discusses their evolutionary origins, physical-chemical properties, X-ray structure determination, and conformational analysis. Although the structures of these lipoprotein classes are similar, they differ in relative proportion of lipids, in the apolipoprotein: lipid ratio and in the apolipoprotein species. The amphipathic α helix plays a pivotal role in the structure and functions of the exchangeable apolipoproteins. Site-directed mutagenesis and other molecular biology-based techniques are available for probing the structural motif. The location and properties of the amphipathic helices in apolipoproteins and the results are compared with recently developed and ever-expanding computer methods for the location and characterization. A variety of structure-function studies, including the activation of lipoprotein lipase, receptor recognition, lecithin-cholesterol acyltransferase (LCAT) activation, and antiviral and anti-inflammatory activities are also discussed.

Mechanisms of Disease: proatherogenic HDL—an evolving field

It is well known that, in large populations, HDL-cholesterol levels are inversely related to the risk of atherosclerotic clinical events; however, in an individual, the predictive value of an HDL-cholesterol level is far from perfect. As a result, other HDL-associated factors have been investigated, including the quality and function of HDL in contradistinction to the level of HDL-cholesterol. Regarding their quality, HDL particles are highly heterogeneous and contain varying levels of antioxidants or pro-oxidants, which results in variation in HDL function. It has been postulated that HDL functions to promote reverse cholesterol transport. Recent studies support this role for HDL but also indicate that HDL is a modulator of systemic inflammation. In the absence of inflammation, HDL has a complement of antioxidant enzymes that work to maintain an anti-inflammatory state. In the presence of systemic inflammation, these antioxidant enzymes can be inactivated and HDL can accumulate oxidized lipids and proteins that make it proinflammatory. Under these conditions the main protein of HDL, apolipoprotein A-I, can be modified by reactive oxygen species. This modification impairs the ability of HDL to promote cholesterol efflux by the ATP-binding cassette transporter A-1 pathway. Animal studies and small-scale human studies suggest that measures of the quality and novel functions of HDL might provide an improved means of identifying subjects at increased risk for atherosclerotic events, compared with the current practice of only measuring HDL-cholesterol levels. The quality and function of HDL are also attractive targets for emerging therapies.

Influenza Infection Promotes Macrophage Traffic Into Arteries of Mice That Is Prevented by D-4F, an Apolipoprotein A-I Mimetic Peptide

Background—We reported that HDL loses its antiinflammatory properties during acute influenza A infection in mice, and we hypothesized that these changes might be associated with increased trafficking of macrophages into the artery wall. The present study tested this hypothesis. Methods and Results—D-4F, an apolipoprotein A-I mimetic peptide, or vehicle in which it was dissolved (PBS) was administered daily to LDL receptor–null mice after a Western diet and after influenza infection. D-4F treatment increased plasma HDL cholesterol and paraoxonase activity compared with PBS and inhibited increases in LDL cholesterol and peak levels of interleukin-6 after infection. Lung viral titers were reduced by 50% in mice receiving D-4F. Injection of female mice with male macrophages, which were detected with real-time polymerase chain reaction to measure the male Sry gene, revealed a marked increase in macrophage traffic into the aortic arch and innominate arteries after infection that was prevented by administration of D-4F. Conclusions—We conclude that loss of antiinflammatory properties of HDL after influenza infection in mice is associated with increased arterial macrophage traffic that can be prevented by administration of D-4F.

apoB-100 has a pentapartite structure composed of three amphipathic alpha-helical domains alternating with two amphipathic beta-strand domains. Detection by the computer program LOCATE.

Due to the great length of apolipoprotein (apo) B-100, the localization of lipid-associating domains in this protein has been difficult. To address this question, we developed a computer program called Locate that searches amino acid sequences to identify potential amphipathic alpha-helixes and beta-strands by using sets of rules for helix and strand termination. A series of model chimeric protein test datasets were created by tandem linking of amino acid sequences of multiple proteins containing four different secondary structural motifs: motif A (exchangeable plasma apolipoproteins); motif G (globular alpha-helical proteins); motif C (coiled-coil alpha-helical proteins); and motif B (beta pleated-sheet proteins). These four test datasets, as well as randomly scrambled sequences of each dataset, were analyzed by Locate using increasingly stringent parameters. Using intermediately stringent parameters under which significant numbers of amphipathic helixes were found only in the unscrambled motif A, two dense clusters of putative lipid-associating amphipathic helixes were located precisely in the middle and at the C-terminal end of apoB-100 (a sparse cluster of class G* helixes is located at the N-terminus). The dense clusters are located between residues 2103 through 2560 and 4061 through 4338 and have densities of 2.4 and 2.2 amphipathic helixes per 100 residues, respectively; under these conditions, motif A has a density of 1.4 amphipathic helixes per 100 residues. These two domains correspond closely to the two major apoB-100 lipid-associated domains at residues 2100 through 2700 and 4100 through 4500 using the principle of releasability of tryptic peptides from trypsin-treated intact low-density lipoprotein. The classes of amphipathic helixes identified within these two putative lipid-associating domains are considerably more diverse than those found in the exchangeable plasma apolipoproteins. Interestingly, apoB-48 terminates at the N-terminal edge of the middle cluster. By using a similar strategy for analysis of amphipathic beta-strands, we discovered that the two gap regions between the three amphipathic helix clusters are highly enriched in putative amphipathic beta-strands, while the three amphipathic helical domains are essentially devoid of this putative lipid-associating motif. We propose, therefore, that apoB-100 has a pentapartite structure, NH2-alpha 1-beta 1-alpha 2-beta 2-alpha 3-COOH, with alpha 1 representing a globular domain.

Structure and function of apolipoprotein A-I and high-density lipoprotein.

Structural biology and molecular modeling have provided intriguing insights into the atomic details of the lipid-associated structure of the major protein component of HDL, apo A-I. For the first time, an atomic resolution map is available for future studies of the molecular interactions of HDL in such biological processes as ABC1-regulated HDL assembly, LCAT activation, receptor binding, reverse lipid transport and HDL heterogeneity. Within the context of this paradigm, the current review summarizes the state of HDL research.

D-4F, an Apolipoprotein A-I Mimetic Peptide, Inhibits the Inflammatory Response Induced by Influenza A Infection of Human Type II Pneumocytes

Background—Evidence suggests that apolipoprotein A-I (apoA-I) and HDL play important roles in modulating inflammation. We previously reported that an apoA-I mimetic peptide, D-4F, reduced inflammatory responses to influenza virus in mice. To further define the antiinflammatory activity of D-4F, a human alveolar type II cell line, A549, was used. Methods and Results—Cells were either uninfected or infected with influenza A in the presence or absence of D-4F. Cells treated with D-4F were more viable, and virus-induced cytokine production was suppressed by D-4F. Caspases associated with cytokine production were activated after infection but suppressed by D-4F treatment. Infected A549 cells showed dramatic increases in cellular phospholipid secretion into the media. When infected cells were incubated with D-4F, secretion of parent nonoxidized, noninflammatory phospholipids was unaltered, but production of proinflammatory oxidized phospholipids was inhibited. Conclusions—Type II pneumocytes respond to influenza A infection by activating caspases and secreting cytokines and cellular phospholipids into the extracellular environment, including oxidized phospholipids that evoke inflammatory responses. D-4F treatment inhibited these events. Our results suggest that apoA-I and apoA-I mimetic peptides such as D-4F are antiinflammatory agents that may have therapeutic potential.

Janus kinase 2 modulates the lipid-removing but not protein-stabilizing interactions of amphipathic helices with ABCA1

ABCA1 mediates the transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Apolipoprotein A-I (apoA-I) interactions with ABCA1-expressing cells elicit several responses, including removing cellular lipids, stabilizing ABCA1 protein, and activating Janus kinase 2 (JAK2). Here, we used synthetic apolipoprotein-mimetic peptides to characterize the relationship between these responses. Peptides containing one amphipathic helix of l- or d-amino acids (2F, D-2F, or 4F) and a peptide containing two helices (37pA) all promoted ABCA1-dependent cholesterol efflux, competed for apoA-I binding to ABCA1-expressing cells, blocked covalent cross-linking of apoA-I to ABCA1, and inhibited ABCA1 degradation. 37pA was cross-linked to ABCA1, confirming the direct binding of amphipathic helices to ABCA1. 2F, 4F, 37pA, and D-37pA all stimulated JAK2 autophosphorylation. Inhibition of JAK2 greatly reduced peptide-mediated cholesterol efflux, peptide binding to ABCA1-expressing cells, and peptide cross-linking to ABCA1, indicating that these processes require an active JAK2. In contrast, apoA-I and peptides stabilized ABCA1 protein even in the absence of an active JAK2, implying that this process is independent of JAK2 and lipid efflux-promoting binding of amphipathic helices to ABCA1. These findings show that amphipathic helices coordinate the activity of ABCA1 by several distinct mechanisms that are likely to involve different cell surface binding sites.

A novel approach to oral apoA-I mimetic therapy

Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR−/−) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.