Fogelman Alan MDivision of Cardiology, Department of Medicine, University of California, Los Angeles School of Medicine, Los Angeles, CA USAPhone : Fax :
Cyclooxygenase 2 (Cox2) total knockout and myeloid knockout (MKO) mice develop Crohn's-like intestinal inflammation when fed cholate-containing high fat diet (CCHF). We demonstrated that CCHF impaired intestinal barrier function and increased translocation of endotoxin, initiating TLR/MyD88-dependent inflammation in Cox2 KO but not WT mice. Cox2 MKO increased pro-inflammatory mediators in LPS-activated macrophages, and in the intestinal tissue and plasma upon CCHF challenge. Cox2 MKO also reduced inflammation resolving lipoxin A4 (LXA4) in intestinal tissue, while administration of an LXA4 analog rescued disease in Cox2 MKO mice fed CCHF. The apolipoprotein A-I (APOA1) mimetic 4F mitigated disease in both the Cox2 MKO/CCHF and piroxicam-accelerated Il10-/- models of inflammatory bowel disease (IBD) and reduced elevated levels of pro-inflammatory mediators in tissue and plasma. APOA1 mimetic Tg6F therapy was also effective in reducing intestinal inflammation in the Cox2 MKO/CCHF model. We further demonstrated that APOA1 mimetic peptides: i) inhibited LPS and oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (oxPAPC) dependent pro-inflammatory responses in human macrophages and intestinal epithelium; and ii) directly cleared pro-inflammatory lipids from mouse intestinal tissue and plasma. Our results support a causal role for pro-inflammatory and inflammation resolving lipids in IBD pathology and a translational potential for APOA1 mimetic peptides for the treatment of IBD.
Exposure to ambient particulate matter (PM) is a risk factor for cardiovascular diseases. The redox-active ultrafine particles (UFPs) promote vascular oxidative stress and inflammatory responses. We hypothesized that UFPs modulated lipid metabolism and anti-oxidant capacity of high density lipoprotein (HDL) with an implication in atherosclerotic lesion size. Fat-fed low density lipoprotein receptor-null (LDLR(-)/(-) mice were exposed to filtered air (FA) or UFPs for 10 weeks with or without administering an apolipoprotein A-I mimetic peptide made of D-amino acids, D-4F. LDLR(-)/(-) mice exposed to UFPs developed a reduced plasma HDL level (P < 0.01), paraoxonase activity (P < 0.01), and HDL anti-oxidant capacity (P < 0.05); but increased LDL oxidation, free oxidized fatty acids, triglycerides, serum amyloid A (P < 0.05), and tumor necrosis factor alpha (P < 0.05), accompanied by a 62% increase in the atherosclerotic lesion ratio of the en face aortic staining and a 220% increase in the cross-sectional lesion area of the aortic sinus (P < 0.001). D-4F administration significantly attenuated these changes. UFP exposure promoted pro-atherogenic lipid metabolism and reduced HDL anti-oxidant capacity in fat-fed LDLR(-)/(-) mice, associated with a greater atherosclerotic lesion size compared with FA-exposed animals. D-4F attenuated UFP-mediated pro-atherogenic effects, suggesting the role of lipid oxidation underlying UFP-mediated atherosclerosis.
OBJECTIVE: To evaluate whether exposure to air pollutants induces oxidative modifications of plasma lipoproteins, resulting in alteration of the protective capacities of high-density lipoproteins (HDLs). APPROACH AND RESULTS: We exposed apolipoprotein E-deficient mice to diesel exhaust (DE) at approximately 250 microg/m(3) for 2 weeks, filtered air (FA) for 2 weeks, or DE for 2 weeks, followed by FA for 1 week (DE+FA). DE led to enhanced lipid peroxidation in the brochoalveolar lavage fluid that was accompanied by effects on HDL functionality. HDL antioxidant capacity was assessed by an assay that evaluated the ability of HDL to inhibit low-density lipoprotein oxidation estimated by 2',7'-dichlorofluorescein fluorescence. HDL from DE-exposed mice exhibited 23,053 +/- 2844 relative fluorescence units, higher than FA-exposed mice (10,282 +/- 1135 relative fluorescence units, P<0.001) but similar to the HDL from DE+FA-exposed mice (22,448 +/- 3115 relative fluorescence units). DE effects on HDL antioxidant capacity were negatively correlated with paraoxonase enzymatic activity, but positively correlated with levels of plasma 8-isoprostanes, 12-hydroxyeicosatetraenoic acid, 13-hydroxyoctadecadienoic acid, liver malondialdehyde, and accompanied by perturbed HDL anti-inflammatory capacity and activation of the 5-lipoxygenase pathway in the liver. CONCLUSIONS: DE emissions induced systemic pro-oxidant effects that led to the development of dysfunctional HDL. This may be one of the mechanisms by which air pollution contributes to enhanced atherosclerosis.
Title: D-4F-mediated reduction in metabolites of arachidonic and linoleic acids in the small intestine is associated with decreased inflammation in low-density lipoprotein receptor-null mice Navab M, Reddy ST, Anantharamaiah GM, Hough G, Buga GM, Danciger J, Fogelman AM Ref: J Lipid Res, 53:437, 2012 : PubMed
To test the hypothesis that intestine is a major site of action for D-4F, LDLR(-/-) mice were fed a Western diet (WD) and administered the peptide subcutaneously (SQ) or orally. Plasma and liver D-4F levels were 298-fold and 96-fold higher, respectively, after SQ administration, whereas peptide levels in small intestine only varied by 1.66 +/- 0.33-fold. Levels of metabolites of arachidonic and linoleic acids known to bind with high affinity to D-4F were significantly reduced in intestine, liver and hepatic bile to a similar degree whether administered SQ or orally. However, levels of 20-HETE, which is known to bind the peptide with low affinity, were unchanged. D-4F treatment reduced plasma serum amyloid A (SAA) and triglyceride levels (P < 0.03) and increased HDL-cholesterol levels (P < 0.04) similarly after SQ or oral administration. Plasma levels of metabolites of arachidonic and linoleic acids significantly correlated with SAA levels (P < 0.0001). Feeding 15-HETE in chow (without WD) significantly increased plasma SAA and triglyceride levels and decreased HDL-cholesterol and paraoxonase activity (P < 0.05), all of which were significantly ameliorated by SQ D-4F (P < 0.05). We conclude that D-4F administration reduces levels of free metabolites of arachidonic and linoleic acids in the small intestine and this is associated with decreased inflammation in LDL receptor deficient mice.
Title: Salutary effects of hemodialysis on low-density lipoprotein proinflammatory and high-density lipoprotein anti-inflammatory properties in patient with end-stage renal disease Vaziri ND, Navab K, Gollapudi P, Moradi H, Pahl MV, Barton CH, Fogelman AM, Navab M Ref: J Natl Med Assoc, 103:524, 2011 : PubMed
End-stage renal disease (ESRD) causes oxidative stress, inflammation, low-density lipoprotein (LDL) oxidation, high-density lipoprotein (HDL) deficiency and accelerated atherosclerosis. Uptake of oxidized LDL by macrophages results in foam cell and plaque formation. HDL mitigates atherosclerosis via reverse cholesterol transport and inhibition of LDL oxidation. ESRD heightens LDL inflammatory activity and suppresses HDL anti-inflammatory activity. The effect of hemodialysis on the LDL and HDL inflammatory properties is unknown. By removing the potential pro-oxidant/proinflammatory uremic toxins, dialysis may attenuate LDL inflammatory and HDL anti-inflammatory properties. Conversely, exposure to dialyzer membrane and tubing and influx of impurities from dialysate can intensify LDL and HDL inflammatory activities. This study examined the effect of hemodialysis on LDL and HDL inflammatory activities. Plasma samples were obtained from 12 normal control and 26 ESRD patients before and after hemodialysis with (16 patients) or without (10 patients) heparinization. HDL and LDL were isolated and tested for monocyte chemotactic activity in cultured endothelial cells. ESRD patients had increased LDL chemotactic activity, reduced HDL anti-inflammatory activity, paraoxonase and glutathione peroxidase levels, and elevated plasma IL-6 before dialysis. Hemodialysis partially improved LDL inflammatory and HDL anti-inflammatory activities and enhanced patients' HDL ability to suppress their LDL inflammatory activity. The salutary effect on LDL inflammatory activity was significantly greater in patients dialyzed with than those without heparin. ESRD heightens LDL inflammatory and impairs HDL anti-inflammatory activities. Hemodialysis partially improves LDL and HDL inflammatory activities. The salutary effects of hemodialysis are in part mediated by heparin, which is known to possess lipolytic and antioxidant properties.
L-4F, an apolipoprotein A-I (apoA-I) mimetic peptide (also known as APL180), was administered daily by either intravenous (IV) infusion for 7 days or by subcutaneous (SC) injection for 28 days in patients with coronary heart disease in two distinct clinical studies. L-4F was well tolerated at all doses tested. Despite achieving plasma levels (mean maximal plasma concentration of 2,907 ng/ml and 395 ng/ml, following IV infusion and SC injection, respectively), that were effective in previously published animal models, treatment with L-4F, as assessed by biomarkers of HDL function such as HDL-inflammatory index (HII), and paraoxonase activity, did not improve. Paradoxically, there was a 49% increase in high-sensitivity C-reactive protein (hs-CRP) levels after seven IV infusions of 30 mg L-4F (P < 0.05; compared with placebo) and a trend for hs-CRP increase in subjects receiving 30 mg SC injection for 28 days. In a subsequent, ex vivo study, addition of L-4F at concentrations of 150, 375, or 1,000 ng/ml to plasma from subjects prior to L-4F treatment resulted in significant dose-dependent HII improvement. In conclusion, in vivo L-4F treatment, delivered by either SC injection or IV infusion, did not improve HDL functional biomarkers despite achieving plasma levels that improved identical biomarkers ex vivo and in animal models.
Title: Is it just paraoxonase 1 or are other members of the paraoxonase gene family implicated in atherosclerosis? Reddy ST, Devarajan A, Bourquard N, Shih DM, Fogelman AM Ref: Curr Opin Lipidol, 19:405, 2008 : PubMed
PURPOSE OF REVIEW: During the past decade, paraoxonase 1, a HDL-associated protein, has been demonstrated to be an important contributor to the antioxidant capacity of HDL. Studies using paraoxonase 1 null mice by gene targeting and transgenic mice corroborated the hypothesis that paraoxonase 1 protects against atherosclerosis. In contrast to paraoxonase 1, the other two members of the paraoxonase gene family, namely paraoxonase 2 and paraoxonase 3, are either undetectable (paraoxonase 2) or detected at very low levels (paraoxonase 3) on HDL, and are considered to participate in intracellular antioxidant mechanisms. In this review, we summarize studies reported in the past 2 years suggesting a protective role for paraoxonase 2 and paraoxonase 3 in the development of atherosclerosis in mice. RECENT FINDINGS: Adenovirus-mediated expression of human paraoxonase 2 or paraoxonase 3 proteins protects against the development of atherosclerosis in apolipoprotein E-deficient mice. Paraoxonase 2-deficient mice develop significantly larger atherosclerotic lesions than their wild-type and heterozygous counterparts on an atherogenic diet despite having lower levels of apolipoprotein B-containing lipoproteins. Atherosclerotic lesions were significantly lower in male hPON3Tg/LDLR null mice than in LDLR null mice on a western diet. SUMMARY: We conclude that, in addition to paraoxonase 1, both paraoxonase 2 and paraoxonase 3 proteins are protective against the development of atherosclerosis in mice. These findings underscore the utility of all members of the paraoxonase gene family as therapeutic targets for the treatment of atherosclerosis.
Atherosclerosis, the underlying cause of cardiovascular disease, is characterized by lipid accumulation, lipoprotein oxidation, and inflammation. Products of the cyclooxygenase (COX) pathway participate in acute and chronic inflammation. The inducible form of COX, COX-2, generates lipid mediators of inflammation that are pro-inflammatory and COX-2-selective inhibitors are potent anti-inflammatory agents. However, clinical data suggest an increased risk of cardiovascular side effects in patients using COX-2-selective inhibitors. In this paper, we sought to determine the effect of COX-2 deficiency on atherosclerosis-related lipoprotein metabolism in mice. We demonstrate that COX-2 deficiency resulted in (i) accumulation of lipids in circulation and liver, (ii) pro-inflammatory properties of HDL as measured by HDL's increased reactive oxygen species (ROS) content, decreased paraoxonase 1 (PON1) activity, decreased serum apoA-1, reduced ability to efflux cholesterol and to prevent LDL oxidizability, and (iii) increased TXB(2) in circulation. Moreover, when placed on an atherogenic diet, COX-2 deficiency resulted in (i) increased lipid deposition in the aorta, (ii) a further dramatic imbalance in circulating eicosanoids, i.e. decreased serum PGI(2) coupled with increased PGE(2) and TXB(2), and (iii) a marked elevation of pro-inflammatory cytokines, TNF and IL-6. Our results suggest, for the first time, that COX-2 deficiency contributes to the pro-atherogenic properties of HDL in mice.
OBJECTIVE: We have previously reported that human paraoxonase 3 (PON3) is an HDL-associated protein capable of preventing LDL oxidation in vitro. The objective of the present study was to determine whether elevated levels of human PON3 in mice could protect against the progression of atherosclerosis in vivo. METHODS AND RESULTS: Twenty-six week-old apolipoprotein E-deficient mice were injected with 3x10(11) particles of adenovirus expressing either GFP alone (AdGFP) or together with human PON3 (AdPON3). Three weeks after injection, lesion area was significantly lower in AdPON3-treated mice compared with AdGFP controls. Serum from AdPON3 mice contained significantly lower levels of lipid hydroperoxides and exhibited an enhanced potential to efflux cholesterol from cholesterol-loaded macrophages. In addition, LDL was less susceptible to oxidation, whereas HDL was more capable of protecting against LDL oxidation. Exogenous human PON3 was not detected in the serum or HDL and more surprisingly we demonstrate that endogenous mouse PON3 is not associated with HDL, suggesting that the antioxidant function of PON3 is at the cellular level in mice. CONCLUSIONS: This study demonstrates for the first time that PON3 enhances the antiatherogenic capacity of serum and protects against the progression of atherosclerosis in vivo.
Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4- to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.
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.
PURPOSE OF REVIEW: To determine the potential clinical utility of high-density lipoprotein-mimetic peptides. RECENT FINDINGS: Oral administration of D-4F together with pravastatin caused lesion regression in old apoE null mice. Administration of D-4F to low-density lipoprotein receptor null mice fed a Western diet reduced the association of myeloperoxidase with apoA-I and reduced the 3-nitrotyrosine content of apoA-I. Oral D-4F improved arterial vasoreactivity independent of apoA-I. Mice genetically lacking apoA-I showed significant improvement in vasoreactivity but, in contrast to mice with apoA-I, did not demonstrate reduced arterial wall thickness after D-4F treatment. In a rat model of diabetes, D-4F administration induced heme oxygenase-1 and extracellular superoxide dismutase, prevented endothelial sloughing, and dramatically improved arterial vasoreactivity. A peptide with 10 D-amino acid residues taken from the sequence of apoJ rendered high-density lipoprotein anti-inflammatory in mice and monkeys, and dramatically reduced atherosclerosis in apoE null mice. Oral administration of tetrapeptides synthesized from either L-amino acids or D-amino acids rendered high-density lipoprotein anti-inflammatory in mice and monkeys, and reduced atherosclerosis in apoE null mice. SUMMARY: Peptides that sequester lipoprotein lipid hydroperoxides release a series of high-density lipoprotein-associated antioxidant enzymes such as paraoxonase from inhibition and protect apoA-I from oxidative damage that would impair cholesterol efflux.
Paraoxonases (PONs) are a family of proteins that may play a significant role in providing relief from both toxic environmental chemicals as well as physiological oxidative stress. Although the physiological roles of the PON family of proteins, PON1, PON2, and PON3, remain unknown, epidemiological, biochemical, and mouse genetic studies of PON1 suggest an anti-atherogenic function for paraoxonases. To determine whether PON2 plays a role in the development of atherosclerosis in vivo, we generated PON2-deficient mice. When challenged with a high fat, high cholesterol diet for 15 weeks, serum levels of high density lipoprotein cholesterol, triglycerides, and glucose were not significantly different between wild-type and PON2-deficient mice. In contrast, serum levels of very low density lipoprotein (VLDL)/low density lipoprotein (LDL) cholesterol were significantly lower (-32%) in PON2-deficient mice compared with wild-type mice. However, despite lower levels of VLDL/LDL cholesterol, mice deficient in PON2 developed significantly larger (2.7-fold) atherosclerotic lesions compared with their wild-type counterparts. Enhanced inflammatory properties of LDL, attenuated anti-atherogenic capacity of high density lipoprotein, and a heightened state of oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages appear to be the main mechanisms behind the larger atherosclerotic lesions in PON2-deficient mice. These results demonstrate that PON2 plays a protective role in atherosclerosis.
Cholesterol can promote inflammation by its ability to stimulate the production of reactive oxygen species that result in the formation of pro-inflammatory oxidised phospholipids. High-density lipoproteins (HDLs) are part of the innate immune response and can be either pro- or anti-inflammatory independently of plasma HDL-cholesterol levels. During systemic inflammation as occurs with atherosclerosis, Apolipoprotein A-I can be altered, reducing its ability to promote reverse cholesterol transport and HDL can become pro-inflammatory. Amphipathic peptides with either a class A amphipathic helix (D-4F) or a class G* amphipathic helix (D-[113-122]apoJ), or even those that are too small to form a helix (KRES and FREL) have some similar characteristics. Their interaction with lipids leads to a reduction in lipoprotein-lipid hydroperoxides that releases HDL-associated antioxidant enzymes, such as paraoxonase, therefore providing antiatherosclerosis and anti-inflammatory activity. In addition, the peptide D-4F stimulates the formation and cycling of pre-beta HDL. These amphipathic peptides appear to have therapeutic potential as oral agents.
Title: Understanding changes in high density lipoproteins during the acute phase response Van Lenten BJ, Reddy ST, Navab M, Fogelman AM Ref: Arterioscler Thromb Vasc Biol, 26:1687, 2006 : PubMed
OBJECTIVE: We tested for synergy between pravastatin and D-4F by administering oral doses of each in combination that were predetermined to be ineffective when given as single agents. METHODS AND RESULTS: The combination significantly increased high-density lipoprotein (HDL)-cholesterol levels, apolipoprotein (apo)A-I levels, paraoxonase activity, rendered HDL antiinflammatory, prevented lesion formation in young (79% reduction in en face lesion area; P<0.0001) and caused regression of established lesions in old apoE null mice (ie, mice receiving the combination for 6 months had lesion areas that were smaller than those before the start of treatment (P=0.019 for en face lesion area; P=0.004 for aortic root sinus lesion area). After 6 months of treatment with the combination, en face lesion area was 38% of that in mice maintained on chow alone; P<0.00004) with a 22% reduction in macrophage content in the remaining lesions (P=0.001), indicating an overall reduction in macrophages of 79%. The combination increased intestinal apoA-I synthesis by 60% (P=0.011). In monkeys, the combination also rendered HDL antiinflammatory. CONCLUSIONS: These results suggest that the combination of a statin and an HDL-based therapy may be a particularly potent treatment strategy.
A peptide containing only 4 amino acid residues (KRES) that is too small to form an amphipathic helix, reduced lipoprotein lipid hydroperoxides (LOOH), increased paraoxonase activity, increased plasma HDL-cholesterol levels, rendered HDL antiinflammatory, and reduced atherosclerosis in apoE null mice. KRES was orally effective when synthesized from either L or D-amino acids suggesting that peptide-protein interactions were not required. Remarkably, changing the order of 2 amino acids (from KRES to KERS) resulted in the loss of all biologic activity. Solubility in ethyl acetate and interaction with lipids, as determined by differential scanning calorimetry, indicated significant differences between KRES and KERS. Negative stain electron microscopy showed that KRES formed organized peptide-lipid structures whereas KERS did not. Another tetrapeptide FREL shared many of the physical-chemical properties of KRES and was biologically active in mice and monkeys when synthesized from either L- or D-amino acids. After oral administration KRES and FREL were found associated with HDL whereas KERS was not. We conclude that the ability of peptides to interact with lipids, remove LOOH and activate antioxidant enzymes associated with HDL determines their antiinflammatory and antiatherogenic properties regardless of their ability to form amphipathic helixes.
Despite identical amino acid composition, differences in class A amphipathic helical peptides caused by differences in the order of amino acids on the hydrophobic face results in substantial differences in antiinflammatory properties. One of these peptides is an apolipoprotein A-I (apoA-I) mimetic, D-4F. When given orally to mice and monkeys, D-4F caused the formation of pre-beta high-density lipoprotein (HDL), improved HDL-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from pro-inflammatory to antiinflammatory. In apolipoprotein E (apoE)-null mice, D-4F increased reverse cholesterol transport from macrophages. Oral D-4F reduced atherosclerosis in apoE-null and low-density lipoprotein (LDL) receptor-null mice. In vitro when added to human plasma at nanomolar concentrations, D-4F caused the formation of pre-beta HDL, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from pro-inflammatory to antiinflammatory. Physical-chemical properties and the ability of various class A amphipathic helical peptides to activate lecithin cholesterol acyltransferase (LCAT) in vitro did not predict biologic activity in vivo. In contrast, the use of cultured human artery wall cells in evaluating these peptides was more predictive of their efficacy in vivo. We conclude that the antiinflammatory properties of different class A amphipathic helical peptides depends on subtle differences in the configuration of the hydrophobic face of the peptides, which determines the ability of the peptides to sequester inflammatory lipids. These differences appear to be too subtle to predict efficacy based on physical-chemical properties alone. However, understanding these physical-chemical properties provides an explanation for the mechanism of action of the active peptides.
OBJECTIVE: To determine the properties of a peptide synthesized from D-amino acids corresponding to residues 113 to 122 in apolipoprotein (apo) J. METHODS AND RESULTS: In contrast to D-4F, D- [113-122]apoJ showed minimal self-association and helicity in the absence of lipids. D-4F increased the concentration of apoA-I with pre-beta mobility in apoE-null mice whereas D- [113-122]apoJ did not. After an oral dose D- [113-122]apoJ more slowly associated with lipoproteins and was cleared from plasma much more slowly than D-4F. D- [113-122]apoJ significantly improved the ability of plasma to promote cholesterol efflux and improved high-density lipoprotein (HDL) inflammatory properties for up to 48 hours after a single oral dose in apoE-null mice, whereas scrambled D- [113-122]apoJ did not. Oral administration of 125 microg/mouse/d of D- [113-122]apoJ reduced atherosclerosis in apoE-null mice (70.2% reduction in aortic root sinus lesion area, P=4.3 x 10(-13); 70.5% reduction by en face analysis, P=1.5 x 10(-6)). In monkeys, oral D- [113-122]apoJ rapidly reduced lipoprotein lipid hydroperoxides (LOOH) and improved HDL inflammatory properties. Adding 250 ng/mL of D-[113-122]apoJ (but not scrambled D- [113-122]apoJ) to plasma in vitro reduced LOOH and increased paraoxonase activity. CONCLUSIONS: Oral D- [113-122]apoJ significantly improves HDL inflammatory properties in mice and monkeys and inhibits lesion formation in apoE-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 microg of D-4F was given orally to apoE-null mice, small cholesterol-containing particles (CCPs) of 7 to 8 nm with pre-beta 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-beta 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-beta 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.
PURPOSE OF REVIEW: Recent publications related to the potential use of apolipoprotein (apo)A-I and apoA-I mimetic peptides in the treatment of atherosclerosis are reviewed. RECENT FINDINGS: A preliminary report indicating that infusion of apoA-IMilano into humans once weekly for 5 weeks caused a significant decrease in coronary artery atheroma volume has sparked great interest in the potential therapeutic use of apoA-I. Recent studies have revealed that HDL quality (e.g. HDL apolipoprotein and lipid content, including oxidized lipids, particle size and electrophoretic mobility, associated enzymatic activities, inflammatory/anti-inflammatory properties, and ability to promote cholesterol efflux) may be more important than HDL-cholesterol levels. Therefore, when developing new strategies to raise HDL-cholesterol concentrations by interfering with HDL metabolism, one must consider the quality of the resulting HDL. In animal models, raising HDL-cholesterol levels by administering oral phospholipids improved both the quantity and quality of HDL and was associated with lesion regression. An apoA-I mimetic peptide, namely 4F synthesized from D-amino acids (D-4F), administered orally to mice did not raise HDL-cholesterol concentrations but promoted the formation of pre-beta HDL containing increased paraoxonase activity, resulting in significant improvements in HDL's anti-inflammatory properties and ability to promote cholesterol efflux from macrophages in vitro. Oral D-4F also promoted reverse cholesterol efflux from macrophages in vivo. SUMMARY: The quality of HDL may be more important than HDL-cholesterol levels. ApoA-I and apoA-I mimetic peptides appear to have significant therapeutic potential in atherosclerosis.
Title: Apparent paradox of low-fat healthy diets increasing plasma levels of oxidized low-density lipoprotein and lipoprotein(a) Navab M, Reddy ST, Van Lenten BJ, Fogelman AM Ref: Arterioscler Thromb Vasc Biol, 24:392, 2004 : PubMed
The effects of apolipoprotein (Apo) AI mimetic peptide synthesized from D- and L-amino acids on atherosclerotic lesion formation were investigated in low-density lipoprotein (LDL) receptor-deficient mice on a Western diet and in apoE null mice. In addition, their effects on the inflammatory changes induced in LDL-receptor mice fed a Western diet following influenza A infection were studied. When apolipoprotein AI mimetic peptides synthesized from either D- or L-amino acids were administered to LDL-receptor null mice, only peptides synthesized from D-amino acids were stable in the circulation and enhanced the ability of high-density lipoprotein (HDL) to protect LDL against oxidation. Administration of the peptide D-4F to LDL-receptor null mice and apoE null mice decreased lesion size. Additionally, in LDL receptor null mice after influenza infection, D-4F treatment increased plasma HDL levels and paraoxonase activity, and inhibited increased in LDL-cholesterol and peak levels of interleukin-6 post-infection. Injection of female mice with male macrophages, and subsequent measurement of the male 'sry' gene, revealed a marked increase in macrophage traffic into the aortic arch after infection that was prevented by administration of D-4F. This indicates that: (i) oral D-4F has powerful anti-atherosclerotic properties, and (ii) the loss of the anti-inflammatory properties of HDL after influenza infection in mice is associated with increased arterial macrophage traffic that can be prevented by administration of D-4F.
Title: Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein Ng CJ, Wadleigh DJ, Gangopadhyay A, Hama S, Grijalva VR, Navab M, Fogelman AM, Reddy ST Ref: Journal of Biological Chemistry, 276:44444, 2001 : PubMed
The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.
Paraoxonase-1 (PON1) is a secreted protein associated primarily with high density lipoprotein (HDL) and participates in the prevention of low density lipoprotein (LDL) oxidation. Two other paraoxonase (PON) family members, namely, PON2 and PON3, have been identified. In this study, we report the cloning and characterization of the human PON3 gene from HepG2 cells. Tissue Northern analysis identifies an approximately 1.3-kb transcript for PON3 primarily in the liver. PON3-specific peptide antibodies detect an approximately 40-kDa protein associated with HDL and absent from LDL. Pretreatment of cultured human aortic endothelial cells with supernatants from HeLa Tet On cell lines overexpressing PON3 prevents the formation of mildly oxidized LDL and inactivates preformed mildly oxidized LDL. In contrast to PON1, PON3 is not active against the synthetic substrates paraoxon and phenylacetate. Furthermore, PON3 expression is not regulated in HepG2 cells by oxidized phospholipids and is not regulated in the livers of mice fed a high-fat atherogenic diet.
Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.
Paraoxonase (PON1) hydrolyses organophosphate insecticides and nerve gases and is responsible for determining the selective toxicity of these compounds in mammals. Human PON1 has two genetic polymorphisms giving rise to amino-acid substitutions at positions 55 and 192. The 192 polymorphism is the major determinant of the PON1 activity polymorphism towards organophosphates. However, the 55 polymorphism also modulates activity. Ex vivo, the PON1 polymorphisms are important in determining the capacity of HDL to protect LDL against oxidative modification in vitro and this may explain the relationship between the PON1 alleles and coronary heart disease in case-control studies. In recent case-control studies serum PON1 concentration and activity were also found to be decreased in coronary heart disease (CHD) independent of the PON1 polymorphism, and in diabetes serum PON1 specific activity decrease is also independent of the PON1 genetic polymorphism. HDL from transgenic mice lacking PON1 fails to protect LDL against oxidative modification. Thus PON1 may be a determinant of resistance to the development of atherosclerosis by protecting lipoproteins against oxidative modification, perhaps by hydrolysing phospholipid and cholesteryl-ester hydroperoxides.
Serum paraoxonase (PON1) is an esterase that is associated with high-density lipoproteins (HDLs) in the plasma; it is involved in the detoxification of organophosphate insecticides such as parathion and chlorpyrifos. PON1 may also confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids present in oxidized low-density lipoproteins (LDLs). To study the role of PON1 in vivo, we created PON1-knockout mice by gene targeting. Compared with their wild-type littermates, PON1-deficient mice were extremely sensitive to the toxic effects of chlorpyrifos oxon, the activated form of chlorpyrifos, and were more sensitive to chlorpyrifos itself. HDLs isolated from PON1-deficient mice were unable to prevent LDL oxidation in a co-cultured cell model of the artery wall, and both HDLs and LDLs isolated from PON1-knockout mice were more susceptible to oxidation by co-cultured cells than the lipoproteins from wild-type littermates. When fed on a high-fat, high-cholesterol diet, PON1-null mice were more susceptible to atherosclerosis than their wild-type littermates.
