Loss of CD226 protects apolipoprotein E-deficient mice from diet-induced atherosclerosis

CD226 is a costimulatory molecule that regulates immune cell functions in T cells, natural killer cells, and macrophages. Because macrophage-derived foam cell formation is a crucial factor contributing to the development of atherosclerosis, we aimed to evaluate the potential roles of CD226 in the pathogenesis of atherosclerosis. The effects of CD226 on atherosclerosis were investigated in CD226 and apolipoprotein E double-knockout (CD226^?/? ApoE^?/?) mice fed with a high-cholesterol atherogenic diet. CD226 expression in macrophages was evaluated using flow cytometry. Histopathological analysis was performed to evaluate the atherosclerotic lesions. Inflammatory cell infiltration was detected using immunofluorescence staining. Bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PEMs) were isolated from the mice and used to explore the mechanism in vitro. The in vivo results indicated that CD226 knockdown protected against atherosclerosis in ApoE^?/? mice, evidenced by reduced plaque accumulation in the brachiocephalic artery, aortic roots, and main aortic tree. CD226 gene-deficient macrophages showed reduced foam cell formation under ox-low density lipoprotein stimulation compared with wild-type (WT) cells. CD226 deficiency also decreased the expression of CD36 and scavenger receptor (SR)-A (responsible for lipoprotein uptake) but increased the expression of ATP-binding cassette transporter A1 and G1 (two transporters for cholesterol efflux). Therefore, loss of CD226 hinders foam cell formation and atherosclerosis progression, suggesting that CD226 is a promising new therapeutic target for atherosclerosis.     

The membrane-binding properties of a class A amphipathic peptide

The membrane-binding properties of a class A amphipathic peptide (18D) were investigated using two different immobilized model membrane systems. The first system involved the use of surface plasmon resonance (SPR) to study the binding of 18D to dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylglycerol (DMPG), which allowed peptide binding to be monitored in real time. The SPR experiments indicated stronger binding of 18D to DMPG than DMPC, which kinetic analysis revealed was due to a faster on-rate. The second model membrane system involved immobilized membrane chromatography in which the binding of 18D to either DMPC or DMPG monolayers covalently linked to silica particles was analysed by elution chromatography. Stronger binding affinity of 18D was also obtained with the negatively charged phosphatidylglycerol (PG) monolayer compared to the phosphatidylcholine (PC) monolayer, which was consistent with the SPR results. Non-linear binding behaviour of 18D to the immobilized lipid monolayers was also observed, which suggests that the peptide undergoes conformational and orientational changes upon binding to the immobilized PC and PG ligands. Significant band broadening was also observed on both monolayers, with larger bandwidths obtained on the PC surface, indicating slower binding and orientation kinetics with the zwitterionic surface. The dependence of logk' on the percentage of methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while at lower temperatures, electrostatic and other polar forces also made a contribution to the affinity of the peptides for the lipid monolayer particularly. Overall, these results demonstrate the complementary use of these two lipid biosensors which allows the role of hydrophobic and electrostatic forces in peptide–membrane interactions to be studied and insight gained into the kinetic factors associated with these interactions.     

Peripheral-specific y2 receptor knockdown protects mice from high-fat diet-induced obesity

Y2 receptors, particularly those in the brain, have been implicated in neuropeptide Y (NPY)-mediated effects on energy homeostasis and bone mass. Recent evidence also indicates a role for Y2 receptors in peripheral tissues in this process by promoting adipose tissue accretion; however their effects on energy balance remain unclear. Here, we show that adult-onset conditional knockdown of Y2 receptors predominantly in peripheral tissues results in protection against diet-induced obesity accompanied by significantly reduced weight gain, marked reduction in adiposity and improvements in glucose tolerance without any adverse effect on lean mass or bone. These changes occur in association with significant increases in energy expenditure, respiratory exchange ratio, and physical activity and despite concurrent hyperphagia. On a chow diet, knockdown of peripheral Y2 receptors results in increased respiratory exchange ratio and physical activity with no effect on lean or bone mass, but decreases energy expenditure without effecting body weight or food intake. These results suggest that peripheral Y2 receptor signaling is critical in the regulation of oxidative fuel selection and physical activity and protects against the diet-induced obesity. The lack of effects on bone mass seen in this model further indicates that bone mass is primarily controlled by non-peripheral Y2 receptors. This study provides evidence that novel drugs that target peripheral rather than central Y2 receptors could provide benefits for the treatment of obesity and glucose intolerance without adverse effects on lean and bone mass, with the additional benefit of avoiding side effects often associated with pharmaceuticals that act on the central nervous system.     

Translocation of the pAntp peptide and its amphipathic analogue AP-2AL

The pAntp peptide, corresponding to the third helix of the homeodomain of the Antennapedia protein, enters by a receptor-independent process into eukaryotic cells. The interaction between the pAntp peptide and the phospholipid matrix of the plasma membrane seems to be the first step involved in the translocation mechanism. However, the mechanism by which the peptide translocates through the cell membrane is still not well established. We have investigated the translocation ability of pAntp through a protein-free phospholipid membrane in comparison with a more amphipathic analogue. We show by fluorescence spectroscopy, circular dichroism, NMR spectroscopy, and molecular modeling that pAntp is not sufficiently helically amphipathic to cross a phospholipid membrane of a model system. Due to its primary sequence related to its DNA binding ability in the Antennapedia homeodomain-DNA complex, the pAntp peptide does not belong to the amphipathic alpha-helical peptide family whose members are able to translocate by pore formation.     

Solution structure of a designed amphipathic antimicrobial synthetic peptide, PGAa

A designed peptide, PGAa showed an excellent antifungal activity as well as an efficient bactericidal activity toward gram-positive, especially in the pathogenic yeast Candida albicans 28838. The solution structures of PGAa have been determined both in 40% TFE/water solution and DPC micelle by CD and NMR spectroscopy. Based on NOEs, vicinal coupling constants, backbone amide exchange rates, and chemical shift indices, PGAa formed a long amphipathic alpha-helical conformation in both TFE and DPC micelle environments, spanning the residues Ile(2)-Ala(19) in TFE and Lys(5)-Ala(19) in DPC micelle, respectively. Solution structures suggested that the hydrophobic residues would interact with the fatty acyl chains of the lipid bilayer, while the positively charged side-chains exposed to aqueous environments. Therefore, we conclude that the alpha-helical structure as well as the highly amphiphatic nature of PGAa peptide may play a critical role in its antimicrobial activity as well as selectivities in different species.     

Hepatocyte-specific lysosomal acid lipase deficiency protects mice from diet-induced obesity but promotes hepatic inflammation

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa^−/−) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa^−/− mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa^−/− mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa^−/− mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.     

The growth suppressor p27(Kip1) protects against diet-induced atherosclerosis.

The molecular basis of atherosclerosis is associated with excessive proliferation of vascular cells. Previous studies have suggested an inverse correlation between the expression of the growth suppressor p27(Kip1) (p27) and cellular proliferation within human atherosclerotic tissue. However, no causal link between diminished p27 expression and atherogenesis has been established. We investigated the effect of p27 inactivation on diet-induced atherogenesis. We find that p27-deficient mice challenged with a high-fat diet for 1 month remain normocholesterolemic and have essentially no visible atheromas. However, when generated in an apolipoprotein E-null genetic background that leads to severe hypercholesterolemia in response to the atherogenic diet, deletion of p27 enhances arterial cell proliferation (approximately fourfold) and accelerates atherogenesis (approximately sixfold) compared with apolipoprotein E-deficient mice with an intact p27 gene. Analysis of apolipoprotein E-null mice bearing only one p27 allele inactivated reveals that a moderate decrease in p27 protein expression in the setting of hypercholesterolemia is sufficient to predispose to atherogenesis. Thus, our study establishes a molecular link between decreased p27 protein expression and atherogenesis in hypercholesterolemic animals.     

Quinoa extract enriched in 20-hydroxyecdysone protects mice from diet-induced obesity and modulates adipokines expression

Besides their well-known effect in the molting control in insects, ecdysteroids are steroid hormones that display potential pharmacologic and metabolic properties in mammals. The most common ecdysteroid, 20-hydroxyecdysone (20E) is found in many plants such as quinoa. The aim of the present study was to investigate the ability of quinoa extract (Q) enriched in 20E supplementation to prevent the onset of diet-induced obesity and to regulate the expression of adipocyte-specific genes in mice. Mice were fed a standard low-fat (LF) or a high-fat (HF) diet with or without supplementation by 20E-enriched Q or pure 20E for 3 weeks. Supplementation with Q reduced adipose tissue development in HF mice without modification of their body weight gain. This adipose tissue-specific effect was mainly associated with a reduced adipocyte size and a decrease in the expression of several genes involved in lipid storage, including lipoprotein lipase and phosphoenolpyruvate carboxykinase. Furthermore, Q-treated mice exhibited marked attenuation of mRNA levels of several inflammation markers (monocyte chemotactic protein-1, CD68) and insulin resistance (osteopontin, plasminogen activator inhibitor-1 (PAI-1)) as compared to HF mice. Q supplementation also reversed the effects of HF-induced downregulation of the uncoupling protein(s) (UCP(s)) mRNA levels in muscle. Similar results were obtained in mice fed a HF diet supplemented with similar amounts of pure 20E, suggesting that the latter accounted for most of the Q effects. Our study indicates that Q has an antiobesity activity in vivo and could be used as a nutritional supplement for the prevention and treatment of obesity and obesity-associated disorders.     

Overexpression of constitutively active PKG-I protects female, but not male mice from diet-induced obesity

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase I (PKG-I) is a multifunctional protein. The direct effects of PKG-I activation on energy homeostasis and obesity development are not well understood. Herein, we generated transgenic mice with expression of the constitutively active PKG-I in adipose tissue as well as in other tissues. Male and female PKG-I overexpressing mice were fed a low-fat (LF) or high-fat (HF) diet for 16 weeks. HF-fed female PKG-I transgenic mice had decreased body weight gain, lower percentage of body fat, and improved glucose tolerance compared to HF-fed wild-type (WT) controls. In contrast, male transgenic PKG-I mice were not resistant to the development of HF-diet-induced obesity, and exhibited similar levels of adiposity and glucose intolerance as HF-fed WT controls. Furthermore, we found that HF-fed female transgenic PKG-I mice had increased energy expenditure and cold-induced adaptive thermogenesis compared to HF-fed WT controls, which was associated with increased expression of uncoupling protein-1 (UCP1) in brown adipose tissue (BAT). In addition, the rates of lipolysis in white adipose tissue (WAT) were also increased in female transgenic PKG-I mice compared to WT controls due to increased phosphorylation of hormone-sensitive lipase (HSL). However, in male mice, adaptive thermogenesis or WAT lipolysis was similar between transgenic PKG-I mice and WT controls. Together, these data demonstrate sex differences in effects of PKG-I activation on the regulation of adipose tissue function and its contribution to diet induced obesity.     

Effect of end group blockage on the properties of a class A amphipathic helical peptide

In a recent classification of biologically active amphipathic α-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J. P., De Loof, H., Dohlman, J. G., Brouillette, C. G., Anantharamaiah, G. M. Proteins 8:103–117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the α-amino terminus and an amide at the α-carboxyl terminus, to obtain Ac-18A-NH₂, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH₂, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the α-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC–water interface is increased only by about 3% (from ?8.0 kcal/mol for 18A to ?8.3 kcal/mol for Ac-18A-NH₂). The Ac-18A-NH₂ has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH₂ more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH₂ peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH₂, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles. © 1993 Wiley-Liss, Inc.     

Solution NMR structure of a model class A (apolipoprotein) amphipathic alpha helical peptide

To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH(2) (acetyl-AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d(3))/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for C(beta)H protons (all the residues except Ala and Val) and gammaCH(3) (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic alpha helical structure of Ac-18A-NH(2) is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix.     

Liver Patt1 deficiency protects male mice from age-associated but not high-fat diet-induced hepatic steatosis

Patt1 is a newly identified protein acetyltransferase that is highly expressed in liver. However, the role of Patt1 in liver is still unclear. We generated Patt1 liver-specific knockout (LKO) mice and mainly measured the effect of hepatic Patt1 deficiency on lipid metabolism. Hepatic Patt1 deficiency in male mice markedly decreases fat mass and dramatically alleviates age-associated accumulation of lipid droplets in liver. Moreover, hepatic Patt1 abrogation in male mice significantly reduces the liver triglyceride and free fatty acid levels, but it has no effect on liver cholesterol level, liver weight, and liver function. Consistently, primary cultured Patt1-deficient hepatocytes are resistant to palmitic acid-induced lipid accumulation, but hepatic Patt1 deficiency fails to protect male mice from high-fat diet-induced hepatic steatosis. Further studies show that hepatic Patt1 deficiency decreases fatty acid uptake, reduces lipid synthesis, and enhances fatty acid oxidation, which may contribute to the attenuated hepatic steatosis in Patt1 LKO mice. These results demonstrate that Patt1 plays an important role in hepatic lipid metabolism and have implications toward resolving age-associated hepatic steatosis.     

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.     

A synthetic VIP peptide analogue inhibits neutrophil recruitment in rat airways in vivo

Currently, there is no effective pharmacotherapy against exaggerated mobilisation of neutrophils in human airway diseases such as chronic obstructive pulmonary disease and asthma. We evaluated the effect of two synthetic vasoactive intestinal peptide (VIP)-like analogues on cytokine-induced neutrophil recruitment in airways in vivo. Recombinant interleukin (IL)-1 beta was administered intratracheally (i.t.) to intubated, spontaneously breathing Sprague-Dawley rats. The rats were pretreated either with a VIP synthetic peptide analogue, a pituitary adenylate cyclase-activating peptide (PACAP)-1-27 synthetic analogue, the beta(2)-adrenoceptor agonist salbutamol or vehicle, systemically or locally. Differential cell counts were performed on bronchoalveolar lavage fluid (BALf) cytospins. Effects on mean arterial blood pressure (MAP) were monitored in separate experiments. Systemic administration of the VIP analogue, the PACAP analogue and salbutamol attenuated the cytokine-induced increase in BALf neutrophil number. Local administration of the VIP analogue and salbutamol, but not the PACAP analogue, also decreased the neutrophil number in BALf. Local administration of the VIP analogue and salbutamol caused a transient decrease in MAP. Systemic or local administration of a synthetic VIP peptide analogue inhibits cytokine-induced neutrophil recruitment in airways in vivo. This action is exerted without severe, sustained cardiovascular side effects, and deserves to be further evaluated in obstructive pulmonary diseases in human.     

Administration of a synthetic TLR4 agonist protects mice from pneumonic tularemia

Francisella tularensis is a Gram-negative intracellular pathogen that causes the zoonosis tularemia. Because F. tularensis LPS causes weak TLR4 activation, we hypothesized that administration of a synthetic TLR4 agonist, aminoalkyl glucosaminide phosphate (AGP), would boost the innate immune system and compensate for reduced TLR4 stimulation. Intranasal administration of AGPs induced intrapulmonary production of proinflammatory cytokines and chemokines. Mice treated with AGPs before and after inhalation of Francisella novicida exhibited augmented cytokine and inflammatory responses to infection; reduced bacterial replication in lung, liver, and spleen; and increased survival, whereas all PBS-treated control mice died within 4 days of infection, all AGP-treated mice showed prolonged time-to-death, and 30-60% of AGP-treated mice survived. The protective effect of AGP was lost in mice lacking IFN-gamma. Long-term survivors developed specific Th1 splenocyte responses and specific Abs dominated by IgG2 isotypes. Survivors were fully protected from rechallenge with aerosolized F. novicida. Thus, preventive administration of AGP successfully modulated innate immune responses to aerosolized F. novicida, leading to protective immunity to pneumonic tularemia. This is the first report of the protective effect of a TLR ligand on resistance to F. novicida-induced pneumonic tularemia.     

A new class of synthetic auxin transport inhibitors

Auxin transport inhibition by a new class of synthetic plant growth regulants, the 2-(3-aryl-5-pyrazolyl)benzoic acids, was examined in bean (Phaseolus vulgaris L.) using the donor-receiver agar cylinder technique. These compounds can be prepared by the dehydrogenation and ring cleavage of compounds like DPX-1840 (2-(4-methoxyphenyl)-3,3adihydro-8H-pyrazolo[5,1-a] isoindol-8-one) which was previously reported (Plant Physiol. 1972. 50: 322-327) to be a potent inhibitor of auxin transport. These new growth regulators inhibit auxin transport more than DPX-1840 does as evidenced by their consistently greater reduction of basipetal auxin transport capacity in bean when incorporated into the receiver agar cylinder or applied foliarly to intact plants. Direct comparisons of the effect of DPX-1840, its dehydrogenation product (2-(4-methoxyphenyl)-8H-pyrazolo [5,1-a]isoindol-8-one), and its open-ring form (2-(3-(4-methoxyphenyl)-5-pyrazolyl) benzoic acid) on auxin transport indicated the following order of activity: ring-open > dehydrogenated form > DPX-1840. DPX-1840-(14)C, applied at 0.5 mg/l to etiolated bean hypocotyl hooks followed by extraction and thin layer chromatography, indicated the biological conversion of DPX-1840 to its open-ring form. Collectively, these results suggest that the biologically active forms of DPX-1840-type compounds are the open-ring (2-(3-aryl-5-pyrazolyl) benzoic acids.     

IL-17A is proatherogenic in high-fat diet-induced and Chlamydia pneumoniae infection-accelerated atherosclerosis in mice

The role of IL-17 in atherogenesis remains controversial. We previously reported that the TLR/MyD88 signaling pathway plays an important role in high-fat diet as well as Chlamydophila pneumoniae infection-mediated acceleration of atherosclerosis in apolipoprotein E-deficient mice. In this study, we investigated the role of the IL-17A in high-fat diet (HFD)- and C. pneumoniae-induced acceleration of atherosclerosis. The aortic sinus plaque and aortic lesion size and lipid composition as well as macrophage accumulation in the lesions were significantly diminished in IL-17A(-/-) mice fed an HFD compared with wild-type (WT) C57BL/6 control mice. As expected, C. pneumoniae infection led to a significant increase in size and lipid content of the atherosclerotic lesions in WT mice. However, IL-17A(-/-) mice developed significantly less acceleration of lesion size following C. pneumoniae infection compared with WT control despite similar levels of blood cholesterol levels. Furthermore, C. pneumoniae infection in WT but not in IL-17A(-/-) mice was associated with significant increases in serum concentrations of IL-12p40, CCL2, IFN-γ, and numbers of macrophages in their plaques. Additionally, in vitro studies suggest that IL-17A activates vascular endothelial cells, which secrete cytokines that in turn enhance foam cell formation in macrophages. Taken together, our data suggest that IL-17A is proatherogenic and that it plays an important role in both diet-induced atherosclerotic lesion development, and C. pneumoniae infection-mediated acceleration of atherosclerotic lesions in the presence of HFD.