Novel molecular imaging of atherosclerosis with gallium-68-labeled apolipoprotein A-I mimetic peptide and positron emission tomography.

BACKGROUND: High-density lipoprotein (HDL) plays a major role in reverse cholesterol transport. Many researchers have been working to enhance the biochemical function of HDL for use in therapy. Although HDL therapy using injections of apolipoprotein (apo)-A-I mimetics, apo A-I Milano or full-length apo A-I is dramatically effective, it is still unclear whether apo A-I or apo A-I mimetics actually enter atherosclerotic plaque and remove cholesterol from the lipid burden. We synthesized a novel 24-amino acid apo A-I mimetic peptide (known as FAMP) that potently removes cholesterol via specific ATP-binding cassette transporter A1. We then investigated the potential of FAMP to image developing plaque lesions in vivo. METHODS AND RESULTS: FAMP was modified with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with gallium-68 ((68)Ga) for noninvasive positron emission tomography (PET) in an animal model (familial hypercholesterolemic myocardial infarction-prone rabbits: WHHL-MI) with atherosclerotic lesions. The (68)Ga-DOTA-FAMP was dramatically taken up by atherosclerotic tissues in the blood vessels and aorta of WHHL-MI rabbits, but not the control rabbits. CONCLUSIONS: An apo A-I mimetic peptide with (68)Ga-DOTA is a promising candidate diagnostic tracer for PET imaging of the atherosclerotic lipid burden and may contribute to the development of a tool for the diagnosis of plaque with PET.

Structure-activity relationships of bacterial outer-membrane permeabilizers based on polymyxin B heptapeptides.

A series of cationic cyclic heptapeptides based on polymyxin B have been synthesized for use as permeabilizers of the outer membrane of Gram-negative bacteria. Only analogs with the Dab(2)-d-Phe(3)-Leu(4)-Xxx(5) sequence (Xxx = Dab or Orn) showed a synergistic bactericidal effect when combined with conventional antibiotics, indicating that the Dab(2) residue plays a critical role in permeation of the outer membrane of Gram-negative bacteria.

Structure-activity relationship of indolicidin, a Trp-rich antibacterial peptide.

A series of Trp and Arg analogs of antibacterial indolicidin (Ind) was synthesized and the antimicrobial and hemolytic activities were investigated. [L(9)]Ind, [L(11)]Ind, [K(8),L(9)]Ind and [K(6, 8),L(9)]Ind showed desirable characteristics, exhibiting negligible hemolytic activity while keeping strong antibacterial activity. The results indicated that the Trp residue at position 11 essentially contributes to both activities and one can not be exchanged for the other, whereas the Trp residues at positions 4 and 9 play important roles in antimicrobial and hemolytic activities, respectively. The Trp residues at positions 6 and 8 play no important roles in biological activities. We then found that the retro analog of Ind showed higher antibacterial activity than Ind against both Gram-positive and Gram-negative bacteria but remarkably lower hemolytic activity than that of Ind.

Histochemical characteristics of soleus muscle in angiotensin-converting enzyme gene knockout mice.

We examined the histochemical characteristics of soleus muscle in the angiotensin-converting enzyme (ACE) gene (Ace in mice, ACE in humans) knockout mice. Serial sections of soleus muscle of wild-type (Ace+/+, n=20) and heterozygous mutant (Ace+/-, n=24) mice were stained for myosin adenosine triphosphatase activity to identify different muscle fiber types. Capillaries were visualized by amylase-periodic acid-Schiff staining. ACE activity in the serum and gastrocnemius muscle was higher in male mice than in female mice. Female and male Ace+/- mice had markedly lower ACE activity in the serum and the gastrocnemius muscle than did female and male Ace+/+ mice, respectively. In both male and female mice, the composition of fiber types (type I and IIa) did not differ significantly between Ace+/+ and Ace+/- mice. There was no significant gender difference in capillary density. Ace+/- mice had significantly more capillaries around type IIa fibers (5.44 +/- 0.18 vs. 5.01 +/- 0.13, p<0.05) than Ace+/+ mice. The differences in the number of capillaries around type I fibers and in the number of capillaries around per fiber (capillary:fiber ratio) between Ace+/- and Ace+/+ mice were not significant (p<0.1). There was no significant difference in the mean cross-sectional area occupied by one capillary and the number of capillaries per fiber area between Ace+/+ and Ace+/- mice. In conclusion, knockout of the Ace gene in mice increased capillary density, as expressed by the mean number of capillaries around type IIa fibers. This finding suggests a possible mechanism for the cardioprotective effects of ACE inhibitors.

Extra amino group-containing gramicidin S analogs possessing outer membrane-permeabilizing activity.

Novel (2S,4R)- and (2S,4S)-4-aminoproline residue-containing analogs of the cyclic decapeptide antibiotic gramicidin S were synthesized, which exhibited marked permeabilizing activity on the outer membrane of gram-negative bacteria.

Improved survival rate after myocardial infarction using an inducible cholesterol efflux (iCE) peptide: FAMP.

BACKGROUND: There have been no previous reports that apolipoprotein (apo) A-I mimetic peptide improves survival rate after myocardial infarction (MI). METHOD AND RESULTS: Male C57Bl/6J mice were subjected to left coronary artery permanent ligation as a model of MI. We synthesized a novel 24-amino acid apoA-I mimetic peptide-type5 (FAMP5), which potently removes cholesterol via specific ATP-binding cassette transporter A1 (ABCA1). FAMP5 was associated with a significantly improved survival rate by protecting against cardiac rupture compared to the control. mRNA levels for eNOS, Gata-4, CTGF and ANP were significantly increased in the hearts of the FAMP5-treated group, while that for MCP-1 decreased. CONCLUSION: This is the first report that high-density lipoprotein (HDL) therapy with FAMP5 improved the survival rate after MI.

A water-soluble selenoxide reagent as a useful probe for the reactivity and folding of polythiol peptides.

A water-soluble selenoxide (DHS(ox)) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHS(ox) would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHS(ox) was extensively studied at pH 4-10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHS(ox) (375 mV at 25 °C) compared to l-cystine supported the high ability of DHS(ox) for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pK a value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTT(red)) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains.

FAMP, a novel apoA-I mimetic peptide, suppresses aortic plaque formation through promotion of biological HDL function in ApoE-deficient mice.

BACKGROUND: Apolipoprotein (apo) A-I is a major high-density lipoprotein (HDL) protein that causes cholesterol efflux from peripheral cells through the ATP-binding cassette transporter A1 (ABCA1), thus generating HDL and reversing the macrophage foam cell phenotype. Pre-ß1 HDL is the smallest subfraction of HDL, which is believed to represent newly formed HDL, and it is the most active acceptor of free cholesterol. Furthermore it has a possible protective function against cardiovascular disease (CVD). We developed a novel apoA-I mimetic peptide without phospholipids (Fukuoka University ApoA-I Mimetic Peptide, FAMP). METHODS AND RESULTS: FAMP type 5 (FAMP5) had a high capacity for cholesterol efflux from A172 cells and mouse and human macrophages in vitro, and the efflux was mainly dependent on ABCA1 transporter. Incubation of FAMP5 with human HDL or whole plasma generated small HDL particles, and charged apoA-I-rich particles migrated as pre-ß HDL on agarose gel electrophoresis. Sixteen weeks of treatment with FAMP5 significantly suppressed aortic plaque formation (scrambled FAMP, 31.3 ± 8.9% versus high-dose FAMP5, 16.2 ± 5.0%; P<0.01) and plasma C-reactive protein and monocyte chemoattractant protein-1 in apoE-deficient mice fed a high-fat diet. In addition, it significantly enhanced HDL-mediated cholesterol efflux capacity from the mice. CONCLUSIONS: A newly developed apoA-I mimetic peptide, FAMP, has an antiatherosclerotic effect through the enhancement of the biological function of HDL. FAMP may have significant atheroprotective potential and prove to be a new therapeutic tool for CVD.