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 g of D-4F was given orally to apoE-null mice, small cholesterolcontaining particles (CCPs) of 7 to 8 nm with pre-␤ mobility and enriched in apoA-I and paraoxonase activity were found in plasma.

Abstract-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-␤ high-density lipoprotein (HDL), improved HDL-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from proinflammatory 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-␤ 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.

Treatment of cells with the macrolide antibiotic bafilomycin A1, an inhibitor of vacuolar (V)-ATPase, or with the lysosomotropic agent chloroquine, has been shown to pharmacologically inhibit autophagy as evidenced by an accumulation of autophagosomes, which in turn causes Bax-dependent apoptosis. However, bafilomycin A1 has also been reported to inhibit chloroquine-induced apoptosis, suggesting a complex interrelationship between these two inhibitors of autophagy. To determine whether the cytoprotective effect of bafilomycin A1 on chloroquine-treated cells was dependent on inhibition of VATPase, we examined the single and combined effects of bafilomycin and chloroquine on cultured cerebellar granule neurons. When added separately, chloroquine or high concentrations of bafilomycin A1 (Ն10 nM) induced a dose-dependent inhibition of autophagy (as measured by an increase in LC3-II, a marker specific for autophagosomes), followed by caspase-3 activation and cell death. When added in combination, bafilomycin A1 potently inhibited chloroquine-induced caspase-3 activity and cell death at concentrations (Յ1 nM) that neither altered vacuolar acidification nor inhibited autophagy. The neuroprotective effects of bafilomycin A1 against chloroquine were substantially greater than those produced by Bax deficiency. Bafilomycin A1-induced neuroprotection seemed to be stimulus-specific, in that staurosporine-induced death was not attenuated by coaddition of bafilomycin A1. Together, these data suggest that in addition to promoting death via inhibition of V-ATPase and autophagy, bafilomycin A1 possesses novel, neuroprotective properties that inhibit Bax-dependent activation of the intrinsic apoptotic pathway resulting from the pharmacological inhibition of autophagy.

Abstract-Previous studies suggest that high-density lipoprotein and apoAI inhibit lipopolysaccharide (LPS)-induced inflammatory responses. The goal of the current study was to test the hypothesis that the apoAI mimetic peptide L-4F exerts antiinflammatory effects similar to apoAI. Pretreatment of human umbilical vein endothelial cells (HUVECs) with LPS induced the adhesion of THP-1 monocytes. Incubation of cells with LPS and L-4F (1 to 50 g/mL) reduced THP-1 adhesion in a concentration-dependent manner. This response was associated with a significant reduction in the synthesis of cytokines, chemokines, and adhesion molecules. L-4F reduced vascular cell adhesion molecule-1 expression induced by LPS or lipid A, whereas a control peptide (Sc-4F) showed no effect. In contrast to LPS treatment, L-4F did not inhibit IL-1␤-or tumor necrosis factor-␣-induced vascular cell adhesion molecule-1 expression. 1 Approximately 50% of patients in intensive care units develop severe sepsis, and the overall mortality rate of all affected patients is 29%. 1 Mortality is attributable, in large part, to the cytotoxic actions of lipopolysaccharide (LPS) (endotoxin), a component of the outer membrane of Gram-negative bacteria. LPS is composed of a core oligosaccharide, a repeating polysaccharide side chain, and the glycolipid moiety lipid A. 2 Proinflammatory and cytotoxic effects of LPS are mediated by lipid A. 3 LPS is released from bacterial membranes into the circulation where it interacts with lipopolysaccharide binding protein (LBP), a member of the superfamily of phospholipid binding proteins. LBP binds to lipid A and mediates the disaggregation of LPS to form an LBP-LPS complex. 4 LBP directs LPS to membrane-associated CD14 receptors (mCD14) on myeloid cells 5 including monocytes and neutrophils. mCD14 is a cell surface-anchored protein that facilitates the binding of LPS and activation of Toll-like receptor (TLR) 4 which acts as the cellular transducer of LPS action. 2,6 Plasma LPS-LBP may also interact with soluble CD14 to form a complex that activates TLRs on endothelial, epithelial, Kuppfer, and other cells. 7 By activating nuclear factor B-dependent signaling mechanisms, LPS stimulates the synthesis/release of inflammatory cytokines, which play an important role in the innate immune response. 8,9 Dysregulation of this response leads to the development of endothelial dysfunction, intravascular coagulation, pulmonary injury, multiple organ failure, and death.The acute-phase response to bacterial infection induces changes in plasma lipoprotein levels that are characterized by Original