Objectives-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; PϽ0.0001) 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 (Pϭ0.019 for en face lesion area; Pϭ0.004 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; PϽ0.00004) with a 22% reduction in macrophage content in the remaining lesions (Pϭ0.001), indicating an overall reduction in macrophages of 79%. The combination increased intestinal apoA-I synthesis by 60% (Pϭ0.011). In monkeys, the combination also rendered HDL antiinflammatory. Key Words: atherosclerosis Ⅲ lipoproteins Ⅲ HDL Ⅲ apoA-I mimetic peptides Ⅲ statins S tatins and apolipoprotein (apo)A-I have similar antiinflammatory properties. 1 Ansell et al 2 reported that the inflammatory/antiinflammatory properties of high-density lipoprotein (HDL) identified patients with coronary heart disease (CHD) or CHD equivalents better than HDL-cholesterol levels. Treatment with 40 mg pravastatin daily for 6 weeks significantly improved the inflammatory/antiinflammatory properties of HDL in these patients. 2 However, even after treatment, 40% to 50% of the patients still had frankly pro-inflammatory HDL. 2 Moreover, after pravastatin only 4 of 26 patients achieved HDL that was antiinflammatory to the degree seen in 24 of 26 age-and gender-matched controls. 2 Conclusions-These
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
Recent evidence indicates that inflammation may significantly contribute to the pathogenesis of Alzheimer’s disease (AD). Since the apo A-I mimetic peptide D-4F has been shown to inhibit atherosclerotic lesion formation and regress already existing lesions (in the presence of pravastatin) and the peptide also decreases brain arteriole inflammation, we undertook a study to evaluate the efficacy of oral D-4F co-administered with pravastatin on cognitive function and amyloid β (Aβ) burden in the hippocampus of APPSwe-PS1ΔE9 mice. Three groups of male mice were administered D-4F and pravastatin, Scrambled D-4F (ScD-4F, a control peptide) and pravastatin in drinking water, while drinking water alone served as control. The escape latency in the Morris Water Maze test was significantly shorter for the D-4F+statin administered animals compared to the other two groups. While the hippocampal region of the brain was covered with 4.2±0.5 and 3.8±0.6% of Aβ load in the control and ScD-4F+statin administered groups, in the D-4F+statin administered group Aβ load was only 1.6±0.1%. Furthermore, there was a significant decrease in the number of activated microglia (p<0.05 vs the other two groups) and activated astrocytes (p<0.05 vs control) upon oral D-4F+statin treatment. Inflammatory markers TNFα and IL-1β levels were decreased significantly in the D-4F+statin group compared to the other two groups (for IL-1β p<0.01 vs the other two groups and for TNF-α p<0.001 vs control) and the expression of MCP-1 were also less in D-4F+statin administered group compared to the other two groups. These results suggest that the apo A-I mimetic peptide inhibits amyloid β deposition and improves cognitive function via exerting anti-inflammatory properties in the brain.
Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders.
Background-These studies were designed to determine whether the dual-domain peptide with a class A amphipathic helix linked to the receptor-binding domain of apolipoprotein (apo) E (Ac-hE-18A-NH 2 ) possesses both antidyslipidemic and antiinflammatory properties. Methods and Results-A single bolus (15 mg/kg IV) of Ac-hE-18A-NH 2 that contains LRKLRKRLLR (141-to 150-residue region of apo E) covalently linked to apo A-I mimetic peptide 18A not only reduced plasma cholesterol levels (baseline, 562Ϯ29.0 mg/dL versus 287.7Ϯ22.0 mg/dL at 18 hours, PϽ0.001) in the Watanabe heritable hyperlipidemic rabbit model but also significantly improved arterial endothelial function. This improvement was associated with a reduction in 2 markers of oxidative stress. First, the plasma lipid hydroperoxide content was reduced significantly, an effect associated with a 5-fold increase in HDL paraoxonase activity. Second, the formation of superoxide anion, a scavenger of nitric oxide, was also significantly reduced in arteries of these animals. Conclusions-Because dyslipidemia and endothelial dysfunction are common features of the atherosclerotic disease process, this unique dual-domain peptide has ideal composite properties that ameliorate key contributory factors to atherosclerosis. (Circulation. 2005;111:3112-3118.)
Apolipoprotein E (apoE) exerts prominent anti-inflammatory effects and undergoes recycling by target cells. We previously reported that the peptide, Ac-hE18A-NH2, composed of the receptor binding domain (LRKLRKRLLR) of apoE covalently linked to the Class A amphipathic peptide 18A, dramatically lowers plasma cholesterol and lipid hydroperoxides and enhances paraoxonase activity in dyslipidemic animal models. The objective of this study was to determine whether this peptide, analogous to apoE, exerts anti-inflammatory effects and undergoes recycling under in vitro conditions. Pulse chase studies using [125I]-Ac-hE18A-NH2 in THP-1 derived macrophages and HepG2 cells showed greater amounts of intact peptide in the cells at later time points indicating recycling of the peptide. Ac-hE18A-NH2 induced a 2.5 fold increase in preβ-HDL in the conditioned media of HepG2 cells. This effect persisted for three days after removal of the peptide from culture medium. Ac-hE18A-NH2 also induced the secretion of cell-surface apoE from THP-1 macrophages. In addition, the peptide increased cholesterol efflux from THP-1 cells by an ABCA-1 independent mechanism. Moreover, Ac-hE18A-NH2 inhibited LPS-induced vascular cell adhesion molecule-1 (VCAM-1) expression, and reduced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). It also reduced the secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) from THP-1 macrophages even when administered post-LPS and abolished the 18-fold increase in LPS-induced mRNA levels for MCP-1 in THP-1 cells. Taken together, these results suggest that addition of the putative apoE receptor-domain to the Class A amphipathic peptide, 18A results in a peptide that, similar to apoE, recycles, thus enabling the potentiation and prolongation of its anti-atherogenic and anti-inflammatory effects. Such a peptide has great potential as a therapeutic agent in the management of atherosclerosis and other inflammatory diseases.
Two homologous apoA-I mimetic peptides, 3F-2 and 3F 14 , differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404 -51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 g/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 ؎ 1000 m 2 , n ؍ 29), whereas 3F 14 does not (lesion area 20,400 ؎ 1000 m 2 , n ؍ 26) compared with control saline administered (19,900 ؎ 1400 m 2 , n ؍ 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F 14 preferentially associates with apoB-containing particles. After intraperitoneal injection of 14 C-labeled peptides, 3F 14 reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F 14 . In contrast, only 3F 14 affects the terminal methyl group of the acyl chain, decreasing the 2 H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F 14 can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.
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