Testing novel apolipoprotein A-I mimetic peptides as a treatment of atherosclerosis
2017-02-06T05:50:53Z (GMT) by
Atherosclerosis is an inflammatory disease that stems from the accumulation of cholesterol in the artery wall. In the initial stages of atherosclerosis, low density lipoproteins (LDL) from the plasma enter and accumulate within the arterial wall and become modified via oxidation. This stimulates the recruitment of inflammatory cells and highlights the early stage of atherosclerosis. High density lipoprotein (HDL) has been shown to be highly atheroprotective by promoting the reverse cholesterol transport pathway (RCT). HDL facilitates the removal of cholesterol from peripheral cells to the liver and biliary excretion. It also has the capacity to inhibit the oxidative modification of LDL, downregulate the expression of adhesion molecules on endothelial cells, and elicit anti-apoptotic and anti-thrombotic properties. These pleiotropic effects of HDL are attributed to apolipoprotein A-I (apoA-I). Epidemiological studies have documented the strong association between low HDL levels and cardiovascular risk. Current therapeutic strategies, such as statins, are only able to reduce the incidence of cardiovascular disease by 30–40%. Even with this treatment, cardiovascular risk is still unacceptably high. Several studies have focused on reducing the levels of pro-atherogenic lipoproteins while increasing the levels of anti-atherogenic lipoproteins HDL, “HDL therapy”. ApoA-I mimetic peptides are a promising type of therapy as they share no sequence homology with apoA-I and can be manipulated to mimic the secondary structure of apoA-I. They can therefore support cholesterol efflux, enhance the anti-inflammatory properties of HDL and reduce the development of atherosclerosis in animal models. This thesis will investigate the optimal structural requirements for the peptides to elicit cholesterol efflux. Two peptides were used as prototypes: 5A and ELK. 5A is a bihelical peptide with two type A amphipathic α-helices connected via a proline residue. The hydrophobicity of the second helix was reduced with the substitution of alanine residues. Four derivatives of 5A were synthesized to test the impact of introducing antioxidant residues (cysteine and histidine) into the 5A prototype. ELK was synthesized using three amino acids: glutamic acid, leucine and lysine. This peptide is bihelical and has two identical Type A amphipathic α-helices with 180° hydrophobic face and neutral charge. The ELK prototype was used to make 16 modifications testing the impact of net charge, size of hydrophobic face, type of helix, proline bridge and asymmetry. The cholesterol efflux efficiency was tested in THP-1 monocytic cells and ATP binding cassette A1 (ABCA1)-dependent efflux specificity was tested in BHK/ABCA1 cells. The findings from this study established the structural features required to construct the “ideal” peptide. This peptide requires a neutral charge, optimal hydrophobicity, large hydrophobic face, two helices connected by a proline residue and it may have antioxidant residues in the second helix, to support cholesterol efflux. The significance of this study was to design apoA-I mimetic peptides for HDL therapy. HDL can inhibit the oxidative modification of LDL and in this study, apoA-I mimetic peptides were utilized to determine what structural features are required for anti-oxidant capacity. LDL oxidation was induced using copper, and the ability of apoA-I mimetic peptides to reduce the rate of oxidation was assessed. A peptide with strong anti-oxidant properties should possess the following structural features: presence of cysteine and/or histidine residues and the type of helix can be type G or type Y. The significance of this finding was to design an apoA-I mimetic with a strong anti-oxidant capacity. HDL can dampen the inflammatory response and therefore slow down the progression of atherosclerosis. This study investigated the optimal features required by apoA-I mimetic peptides to reduce CD11b expression on activated monocytes, as well as VCAM-1 expression on endothelial cells. The findings from this study suggested that different structural features are responsible for anti-inflammatory effects. The optimal structural features for CD11b inhibition in monocytes are an asymmetrical peptide with a type A helix, a hydrophobic face with <180° and a neutral or negative charge. In comparison, VCAM inhibition requires the following structural features: larger hydrophobic face, positive or neutral charge, and the resulting peptide can contain a cysteine residue. The differential effects on systemic and local anti-inflammatory functions were investigated using apoA-I mimetic peptides but provides an insight into the anti-inflammatory properties of HDL. ApoA-I elicits a plethora of anti-atherogenic functions, and this study attempted to create a single “super-peptide” which was strong in cholesterol efflux and anti-oxidant capacity. A mixture of the two most active peptides (ELK-2A2K2E and 5A-C1) was created and tested for its ability to surpass the anti-oxidant and efflux capacity of apoA-I. The mixture of these two peptides also surpassed the most active peptides in the model of local and systemic inflammation. The most active peptide in cholesterol efflux was tested in an animal model of atherosclerosis. This ELK-2A2K2E peptide had a significant affect on the reduction of circulating pro-atherogenic lipoproteins, which was reflected in reduced inflammatory markers and macrophage infiltration in plaque lesions. Based on the findings from these studies, apoA-I mimetic peptides could potentially be useful in the treatment and prevention of cardiovascular–related diseases.