Using adenovirus-mediated gene transfer to study the effect of myeloperoxidase on plasma lipid levels, HDL structure and functionality in mice expressing human apoA-I forms.

Apolipoprotein A-I (apoA-I), the main protein component of High-Density Lipoprotein (HDL), is modified in plasma and the arterial wall by various enzymes. Myeloperoxidase (MPO), a leukocyte-derived peroxidase, is highly expressed during inflammation and associates with HDL reducing its functionality and contributing to atherosclerosis. In the present study we sought to explore further the effect of MPO on HDL structure and functionality in vivo using adenovirus-mediated gene transfer of human MPO combined with human apoA-I forms containing substitutions at MPO-sensitive sites or wild type apoA-I. We found that overexpression of MPO in mice significantly increased plasma apoA-I and HDL levels without affecting the expression of genes involved in HDL biogenesis or catabolism in the liver. Overexpression of MPO in the liver reduced the expression of pro-inflammatory genes and increased or did not affect the expression of anti-inflammatory genes suggesting that MPO had no toxic effects in this organ. In the plasma of mice overexpressing MPO, no significant alterations in HDL size or electrophoretic mobility was observed with the exception of mice expressing apoA-I (M148A) which showed enriched pre-ß relative to α HDL particles, suggesting that the apoA-I (M148A) mutation may interfere with HDL remodelling. Overexpression of MPO was associated with reduced anti-oxidant capacity of HDL particles in all mice. Interestingly, HDL particles bearing apoA-I (Y192A) showed enhanced ABCA1-dependent cholesterol efflux from macrophages which was not affected by MPO and these mice had reduced levels of LDL-c. These findings provide new insights on the role of specific amino acid residues of apoA-I in HDL structure and function following modification by MPO. This knowledge may facilitate the development of novel therapies based on improved HDL forms for patients with chronic diseases that are characterized by dysfunctional HDL.

Reconstituted HDL-apoE3 promotes endothelial cell migration through ID1 and its downstream kinases ERK1/2, AKT and p38 MAPK.

INTRODUCTION: Atherosclerotic Coronary Artery Disease (ASCAD) is the leading cause of mortality worldwide. Novel therapeutic approaches aiming to improve the atheroprotective functions of High Density Lipoprotein (HDL) include the use of reconstituted HDL forms containing human apolipoprotein A-I (rHDL-apoA-I). Given the strong atheroprotective properties of apolipoprotein E3 (apoE3), rHDL-apoE3 may represent an attractive yet largely unexplored therapeutic agent. OBJECTIVE: To evaluate the atheroprotective potential of rHDL-apoE3 starting with the unbiased assessment of global transcriptome effects and focusing on endothelial cell (EC) migration as a critical process in re-endothelialization and atherosclerosis prevention. The cellular, molecular and functional effects of rHDL-apoE3 on EC migration-associated pathways were assessed, as well as the potential translatability of these findings in vivo. METHODS: Human Aortic ECs (HAEC) were treated with rHDL-apoE3 and total RNA was analyzed by whole genome microarrays. Expression and phosphorylation changes of key EC migration-associated molecules were validated by qRT-PCR and Western blot analysis in primary HAEC, Human Coronary Artery ECs (HCAEC) and the human EA.hy926 EC line. The capacity of rHDL-apoE3 to stimulate EC migration was assessed by wound healing and transwell migration assays. The contribution of MEK1/2, PI3K and the transcription factor ID1 in rHDL-apoE3-induced EC migration and activation of EC migration-related effectors was assessed using specific inhibitors (PD98059: MEK1/2, LY294002: PI3K) and siRNA-mediated gene silencing, respectively. The capacity of rHDL-apoE3 to improve vascular permeability and hypercholesterolemia in vivo was tested in a mouse model of hypercholesterolemia (apoE KO mice) using Evans Blue assays and lipid/lipoprotein analysis in the serum, respectively. RESULTS: rHDL-apoE3 induced significant expression changes in 198 genes of HAEC mainly involved in re-endothelialization and atherosclerosis-associated functions. The most pronounced effect was observed for EC migration, with 42/198 genes being involved in the following EC migration-related pathways: 1) MEK/ERK, 2) PI3K/AKT/eNOS-MMP2/9, 3) RHO-GTPases, 4) integrin. rHDL-apoE3 induced changes in 24 representative transcripts of these pathways in HAEC, increasing the expression of their key proteins PIK3CG, EFNB2, ID1 and FLT1 in HCAEC and EA.hy926 cells. In addition, rHDL-apoE3 stimulated migration of HCAEC and EA.hy926 cells, and the migration was markedly attenuated in the presence of PD98059 or LY294002. rHDL-apoE3 also increased the phosphorylation of ERK1/2, AKT, eNOS and p38 MAPK in these cells, while PD98059 and LY294002 inhibited rHDL-apoE3-induced phosphorylation of ERK1/2, AKT and p38 MAPK, respectively. LY had no effect on rHDL-apoE3-mediated eNOS phosphorylation. ID1 siRNA markedly decreased EA.hy926 cell migration by inhibiting rHDL-apoE3-triggered ERK1/2 and AKT phosphorylation. Finally, administration of a single dose of rHDL-apoE3 in apoE KO mice markedly improved vascular permeability as demonstrated by the reduced concentration of Evans Blue dye in tissues such as the stomach, the tongue and the urinary bladder and ameliorated hypercholesterolemia. CONCLUSIONS: rHDL-apoE3 significantly enhanced EC migration in vitro, predominantly via overexpression of ID1 and subsequent activation of MEK1/2 and PI3K, and their downstream targets ERK1/2, AKT and p38 MAPK, respectively, and improved vascular permeability in vivo. These novel insights into the rHDL-apoE3 functions suggest a potential clinical use to promote re-endothelialization and retard development of atherosclerosis.

Advances in biological therapies for dyslipidemias and atherosclerosis.

Atherosclerosis is a multifactorial disease influenced by genetics, lifestyle and environmental factors. Despite therapeutic advances that reduce the risk of cardiovascular events, atherosclerosis-related diseases remain the leading cause of mortality worldwide. Precise targeting of genes involved in lipoprotein metabolism is an emerging approach for atherosclerosis prevention and treatment. This article focuses on the latest developments, clinical potential and current challenges of monoclonal antibodies, vaccines and genome/transcriptome modification strategies, including antisense oligonucleotides, genome/base editing and gene therapy. Multiple lipid lowering biological therapies have already been approved by the FDA with impressive results to date, while many more promising targets are being pursued in clinical trials or pre-clinical animal models.

Transcriptome analysis of the adipose tissue in a mouse model of metabolic syndrome identifies gene signatures related to disease pathogenesis.

The white adipose tissue (WAT) contributes to the metabolic imbalance observed in obesity and the metabolic syndrome (MetS) by mechanisms that are poorly understood. The aim of this study was to monitor changes in the transcriptome of epididymal WAT during the development of MetS. ApoE3L.CETP mice were fed a high fat (HFD) or a low-fat (LFD) diet for different time periods. Adipose RNA was analyzed by microarrays. We found an increasing number of differentially expressed transcripts during MetS development. In mice with MetS, 1396 transcripts were differentially expressed including transcripts related to immune/inflammatory responses and extracellular matrix enzymes, suggesting significant inflammation and tissue remodeling. The top list of pathways included focal adhesion, chemokine, B and T cell receptor and MAPK signaling. The data identify for the first time adipose gene signatures in apoE3L.CETP mice with diet-induced MetS and might open new avenues for investigation of potential biomarkers or therapeutic targets.

Current and Emerging Reconstituted HDL-apoA-I and HDL-apoE Approaches to Treat Atherosclerosis.

Atherosclerosis affects millions of people worldwide. However, the wide variety of limitations in the current therapeutic options leaves much to be desired in future lipid-lowering therapies. For example, although statins, which are the first-line treatment for coronary heart disease (CHD), reduce the risk of cardiovascular events in a large percentage of patients, they lead to optimal levels of low density lipoprotein-cholesterol (LDL-C) in only about one-third of patients. A new promising research direction against atherosclerosis aims to improve lipoprotein metabolism. Novel therapeutic approaches are being developed to increase the levels of functional high density lipoprotein (HDL) particles. This review aims to highlight the atheroprotective potential of the in vitro synthesized reconstituted HDL particles containing apolipoprotein E (apoE) as their sole apolipoprotein component (rHDL-apoE). For this purpose, we provide: (1) a summary of the atheroprotective properties of native plasma HDL and its apolipoprotein components, apolipoprotein A-I (apoA-I) and apoE; (2) an overview of the anti-atherogenic functions of rHDL-apoA-I and apoA-I-containing HDL, i.e., natural HDL isolated from transgenic Apoa1-/- × Apoe-/- mice overexpressing human apoA-I (HDL-apoA-I); and (3) the latest developments and therapeutic potential of HDL-apoE and rHDL-apoE. Novel rHDL formulations containing apoE could possibly present enhanced biological functions, leading to improved therapeutic efficacy against atherosclerosis.