ABSTRACT
Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (K(m)=2.1±0.3µg/mL vs. 0.59±0.06µg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug.
Subject(s)
Apolipoprotein A-I/metabolism , Atherosclerosis/metabolism , Cardiovascular Agents/metabolism , Coronary Disease/metabolism , Endothelial Cells/metabolism , Recombinant Proteins/metabolism , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/metabolism , Animals , Aorta/cytology , Aorta/drug effects , Aorta/metabolism , Apolipoprotein A-I/chemistry , Apolipoprotein A-I/pharmacology , Atherosclerosis/drug therapy , Atherosclerosis/pathology , Cardiovascular Agents/chemistry , Cardiovascular Agents/pharmacology , Cattle , Coronary Disease/drug therapy , Coronary Disease/pathology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Gene Silencing , Humans , Kinetics , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/metabolism , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Phosphatidylcholines/metabolism , Protein Binding , Protein Transport/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacologyABSTRACT
Here, we identify ADP-ribosylation factor (ARF)-like 7 (ARL7) as the only ARF- and ARL-family member whose mRNA-expression is induced by liver X-receptor/retinoid X-receptor agonists or cholesterol loading in human macrophages. Moreover, subcellular distribution of mutant and wild type ARL7-enhanced green fluorescent protein (EGFP) supports that ARL7 may be involved in a vesicular transport step between a perinuclear compartment and the plasma membrane. Therefore, we investigated the effect of ARL7 over-expression on the cholesterol secretory pathway. We found that expression of wild type and dominant active ARL7-EGFP stimulated the rate of apolipoprotein AI-specific cholesterol efflux 1.7- and 2.8-fold. In contrast, expression of the dominant negative form of ARL7-EGFP led to approximately 50% inhibition of cholesterol efflux. This data is consistent with a model in which ARL7 is involved in transport between a perinuclear compartment and the plasma membrane apparently linked to the ABCA1-mediated cholesterol secretion pathway.
