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Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro.

Hafiane A, Bielicki JK, Johansson JO, Genest J - PLoS ONE (2015)

Bottom Line: We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis.CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1.When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Laboratories Laboratory, Research Institute of the McGill University Health Centre, Montréal, Québec H4A 3J1, Canada.

ABSTRACT
Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic.

No MeSH data available.


Related in: MedlinePlus

Dynamics of transfer and redistribution of BHK-ABCA1 cell-derived cholesterol content of nHDL-CS-6253.nHDL-CS-6253 particles were labelled with cell derived 3[H]cholesterol and incubated with normolipidemic plasma for various time periods at 37°C (1μg peptide: 10μg plasma apo A-I). A. Characterization of lipid particles released to the medium in the presence of CS-6253. 3[H]-cholesterol-labelled BHK cells expressing ABCA1 or not were incubated in the presence of 0.96 μM apo A-I (solid line) or CS-6253 (dashed line) for 45min at 37°C. Concentrated medium from cells was analysed by FPLC, and radioactivity associated with each fraction was determined. B. Transfer of total 3[H]cholesterol from 3[H]nHDL-CS-6253 to plasma lipoproteins. nHDL-CS-6253 or nHDL-apo A-I were generated after incubation (4h) of BHK cells expressing ABCA1 with increased doses of lipid free CS-6253 or apo A-I. Media cell culture containing 3[H]-nHDL-particles was incubated in human plasma for 1h, 37°C. After ApoB precipitation, fractions were dialysed and counted for radioactivity. Kinetic parameters for cholesterol transfer to apoB particles in plasma: nHDL-CS-6253 (439±0.20μM), Vmax (439± 19.74%cpm/μl) vs nHDL-apo A-I (0.37 ± 0.035), Vmax (517±24.45).C. Effects of ex vivo incubation of CS-6253 on plasma cholesterol lipoproteins. FPLC cholesterol profiles (OD 490 nm) of plasma apo A-I: peptide ratio (1:1) (dashed line) and plasma apo A-I: peptide ratios (1:0; i.e. apo A-I alone) (solid line) are obtained after 5 min incubation at 37°C. In all experiment counts were made in triplicate, (*P<0.05 by Student‘s t-test). For reference purposes, the inset shows an FPLC profile of normal plasma. D. Effect of ex vivo incubation of CS-6253 on plasma lipoproteins after separation by ND-PAGGE (5–35%) electrophoresis. CS-6253 (0.96 μM final dose) was incubated for 1h at 37°C with HDL, LDL or VLDL isolated by ultracentrifugation or with human plasma and then separated by electrophoresis at concentration of 1 mg/protein/mL. Gel is followed by Western blot analysis with anti-CS-6253 antibody. Internal control was run on the left of the gel and detected by Ponceau S.
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pone.0131997.g006: Dynamics of transfer and redistribution of BHK-ABCA1 cell-derived cholesterol content of nHDL-CS-6253.nHDL-CS-6253 particles were labelled with cell derived 3[H]cholesterol and incubated with normolipidemic plasma for various time periods at 37°C (1μg peptide: 10μg plasma apo A-I). A. Characterization of lipid particles released to the medium in the presence of CS-6253. 3[H]-cholesterol-labelled BHK cells expressing ABCA1 or not were incubated in the presence of 0.96 μM apo A-I (solid line) or CS-6253 (dashed line) for 45min at 37°C. Concentrated medium from cells was analysed by FPLC, and radioactivity associated with each fraction was determined. B. Transfer of total 3[H]cholesterol from 3[H]nHDL-CS-6253 to plasma lipoproteins. nHDL-CS-6253 or nHDL-apo A-I were generated after incubation (4h) of BHK cells expressing ABCA1 with increased doses of lipid free CS-6253 or apo A-I. Media cell culture containing 3[H]-nHDL-particles was incubated in human plasma for 1h, 37°C. After ApoB precipitation, fractions were dialysed and counted for radioactivity. Kinetic parameters for cholesterol transfer to apoB particles in plasma: nHDL-CS-6253 (439±0.20μM), Vmax (439± 19.74%cpm/μl) vs nHDL-apo A-I (0.37 ± 0.035), Vmax (517±24.45).C. Effects of ex vivo incubation of CS-6253 on plasma cholesterol lipoproteins. FPLC cholesterol profiles (OD 490 nm) of plasma apo A-I: peptide ratio (1:1) (dashed line) and plasma apo A-I: peptide ratios (1:0; i.e. apo A-I alone) (solid line) are obtained after 5 min incubation at 37°C. In all experiment counts were made in triplicate, (*P<0.05 by Student‘s t-test). For reference purposes, the inset shows an FPLC profile of normal plasma. D. Effect of ex vivo incubation of CS-6253 on plasma lipoproteins after separation by ND-PAGGE (5–35%) electrophoresis. CS-6253 (0.96 μM final dose) was incubated for 1h at 37°C with HDL, LDL or VLDL isolated by ultracentrifugation or with human plasma and then separated by electrophoresis at concentration of 1 mg/protein/mL. Gel is followed by Western blot analysis with anti-CS-6253 antibody. Internal control was run on the left of the gel and detected by Ponceau S.

Mentions: To characterize cholesterol content in particles released from BHK cells expressing ABCA1 in media and plasma; lipoproteins profiles were determined by FPLC. After radiolabelling cells with 3[H]cholesterol for 24 hours, efflux medium (45 min) was collected, concentrated and analyzed. The elution profiles of 3[H]cholesterol are shown in (Fig 6A). In the presence of CS-6253, BHK cells produced a larger peak than apo A-I (fractions: 45–55) (Fig 6A), associated with an increase in the size of nHDL-CS-6253. nHDL-CS-6253 was confirmed by Western blot (data not shown) similar to nHDL-apo A-I [38].


Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro.

Hafiane A, Bielicki JK, Johansson JO, Genest J - PLoS ONE (2015)

Dynamics of transfer and redistribution of BHK-ABCA1 cell-derived cholesterol content of nHDL-CS-6253.nHDL-CS-6253 particles were labelled with cell derived 3[H]cholesterol and incubated with normolipidemic plasma for various time periods at 37°C (1μg peptide: 10μg plasma apo A-I). A. Characterization of lipid particles released to the medium in the presence of CS-6253. 3[H]-cholesterol-labelled BHK cells expressing ABCA1 or not were incubated in the presence of 0.96 μM apo A-I (solid line) or CS-6253 (dashed line) for 45min at 37°C. Concentrated medium from cells was analysed by FPLC, and radioactivity associated with each fraction was determined. B. Transfer of total 3[H]cholesterol from 3[H]nHDL-CS-6253 to plasma lipoproteins. nHDL-CS-6253 or nHDL-apo A-I were generated after incubation (4h) of BHK cells expressing ABCA1 with increased doses of lipid free CS-6253 or apo A-I. Media cell culture containing 3[H]-nHDL-particles was incubated in human plasma for 1h, 37°C. After ApoB precipitation, fractions were dialysed and counted for radioactivity. Kinetic parameters for cholesterol transfer to apoB particles in plasma: nHDL-CS-6253 (439±0.20μM), Vmax (439± 19.74%cpm/μl) vs nHDL-apo A-I (0.37 ± 0.035), Vmax (517±24.45).C. Effects of ex vivo incubation of CS-6253 on plasma cholesterol lipoproteins. FPLC cholesterol profiles (OD 490 nm) of plasma apo A-I: peptide ratio (1:1) (dashed line) and plasma apo A-I: peptide ratios (1:0; i.e. apo A-I alone) (solid line) are obtained after 5 min incubation at 37°C. In all experiment counts were made in triplicate, (*P<0.05 by Student‘s t-test). For reference purposes, the inset shows an FPLC profile of normal plasma. D. Effect of ex vivo incubation of CS-6253 on plasma lipoproteins after separation by ND-PAGGE (5–35%) electrophoresis. CS-6253 (0.96 μM final dose) was incubated for 1h at 37°C with HDL, LDL or VLDL isolated by ultracentrifugation or with human plasma and then separated by electrophoresis at concentration of 1 mg/protein/mL. Gel is followed by Western blot analysis with anti-CS-6253 antibody. Internal control was run on the left of the gel and detected by Ponceau S.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514675&req=5

pone.0131997.g006: Dynamics of transfer and redistribution of BHK-ABCA1 cell-derived cholesterol content of nHDL-CS-6253.nHDL-CS-6253 particles were labelled with cell derived 3[H]cholesterol and incubated with normolipidemic plasma for various time periods at 37°C (1μg peptide: 10μg plasma apo A-I). A. Characterization of lipid particles released to the medium in the presence of CS-6253. 3[H]-cholesterol-labelled BHK cells expressing ABCA1 or not were incubated in the presence of 0.96 μM apo A-I (solid line) or CS-6253 (dashed line) for 45min at 37°C. Concentrated medium from cells was analysed by FPLC, and radioactivity associated with each fraction was determined. B. Transfer of total 3[H]cholesterol from 3[H]nHDL-CS-6253 to plasma lipoproteins. nHDL-CS-6253 or nHDL-apo A-I were generated after incubation (4h) of BHK cells expressing ABCA1 with increased doses of lipid free CS-6253 or apo A-I. Media cell culture containing 3[H]-nHDL-particles was incubated in human plasma for 1h, 37°C. After ApoB precipitation, fractions were dialysed and counted for radioactivity. Kinetic parameters for cholesterol transfer to apoB particles in plasma: nHDL-CS-6253 (439±0.20μM), Vmax (439± 19.74%cpm/μl) vs nHDL-apo A-I (0.37 ± 0.035), Vmax (517±24.45).C. Effects of ex vivo incubation of CS-6253 on plasma cholesterol lipoproteins. FPLC cholesterol profiles (OD 490 nm) of plasma apo A-I: peptide ratio (1:1) (dashed line) and plasma apo A-I: peptide ratios (1:0; i.e. apo A-I alone) (solid line) are obtained after 5 min incubation at 37°C. In all experiment counts were made in triplicate, (*P<0.05 by Student‘s t-test). For reference purposes, the inset shows an FPLC profile of normal plasma. D. Effect of ex vivo incubation of CS-6253 on plasma lipoproteins after separation by ND-PAGGE (5–35%) electrophoresis. CS-6253 (0.96 μM final dose) was incubated for 1h at 37°C with HDL, LDL or VLDL isolated by ultracentrifugation or with human plasma and then separated by electrophoresis at concentration of 1 mg/protein/mL. Gel is followed by Western blot analysis with anti-CS-6253 antibody. Internal control was run on the left of the gel and detected by Ponceau S.
Mentions: To characterize cholesterol content in particles released from BHK cells expressing ABCA1 in media and plasma; lipoproteins profiles were determined by FPLC. After radiolabelling cells with 3[H]cholesterol for 24 hours, efflux medium (45 min) was collected, concentrated and analyzed. The elution profiles of 3[H]cholesterol are shown in (Fig 6A). In the presence of CS-6253, BHK cells produced a larger peak than apo A-I (fractions: 45–55) (Fig 6A), associated with an increase in the size of nHDL-CS-6253. nHDL-CS-6253 was confirmed by Western blot (data not shown) similar to nHDL-apo A-I [38].

Bottom Line: We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis.CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1.When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Laboratories Laboratory, Research Institute of the McGill University Health Centre, Montréal, Québec H4A 3J1, Canada.

ABSTRACT
Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic.

No MeSH data available.


Related in: MedlinePlus