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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

Effect of CS-6253 treatment on cholesterol efflux.A. Time-course activity of ABCA1-mediated cholesterol efflux to lipid-free CS-6253 (30 μg/ml) and apo A-I (10 μg/ml), using BHK cells expressing ABCA1, and control cells (unstimulated BHK-ABCA1 and BHK-mock cells). Cholesterol efflux from either ABCA1 cells or control cells to apo A-I (closed circles), and CS-6253 (closed triangles) was determined at the indicated time points. B. Dose dependent ABCA1 mediated cholesterol efflux in BHK-ABCA1 induced with mifepristone. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK-ABCA1 cells to apo A-I: Km = 4.53±0.67 μg/ml (0.15±0.02 μM), Vmax = 14.85±0.02% efflux/4h, and relative catalytic efficiency: Vmax/Km = 3.27. CS-6253: Km = 2.27±0.16 μg/ml (0.73±0.05 μM), Vmax = 15.25±0.05% efflux/4h, and relative catalytic efficiency: Vmax/Km = 6.71. ATI-5261: Km = 1.04 ± 0.16 μg/ml (0.37 ± 0.04 μM), Vmax = 14.48 ± 0.29% efflux/ h, and relative catalytic efficiency: Vmax/Km = 13.92. Apo E: Km = 10.30±2.84 μg/ml (0.21±0.05 μM), Vmax = 11.45±1.16% efflux/4h, and relative catalytic efficiency: Vmax/Km = 1.11. C. Ability of CS-6253 to stimulate cholesterol efflux from human macrophages THP-1. Foam cells were incubated with CS-6253 or apoA-I at equimolar ratio (0.96 μM) for 4h. Cholesterol efflux induced by CS-6253 and apoA-I were compared to control cells incubated alone. P<0.001 by Student’s t-test. D. Membrane ABCA1 levels assessment by Western blotting assays. ABCA1 was detected from THP-1 foam cells lysis at 4°C with lysis buffer containing 0.5% n-dodecylmaltoside in the presence of a protease inhibitor mixture followed by low speed centrifugation to remove cell debris. Protein concentration was determined by standard assay (Bio-Rad). The supernatants were then separated by SDS-PAGE (4–22.5%) in duplicate. After electrophoresis, ABCA1 was detected by an anti-ABCA1 antibody. GAPDH was used as loading control. E. Quantification of FC and CE in foam cells after interaction with CS-6253 and apoA-I. After a period of 4h cholesterol efflux, THP-1 foam cell lipids were extracted with hexane: isopropanol (3v/2v) as under ‘‘Material and Methods’. 3[H]-FC and 3[H]-CE were separated by TLC and located by exposure to iodine vapor, and were scraped off into liquid scintillating vials and assayed for radioactivity. Results are from a single experiment using triplicate wells and the mean (±SD) is presented. P<0.0001 versus control sample untreated THP-1 cells for cholesterol efflux. P<0.05 versus FC or CE in untreated cells, by Student’s t-test.
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pone.0131997.g002: Effect of CS-6253 treatment on cholesterol efflux.A. Time-course activity of ABCA1-mediated cholesterol efflux to lipid-free CS-6253 (30 μg/ml) and apo A-I (10 μg/ml), using BHK cells expressing ABCA1, and control cells (unstimulated BHK-ABCA1 and BHK-mock cells). Cholesterol efflux from either ABCA1 cells or control cells to apo A-I (closed circles), and CS-6253 (closed triangles) was determined at the indicated time points. B. Dose dependent ABCA1 mediated cholesterol efflux in BHK-ABCA1 induced with mifepristone. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK-ABCA1 cells to apo A-I: Km = 4.53±0.67 μg/ml (0.15±0.02 μM), Vmax = 14.85±0.02% efflux/4h, and relative catalytic efficiency: Vmax/Km = 3.27. CS-6253: Km = 2.27±0.16 μg/ml (0.73±0.05 μM), Vmax = 15.25±0.05% efflux/4h, and relative catalytic efficiency: Vmax/Km = 6.71. ATI-5261: Km = 1.04 ± 0.16 μg/ml (0.37 ± 0.04 μM), Vmax = 14.48 ± 0.29% efflux/ h, and relative catalytic efficiency: Vmax/Km = 13.92. Apo E: Km = 10.30±2.84 μg/ml (0.21±0.05 μM), Vmax = 11.45±1.16% efflux/4h, and relative catalytic efficiency: Vmax/Km = 1.11. C. Ability of CS-6253 to stimulate cholesterol efflux from human macrophages THP-1. Foam cells were incubated with CS-6253 or apoA-I at equimolar ratio (0.96 μM) for 4h. Cholesterol efflux induced by CS-6253 and apoA-I were compared to control cells incubated alone. P<0.001 by Student’s t-test. D. Membrane ABCA1 levels assessment by Western blotting assays. ABCA1 was detected from THP-1 foam cells lysis at 4°C with lysis buffer containing 0.5% n-dodecylmaltoside in the presence of a protease inhibitor mixture followed by low speed centrifugation to remove cell debris. Protein concentration was determined by standard assay (Bio-Rad). The supernatants were then separated by SDS-PAGE (4–22.5%) in duplicate. After electrophoresis, ABCA1 was detected by an anti-ABCA1 antibody. GAPDH was used as loading control. E. Quantification of FC and CE in foam cells after interaction with CS-6253 and apoA-I. After a period of 4h cholesterol efflux, THP-1 foam cell lipids were extracted with hexane: isopropanol (3v/2v) as under ‘‘Material and Methods’. 3[H]-FC and 3[H]-CE were separated by TLC and located by exposure to iodine vapor, and were scraped off into liquid scintillating vials and assayed for radioactivity. Results are from a single experiment using triplicate wells and the mean (±SD) is presented. P<0.0001 versus control sample untreated THP-1 cells for cholesterol efflux. P<0.05 versus FC or CE in untreated cells, by Student’s t-test.

Mentions: Previous studies indicated that apolipoprotein peptides can act as ABCA1-dependent acceptors for cellular cholesterol efflux [7–9, 32]. At high concentrations, other peptides displayed detergent-like properties and could extract cholesterol from cells independently of ABCA1 [33]. Notably, Remaley et al. demonstrated that apolipoprotein peptides composed of D-amino acids (10μg/ml) can extract cholesterol in ABCA1-dependent and independent fashions [8]. This suggests that these peptides may cause non-specific side effects compared to apo A-I [34]. To address this issue we tested our peptide at a concentration of 30μg/ml (9.69 μM) in a time- dependent manner to ensure that cholesterol efflux was ABCA1-dependent (Fig 2A). Mock-transfected BHK cells and non-stimulated BHK-ABCA1 cells were used as controls. CS-6253-mediated cholesterol efflux reached saturation rapidly after ~ 6 h incubation period. In contrast, apo A-I-mediated cholesterol efflux continues to increase (Fig 2A). There was no significant cholesterol efflux to CS-6253 from control cells similarly to apo A-I.


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)

Effect of CS-6253 treatment on cholesterol efflux.A. Time-course activity of ABCA1-mediated cholesterol efflux to lipid-free CS-6253 (30 μg/ml) and apo A-I (10 μg/ml), using BHK cells expressing ABCA1, and control cells (unstimulated BHK-ABCA1 and BHK-mock cells). Cholesterol efflux from either ABCA1 cells or control cells to apo A-I (closed circles), and CS-6253 (closed triangles) was determined at the indicated time points. B. Dose dependent ABCA1 mediated cholesterol efflux in BHK-ABCA1 induced with mifepristone. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK-ABCA1 cells to apo A-I: Km = 4.53±0.67 μg/ml (0.15±0.02 μM), Vmax = 14.85±0.02% efflux/4h, and relative catalytic efficiency: Vmax/Km = 3.27. CS-6253: Km = 2.27±0.16 μg/ml (0.73±0.05 μM), Vmax = 15.25±0.05% efflux/4h, and relative catalytic efficiency: Vmax/Km = 6.71. ATI-5261: Km = 1.04 ± 0.16 μg/ml (0.37 ± 0.04 μM), Vmax = 14.48 ± 0.29% efflux/ h, and relative catalytic efficiency: Vmax/Km = 13.92. Apo E: Km = 10.30±2.84 μg/ml (0.21±0.05 μM), Vmax = 11.45±1.16% efflux/4h, and relative catalytic efficiency: Vmax/Km = 1.11. C. Ability of CS-6253 to stimulate cholesterol efflux from human macrophages THP-1. Foam cells were incubated with CS-6253 or apoA-I at equimolar ratio (0.96 μM) for 4h. Cholesterol efflux induced by CS-6253 and apoA-I were compared to control cells incubated alone. P<0.001 by Student’s t-test. D. Membrane ABCA1 levels assessment by Western blotting assays. ABCA1 was detected from THP-1 foam cells lysis at 4°C with lysis buffer containing 0.5% n-dodecylmaltoside in the presence of a protease inhibitor mixture followed by low speed centrifugation to remove cell debris. Protein concentration was determined by standard assay (Bio-Rad). The supernatants were then separated by SDS-PAGE (4–22.5%) in duplicate. After electrophoresis, ABCA1 was detected by an anti-ABCA1 antibody. GAPDH was used as loading control. E. Quantification of FC and CE in foam cells after interaction with CS-6253 and apoA-I. After a period of 4h cholesterol efflux, THP-1 foam cell lipids were extracted with hexane: isopropanol (3v/2v) as under ‘‘Material and Methods’. 3[H]-FC and 3[H]-CE were separated by TLC and located by exposure to iodine vapor, and were scraped off into liquid scintillating vials and assayed for radioactivity. Results are from a single experiment using triplicate wells and the mean (±SD) is presented. P<0.0001 versus control sample untreated THP-1 cells for cholesterol efflux. P<0.05 versus FC or CE in untreated cells, by Student’s t-test.
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Related In: Results  -  Collection

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pone.0131997.g002: Effect of CS-6253 treatment on cholesterol efflux.A. Time-course activity of ABCA1-mediated cholesterol efflux to lipid-free CS-6253 (30 μg/ml) and apo A-I (10 μg/ml), using BHK cells expressing ABCA1, and control cells (unstimulated BHK-ABCA1 and BHK-mock cells). Cholesterol efflux from either ABCA1 cells or control cells to apo A-I (closed circles), and CS-6253 (closed triangles) was determined at the indicated time points. B. Dose dependent ABCA1 mediated cholesterol efflux in BHK-ABCA1 induced with mifepristone. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK-ABCA1 cells to apo A-I: Km = 4.53±0.67 μg/ml (0.15±0.02 μM), Vmax = 14.85±0.02% efflux/4h, and relative catalytic efficiency: Vmax/Km = 3.27. CS-6253: Km = 2.27±0.16 μg/ml (0.73±0.05 μM), Vmax = 15.25±0.05% efflux/4h, and relative catalytic efficiency: Vmax/Km = 6.71. ATI-5261: Km = 1.04 ± 0.16 μg/ml (0.37 ± 0.04 μM), Vmax = 14.48 ± 0.29% efflux/ h, and relative catalytic efficiency: Vmax/Km = 13.92. Apo E: Km = 10.30±2.84 μg/ml (0.21±0.05 μM), Vmax = 11.45±1.16% efflux/4h, and relative catalytic efficiency: Vmax/Km = 1.11. C. Ability of CS-6253 to stimulate cholesterol efflux from human macrophages THP-1. Foam cells were incubated with CS-6253 or apoA-I at equimolar ratio (0.96 μM) for 4h. Cholesterol efflux induced by CS-6253 and apoA-I were compared to control cells incubated alone. P<0.001 by Student’s t-test. D. Membrane ABCA1 levels assessment by Western blotting assays. ABCA1 was detected from THP-1 foam cells lysis at 4°C with lysis buffer containing 0.5% n-dodecylmaltoside in the presence of a protease inhibitor mixture followed by low speed centrifugation to remove cell debris. Protein concentration was determined by standard assay (Bio-Rad). The supernatants were then separated by SDS-PAGE (4–22.5%) in duplicate. After electrophoresis, ABCA1 was detected by an anti-ABCA1 antibody. GAPDH was used as loading control. E. Quantification of FC and CE in foam cells after interaction with CS-6253 and apoA-I. After a period of 4h cholesterol efflux, THP-1 foam cell lipids were extracted with hexane: isopropanol (3v/2v) as under ‘‘Material and Methods’. 3[H]-FC and 3[H]-CE were separated by TLC and located by exposure to iodine vapor, and were scraped off into liquid scintillating vials and assayed for radioactivity. Results are from a single experiment using triplicate wells and the mean (±SD) is presented. P<0.0001 versus control sample untreated THP-1 cells for cholesterol efflux. P<0.05 versus FC or CE in untreated cells, by Student’s t-test.
Mentions: Previous studies indicated that apolipoprotein peptides can act as ABCA1-dependent acceptors for cellular cholesterol efflux [7–9, 32]. At high concentrations, other peptides displayed detergent-like properties and could extract cholesterol from cells independently of ABCA1 [33]. Notably, Remaley et al. demonstrated that apolipoprotein peptides composed of D-amino acids (10μg/ml) can extract cholesterol in ABCA1-dependent and independent fashions [8]. This suggests that these peptides may cause non-specific side effects compared to apo A-I [34]. To address this issue we tested our peptide at a concentration of 30μg/ml (9.69 μM) in a time- dependent manner to ensure that cholesterol efflux was ABCA1-dependent (Fig 2A). Mock-transfected BHK cells and non-stimulated BHK-ABCA1 cells were used as controls. CS-6253-mediated cholesterol efflux reached saturation rapidly after ~ 6 h incubation period. In contrast, apo A-I-mediated cholesterol efflux continues to increase (Fig 2A). There was no significant cholesterol efflux to CS-6253 from control cells similarly to apo A-I.

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