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Mohamad Navab, Ph.D.An Oral ApoJ Peptide Renders HDL
Anti-inflammatory in Mice and Monkeys and Dramatically Reduces
Atherosclerosis in ApoE Null Mice
ApoA-I Peptide mimetics- An emerging strategy for CVD prevention is the development of apoA-I mimetic peptides. This strategy is based on the hypothesis that apoA-I mimetic peptides could be capable of stimulating the first step in the RCT pathway in a manner similar to native apoA-I, which is 243 amino acid residues in length. In comparison, the apoA-I mimetic peptide D-4F comprises only 18 amino acids.
-D-4F administered orally to mice led to the formation of pre-β HDL, increased cholesterol efflux from macrophages and enrichment of HDL with apoA-I and paraoxonase. -
18 AA peptideAc-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
D-4F
-D-4F administered orally to mice led to the formation of pre-β HDL, increased cholesterol efflux from macrophages and enrichment of HDL with apoA-I and paraoxonase.
- It markedly reduced lesion formation on LDL r-/- mice on Western diet and in apoE-/- mice
18 AA peptideAc-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2
D-4F
D-4F
Similarly, when added to human plasma at nanomolar concentrations in an in vitro system, D-4F caused:- the movement of apoA-I to smaller particles, - reduction of lipid hydroperoxides, -increase of paraoxonase activity and -conversion of proinflammatory HDL to anti-inflammatory HDL.
-D-4F in humans.
D-4F
Similarly, when added to human plasma at nanomolar concentrations in an in vitro system, D-4F caused:- the movement of apoA-I to smaller particles, - reduction of lipid hydroperoxides, -increase of paraoxonase activity and -conversion of proinflammatory HDL to anti-inflammatory HDL.
-D-4F in humans: Phase 1, a, b, c
Proposed Mechanism of Action of D-4F
LOOH & Ox-PAPC
and cholesterol Cholesterol Phospholipids
Pre- HDL
ABCA1
HDLApoA-I cycles
through pre- HDL
Chol
este
rol
ABCA
1
Phos
phol
ipid
sD-4F
Proposed Mechanism of Action of D-4F
LOOH & Ox-PAPC
and cholesterol
POVPC m/z 594.3
PGPC m/z 610.2
PEIPC m/z 828.6
CE-OOH
HPETEHPODE
MM-LDL
O
O
PAPC m/z 782.4
LDL
CH3(CH2 )14 CH2
Phosphorylcholine -
12-LO
Chol.18:2
CH2
HC
O
O -CH3(CH2 )14 CH2
Phosphorylcholine - CH2
HC
O
O -
CH3(CH2 )14 CH2
Phosphorylcholine CH2
HC
O
O
CH3(CH2 )14 CH2
Phosphorylcholine - CH2
HC
O
O -
O
O
OH
OO
O OH
O O
12-LOHPETEHPODEHPETEHPODE
O O
O
POVPC m/z 594.3
PGPC m/z 610.2
PEIPC m/z 828.6
Cholesterol Phospholipids
Pre- HDL
ABCA1
HDLApoA -I cycles through pre - HDL
Chol
este
rol
ABCA
1
Phos
phol
ipid
sD-4F
Proposed Mechanism of Action of D-4F
LOOH & Ox-PAPCand cholesterol
POVPC m/z 594.3
PGPC m/z 610.2
PEIPC m/z 828.6
CE-OOH
HPETEHPODE
MM-LDL
O
O
PAPC m/z 782.4
LDL
CH3(CH2 )14 CH2
Phosphorylcholine -
12-LO
Chol.18:2
CH2
HC
O
O -CH3(CH2 )14 CH2
Phosphorylcholine - CH2
HC
O
O -
CH3(CH2 )14 CH2
Phosphorylcholine CH2
HC
O
O
CH3(CH2 )14 CH2
Phosphorylcholine - CH2
HC
O
O -
O
O
OH
OO
O OH
O O
12-LOHPETEHPODEHPETEHPODE
O O
O
POVPC m/z 594.3
PGPC m/z 610.2
PEIPC m/z 828.6
Cholesterol Phospholipids
Pre- HDL
ABCA1
HDLApoA-I cycles
through pre- HDL
Chol
este
rol
ABCA
1
Phos
phol
ipid
sD-4F
Proposed Mechanism of Action of D-4FLOOH & Ox-PAPCinactivated and/or cleared and reverse cholesterol transport is also increased
LOOH & Ox-PAPCand cholesterol
POVPC m/z 594.3
PGPC m/z 610.2
PEIPC m/z 828.6
CE-OOH
HPETEHPODE
MM-LDL
O
O
PAPC m/z 782.4
LDL
CH3(CH2 )14 CH2
Phosphorylcholine -
12-LO
Chol.18:2
CH2
HC
O
O -CH3(CH2 )14 CH2
Phosphorylcholine - CH2
HC
O
O -
CH3(CH2 )14 CH2
Phosphorylcholine CH2
HC
O
O
CH3(CH2 )14 CH2
Phosphorylcholine - CH2
HC
O
O -
O
O
OH
OO
O OH
O O
12-LOHPETEHPODEHPETEHPODE
O O
O
POVPC m/z 594.3
PGPC m/z 610.2
PEIPC m/z 828.6
Cholesterol Phospholipids
Pre- HDL
ABCA1
HDLApoA-I cycles
through pre- HDL
Chol
este
rol
ABCA
1
Phos
phol
ipid
sD-4F
D-4F Plus Pravastatin Increased ApoA-I in both Alpha and Pre-beta HDL Particles
CHOW D-4F
PRAVASTATIN D-4F+PRAVASTATINPre- alpha
Lesion Prevention
0
50,000
100,000
150,000
200,000
250,000
300,000
m2 /
Sect
ion
CHOW D-4F PRAVASTATIND-4F+
PRAVASTATIN
A Comparison of 4F and [113-122]apoJ
Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 4F (18 aa)
Ac- L-V-G-R-Q-L-E-E-F-L-NH2 [113-122]apoJ (10aa)
D-[113-122]apoJ Increases Paraoxonase 1 Activity in ApoE Null Mice
PON Activity per unit
Cholesterol
0.00
0.50
1.00
1.50
2.00
2.50
3.00
CHOW HDL
D-[113-122]apoJ HDL
0.00
0.50
1.00
1.50
2.00
2.50
3.00
CHOW D-[113-122]apoJ HDL
p=0.0001
D-[113-122]apoJ Renders HDL Anti-
inflammatory in ApoE Null Mice
NoAddition
Cont.LDL
Cont. LDL +
HDL
0
2
4
6
8
Water
Mig
rate
dM
onoc
ytes
/Fie
ld
Autologous HDL+ LDL from treated ApoE Null Mice
*
Assay Controls
D [113-122] apoJNoAddition
Cont.LDL
Cont. LDL +
HDL
0
2
4
6
8
Assay Controls
- -
Comparison of D-4F and D-[113-122]apoJ
8 Br - cAMP-Induced Efflux (ABCA1-Mediated)
0
10
20
30
40
50
% E
fflux
HDL D-[113-122]apoJ
8 Br - -Induced Efflux (ABCA1-Mediated)
VehicleStd.
(5% plasma)
D-4FD-4F
D-[113-122]apoJ Decreases Lesions in ApoE Null Mice (Aortic Root Sinus)
0
100,000
200,000
300,000
400,000
500,000
CHOWn=21
CHOW+D-[113-122]apoJ
n=23
395893+63371
117685+107571
p=4.3x 10-13
70.2%
Aorti
c sin
us le
sion
area
CHOWn=21
CHOW+D-[113-122]apoJ
n=23
395893+63371
117685+107571
p=4.3x 10-13
m2/section
D-[113-122]apoJ Decreases Lesions in ApoE Null Mice (En Face)
0
1.0
2.0
3.0
4.0
CHOWn=27
CHOW +D-[113-122]apoJ
n=23
% S
URFA
CE A
REA
WIT
H LE
SION
S
1.7+/-0.8
0.5+/-0.5
70.5%
p=1.5x10-6
D-[113-122]apoJ Decreases Lipoprotein Lipid Hydroperoxides in Monkeys
0
100
200
300
400
Time 0 3 hrs
HDL
0
400
800
1200
Time 0 3 hrs
LDLp=0.001
ng L
OO
H p
er 5
0 g
cho
lest
erol
ng L
OO
H p
er 5
0 g
cho
lest
erol
p=0.001
0
100
200
300
400
0
100
200
300
400
Time 0 3 hrs
HDL
0
400
800
1200
0
400
800
1200
Time 0 3 hrs
LDL
g ch
oles
tero
l
g ch
oles
tero
l
D-[113-122]apoJ Renders HDL Anti-inflammatory in Monkeys
Mig
rate
d M
onoc
ytes
/Fie
ld
0
5
10
15
20
0
5
10
15
20
No Addition
LDL+ Monkey HDL at Time 0
Monkey # 1 2 3 4 1 2 3 4 Std. LDL
LDL+ Std. HDL
Assay ControlsLDL+ Monkey HDL at 3 hrs
P<0.001
A Comparison of ApoA-I, 4F, [113-122]apoJ, and Tetrapeptides
ApoA-I (243 aa)
Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 4F (18 aa)
Ac- L-V-G-R-Q-L-E-E-F-L-NH2 [113-122]apoJ (10aa)
Boc-K(eBoc)-R-E-S(tBu)-OtBu KRES (4aa)
Boc-F-R-E-L-OtBu FREL (4aa)
Making the Good Cholesterol Better TM
Oral Amphipathic Peptides with Anti-atherosclerotic Properties
•D-[113-122]apoJ is one of three classes of peptides that associate with HDL after an oral dose
•Peptides with a Class A amphipathic helix (D-4F)•Peptides with a G* amphipathic helix (D-[113-122]apoJ)•Tetrapeptides that are too small to form a helix (KRES; FREL)
Exploring the Differences in the Association
of the Peptides with HDL
•We hypothesized that given the differing temporal association of the peptids with HDL a combination of the three compounds ( D-4F and D-[113-122]apoJ and FREL) might extend the anti-inflammatory properties of HDL after a single oral dose.
HDL Inflammatory Properties 6 Hours After Dosing
0
5
10
15
20
25
F+J F+J
0.5 D- 4F
Mig
rate
d M
onoc
ytes
/Fie
ld
F+J 1.0 D- 4F
p<0.001p<0.001
LDL+HDLNo Addition
Std LDL
+Std HDL
24 Hours After Dosing
0
5
10
15
Mig
rate
d M
onoc
ytes
/Fie
ld
No Addition
Std LDL
+Std HDL F+J
F+J
0.5 D -4F
F+J
1.0 D - 4F
p<0.001p<0.001
LDL+HDL
48 Hours After Dosing
0
5
10
15
20
25
30
Mig
rate
d M
onoc
ytes
/Fie
ld p<0.001p<0.01
No Addition
Std LDL
+Std HDL F+J
F+J
0.5 D- 4F
F+J
1.0 D- 4F
LDL+HDL
72 Hours After Dosing
0
5
10
15
20
25
30
Mig
rate
d M
onoc
ytes
/Fie
ld
No + HDL + HDL + HDL+F+J + HDL + HDL+F+J
p<0.001 p<0.001
No Addition
Std LDL
+Std HDL F+J
F+J
0.5 D- 4F
F+J
1.0 D- 4F
D-4F versus D-[113-122]apoJ versus Tetrapeptides
Property D-4F 113-122 TetrapeptidesStimulatePre- HDL Yes No NoStim HDLChol Efflux Yes Yes NoStim. ReverseChol Transport Yes ND NoReduceLOOH Yes Yes YesIncrease PON Yes Yes YesImprove HDL Yes Yes YesInflammatoryProperties
D-4F versus D-[113-122]apoJ versus TetrapeptidesProperty D-4F 113-122 TetrapeptidesImproveLDL-MCA Yes Yes YesPrevent Lesions ~75% ~70% ~50%L-Active Orally No Not likely YesStatin Synergy Yes ND NDRegress Lesions Yes ND ND
Summary
•Oral amphipathic peptides from different classes associate with HDL differently but share certain common properties:
•Reduce HDL-lipid hydroperoxides•Increase HDL-anti-oxidant enzymes (e.g. Paraoxonase)•Render HDL anti-inflammatory
•These peptides provide unique tools for dissecting and understanding HDL metabolism and its relationship to the innate immune system.