RPE cell survival (% of DMSO-A2E) 100 · INVOLVEMENT OF PEROXISOME PROLIFERATOR ACTIVATOR RECEPTORS...
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INVOLVEMENT OF PEROXISOME PROLIFERATOR ACTIVATOR RECEPTORS (PPARs) IN THE PHOTOPROTECTIVE ACTIVITY OF BIO201 Introduction According to Herzlich et al. (PPAR Research, 2008, article ID389507), PPARs play a key role in the protection of RPE cells against AMD. There is increasing evidence for the involvement of PPARs in the protection against the development of Age-Related Macular Degeneration (AMD). PPAR receptors comprise three sub-types of nuclear receptors (PPARα, PPARβ/δ and PPARγ) involved in the control of lipid and carbohydrate metabolism, as well as inflammatory processes. They are characterized by a large ligand-binding pocket and therefore accommodate diverse types of endogenous or exogenous ligands. BIO201 is a pharmaceutical grade preparation of the di-apo-carotenoid norbixin. It has been formulated for improving its stability and per os bioavailability as Macuneos. Biophytis will start a phase 1 clinical trial in 2017 with Macuneos as an oral treatment for dry AMD. The mechanism by which BIO201 exerts in vitro and in vivo protective effects on RPE was studied. Materials and Methods The photoprotective effect of BIO201 was tested in an in vitro model of phototoxicity of porcine primary RPE cells challenged with A2E then blue-light illuminated. Cell survival was measured 24h after illumination. BIO201 protective activity was then characterized by a pharmacological approach employing selective agonists and antagonists of the different PPAR subtypes. In order to study the antioxidant and anti-apoptotic properties of BIO201, Arpe-19 cells were treated with antimycin A and PENAO, two pro-oxidant compounds, and ROS and PARP cleavage were measured by flow cytometry (DCFDA) and Western Blot, respectively, in the presence or the absence of BIO201 (30 µM). To determine whether BIO201 was able to activate the 3 PPAR isoforms, Cv-1 cells were transfected with luciferase reporter plasmid (pG5-TK-pGL3) in the presence of pGal4hPPAR x (the vector expressed chimeric proteins containing the Gal4 DNA-binding domain fused to a human PPAR ligand-binding domain coding sequence) expression vector, and luciferase was measured after 16h contact with various concentrations of BIO201 (or other substances). The expression level of the PPARs was assessed by Western Blot. Statistical analyses were performed by one-way ANOVA followed by Dunnett's test. Fontaine V. 1 , Monteiro E 1 ., Fournie M 1 ., Bonnard B 1 ., On S. 2 , Serova M. 2 , Balducci C. 2 , Guibout L. 2 , Sahel J.-A. 1 , Veillet S. 2 , Dilda P. 2 and Lafont R. 2 1 Sorbonne Universités, UPMC Univ Paris 06, INSERM U968, CNRS UMR 7210 - Institut de la Vision, 75012 Paris, France 2 Biophytis, Université Pierre et Marie Curie, 4 place Jussieu, 75005 Paris, France Conclusions Contacts: [email protected] [email protected] Results Financial disclosure: Financial support came from Biophytis # F070 BIO201, PPARa and PPARb/d agonists protect RPE cells against A2E-mediated phototoxicity BIO201 protection is partially reversed by PPARb/d and PPARg antagonists DMSO - A2E DMSO + A2E BIO201 + A2E BIO201 + A2E + MK BIO201 + A2E + GSK BIO201 + A2E + T 0 50 100 RPE cell survival (% of DMSO-A2E) ** **** DMSO - A2E DMSO + A2E BIO20 1 + A2E SUL + A2E GW + A2E RGZ + A2E TGZ + A2E 0 50 100 RPE cell survival (% of DMSO-A2E) **** **** **** **** ** seeding + BIO201 or agonists - 72h - 48h - 19h + A2E survival 24h 0h seeding + BIO201 - 72h - 48h - 19h + A2E + antagonists - 1h survival 24h 0h **p<0,01 ****p<0,0001 compared to DMSO + A2E **p<0,01 ****p<0,0001 compared to BIO201 + A2E Molecule Abbreviation Function Conc. (μM) sulindac SUL PPARa agonist 200 GW0742 GW PPARb/d agonist 30 rosiglitazone RGZ PPARg agonist 20 troglitazone TGZ PPARa, g dual agonist 20 MK886 MK PPARa antagonist 5 GSK3787 GSK PPARb/d antagonist 1 T0070907 T PPARg antagonist 10 BIO201, as well as sulindac, GW0742 and troglitazone significantly protect RPE cells against A2E-indiced phototoxicity. Rosiglitazone, a specific PPARg agonist, shows no photo-protective activity. BIO201 protective activity is partially reversed by PPARa and PPARb/d antagonists. BIO201 activates hPPARa and, even more efficiently, hPPARb/d 0,00E+00 3,00E-08 1,00E-07 3,00E-07 1,00E-06 3,00E-06 1,00E-05 3,00E-05 1,00E-04 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 109% 121% 158% * 231% *** 285% *** 321% *** 358% *** 212% *** 100% BIO201 (M) Luciferase assay (Arbitrary units) PPARa 0,00E+00 3,00E-08 1,00E-07 3,00E-07 1,00E-06 3,00E-06 1,00E-05 3,00E-05 1,00E-04 0.000 0.002 0.004 0.006 0.008 97%109% 176% 358% 394% 637% *** 1085% *** 91% 100% Troglitazone (M) Luciferase assay (Arbitrary units) PPARa 0,00E+00 1,00E-08 1,00E-07 3,00E-07 1,00E-06 3,00E-06 1,00E-05 3,00E-05 1,00E-04 0.0 0.2 0.4 0.6 0.8 86%113%123% 135% 281% 1727% 5219% *** 8851% *** 100% BIO201 (M) Luciferase assay (Arbitrary units) PPARb/d 0,00E+00 3,00E-08 1,00E-07 3,00E-07 1,00E-06 3,00E-06 1,00E-05 3,00E-05 1,00E-04 0.0000 0.0005 0.0010 0.0015 125% 16% 50% 52% 61% 116% 207% 202% 100% BIO201 (M) Luciferase assay (Arbitrary units) PPARg BIO201 is able to significantly trans-activate PPARa (C ≥ 0.3 µM) and PPARb/d (C ≥ 3 µM), but it does not trans-activate PPARg. Troglitazone, which is in fact a dual PPARa/g agonist indeed activates PPARa (C ≥ 1 µM). This result suggests that the photoprotective effect of troglitazone on RPE cells is mediated by its interaction with PPARa. These experiments however do not prove a direct binding of BIO201 to PPARs. *p<0,05 **p<0,001 compared to not treated BIO201 reduces ROS production and abolishes PARP cleavage in Arpe-19 cells BIO201 (30 µM) significantly protects ARPE-19 cells against ROS induced by PENAO (A, B, C) or Antimycin A (D, E, F). *p<0,05, **p<0,01 ***p<0,00, Mann-Whitney test BIO201 (30 µM) decreases c- PARP expression induced by Antimycin A in ARPE-19 cells, suggesting the protection from ROS-induced apoptosis. BIO201 counteracts PPARa protein degradation induced by A2E DMSO DMSO + A2E BIO201 + A2E GW + A2E RGZ + A2E TGZ + A2E 0.0 0.5 1.0 1.5 protein relative expression **** **** ** **p<0,01 ****p<0,0001 compared to DMSO + A2E seeding + BIO201 or agonists - 72h - 48h - 19h + A2E 0h PPAR analysis (western blot) We observe here for the first time a rapid and profound reduction of PPARa protein expression upon A2E treatment. This is abolished when RPE cells are treated with BIO201 before A2E exposure. GW0742 and troglitazone, two photoprotective PPAR agonists, display similar patterns. On the contrary, rosiglitazone, which is lacking cytoprotective activity, is unable to sustain PPARa protein expression. PPARa expression is not modified by BIO201 or PPAR agonists when used alone. PPARb/d and g protein expression were not affected by A2E treatment. - Both photoprotection and transactivaction results indicate that BIO201’s beneficial effect on RPE cells is linked with PPARα and/or PPARβ/δ activation. - BIO201 inhibits ROS production and apoptosis. - Prevention of PPARα protein degradation appears to be a key feature in BIO201 photoprotective activity. - Further experiments are underway with BIO201 (i) to better understand the crosstalk between the PPARs and (ii) to elucidate the signaling pathways involved in RPE cell survival. NEW THERAPEUTICS FOR AGING DISEASES
Transcript of RPE cell survival (% of DMSO-A2E) 100 · INVOLVEMENT OF PEROXISOME PROLIFERATOR ACTIVATOR RECEPTORS...
INVOLVEMENT OF PEROXISOME PROLIFERATOR ACTIVATOR RECEPTORS (PPARs) IN THE PHOTOPROTECTIVE ACTIVITY OF BIO201
IntroductionAccording to Herzlich et al. (PPAR Research, 2008, article ID389507), PPARsplay a key role in the protection of RPE cells against AMD.
There is increasing evidence for the involvement of PPARs in the protectionagainst the development of Age-Related Macular Degeneration (AMD). PPARreceptors comprise three sub-types of nuclear receptors (PPARα, PPARβ/δ andPPARγ) involved in the control of lipid and carbohydrate metabolism, as well asinflammatory processes. They are characterized by a large ligand-binding pocketand therefore accommodate diverse types of endogenous or exogenous ligands.BIO201 is a pharmaceutical grade preparation of the di-apo-carotenoid norbixin.It has been formulated for improving its stability and per os bioavailability asMacuneos. Biophytis will start a phase 1 clinical trial in 2017 with Macuneos asan oral treatment for dry AMD. The mechanism by which BIO201 exerts in
vitro and in vivo protective effects on RPE was studied.
Materials and MethodsThe photoprotective effect of BIO201 was tested in an in vitro model ofphototoxicity of porcine primary RPE cells challenged with A2E then blue-lightilluminated. Cell survival was measured 24h after illumination. BIO201 protectiveactivity was then characterized by a pharmacological approach employingselective agonists and antagonists of the different PPAR subtypes.In order to study the antioxidant and anti-apoptotic properties of BIO201, Arpe-19cells were treated with antimycin A and PENAO, two pro-oxidant compounds, andROS and PARP cleavage were measured by flow cytometry (DCFDA) andWestern Blot, respectively, in the presence or the absence of BIO201 (30 µM).To determine whether BIO201 was able to activate the 3 PPAR isoforms, Cv-1cells were transfected with luciferase reporter plasmid (pG5-TK-pGL3) in thepresence of pGal4hPPARx (the vector expressed chimeric proteins containing theGal4 DNA-binding domain fused to a human PPAR ligand-binding domain codingsequence) expression vector, and luciferase was measured after 16h contact withvarious concentrations of BIO201 (or other substances).The expression level of the PPARs was assessed by Western Blot.Statistical analyses were performed by one-way ANOVA followed by Dunnett'stest.
Fontaine V.1, Monteiro E1., Fournie M1., Bonnard B1., On S.2, Serova M.2, Balducci C.2, Guibout L.2, Sahel J.-A.1, Veillet S.2, Dilda P.2 and Lafont R.2
1 Sorbonne Universités, UPMC Univ Paris 06, INSERM U968, CNRS UMR 7210 - Institut de la Vision, 75012 Paris, France2 Biophytis, Université Pierre et Marie Curie, 4 place Jussieu, 75005 Paris, France
Conclusions
Contacts: [email protected] [email protected]
Results
Financial disclosure: Financial support came from Biophytis
# F070
BIO201, PPARa and PPARb/d agonists
protect RPE cells against A2E-mediated
phototoxicity
BIO201 protection is partially reversed
by PPARb/d and PPARg antagonists
DMSO - A2E
DMSO + A2E
BIO201 + A
2E
BIO201 + A
2E + MK
BIO201 + A
2E + GSK
BIO201 + A
2E + T0
50
100
RP
E c
ell s
urv
ival (%
of
DM
SO
-A2E
)
******
DMSO - A2E
DMSO + A2E
BIO201 + A
2E
SUL + A2E
GW + A
2E
RGZ + A2E
TGZ + A2E
0
50
100
RP
E c
ell s
urv
ival (%
of
DM
SO
-A2E
)
****
******** ****
**
seeding
+ BIO201 or agonists
- 72h
- 48h
- 19h
+ A2E
survival
24h 0h
seeding
+ BIO201
- 72h
- 48h
- 19h
+ A2E
+ antagonists
- 1h
survival
24h 0h
**p<0,01 ****p<0,0001 compared to DMSO + A2E**p<0,01 ****p<0,0001 compared to BIO201 + A2E
Molecule Abbreviation Function Conc.
(µM)
sulindac SUL PPARa agonist 200GW0742 GW PPARb/d agonist 30rosiglitazone RGZ PPARg agonist 20troglitazone TGZ PPARa, g dual agonist 20MK886 MK PPARa antagonist 5GSK3787 GSK PPARb/d antagonist 1T0070907 T PPARg antagonist 10
BIO201, as well as sulindac, GW0742and troglitazone significantly protect RPEcells against A2E-indiced phototoxicity.Rosiglitazone, a specific PPARg agonist,shows no photo-protective activity.BIO201 protective activity is partiallyreversed by PPARa and PPARb/d
antagonists.
BIO201 activates hPPARa and, even more efficiently, hPPARb/d
BIO201
0,00
E+0
0
3,00
E-0
8
1,00
E-0
7
3,00
E-0
7
1,00
E-0
6
3,00
E-0
6
1,00
E-0
5
3,00
E-0
5
1,00
E-0
4
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
109%121%
158%*
231%***
285%***
321%***
358%***
212%***
100%
BIO201 (M)
Lu
cifera
se a
ssay (
Arb
itra
ry u
nit
s)
PPARa Troglitazone
0,00
E+0
0
3,00
E-0
8
1,00
E-0
7
3,00
E-0
7
1,00
E-0
6
3,00
E-0
6
1,00
E-0
5
3,00
E-0
5
1,00
E-0
4
0.000
0.002
0.004
0.006
0.008
97%109%176%
358%394%
637%***
1085%***
91%100%
Troglitazone (M)
Lu
cifera
se a
ssay (
Arb
itra
ry u
nit
s) PPARa
BIO201
0,00
E+00
1,00
E-08
1,00
E-07
3,00
E-07
1,00
E-06
3,00
E-06
1,00
E-05
3,00
E-05
1,00
E-04
0.0
0.2
0.4
0.6
0.8
86%113%123%135%281%
1727%
5219%***
8851%***
100%
BIO201 (M)
Lu
cifera
se a
ssay (
Arb
itra
ry u
nit
s)
PPARb/d BIO201
0,00
E+0
0
3,00
E-0
8
1,00
E-0
7
3,00
E-0
7
1,00
E-0
6
3,00
E-0
6
1,00
E-0
5
3,00
E-0
5
1,00
E-0
4
0.0000
0.0005
0.0010
0.0015
125%
16% 50%52% 61%
116%
207%202%
100%
BIO201 (M)
Lu
cifera
se a
ssay (
Arb
itra
ry u
nit
s)
PPARg
BIO201 is able tosignificantly trans-activatePPARa (C ≥ 0.3 µM) andPPARb/d (C ≥ 3 µM), butit does not trans-activatePPARg.
Troglitazone, which is infact a dual PPARa/gagonist indeed activatesPPARa (C ≥ 1 µM). Thisresult suggests that thephotoprotective effect oftroglitazone on RPE cellsis mediated by itsinteraction with PPARa.
These experimentshowever do not prove adirect binding of BIO201to PPARs.
*p<0,05 **p<0,001 compared to not treated
BIO201 reduces ROS production and abolishes PARP cleavage in
Arpe-19 cells
BIO201 (30 µM) significantly protectsARPE-19 cells against ROS induced byPENAO (A, B, C) or Antimycin A (D, E, F).
*p<0,05, **p<0,01 ***p<0,00, Mann-Whitney test
BIO201 (30 µM) decreases c-PARP expression induced byAntimycin A in ARPE-19 cells,suggesting the protection fromROS-induced apoptosis.
BIO201 counteracts PPARa protein degradation induced by A2E
DMSO
DMSO + A2E
BIO201 + A
2E
GW + A
2E
RGZ + A2E
TGZ + A2E
0.0
0.5
1.0
1.5
pro
tein
rela
tive e
xp
ressio
n
**** ******
**p<0,01 ****p<0,0001 compared to DMSO + A2E
seeding
+ BIO201 or agonists
- 72h
- 48h
- 19h
+ A2E
0h
PPAR analysis (western blot)
We observe here for the first time a rapidand profound reduction of PPARa proteinexpression upon A2E treatment. This isabolished when RPE cells are treated withBIO201 before A2E exposure. GW0742and troglitazone, two photoprotective PPARagonists, display similar patterns. On thecontrary, rosiglitazone, which is lackingcytoprotective activity, is unable to sustainPPARa protein expression. PPARa
expression is not modified by BIO201 orPPAR agonists when used alone.PPARb/d and g protein expression were notaffected by A2E treatment.
- Both photoprotection and transactivaction results indicate thatBIO201’s beneficial effect on RPE cells is linked with PPARα and/orPPARβ/δ activation.
- BIO201 inhibits ROS production and apoptosis.- Prevention of PPARα protein degradation appears to be a key feature
in BIO201 photoprotective activity.- Further experiments are underway with BIO201 (i) to better
understand the crosstalk between the PPARs and (ii) to elucidate thesignaling pathways involved in RPE cell survival.
NEW THERAPEUTICS FOR AGING DISEASES
