U.S. patent application number 16/159408 was filed with the patent office on 2019-07-04 for treating ocular neovascularization.
The applicant listed for this patent is Massachusetts Eye and Ear Infirmary. Invention is credited to Joan W. Miller, Kimio Takeuchi, Demetrios Vavvas.
Application Number | 20190201430 16/159408 |
Document ID | / |
Family ID | 53494054 |
Filed Date | 2019-07-04 |
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United States Patent
Application |
20190201430 |
Kind Code |
A1 |
Vavvas; Demetrios ; et
al. |
July 4, 2019 |
TREATING OCULAR NEOVASCULARIZATION
Abstract
Methods of treating ocular neovascularization, e.g., associated
with wet age-related macular degeneration (AMD), using activators
of AMP-activated protein kinase (AMPK) and/or of Phosphatase and
tensin homolog deleted on chromosome 10 (PTEN).
Inventors: |
Vavvas; Demetrios; (Boston,
MA) ; Miller; Joan W.; (Winchester, MA) ;
Takeuchi; Kimio; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Eye and Ear Infirmary |
Boston |
MA |
US |
|
|
Family ID: |
53494054 |
Appl. No.: |
16/159408 |
Filed: |
October 12, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15108751 |
Jun 28, 2016 |
10143703 |
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PCT/US2015/010046 |
Jan 2, 2015 |
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16159408 |
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61922964 |
Jan 2, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 19/052 20130101;
A61K 31/6615 20130101; A61K 31/7056 20130101; A61K 31/4439
20130101; A61K 31/616 20130101; A61K 31/155 20130101; A61K 45/06
20130101; A61K 9/0048 20130101; A61K 31/515 20130101; C07F 9/117
20130101; A61P 27/02 20180101; A61K 31/436 20130101; A61K 31/683
20130101; A61K 31/519 20130101; A61K 31/7004 20130101 |
International
Class: |
A61K 31/7056 20060101
A61K031/7056; A61K 45/06 20060101 A61K045/06; A61K 9/00 20060101
A61K009/00; A61K 31/436 20060101 A61K031/436; A61K 31/7004 20060101
A61K031/7004; A61K 31/6615 20060101 A61K031/6615; A61K 31/616
20060101 A61K031/616; A61K 31/683 20060101 A61K031/683; A61K 31/515
20060101 A61K031/515; A61K 31/4439 20060101 A61K031/4439; A61K
31/155 20060101 A61K031/155; C07F 9/117 20060101 C07F009/117; C07H
19/052 20060101 C07H019/052; A61K 31/519 20060101 A61K031/519 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant
No. EY014104 awarded by the National Eye Institute of the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
1-17. (canceled)
18. A method of treating ocular neovascularization characterized by
surface, corneal, retinal, choroidal, uveal, or iris
neovascularization in a mammal, the method comprising: identifying
a mammal in need of reduced or delayed ocular neovascularization;
and administering to the mammal an effective amount of one or both
of: (i) an amp-activated protein kinase (AMPK) activator, or (ii) a
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)
activator, in combination with one or more anti-VEGF therapies.
19. The method of claim 18, wherein the one or more anti-VEGF
therapies is a monoclonal antibody or aptamer that binds and
inhibits VEGF.
20. The method of claim 19, wherein the one or more anti-VEGF
therapies is selected from the group consisting of Avastin
(Bevacizumab), Lucentis (Ranibizumab), Eylea (Aflibercept), Zaltrap
(Aflibercept), and Macugen (Pegaptanib).
21. The method of claim 18, wherein the mammal has retinopathy,
symptoms associated with microangiopathy, neovascular glaucoma,
corneal graft rejection, glaucoma, herpetic and infectious
keratitis, ocular ischemia, neovascular glaucoma, corneal, uveal
and iris neovascularization, orbital and eyelid tumors, Stevens
Johnson Syndrome, ocular cicatricial pemphigoid, wounds or other
injuries, and ocular surface diseases in a mammal.
22. The method of claim 21, wherein the mammal has a retinopathy
selected from the group consisting of retinopathy of prematurity
(ROP); retina vein occlusion; sickle cell retinopathy; Stargardt's
disease; choroidal neovascularization; and radiation
retinopathy.
23. The method of claim 21, wherein the mammal has an injury that
is a chemical injury due to exposure to irritants, acids or
bases.
24. The method of claim 18, wherein the mammal has endophthalmitis,
macular edema, conjunctivitis, episcleritis, keratitis, optic
neuritis, orbital pseudotumor, retinal vasculitis, or
scleritis.
25. The method of claim 18, comprising administering an AMPK
activator selected from the group consisting of guanidine;
galegine; phenobarbital; A-769662; PT1; and salicylate.
26. The method of claim 18, comprising administering a PTEN
activator selected from the group consisting of
di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2) and PI(5)P.
27. The method of claim 18, comprising administering an AMPK
activator selected from the group consisting of
5-Aminoimidazole-4-carboxamide riboside (AICA riboside or AICAR);
ZMP; guanidine; galegine; metformin (dimethylbiguanide); phemformin
(phenethylbiguanide); pemetrexed; pioglitazone; troglitazone;
phenobarbital; A-769662; PT1; and salicylate.
28. The method of claim 18, comprising administering a PTEN
activator selected from the group consisting of
di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2) and PI(5)P.
29. A method of treating wet age-related macular degeneration (AMD)
in a mammal, the method comprising: identifying a mammal who has
wet AMD; and administering to the mammal a therapeutically
effective amount of one or both of: (i) an amp-activated protein
kinase (AMPK) activator, or (ii) a Phosphatase and tensin homolog
deleted on chromosome 10 (PTEN) activator, in combination with one
or more anti-VEGF therapies.
30. The method of claim 29, wherein the one or more anti-VEGF
therapies is a monoclonal antibody or aptamer that binds and
inhibits VEGF.
31. The method of claim 30, wherein the one or more anti-VEGF
therapies is selected from the group consisting of Avastin
(Bevacizumab), Lucentis (Ranibizumab), Eylea (Aflibercept), Zaltrap
(Aflibercept), and Macugen (Pegaptanib).
32. The method of claim 29, wherein the mammal has retinopathy,
symptoms associated with microangiopathy, neovascular glaucoma,
corneal graft rejection, glaucoma, herpetic and infectious
keratitis, ocular ischemia, neovascular glaucoma, corneal, uveal
and iris neovascularization, orbital and eyelid tumors, Stevens
Johnson Syndrome, ocular cicatricial pemphigoid, wounds or other
injuries, and ocular surface diseases in a mammal.
33. The method of claim 32, wherein the mammal has a retinopathy
selected from the group consisting of retinopathy of prematurity
(ROP); retina vein occlusion; sickle cell retinopathy; Stargardt's
disease; choroidal neovascularization; and radiation
retinopathy.
34. The method of claim 32, wherein the mammal has an injury that
is a chemical injury due to exposure to irritants, acids or
bases.
35. The method of claim 29, wherein the mammal has endophthalmitis,
macular edema, conjunctivitis, episcleritis, keratitis, optic
neuritis, orbital pseudotumor, retinal vasculitis, or
scleritis.
36. The method of claim 29, comprising administering an AMPK
activator selected from the group consisting of guanidine;
galegine; phenobarbital; A-769662; PT1; and salicylate.
37. The method of claim 29, comprising administering a PTEN
activator selected from the group consisting of
di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2) and PI(5)P.
38. The method of claim 29, comprising administering an AMPK
activator selected from the group consisting of
5-Aminoimidazole-4-carboxamide riboside (AICA riboside or AICAR);
ZMP; guanidine; galegine; metformin (dimethylbiguanide); phemformin
(phenethylbiguanide); pemetrexed; pioglitazone; troglitazone;
phenobarbital; A-769662; PT1; and salicylate.
39. The method of claim 29, comprising administering a PTEN
activator selected from the group consisting of
di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2) and PI(5)P.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/108,751, filed Jun. 28, 2016, which is a
371 U.S. National Phase Application of PCT/US2015/010046, filed on
Jan. 2, 2015, which claims the benefit of U.S. Patent Application
Ser. No. 61/922,964, filed on Jan. 2, 2014. The entire contents of
the foregoing are hereby incorporated by reference.
TECHNICAL FIELD
[0003] This invention relates to methods of treating ocular
neovascularization using activators of AMP-activated protein kinase
(AMPK) and/or of Phosphatase and tensin homolog deleted on
chromosome 10 (PTEN).
BACKGROUND
[0004] Age-related macular degeneration (AMD) is the primary cause
of blindness in elderly individuals of industrialized countries
(Lim et al. (2012) Lancet. 379, 1728-1738; Zhang et al. (2012) Nat.
Rev. Drug Discov. 11, 541-559), and has a projected 50% increase by
the year 2020 in the United States (Friedman et al. (2004) Arch.
Ophthalmol. 122, 564-572). There is an urgent need for new
pharmacological interventions that are safe over the long term for
the treatment or prevention of AMD.
SUMMARY
[0005] The studies described herein connect AMPK activation to two
VEGF-mediated pathological processes in ocular
neovascularization--vascular tube formation and vascular leakage.
Examples 1 and 2 describe AMPK activation as inhibiting vascular
tube formation and also as inhibiting vascular leakage in in vitro
experiments. Thus, in one embodiment the present invention includes
the use of an AMPK activator (e.g., AICAR) to treat pathological
ocular neovascularization, e.g., in AMD. In addition, Example 2
demonstrates that AMPK activation inhibits VEGF-induced tube
formation through PTEN dependent dephosphorylation of Akt; thus, in
another aspect the invention provides methods for reducing
VEGF-induced neovascularization in vivo, e.g., neovascularization
associated with AMD, by administering a PTEN activator.
[0006] Thus in a first aspect the invention provides methods for
reducing or delaying ocular neovascularization in a mammal, the
method comprising:
identifying a mammal in need of reduced or delayed ocular
neovascularization; and administering to the mammal an effective
amount of an amp-activated protein kinase (AMPK) activator and/or
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)
activator sufficient to reduce or delay ocular neovascularization
in the mammal.
[0007] In another aspect the invention provides methods for
treating wet age-related macular degeneration (AMD) in a mammal,
the method comprising:
identifying a mammal who has wet AMD; and administering to the
mammal a therapeutically effective amount of an amp-activated
protein kinase (AMPK) activator and/or Phosphatase and tensin
homolog deleted on chromosome 10 (PTEN) activator.
[0008] In another aspect the invention provides an amp-activated
protein kinase (AMPK) activator and/or Phosphatase and tensin
homolog deleted on chromosome 10 (PTEN) activator to reduce ocular
neovascularization in a mammal.
[0009] In another aspect the invention provides for the use of an
amp-activated protein kinase (AMPK) activator and/or Phosphatase
and tensin homolog deleted on chromosome 10 (PTEN) activator in the
manufacture of a medicament to reduce IOP in a mammal.
[0010] In some embodiments, the mammal has wet age-related macular
degeneration, retinopathy (selected from a group comprising of:
retinopathy of prematurity (ROP); diabetic retinopathy; retina vein
occlusion; sickle cell retinopathy; Stargardt's disease; choroidal
neovascularization, radiation retinopathy), symptoms associated
with microangiopathy, neovascular glaucoma, corneal graft
rejection, glaucoma, herpetic and infectious keratitis, ocular
ischemia, neovascular glaucoma, corneal, uveal and iris
neovascularization, orbital and eyelid tumors, Stevens Johnson
Syndrome, ocular cicatricial pemphigoid, wounds or other injuries
(e.g., chemical injuries due to exposure to irritants, acids or
bases), and ocular surface diseases.
[0011] In some embodiments, the disorder is characterized by
surface, corneal, retinal, choroidal, uveal, or iris
neovascularization.
[0012] In some embodiments, the mammal has endophthalmitis (e.g.,
the endogenous form and the exogenous form), macular edema (e.g.,
macular edema that occurs as a result of age-related macular
degeneration, cataract surgery, diabetes, drug toxicity, eye
injury, retinal vein occlusion (e.g., central retinal vein
occlusion (CRVO) and branch retinal vein occlusion), or other
inflammatory eye diseases, e.g., pseudophakic macular edema),
conjunctivitis, episcleritis, keratitis, optic neuritis, orbital
pseudotumor, retinal vasculitis, scleritis, and uveitis (e.g., (i)
uveitis associated with sepsis (e.g., LPS-induced uveitis); (ii)
autoimmune uveitis (e.g., uveitis associated with lupus); or (iii)
uveitis associated with type II, type III, type IV, or type V
hypersensitivity reactions).
[0013] In some embodiments, the AMPK activator is selected from the
group consisting of 5-Aminoimidazole-4-carboxamide riboside (AICA
riboside or AICAR); ZMP; guanidine; galegine; metformin
(dimethylbiguanide); phemformin (phenethylbiguanide); antifolate
drugs that inhibit AICAR transformylase (e.g., methotrexate,
pemetrexed); thiazolidinediones (e.g., rosiglitazone, pioglitazone,
or troglitazone); 2-Deoxyglucose (2DG); phenobarbital; A-769662;
PT1; and salicylate.
[0014] In some embodiments, the PTEN activator is selected from the
group consisting of di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2
and PI(5)P; PPARgamma agonists such as rosiglitazone; and mTOR
inhibitors including rapamycin and its chemical analogues such as
temsirolimus, everolimus, and AP-2357.
[0015] In some embodiments, the AMPK activator and/or PTEN
activator is administered in combination with another treatment
such as anti VEGF therapies, non-steroidal or steroidal
anti-inflammatory treatments, or neuroprotective treatments.
[0016] In another aspect the invention provides a pharmaceutical
composition comprising a PTEN activator formulated for ocular
administration. In some embodiments, the composition is formulated
for topical ocular administration, e.g., as eye drops, topical eye
cream, or topical eye lotion. In some embodiments, the formulation
comprises microcapsules, microemulsions, or nanoparticles.
[0017] In another aspect the invention provides container for
drop-wise dispensation of a pharmaceutical composition into the eye
of a subject, the container having disposed therein an amount of a
PTEN activator.
[0018] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0019] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0020] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0021] FIG. 1. AICAR suppresses phosphorylation of caveolin-1 and
c-Abl, and albumin endocytosis under oxidative stress.
[0022] FIG. 1A, Cells were cultured in AICAR (2 mM) containing
medium for 2 h, and then stimulated with each different
concentration (0, 0.5, 1.0, 2.0 mM) of H.sub.2O.sub.2 for 30 min.
The amounts of p-caveolin-1 and p-c-Abl in HUVEC were then examined
by western blotting.
[0023] FIG. 1B, Densitometry of p-caveolin-1 in FIG. 1A.
[0024] FIG. 1C, Densitometry of p-c-Abl in FIG. 1A.
[0025] FIG. 1D, Albumin endocytosis assay. Cells were exposed to
Alexa555 conjugated BSA. a, control (untreated cells), b,
H.sub.2O.sub.2 (2 mM) stimulation for 30 min, c, Pretreated with
AICAR (2 mM) for 2 h, d, Pretreated with AICAR for 2 h followed by
H.sub.2O.sub.2 (2 mM) stimulation for 30 min. BSA conjugated with
Alexa 555 (red), p-caveolin (green), VE-cadherin (blue). Scale
bar=50 .mu.m.
[0026] FIG. JA, Representative blots are shown. *, P<0.01.
[0027] FIG. 2. AICAR inhibits H.sub.2O.sub.2 induced
phosphorylation of caveolin-1 via activation of AMPK.
[0028] FIG. 2A, Cells were treated with each concentration of AICAR
for 2 h.
[0029] FIG. 2B, Cells were treated with each concentration of DPY
for 1 h, and then stimulated with 2 mM of AICAR for 2 h.
[0030] FIG. 2C, Cells were treated with Adenosine transporter
inhibitor DPY (8 .mu.M) for 1 h, and then stimulated with 2 mM of
AICAR for 2 h, followed by H.sub.2O.sub.2 (2 mM) stimulation for 30
min.
[0031] FIG. 2D, Densitometry of p-caveolin-1 in FIG. 2 C.
[0032] FIG. 2E, Densitometry of p-c-Abl in FIG. 2 C.
[0033] FIG. 2F, Cells were treated with each concentration of
adenosine kinase inhibitor IODO for 1 h, and then stimulated with 2
mM of AICAR for 2 h.
[0034] FIG. 2G, Cells were treated with 0.4 .mu.M of IODO for 1 h,
and then stimulated with 2 mM of AICAR for 2 h, followed by
H.sub.2O.sub.2 (2 mM) stimulation for 30 min.
[0035] FIG. 2H, Densitometry of p-caveolin-1 in FIG. 2 G.
[0036] FIG. 2I, Densitometry of p-c-Abl in FIG. 2 G.
[0037] FIG. 2A-FIG. 2E, Representative blots are shown. *,
P<0.01.
[0038] FIG. 3. Both AMPK.alpha.1 and .alpha.2 isoforms are required
for AICAR inhibition of caveolin-1 phosphorylation under oxidative
stress.
[0039] FIG. 3A and FIG. 3D, The amounts of p-caveolin-1 and p-c-Abl
in HUVEC were examined by western blotting. Cells were transfected
with siRNA against AMPK.alpha.1 (A) or .alpha.2 (D). Three days
after transfection, cells were stimulated with 2 mM of AICAR for 2
h, followed by H.sub.2O.sub.2 (2 mM) stimulation for 30 min.
[0040] FIG. 3B, Densitometry of p-caveolin-1 in FIG. 3 A.
[0041] FIG. 3C, Densitometry of p-c-Abl in FIG. 3 A.
[0042] FIG. 3E, Densitometry of p-caveolin-1 in FIG. 3 D.
[0043] FIG. 3F, Densitometry of p-c-Abl in FIG. 3 D.
[0044] FIG. 3A and FIG. 3B, Representative blots are shown. *,
P<0.01.
[0045] FIG. 4. AMPK mediates AICAR effects on c-Abl and caveolin1
phosphorylation
[0046] FIG. 4A, Cells were co-transfected with both AMPK.alpha.1
and .alpha.2 siRNAs (two independent oligos) (.alpha.1
(PRKAA1)-CCAUACCCUUGAUGAAUUA (SEQ ID NO:1), .alpha.2
(PRKAA2)-CGACUAAGCCCAAAUCUUU (SEQ ID NO:2)). Three days after
transfection, cells were stimulated with 2 mM of AICAR for 2 h,
followed by H.sub.2O.sub.2 (2 mM) stimulation for 30 min. The
amounts of p-c-Abl, p-caveolin-1 were examined by western
blotting.
[0047] FIG. 4B, Densitometry of p-c-Abl in FIG. 4 A.
[0048] FIG. 4C, Densitometry of p-caveolin-1 in FIG. 4 A.
[0049] FIG. 4D, Cells were co-transfected with both AMPK.alpha.1
and .alpha.2 siRNAs (two independent oligos). (.alpha.1'
(PRKAA1)-GCCCAGAGGUAGAUAUAUG (SEQ ID NO:3), .alpha.2'
(PRKAA2)-GAGCAUGUACCUACGUUAU (SEQ ID NO:4)). Three days after
transfection, cells were stimulated with 2 mM of AICAR for 2 h,
followed by H.sub.2O.sub.2 (2 mM) stimulation for 30 min. The
amounts of p-c-Abl, p-caveolin-1 were examined by western
blotting.
[0050] FIG. 4E, Densitometry of p-c-Abl in FIG. 4 D.
[0051] FIG. 4F, Densitometry of p-caveolin-1 in FIG. 4 D.
[0052] FIG. 4A and FIG. 4D, Representative blots are shown. *,
P<0.01; NS, not significant.
[0053] FIG. 5. Inhibitory effect of AMPK on caveolin-1
phosphorylation under oxidative stress is dependent on c-Abl.
[0054] FIG. 5A, Cells were treated with 10 or 20 uM of imatinib
mesylate for 24, 48 or 72 h before stimulation with H.sub.2O.sub.2
(2 mM) for 30 min.
[0055] FIG. 5B, Densitometry of p-caveolin-1 in FIG. 5 A.
[0056] FIG. 5C, Densitometry of p-c-Abl in FIG. 5 A.
[0057] FIG. 5D, Cells were transfected with siRNA against c-Abl.
Three days after transfection, cells were stimulated with 2 mM of
AICAR for 2 h, followed by stimulation with H.sub.2O.sub.2 (2 mM)
for 30 min.
[0058] FIG. 5E, Densitometry of p-caveolin-1 in FIG. 4, B.
[0059] FIG. 5A and FIG. 5D, Representative blots are shown. *,
P<0.01; NS, not significant.
[0060] FIG. 6. AICAR inhibits caveolin-1 phosphorylation under
oxidative stress by suppressing the dissociation between prdx1 and
c-Abl.
[0061] FIG. 6A, Cells were transfected with siRNA against prdx1.
Three days after transfection, cells were stimulated with 2 mM of
AICAR for 2 h, followed by H.sub.2O.sub.2 (2 mM) stimulation for 30
min. The amounts of p-c-Abl, p-caveolin-1 were examined by western
blotting.
[0062] FIG. 6B, Densitometry of p-caveolin-1 in FIG. 6 A.
[0063] FIG. 6C, Densitometry of p-c-Abl in FIG. 6 A.
[0064] FIG. 6D, Cells were stimulated with 2 mM of AICAR for 2 h,
followed by H.sub.2O.sub.2 (2 mM) stimulation for 30 min. After
total cell lysates of each group were collected, the interaction
between prdx1 and c-Abl was examined by immunoprecipitation with
anti-prdx1 antibody. Immunoprecipitates were then subjected to
immunoblotting using anti-c-Abl antibody.
[0065] FIG. 6E, Densitometry of p-c-Abl in FIG. 6 D.
[0066] FIG. 6F, Cells were transfected with siRNA against
AMPK.alpha.1 or .alpha.2. Three days after transfection, cells were
stimulated with 2 mM of AICAR for 2 h, followed by H.sub.2O.sub.2
(2 mM) stimulation for 30 min. After total cell lysates of each
group were collected, the interaction between c-Abl and prdx1 was
examined by immunoprecipitation with anti-prdx1 antibody.
Immunoprecipitates were then subjected to immunoblotting using
anti-c-Abl antibody.
[0067] FIG. 6G, Densitometry of c-Abl in FIG. 5 F.
[0068] FIG. 6H, Co-immunoprecipitation experiments with pull-down
using anti-AMPK antibodies showing that AMPK does not directly
associate with the c-abl/prdx1 complex.
[0069] FIGS. 6A, 6D and 6F, Representative blots are shown. *,
P<0.01; NS, not significant.
[0070] FIG. 7. Proposed model for the mechanism by which AMPK
suppresses caveolin-1 phosphorylation and endocytosis under
oxidative stress.
[0071] AMPK negatively regulates caveolin-1 phosphorylation by
suppressing the dissociation between c-Abl and prdx-1.
[0072] FIG. 8. AICAR inhibits VEGF-induced vascular tube formation
in an in-vitro model through VEGFR2 independent mechanisms.
[0073] FIG. 8A: Morphological changes of HUVECs in the presence of
VEGF (12.5 ng/mL) and AICAR. The culture conditions of each group
in descending order were VEGF (-), AICAR (-), Suramin (-); VEGF
(+), AICAR (-), Suramin (-); VEGF (+), AICAR (0.25 mM), Suramin
(-); VEGF (+), AICAR (0.5 mM), Suramin (-); VEGF (+), AICAR (1.0
mM), Suramin (-); VEGF (+), AICAR (2.0 mM), Suramin (-); VEGF (+),
AICAR (-), Suramin (50 .mu.M). Bar equals 500 .mu.m.
[0074] FIG. 8B: Statistical analysis performed to evaluate the tube
length. *, p<0.01.
[0075] FIG. 8C: HUVECs were cultured in AICAR (2 mM)-containing
medium for 2 h, and then after the medium was changed, they were
stimulated with VEGF (12.5 ng/mL) for 0 to 60 min. The amounts of
(p-)VEGFR2 and (p-)Akt in the HUVECs were then examined by western
blotting.
[0076] FIG. 8D: Densitometry of p-VEGFR2 in panel C
[0077] FIG. 8E: Densitometry of p-Akt in panel C
[0078] FIG. 8F: Cells were stimulated with 2 mM of AICAR for 2 h,
followed by VEGF (12.5 ng/mL) stimulation for 10 min. After total
cell lysates of each group were collected, the interaction between
VEGFR2 and caveolin-1 was examined by immunoprecipitation with
anti-caveolin-1 antibody. Immunoprecipitates were then subjected to
immunoblotting using anti-VEGFR2 antibody.
[0079] FIG. 8G: Densitometry of p-VEGFR2 in panel F
[0080] FIG. 8C and FIG. 8F: Representative blots are shown. *,
p<0.01; NS, not significant.
[0081] FIG. 9. AICAR Activation of AMPK leads to PTEN dependent
dephosphorylation of Akt.
[0082] FIG. 9A: HUVECs were treated with Adenosine Kinase Inhibitor
IODO (0.1 .mu.M) for 60 min, and then stimulated with AICAR (2 mM)
in the presence or absence of VEGF.
[0083] FIG. 9B: Densitometry of p-AMPK in panel A.
[0084] FIG. 9C: Densitometry of p-Akt in panel A.
[0085] FIG. 9D: Densitometry of p-PTEN in panel A.
[0086] FIG. 9E: Cells were transfected with siRNA against PTEN.
Three days after transfection, cells were stimulated with 2 mM of
AICAR for 2 h, followed by VEGF (12.5 ng/mL) stimulation for 10
min.
[0087] FIG. 9F: Densitometry of p-Akt in panel E.
[0088] FIG. 9A and FIG. 9E: Representative blots are shown. *,
p<0.01; NS, not significant.
[0089] FIG. 10. AICAR inhibits VEGF-induced albumin endocytosis and
leakage in HUVECs in an in vitro model.
[0090] FIG. 10A: After a cell monolayer was formed, each chamber
was treated with 2 mM of AICAR for 2 h, followed by VEGF (12.5
ng/mL) stimulation for 10 min. The fluorescent density was measured
by spectrofluorometry. *, p<0.01.
[0091] FIG. 10B: The expression of (p-)caveolin-1 was also examined
by immunofluorescence. (a): control (untreated cells), (b): VEGF
(12.5 ng/mL) stimulation for 10 min, (c) pretreated with AICAR (2
mM) for 2 h, (d) pretreated with AICAR for 2 h followed by VEGF
(12.5 ng/mL) stimulation for 10 min. Bar=50 .mu.m.
[0092] FIG. 11. AICAR inhibits VEGF-induced c-Abl, and caveolin-1
phosphorylation.
[0093] FIG. 11A: HUVECs were cultured in AICAR (2 mM)-containing
medium for 2 h, and then after the medium was changed, they were
stimulated with VEGF (12.5 ng/mL) for 0 to 60 min. The amounts of
(p-)caveolin-1 and (p-)c-Abl in the HUVECs were then examined by
western blotting.
[0094] FIG. 11B: Densitometry of p-c-Abl in panel A.
[0095] FIG. 11C: Densitometry of p-caveolin-1 in panel A.
[0096] FIG. 11A: Representative blots are shown. *, p<0.01.
[0097] FIG. 12. AICAR suppresses VEGF-induced caveolin-1, c-Abl and
Akt phosphorylation likely via AMPK.
[0098] FIG. 12A: Cells were treated with each concentration of
AICAR for 2 h.
[0099] FIG. 12B: Cells were treated with each concentration of DPY
for 1 h, and then stimulated with 2 mM of AICAR for 2 h.
[0100] FIG. 12C: Cells were treated with 8 .mu.M of DPY for 1 h,
and then stimulated with 2 mM of AICAR for 2 h, followed by VEGF
(12.5 ng/mL) stimulation for 10 min.
[0101] FIG. 12D: Cells were treated with each concentration of IODO
for 1 h, and then stimulated with 2 mM of AICAR for 2 h.
[0102] FIG. 12E: Cells were treated with 0.4 .mu.M of IODO for 1 h,
and then stimulated with 2 mM of AICAR for 2 h, followed by VEGF
(12.5 ng/mL) stimulation for 10 min.
[0103] FIG. 12F: Densitometry of p-Akt in panel D.
[0104] FIG. 12G: Densitometry of p-c-Abl in panel D.
[0105] FIG. 12H: Densitometry of p-caveolin-1 in panel D.
[0106] FIG. 12I: Densitometry of p-Akt in panel E.
[0107] FIG. 12J: Densitometry of p-c-Abl in panel E.
[0108] FIG. 12K: Densitometry of p-caveolin-1 in panel E.
[0109] FIG. 12A-E: Representative blots are shown. *,
p<0.01.
[0110] FIG. 13. Both AMPK.alpha.1 and .alpha.2 isoforms are
required for AICAR inhibition of VEGF dependent caveolin-1, c-Abl
and Akt phosphorylation.
[0111] FIG. 13A and FIG. 13F: The amounts of p-caveolin-1 and
p-c-Abl in HUVECs were examined by western blotting. Cells were
transfected with siRNA against AMPK.alpha.1 (A) or .alpha.2 (B).
Three days after transfection, cells were stimulated with 2 mM of
AICAR for 2 h, followed by VEGF (12.5 ng/mL) stimulation for 10
min.
[0112] FIG. 13B: Densitometry of p-Akt in panel A.
[0113] FIG. 13C: Densitometry of p-c-Abl in panel A.
[0114] FIG. 13D: Densitometry of p-caveolin-1 in panel A.
[0115] FIG. 13E: Densitometry of p-PTEN in panel A.
[0116] FIG. 13G, Densitometry of p-Akt in panel B.
[0117] FIG. 13H, Densitometry of p-c-Abl in panel B.
[0118] FIG. 13I, Densitometry of p-caveolin-1 in panel B.
[0119] FIG. 13J, Densitometry of p-PTEN in panel B.
[0120] FIG. 13A and FIG. 13F: Representative blots are shown.
P<0.01.
[0121] FIG. 14. c-Abl is required for VEGF dependent caveolin-1
phosphorylation.
[0122] FIG. 14A: Cells were treated with 10 or 20 .mu.M of imatinib
mesylate for 24, 48 or 72 h before stimulation with VEGF (12.5
ng/mL) for 10 min.
[0123] FIG. 14B: Densitometry of p-VEGFR2 in panel A.
[0124] FIG. 14C: Densitometry of p-caveolin-1 in panel A.
[0125] FIG. 14D: Densitometry of p-c-Abl in panel A.
[0126] FIG. 14E: Cells were transfected with siRNA against c-Abl.
Three days after transfection, cells were stimulated with VEGF
(12.5 ng/mL) for 10 min.
[0127] FIG. 14F: Densitometry of p-caveolin-1 in panel B.
[0128] FIG. 14G: Densitometry of p-VEGFR2 in panel B.
[0129] FIG. 14A and FIG. 14E: Representative blots are shown. *,
p<0.01; NS, not significant.
[0130] FIG. 15. AICAR mediated AMPK activation inhibits VEGF
dependent caveolin-1 phosphorylation by suppressing the
dissociation between prdx1 and c-Abl.
[0131] FIG. 15A: Cells were transfected with siRNA against prdx1.
Three days after transfection, cells were stimulated with 2 mM of
AICAR for 2 h, followed by VEGF (12.5 ng/mL) stimulation for 10
min. The amounts of p-c-Abl and p-caveolin-1 were examined by
western blotting.
[0132] FIG. 15B: Densitometry of p-c-Abl in panel A.
[0133] FIG. 15C: Densitometry of p-caveolin-1 in panel A.
[0134] FIG. 15D: Cells were stimulated with 2 mM of AICAR for 2 h,
followed by VEGF (12.5 ng/mL) stimulation for 10 min. After the
total cell lysates of each group were collected, the interaction
between prdx1 and c-Abl was examined by immunoprecipitation with
anti-prdx1 antibody. Immunoprecipitates were then subjected to
immunoblotting using anti-c-Abl antibody.
[0135] FIG. 15E: Densitometry of c-Abl in panel D.
[0136] FIG. 15F: Cells were transfected with siRNA against
AMPK.alpha.1 or .alpha.2. Three days after transfection, cells were
stimulated with 2 mM of AICAR for 2 h, followed by VEGF (12.5
ng/mL) stimulation for 10 min. After total cell lysates of each
group were collected, the interaction between c-Abl and prdx1 was
examined by immunoprecipitation with anti-prdx1 antibody.
Immunoprecipitates were then subjected to immunoblotting using
anti-c-Abl antibody.
[0137] FIG. 15G: Densitometry of c-Abl in panel F.
[0138] FIGS. 15A, 15D, and 15F: Representative blots are shown. *,
P<0.01; NS, not significant.
[0139] FIG. 16. Proposed model for the mechanism by which AMPK
activator AICAR suppresses VEGF induced angiogenesis and caveolin-1
dependent trancytosis.
[0140] AMPK negatively regulates Akt and caveolin-1 phosphorylation
by activating PTEN and suppressing the dissociation between c-Abl
and prdx1.
DETAILED DESCRIPTION
[0141] Using multiple biochemical and molecular biology techniques,
the present inventors have identified AMPK as a novel negative
regulator of VEGF-induced caveolin-1 and Akt phosphorylation in
HUVECs contributing to the suppression of VEGF induced tube
formation and vascular endothelial cell permeability. These effects
are mediated in part by PTEN dephosphorylation of Akt and AMPK
dependent stabilization of c-Abl/Prdx1 complex. Thus, the present
disclosure includes methods for reducing or delaying ocular
neovascularization by administering one or both of an AMPK
activators or a PTEN activator.
[0142] AMPK
[0143] AMP-activated protein kinase (AMPK) is a serine/threonine
kinase that regulates energy homeostasis and metabolic stress
(Hardie and Hawley, (2001) Bioessays 23, 1112-1119). AMPK acts as a
sensor of cellular energy status and maintains the balance between
ATP production and consumption. In mammals, AMPK exists as a
heterotrimer with .alpha., .beta., and .gamma. subunits, each of
which is encoded by two or three genes (.alpha.1, .alpha.2,
.beta.1, .beta.2, .gamma.1, .gamma.2, and .gamma.3). The a subunit
possesses catalytic activity, whereas the .beta. and .gamma.
subunits are regulatory and maintain the stability of the
heterotrimer complex. The importance of AMPK.alpha. is illustrated
by the finding that dual deficiency of AMPK.alpha.1 and
AMPK.alpha.2 results in an embryonic-lethal phenotype (Viollet et
al. (2009) Front Biosci 14, 19-44).
[0144] Prior studies suggest that AMPK has a much wider range of
functions, including the regulation of cell growth, cell
proliferation, cell polarity, and autophagy (Wang et al. (2009)
Acta Physiol (Oxf) 196, 55-63; Theodoropoulou et al. (2010) FASEB J
24, 2620-2630) and activation of PTEN (Phosphatase and tensin
homolog deleted on chromosome 10) (Kim and Choi, (2012) Biochem
Biophys Res Commun 425, 866-872), which negatively regulates the
activity of this VEGF/PI3K/Akt (Myers et al. (1997) Proc Natl Acad
Sci USA 94, 9052-9057; Tamura, et al. (1998) Science 280,
1614-1617). In addition, we have demonstrated that activation of
AMPK inhibits retinoblastoma cell proliferation, tumor growth and
angiogenesis, ocular inflammation, and MMP-9 expression
(Theodoropoulou et al., 2010; Theodoropoulou et al., (2013) PLoS
One 8, e52852; Suzuki et al., (2011) Invest Ophthalmol Vis Sci 52,
6565-6571; Suzuki et al., (2012) Invest Ophthalmol Vis Sci 53,
4158-4169; Morizane et al., (2011) J Biol Chem 286, 16030-16038).
Because these functions of AMPK are closely linked to the vascular
hyper-permeability and angiogenesis induced by stress, we
hypothesized that AICAR activation of AMPK has an inhibitory effect
on VEGF induced vascular permeability and angiogenesis. Indeed, a
recent study reported that AMPK protects a paracellular pathway by
supporting the adherent junction proteins of N-cadherin and
VE-cadherin (Creighton et al., (2011) FASEB J 25, 3356-3365), and
there have been conflicting studies on the role of AMPK in
angiogenesis (Ahluwalia and Tarnawski, (2011) J Physiol Pharmacol
62, 583-587; Stahmann et al., (2010) J Biol Chem 285, 10638-10652;
Peyton et al., (2012) J Pharmacol Exp Ther 342, 827-834). Thus, the
present study examined the role of AMPK in the transcellular
pathway and phosphorylation of caveolin-1 as well as angiogenesis
under VEGF stimulation.
[0145] The present study identified AICAR as a novel chemical
inhibitor of VEGF induced Akt, c-Abl and caveolin-1
phosphorylation. Provided herein is evidence that the AMPK
activator AICAR suppresses tube formation (angiogenesis) in an in
vitro assay by inhibiting Akt phosphorylation, likely due to
activation of PTEN. In addition AMPK activation by AICAR suppresses
VEGF induced endocytosis and leakage by inhibiting caveolin-1
phosphorylation and stabilizing Prdx1/c-Abl complex. These results
reveal the suppressive role of AMPK in VEGF-induced caveolin-1,
c-Abl and Akt phosphorylation. The possibility of caveolin-1
phosphorylation as a therapeutic target for VEGF-mediated vascular
diseases was not described prior to the present study. In addition,
the inhibitory effect of AICAR on angiogenesis has not been prior
studied, though the present inventors observed a decrease in tumor
vessel formation in AICAR-treated retinoblastoma xenografts
(Theodoropoulou et al. (2013) PLoS One 8, e52852).
[0146] In other studies AICAR and activation of AMPK has been
related with cytoprotection and stimulation of angiogenesis in
situations of ischemia/re-perfusion injury or hypoxia (Russell et
al., (2004) J Clin Invest 114, 495-503; Nagata, et al., (2003) J
Biol Chem 278, 31000-31006; Ouchi et al., (2005) Circ Res 96,
838-846). Yet Zou et al. ((2003) J Biol Chem 278, 34003-34010) and
Nagata et al. (2003) did not observe a positive role of AMPK in
VEGF-mediate angiogenesis under normoxic conditions. In other
studies (Reihill et al., (2011) Vasc Cell 3, 9), despite the
apparent requirement for AMPK in VEGF-stimulated endothelial cell
proliferation, activation of AMPK with AICAR, A769662 or Ad.AMPK-CA
suppressed endothelial proliferation in the absence of VEGF and may
relate to the cell cycle inhibition effects of AMPK. The in vitro
study described herein and an in vivo study with retinoblastoma
related angiogenesis (Theodoropoulou et al. (2013) PLoS ONE 8(1):
e52852) shows that the AMPK activator AICAR is related with
anti-angiogenesis properties and may be related to its
anti-proliferative effects. Recently, Zhou et al. ((2011) Oncogene
30, 1892-1900) reported that AMPK upregulates TNF SF15, a cytokine
that exerts a potent inhibitory effect on vascular endothelial
cells and tumor angiogenesis. It is also possible that the various
effects of AICAR depend on the specific cell type, cellular events
following external stimuli, paracrine effects and/or
downstream-regulated pathways.
[0147] PTEN
[0148] Phosphatase and tensin homologue deleted on chromosome 10
(PTEN), which has been identified as a tumor suppressor (see Li et
al., J Cell. Biochem. 102:1368, 2007), is a phospholipid
phosphatase that converts PI(3,4,5)P3 to PI(4,5)P2 (PIP3 to PIP2).
This action opposes the phosphatidylinositol 3-kinases (PI3Ks), a
large family of proteins activated by numerous cellular processes
(including growth factor signalling) and activate the Akt protein
via PIP3. Akt then directly or indirectly activates a number of
other proteins including mammalian target of rapamycin (mTOR) which
leads to protein synthesis, enhancing cell proliferation and cell
survival (Jiang et al., Biochim. Biophys. Acta 1784:150, 2008).
PTEN thus controls and down-regulates this survival pathway by
reducing levels of PIP3. PTEN also possesses
phosphatase-independent tumor suppressive functions. See, e.g.,
WO2009126842A1 and US20070280918.
[0149] AMPK and PTEN
[0150] The effects of AICAR in inhibiting tube formation in the
present studies appear to be downstream of VEGFR2, since AICAR
pretreatment has no influence on VEGFR2 phosphorylation (FIG. 8
C,D) or on VEGFR2 dissociation from caveolin-1 (FIG. 8 F,G). It is
well known that VEGF regulates the activity of Akt pathway and that
PTEN is a negative regulator of that pathway (Myers et al. (1997)
and Tamura et al. (1998)). In addition, Kim et al. (2012) reported
that AMPK can induce PTEN phosphorylation in vascular smooth muscle
cells. In the present study, AICAR administration lead to
concomitant activation of PTEN in an AMPK dependent fashion and a
subsequent Akt de-phosphorylation (FIGS. 9 A-D and 6) and thus it
was hypothesized that AICAR activation of AMPK suppresses VEGF
mediated tube formation via PTEN de-phosphorylation of Akt. This
finding of AICAR and AMPK effects on Akt differ somewhat from the
findings of Levine et al. ((2007) J Biol Chem 282, 20351-20364)
which show that siRNA downregulation of AMPK al suppresses overall
phospho-Akt. In that study although VEGF stimulation of Akt
phosphorylation was blunted it was not completely abolished and
showed at least a 2.5 fold activation.
[0151] VEGF
[0152] VEGF is a key regulator of angiogenesis, and it controls the
proliferation, migration, differentiation, and survival of
endothelial cells through binding to VEGF receptor-2 (VEGFR2)
(Shibuya et al., (2006) Exp Cell Res 312, 549-560). VEGFR2 is a
receptor tyrosine kinase that autophosphorylates and initiates a
variety of signaling pathways, including the phospholipase
C.gamma./protein kinase C/Ca.sup.2+ pathway and the
phosphoinositide 3-kinase/Akt pathway (Holmes et al., (2007) Cell
Signal 19, 2003-2012; Olsson et al., (2006) Nat Rev Mol Cell Biol
7, 359-371). Over-expression of VEGF can induce pathological
endothelial cell permeability and angiogenesis via Akt
phosphorylation at Ser473 in the diseases such as cancer, diabetic
retinopathy and age-related macular degeneration (Olson et al.,
(2006) Nat Rev Mol Cell Biol 7, 359-371; Komarova and Malik, (2010)
Annu Rev Physiol 72, 463-493; Bates, (2010) Cardiovasc Res 87,
262-271; Bates and Harper, (2002) Vascul Pharmacol 39, 225-237),
and it leads to the disorder of vessel fenestrations, tight
junctions and adherent junctions (in the paracellular pathway) and
in the transcellular pathway ((Olson et al., (2006)). Passage of
small proteins such as albumin has been attributed to the
VEGF-induced formation of caveolae, the assembly of caveolae into
vesiculovacuolar organelles (VVOs), and/or the induction of
trans-endothelial pores. Feng et al. ((1999) Invest Ophthalmol Vis
Sci 40, 157-167) reported that a VEGF-induced increase in the
permeability of the cell membrane was mediated by caveolae, and
Zhao et al. ((2011) J Mol Neurosci 44, 122-129) reported that VEGF
increased the permeability through a caveolae-mediated
transcellular pathway in a blood-tumor barrier. It is also known
that VEGFR2 colocalizes with VEGFR2 in the caveolae (Holmes et al.,
(2007) Cell Signal 19, 2003-2012; Labrecque et al., (2003) Mol Biol
Cell 14, 334-347; Tahir et al., (2009) Cancer Biol Ther 8,
2286-2296).
[0153] Methods of Treatment
[0154] The methods described herein include methods for the
treatment of disorders associated with ocular neovascularization.
In some embodiments, the disorder is choroidal, retinal, or surface
neovascularization; vasoproliferative ocular tumours; or
inflammation and vascular leakage conditions.
[0155] In some embodiments, the disorder will stem from
overformation of blood vessels, or formation of blood vessels in an
unwanted area, e.g., in the avascular regions of the eye, e.g.,
retinopathies, or in a tumor, e.g., a cancerous or benign tumor.
For example, the ophthalmological disorder can be age-related
macular degeneration (AMD), where new blood vessels grow under the
retina, or retinopathy, e.g., diabetic retinopathy, where abnormal
vessels grow on top of the retina. Other ophthalmological disorders
include retinopathy (e.g., is selected from a group comprising of:
retinopathy of prematurity (ROP); diabetic retinopathy; retina vein
occlusion; sickle cell retinopathy; Stargardt's disease; choroidal
neovascularization, radiation retinopathy), microangiopathy,
neovascular glaucoma, corneal graft rejection, glaucoma, herpetic
and infectious keratitis, ocular ischemia, neovascular glaucoma,
corneal, uveal and iris neovascularization, orbital and eyelid
tumors, Stevens Johnson Syndrome, ocular cicatricial pemphigoid,
wounds or other injuries (e.g., chemical injuries due to exposure
to irritants, acids or bases), and ocular surface diseases. The
disorder can be characterized by, for example, corneal, retinal,
choroidal, uveal, or iris neovascularization.
[0156] In some embodiments, the disorder is associated with
choroidal neovascularization (CNV), e.g., choroidal
neovascularization secondary to, for example, the neovascular (wet)
form of age-related macular degeneration (AMD), pathologic myopia,
or ocular histoplasmosis syndrome. In some embodiments, the
disorder is associated with retinal neovascularization (e.g.,
proliferative diabetic retinopathy). In some embodiments, the
disorder is associated with surface neovascularization (e.g.,
secondary to a chemical or other injury, or Stevens-Johnson
syndrome).
[0157] In some embodiments, the disorder is associated with tumor
neovascularization, e.g., vasoproliferative ocular tumours (e.g.,
neoplastic and benign retinal vascular tumors such as retinal
capillary hemangioma, hemangioblastomas, cavernous hemangiomas,
Racemose Hemangioma (Wyburn-Mason Syndrome), Retinal
Vasoproliferative Tumors, and tumors associated with Von
Hippel-Lindau (VHL) disease; or choroidal vascular tumors including
circumscribed choroidal hemangiomas and diffuse choroidal
hemangiomas). See, e.g., Turell and Singh, Middle East Afr J
Ophthalmol. 2010 July-September; 17(3): 191-200.
[0158] In addition, the methods described herein include methods
for the treatment of disorders associated with inflammation or
"leaky" vasculature. Ocular inflammatory conditions that may be
treated with the methods described herein include, but are not
limited to, endophthalmitis (e.g., the endogenous form and the
exogenous form), macular edema (e.g., macular edema that occurs as
a result of age-related macular degeneration, cataract surgery,
diabetes, drug toxicity, eye injury, retinal vein occlusion (e.g.,
central retinal vein occlusion (CRVO) and branch retinal vein
occlusion), or other inflammatory eye diseases, e.g., pseudophakic
macular edema), conjunctivitis, episcleritis, keratitis, optic
neuritis, orbital pseudotumor, retinal vasculitis, scleritis, and
uveitis (e.g., (i) uveitis associated with sepsis (e.g.,
LPS-induced uveitis); (ii) autoimmune uveitis (e.g., uveitis
associated with lupus); or (iii) uveitis associated with type II,
type III, type IV, or type V hypersensitivity reactions). See,
e.g., WO2011133964 and WO2013003467.
[0159] Generally, the methods include administering a
therapeutically effective amount of one or more of an AMPK
activator, a PTEN activator, or both, to a subject who is in need
of, or who has been determined to be in need of, such
treatment.
[0160] Examples of routes of administration include systemic
parenteral, e.g., intravenous, intraperitoneal, intradermal, or
subcutaneous; local to the eye, e.g., topical, intravitreal,
intraocular, intraorbital, periorbital, subconjuctival, subretinal,
subtenons or transscleral; and systemic oral administration. In
some embodiments, intraocular administration or administration by
eye drops, ointments, creams, gels, or lotions may be used, inter
alia. In some embodiments, the AMPK or PTEN activator is
administered systemically, e.g., orally; in preferred embodiments,
the AMPK or PTEN activator is administered to the eye, e.g., via
topical (eye drops, lotions, or ointments) administration, or by
local injection, e.g., periocular or intravitreal injection; see,
e.g., Gaudana et al., AAPS J. 12(3):348-360 (2010); Fischer et al.,
Eur J Ophthalmol. 21 Suppl 6:S20-6 (2011). Administration may be
provided as a periodic bolus (for example, intravitreally or
intravenously) or as continuous infusion from an internal reservoir
(for example, from an implant disposed at an intra- or extra-ocular
location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242)) or from an
external reservoir (for example, from an intravenous bag, or a
contact lens slow release formulation system). The AMPK or PTEN
activator may be administered locally, for example, by continuous
release from a sustained release drug delivery device immobilized
to an inner wall of the eye or via targeted transscleral controlled
release into the choroid (see, for example, PCT/US00/00207,
PCT/US02/14279, PCT/US2004/004625, Ambati et al. (2000) Invest.
Ophthalmol. Vis. Sci. 41:1181-1185, and Ambati et al (2000) Invest.
Ophthalmol. Vis. Sci. 41:1186-1191). A variety of devices suitable
for administering agents locally to the inside of the eye are known
in the art. See, for example, U.S. Pat. Nos. 6,251,090, 6,299,895,
6,416,777, 6,413,540, and 6,375,972, and PCT/US00/28187.
[0161] In some embodiments, the treatment is administered to a
subject who has been diagnosed with a disorder associated with
ocular neovascularization; such a diagnosis can be made by a
skilled practitioner using known methods and ordinary skill. In
some embodiments, the methods include a step of diagnosing or
identifying or selecting a subject with a disorder associated with
ocular neovascularization, or identifying or selecting a subject
based on the presence or a diagnosis of a disorder associated with
ocular neovascularization.
[0162] As used in this context, to "treat" means to ameliorate at
least one symptom of the disorder associated with ocular
neovascularization. Often, pathological ocular neovascularization
results in a loss of visual acuity; thus, a treatment can result in
a reduction in ocular vascularity and a return or approach to
normal sight. Administration of a therapeutically effective amount
of a compound described herein for the treatment of a condition
associated with ocular neovascularization will result in decreased
levels or rate of ocular neovascularization (which can prevent or
delay the progression or onset of loss of visual acuity), or a
regression in ocular vascularity.
[0163] The methods described herein include the manufacture and use
of pharmaceutical compositions, which include compounds identified
by a method described herein as active ingredients. Also included
are the pharmaceutical compositions themselves.
[0164] Pharmaceutical compositions typically include a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes saline, solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration.
[0165] Pharmaceutical compositions are typically formulated to be
compatible with its intended route of administration. Examples of
routes of administration include systemic (e.g., parenteral and
oral) and local (ocular, e.g., intravitreal or topical)
administration. Thus also within the scope of the present
disclosure are compositions comprising the AMPK or PTEN activators
described herein in a formulation for administration for the eye,
e.g., in eye drops, lotions, creams, e.g., comprising
microcapsules, microemulsions, or nanoparticles. Methods of
formulating suitable pharmaceutical compositions for ocular
delivery are known in the art, see, e.g., Losa et al.,
Pharmaceutical Research 10:1 (80-87 (1993); Gasco et al., J. Pharma
Biomed Anal., 7(4):433-439 (1989); Fischer et al., Eur J
Ophthalmol. 21 Suppl 6:S20-6 (2011); and Tangri and Khurana, Intl J
Res Pharma Biomed Sci., 2(4):1541-1442 (2011).
[0166] General methods of formulating suitable pharmaceutical
compositions are known in the art, see, e.g., Remington: The
Science and Practice of Pharmacy, 21st ed., 2005; and the books in
the series Drugs and the Pharmaceutical Sciences: a Series of
Textbooks and Monographs (Dekker, NY). For example, solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0167] Pharmaceutical compositions suitable for injectable use can
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and
gelatin.
[0168] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle, which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying, which yield a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0169] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0170] Systemic administration of a therapeutic compound as
described herein can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0171] The pharmaceutical compositions can be included in a
container, pack, or dispenser (e.g., eye drop bottle) together with
instructions for administration. In some embodiments, the
compositions are provided lyophilized or dry, and the kit includes
saline for making a solution comprising the AMPK or PTEN
activator(s).
[0172] Age-Related Macular Degeneration
[0173] Advanced AMD is characterized as "atrophic" or
"neovascular," the former showing loss of outer retinal layers, and
the latter the presence of choroidal neovascularization
(CNV)..sup.8 Neovascular (or "wet") AMD is defined by the formation
of abnormal blood vessels that grow from the choroidal vasculature,
through breaks in Bruch's membrane, toward the outer retina.sup.1.
These blood vessels are immature in nature and leak fluid below or
within the retina..sup.9 The two forms of AMD can occur together
and share pathologies of cell death and fibroglial
replacement..sup.10,11 Neovascular AMD accounts for 10 to 15% of
AMD cases, develops abruptly, and rapidly leads to substantial loss
of vision..sup.9,12 Although growth factors appear to play an
important role in the late stage of neovascular AMD progression,
they likely do not contribute to the underlying cause of the
disease. Current standard of care for patients with CNV involves
targeting the proangiogenic and permeability molecule vascular
endothelial growth factor-A (VEGF)..sup.13-15 However, although
anti-VEGF therapy blocks vascular permeability and angiogenesis, it
does not lead to complete vascular regression..sup.14 Moreover, in
patients treated with VEGF antagonists, substantial vision
improvement occurs in only one-third, with one-sixth of treated
patients still progressing to legal blindness..sup.13,15 Thus,
there is an urgent need for safe nutritional or pharmacological
interventions for the treatment and ideally the prevention of
AMD.
[0174] PTEN Activators
[0175] The methods described herein can include the administration
of a therapeutically effective amount of one or more PTEN
activators. PTEN agonists or activators are agents that directly
stimulate the expression of PTEN in a cell, or directly stimulates
the activity of PTEN; such agonists include
di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2 and PI(5)P (but
PI(4)P, PI(3,4)P2, and PI(3,5)P2 do not activate PTEN).
Alternatively or in addition, any of the methods described herein
can include the administration of PPARgamma agonists such as
rosiglitazone (Patel et al., (2001) Current. Biol. 11:764-8), or a
compound that down regulates the PI3K/Akt/mTOR pathway, e.g., an
inhibitor of mTOR, which is considered herein to be a PTEN agonist.
Preferred PTEN agonists/mTOR inhibitors for use in the methods
described herein include rapamycin (Rapamune.RTM., sirolimus, ATC
code L04AA10 commercially available from Wyeth) and its chemical
analogues such as CCI-779 (temsirolimus, Anatomical Therapeutic
Chemical (ATC) code L01XE09, commercially available from Wyeth),
RAD-001 (everolimus, ATC code L04AA18. commercially available from
Novartis) and AP-2357 (Granville et al, op. cit.). Other agonists
include zinc finger proteins or nucleic acids encoding the same
that bind to and activate transcription of PTEN (see, e.g., WO
00/00388). Other PTEN agonists are described in US20070280918.
Whereas proteins are typically administered parenterally, e.g.
intravenously, small molecules may be administered parenterally or
orally.
[0176] AMPK Activators
[0177] The methods described herein can include the administration
of a therapeutically effective amount of one or more AMPK
activators. A number of small molecule inhibitors of AMPK are known
in the art, including C24 (Li et al., Toxicol Appl Pharmacol. 2013
Dec. 1; 273(2):325-34); A-769662
(4-hydroxy-3-[4-(2-hydroxyphenyl)phenyl]-6-oxo-7H-thieno[2,3-b]pyridine-5-
-carbonitrile; Cool et al., Cell Metab. 2006 June; 3(6):403-16);
D942 (5-[3-[4-[2-(4-fluorophenyl)ethoxy]phenyl]propyl]
furan-2-carboxylic acid); ZLN024 (see FIG. 1A of Zhang et al., PLoS
ONE 8(8): e72092 (2013)). Other known AMPK activators include drugs
such as 5-Aminoimidazole-4-carboxamide riboside (AICA riboside or
AICAR); AICA ribotide (ZMP); guanidine; galegine; metformin
(dimethylbiguanide); phemformin (phenethylbiguanide); antifolate
drugs that inhibit AICAR transformylase (e.g., methotrexate,
pemetrexed); thiazolidinediones (e.g., rosiglitazone, pioglitazone,
or troglitazone); 2-Deoxyglucose (2DG); phenobarbital; PT1; and
salicylate. See, e.g., Hardie et al. (2012) Chem. Biol.
19:1222-1236; Hawley et al. (2012) Science 336:918-922. In
addition, AMPK activators are described in the following: U.S. Pat.
No. 8,604,202B2 (Merck); U.S. Pat. No. 8,592,594B2 (Roche); U.S.
Pat. No. 8,586,747B2 (Roche); U.S. Pat. No. 8,563,746B2 (Merck);
U.S. Pat. No. 8,546,427B2 (Roche); U.S. Pat. No. 8,563,729B2
(Merck); U.S. Pat. No. 8,394,969B2 (Merck); U.S. Pat. No.
8,329,914B2 (Merck); U.S. Pat. No. 8,329,738B2 (Merck);
US20120172333A1 (GSK); US20110060001A1 (Merck); US20090105293A1
(Merck); EP2519527B1 (Poxel); and WO2010073011A2 (Betagenon).
[0178] Combination Therapies
[0179] In some embodiments, the methods described herein are
administered in combination with another therapy. Thus, the methods
can optionally include administration (e.g., in the same
composition, or separately but during the same time frame as the
administration of an AMPK activator, a PTEN activator, or both) of
one or more additional therapies or active agents. For example, the
present methods can be used in combination with other established
treatments such as anti VEGF therapies, non-steroidal or steroidal
anti-inflammatory treatments, or neuroprotective treatments. For
example, to treat inflammatory disease, corticosteroids,
antimetabolites, cycloplegics, and biologics can be used in
combination with an AMPK activator, a PTEN activator, or both, to
control the inflammatory process.
[0180] In some embodiments a neuroprotective treatment is
administered in combination with an AMPK activator, a PTEN
activator, or both; a neuroprotective treatment can include, for
example, administration of a hydrophilic bile acid (e.g., a
ursodeoxycholic acid (UDCA) or a tauroursodeoxycholic acid
(TUDCA)), e.g., as described in WO 2013025840 A1; administration of
a necrosis inhibitor, e.g., RIP-3 kinase inhibitor, e.g., a
necrostatin, e.g., necrostatin-1, alone or combined with an
apoptotic inhibitor (e.g., a pan-caspase inhibitor, e.g., Z-VAD
and/or IDN-6556), as described in WO2012061045 and
WO2011133964.
[0181] In some embodiments, one or more anti-VEGF therapies are
administered in combination with an AMPK activator, a PTEN
activator, or both; anti-VEGF therapies are known in the art and
include Avastin (Bevacizumab) monoclonal antibody that inhibits
VEGF-A; Lucentis (Ranibizumab) monoclonal Fab antibody fragment
that inhibits VEGF-A; Eylea (Aflibercept) fusion protein that binds
VEGF-A, VEGF-B and PGF; Zaltrap (Aflibercept used for cancer
treatment); and Macugen (Pegaptanib) aptamer that binds VEGF. See,
e.g., US20130209570 (Carasquillo, Miller, MEEI).
EXAMPLES
[0182] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1. AMPK Inhibits Oxidative Stress Induced Caveolin-1
Phosphorylation and Endocytosis by Suppressing the Dissociation
Between c-Abl and Prdx1 in Endothelial Cells
[0183] This Example demonstrates that activation of AMPK inhibits
oxidative stress induced caveolin-1 phosphorylation and endocytosis
and this effect is mediated in part by stabilizing the interaction
between c-Abl and prdx-1.
[0184] Materials and Methods
[0185] The following materials and methods were used in Example
1.
[0186] Materials--
[0187] Antibodies for (p-) caveolin-1, (p-) c-Abl, Peroxiredoxin I
(Prdx1), (p-) AMPK, AMPK.alpha.1, AMPK.alpha.2, and VE-cadherin
were purchased from Cell Signaling Technologies (Beverly, Mass.).
Antibodies for .beta.-actin and p-caveolin-1 (for
immunofluorescence) were obtained from Abcam (Cambridge, Mass.) and
R&D Systems (Minneapolis, Minn.), respectively. Secondary
antibodies of Alexa Flour 488 goat anti mouse IgG and Alexa Flour
647 goat anti rabbit IgG were purchased from Invitrogen (Carlsbad,
Calif.). 5-amino-4-imidazole carboxamide riboside (AICAR); a
pharmacological activator of AMPK, was purchased from Toronto
Research Chemicals (Toronto, ON, Canada). Hydrogen Peroxide
(H.sub.2O.sub.2), 5-Iodotubericidin (IODO) and dipyridamole (DPY)
were purchased from Sigma (St. Louis, Mo.). Imatinib mesylate,
c-Abl inhibitor, was purchased from Cayman Chemicals (Ann Arbor,
Mich.). SiRNAs targeting c-Abl, AMPK.alpha.1, AMPK.alpha.2 and
Prdx1, and control siRNA were purchased from Thermoscientific
(Rockford, Ill.).
[0188] Cell Culture--
[0189] HUVECs (Lonza, Walkersville, Md.) were cultured in
Endothelial Growth Medium (EGM, Lonza, Walkersville, Md.). For all
experiments, cells were grown at 37.degree. C. in a humidified
atmosphere of 5% CO2 and 95% air. Experiments were performed on
cells below passage 3 to 6 grown to 80-90% confluence.
[0190] Protein Extraction and Western Blotting--
[0191] Protein extraction and western blotting were carried out as
described previously (Morizane et al. JBC). Densitometric analysis
of bands was performed using ImageJ software. Lane-loading
differences were normalized by .beta.-actin.
[0192] Immunoprecipitation--
[0193] Immunoprecipitation was performed with the Universal
Magnetic Co-IP Kit (Active Motif North America, Carlsbad, Calif.),
according to the manufacturer's instruction.
[0194] siRNA--
[0195] Cells were transfected with siRNAs using Nucleofection kit
(Amaxa Biosysteins, Gaithersburg, Md.), following the
manufacturer's protocol. The medium was changed at 6 h after
transfection. The down-regulation of each protein was evaluated at
3 days after nucleofection.
[0196] Albumin Endocytosis Assay--
[0197] After serum starvation for overnight, HUVECs were pretreated
with AICAR (2 mM) for 2 h, and then stimulated with H.sub.2O.sub.2
(2 mM) for 30 min. We added BSA conjugated with Alexa 555 (50
.mu.g/ml, Life Technologies, Gaithersburg, Md.) in the medium
during the experiment. Cells on coverslips were washed three times
with cold TBS and fixed in 100% methanol at -20.degree. C. for 15
min. Cells were then permeabilized in 0.3% Triton X-100, 0.15% BSA
in TBS with 0.05% Tween 20 (TBST) for 15 min at room temperature
and blocked with 0.5% skim milk in TBST for 60 min at room
temperature. Cells were incubated in p-caveolin-1 antibody diluted
1:200 and VE-cadherin antibody diluted 1:400 for overnight at
4.degree. C., and then incubated for 2 h in secondary antibody
diluted 1:300. Cells were then rinsed three times in TBST before
mounting in Toto3 (Life Technologies, Gaithersburg, Md.). Images
were acquired with confocal microscope, Leica TCS SP2 spectral
confocal laser scanning microscope (Leica Microsystems, Wetzlar,
Germany).
[0198] Statistical Analysis--
[0199] All experiments were repeated a minimum of three times. All
data were expressed as means.+-.S.D. Statistical differences were
analyzed by the unpaired. Student's t test. Differences were
considered significant at P<0.05.
[0200] 1.1 AICAR Suppresses Oxidative Stress Induced
Phosphorylation of Caveolin-1 and c-Abl.
[0201] It has been already reported that caveolin-1 is
phosphorylated on tyrosine 14 under hyperosmotic shock and
oxidative stress (17,18) and that c-Abl, which is an upstream
kinase of caveolin-1, is required for oxidative stress-induced
phosphorylation of caveolin-1 (19). To study the effect of
oxidative stress on the phosphorylation of caveolin-1 and c-Abl in
HUVEC, we exposed HUVEC to H.sub.2O.sub.2 and determined the
phosphorylation by western blotting. Incubation with H.sub.2O.sub.2
resulted in the phosphorylation of both caveolin-1 and c-Abl
dose-dependently (FIG. 1A). To investigate whether AMPK activation
inhibits oxidative stress induced phosphorylation of caveolin-1 and
c-abl, we pretreated HUVEC with a pharmacological activator of
AMPK, AICAR, prior to H.sub.2O.sub.2 exposure. As shown in FIGS.
1A, B and C), AICAR significantly suppressed the phosphorylation of
both caveolin-1 and c-Abl. Caveolin-1 is the main component of the
caveolae plasma membranes and involved in receptor-independent
endocytosiss (2,20). To determine the effect of H.sub.2O.sub.2 and
AICAR on the endocytosis, we evaluated the amount of
fluorescein-conjugated albumin endocytosed by HUVEC. Exposure to
H.sub.2O.sub.2 resulted in the elevation of albumin endocytosis
together with caveolin-1 phosphorylation (FIG. 1D). By contrast,
pretreatment by AICAR suppressed both endocytosis and caveolin-1
phosphorylation (FIG. 1D).
[0202] 1.2 AICAR Inhibits H.sub.2O.sub.2 Induced Phosphorylations
of Caveolin-1 Via Activation of AMPK.
[0203] It has been reported that AICAR has several effects
independent of AMPK pathway (21-24). To determine the effect of
AICAR on AMPK phosphorylation in HUVEC, we investigated
phosphorylation of AMPK after AICAR administration by western
blotting. As shown in FIG. 2A, AICAR phosphorylated AMPK
dose-dependently. We next used 2 different inhibitors of AICAR, DPY
and IODO, to exclude the possibility that the inhibitory effect of
AICAR on caveolin-1 phosphorylation was caused by mechanisms other
than AMPK activation. DPY blocks adenosine transporters and
prevents uptake of AICAR into the cells (11,25). IODO inhibits
adenosine kinase in the cell and prevents conversion of AICAR to
ZMP, which activates AMPK (11,25). Pretreatment with DPY or IODO
inhibited AICAR induced AMPK phosphorylation dose-dependently
(FIGS. 2, B and F). Furthermore, pretreatment with DPY or IODO
prior to H.sub.2O.sub.2 exposure significantly restored the
inhibitory effect of AICAR on phosphorylation of both caveolin-1
and c-Abl (FIG. 2). These results indicate that ZMP accumulation
through both transport and phosphorylation of AICAR is required for
the suppression of caveolin-1 phosphorylation, suggesting that AMPK
activation is a key process for the inhibitory effect of AICAR.
[0204] 1.3 Both AMPK.alpha.1 and .alpha.2 Isoforms are Required for
the Inhibition of Caveolin-1 Phosphorylation Under Oxidative
Stress.
[0205] The catalytic subunit of AMPK, AMPK.alpha., has two isoforms
(i.e. AMPK.alpha.1 and .alpha.2), which show differential
tissue-specific expression (8,9,15). To determine the role of both
isoforms in the inhibitory effect of AMPK on caveolin-1
phosphorylation under oxidative stress, we used RNA interference
technology to knock down AMPK.alpha.1 or .alpha.2 in HUVEC.
Knockdown of either isoform of AMPK.alpha. abolished the inhibitory
effect of AICAR on H.sub.2O.sub.2 induced phosphorylation of
caveolin-1 and c-Abl (FIG. 3). Knockdown of both AMPK isoforms with
two different siRNA oligos showed similar results (FIG. 4). These
results suggest that both AMPK.alpha.1 and .alpha.2 isoforms are
required to inhibit caveolin-1 phosphorylation under oxidative
stress.
[0206] 1.4 Inhibitory Effect of AMPK on Caveolin-1 Phosphorylation
Under Oxidative Stress is Dependent on c-Abl.
[0207] Next, to determine the role of c-Abl in the oxidative stress
induced phosphorylation of caveolin-1, we utilized a c-Abl
inhibitor, imatinib mesylate (26,27). As shown in FIGS. 5 A, B and
C, imatinib mesylate inhibited H.sub.2O.sub.2 induced
phosphorylation of both caveolin-1 and c-Abl dose- and
time-dependently, indicating that c-Abl is an upstream kinase of
caveolin-1 in HUVEC. We next investigated the role of c-Abl in the
inhibitory effect of AICAR on caveolin-1 phosphorylation by knock
down c-Abl with siRNA. Deletion of c-Abl resulted in the
significant decrease in caveolin-1 phosphorylation after
H.sub.2O.sub.2 exposure (FIGS. 5 D and E). Furthermore,
pretreatment with AICAR prior to H.sub.2O.sub.2 exposure did not
change caveolin-1 phosphorylation significantly, suggesting that
inhibitory effect of AICAR on caveolin-1 phosphorylation under
oxidative stress is dependent on c-Abl (FIGS. 5 D and E).
[0208] 1.5 Prdx1 is Indispensable for the Inhibitory Effect of AMPK
on the H.sub.2O.sub.2 Induced Phosphorylation of Caveolin-1.
[0209] Prdx1, one of the antioxidant enzymes, plays a protective
role in cells against oxidative stress. In cytoplasm, prdx1 exists
as a protein complex with c-Abl-SH domain (28-31), and protects
c-Abl from phosphorylation (32). Under oxidative stress, oxidant
dissociates the protein-protein interaction and phosphorylates
liberated c-Abl. To investigate the role of prdx1 in the inhibitory
effect of AICAR on caveolin-1 phosphorylation, we knocked down
prdx1 in HUVEC with siRNA and determined the level of caveolin-1
phosphorylation by western blotting. As shown in FIGS. 6 A, B and
C, knockdown of prdx1 resulted in increased phosphorylation of both
caveolin-1 and c-Abl after H.sub.2O.sub.2 exposure. Furthermore,
lack of prdx1 abolished the inhibitory effect of AICAR on the
H.sub.2O.sub.2 induced phosphorylation of both caveolin-1 and
c-Abl. These results indicate that prdx1 is indispensable for the
inhibitory effect of AMPK on the H.sub.2O.sub.2 induced
phosphorylation of caveolin-1.
[0210] 1.6 AMPK Inhibits Caveolin-1 Phosphorylation Under Oxidative
Stress by Suppressing the Dissociation Between Prdx1 and c-Abl.
[0211] To investigate the relationship between AMPK and protein
interaction of c-Abl and prdx1, we performed co-immunoprecipitation
experiments. As shown in FIG. 6D, oxidative stress resulted in the
dissociation between prdx1 and c-Abl. The dissociation was
inhibited by treatment with AICAR prior to H.sub.2O.sub.2. In
contrast, treatment with IODO prior to H.sub.2O.sub.2 and AICAR
restored the dissociation, indicating AICAR inhibits the
H.sub.2O.sub.2 induced dissociation between c-Abl and prdx1. To
confirm this, we further conducted co-immunoprecipitation for the
cell lysates from HUVEC lacking AMPK.alpha.1 or .alpha.2. Deletion
of either AMPK.alpha.1 or .alpha.2 isoform decreased the inhibitory
effect of AICAR on the dissociation between c-Abl and prdx1 (FIG. 6
F, G). These results indicated that activation of AMPK inhibits
caveolin-1 phosphorylation under oxidative stress by suppressing
the dissociation between prdx1 and c-Abl.
[0212] 1.7 AMPK is not Detected in the Prdx1/c-Abl Complex.
[0213] To further investigate the mechanism, we asked if AMPK
directly associates with the c-abl/prdx1 complex.
Co-immunoprecipitation experiments (FIG. 6 H), failed to show any
direct association. This could be because the association is very
weak or because the effects of AMPK on the prdx1/c-abl complex are
indirect.
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Example 2. The AMPK Agonist AICAR Suppresses VEGF Stimulated Tube
Formation, Transcytosis, Endocytosis, Caveolin-1 Phosphorylation
and Prdx1/c-Abl Dissociation
[0266] This Example shows that AMPK activation inhibits VEGF tube
formation through PTEN dependent dephosphorylation of Akt and
suppresses VEGF induced caveolin-1 phosphorylation through
stabilization of c-alb/prdx1 complex.
[0267] Materials and Methods
[0268] The following materials and methods were used in Example
2.
[0269] Materials
[0270] Antibodies for (p-)VEGFR2, (p-)caveolin-1, (p-)c-Abl,
(p-)Akt, (p-) PTEN, peroxiredoxin I (Prdx1), (p-)AMPK,
AMPK.alpha.1, AMPK.alpha.2 and VE-cadherin were purchased from Cell
Signaling Technologies (Beverly, Mass.). Antibodies for
.beta.-actin was purchased from Abcam (Cambridge, Mass.). CD31 and
p-caveolin-1 (for immunofluorescence) were obtained from R&D
Systems (Minneapolis, Minn.). Secondary antibodies of Alexa Fluor
488 goat anti-mouse IgG and Alexa Fluor 647 goat anti-rabbit IgG
were purchased from Invitrogen (Carlsbad, Calif.).
5-amino-4-imidazole carboxamide riboside (AICAR), a pharmacological
activator of AMPK, was purchased from Toronto Research Chemicals
(Toronto, ON, Canada). 5-Iodotubericidin (IODO), dipyridamole (DPY)
and suramin were purchased from Sigma (St. Louis, Mo.). VEGF was
purchased from R&D Systems. Imatinib mesylate, a c-Abl
inhibitor, was purchased from Cayman Chemicals (Ann Arbor, Mich.).
The in vitro vascular permeability assay kit was from Millipore
(Beverly, Mass., USA). SiRNAs targeting c-Abl, AMPK.alpha.1 and
.alpha.2 and Prdx1, PTEN and control siRNA were purchased from
Thermoscientific (Rockford, Ill.).
[0271] Cell Cultures.
[0272] Human umbilical vein endothelial cells (HUVECs) were
cultured in endothelial growth medium (EGM, Lonza, Walkersville,
Md.). Normal human dermal fibroblasts (NHDFs) were cultured in
fibroblast basal medium (ATCC, Rockville, Md.). Cells were grown at
37.degree. C. in a humidified atmosphere of 95% air, 5% CO2.
Experiments were performed on cells below passage 3 to 6 grown to
80%-90% confluence.
[0273] In Vitro Angiogenesis (Tube Formation) Assay.
[0274] To evaluate the effects of AICAR on angiogenesis, HUVECs
were cocultured with NHDFs in a 24-well plate with or without VEGF
and AICAR. NHDF cells were seeded at 1.0.times.10.sup.5 cells/well
and then cultured for 2 wks to form a fibroblast cell sheet. HUVECs
were then seeded at 5.0.times.10.sup.3 cells/well on the sheets,
and the next day, AICAR (2 mM) and VEGF (12.5 ng/mL) were added to
each well. Suramin (50 .mu.M) was used as the inhibitor of VEGF. At
3, 7 and 10 days after treatment, cells were fixed at -20.degree.
C. in ethanol and acetone (1:1). Subsequently, cells were blocked
with 1% bovine serum albumin (BSA) in phosphate-buffered saline for
30 min at room temperature, and then incubated with primary rabbit
anti-human CD31 antibody overnight at 4.degree. C. After the cells
were washed with Tris-buffered saline (TBS), Alexa Flour 488 goat
anti-rabbit IgG was applied for 2 h at room temperature. The tube
length was quantified using a Kurabo Angiogenesis Image Analyzer
(imaging software; Kurabo, Osaka, Japan).
[0275] In Vitro Vascular Permeability Assay.
[0276] We conducted the in vitro vascular permeability assay using
Alexa Flour 555-labeled bovine serum albumin (BSA) (Invitrogen) and
an In Vitro Vascular Permeability Assay kit (Millipore). According
to the manufacturer's instructions, HUVECs were seeded at a density
of 1.times.10.sup.5 on a collagen-coated polystyrene filter. After
a confluent monolayer was formed, each chamber was treated with
AICAR or stimulated with VEGF. To measure endothelial permeability,
100 .mu.L of Alexa Flour 555-BSA solution (0.050 mg/mL) was added
into the insert and incubated for 10 min, and then the insert was
removed and 100 .mu.L medium was collected from the bottom
chamber.
[0277] The fluorescent density of samples was analyzed on a
SPECTRAmax GEMINI XS Microplate Spectrofluorometer (Molecular
Devices, Sunnyvale, Calif.) at excitation and emission wavelengths
of 555 nm and 565 nm, respectively.
[0278] Albumin Endocytosis Assay.
[0279] After overnight serum starvation, HUVECs were pretreated
with AICAR (2 mM) for 2 h and then stimulated with VEGF (12.5
ng/mL) for 10 min. We added BSA conjugated with Alexa 555 (50
.mu.g/mL, Life Technologies, Gaithersburg, Md.) in the medium
during the experiment. Cells on coverslips were washed three times
with cold TBS and fixed in 100% methanol at -20.degree. C. for 15
min. Cells were then permeabilized in 0.3% Triton X-100, 0.15% BSA
in TBS with 0.05% Tween 20 (TBST) for 15 min at room temperature,
and blocked with 0.5% skim milk in TBST for 60 min at room
temperature. Cells were incubated in p-caveolin-1 antibody diluted
1:200 and VE-cadherin antibody diluted 1:400 overnight at 4.degree.
C., and then incubated for 2 h in secondary antibody diluted 1:300.
Cells were then rinsed three times in TBST before mounting in Toto3
(Life Technologies). Images were acquired with a Leica TCS SP2
spectral confocal laser scanning microscope (Leica Microsystems,
Wetzlar, Germany).
[0280] Protein Extraction and Western Blotting.
[0281] Protein extraction and Western blotting were carried out as
described previously (Morizane et al. JBC (26)). We conducted a
densitometric analysis of bands using ImageJ software. Lane-loading
differences were normalized by .beta.-actin.
[0282] Immunoprecipitation.
[0283] Immunoprecipitation was performed with the Universal
Magnetic Co-IP Kit (Active Motif North America, Carlsbad, Calif.)
according to the manufacturer's instruction.
[0284] Small Interfering RNA.
[0285] Cells were transfected with siRNAs using a Nucleofection kit
(Amaxa Biosystems, Gaithersburg, Md.), following the manufacturer's
protocol. The medium was changed 6 h after transfection. The
down-regulation of each protein was evaluated 3 days after
nucleofection.
[0286] Statistical Analysis.
[0287] Data are expressed as means.+-.SDs. Statistical analyses
were performed using the unpaired Student's t-test. Differences
were considered significant at p<0.01 or 0.05.
[0288] 2.1 AMPK Activator AICAR Inhibits VEGF-Induced Vascular Tube
Formation in an in-Vitro Model Through VEGFR2 Independent
Mechanisms.
[0289] Co-culture of HUVECs and NHDFs in the presence of VEGF
results in significant vascular tube formation. Addition of AICAR
(0.25, 0.5, 1.0, 2.0 mM), led to a dose-dependent inhibition of
tube formation (FIG. 8A,B). Phosphorylation of VEGFR2 and Akt
(31-33) by VEGF are key steps of angiogenesis in addition to VEGF
induced dissociation of VEGFR2 from Caveolin-1 in the caveolae (7).
As seen in FIG. 8C-G, AICAR pretreatment had no effect on VEGFR2
phosphorylation or its dissociation from caveolin-1 upon VEGF
stimulation, however it significantly suppressed and delayed VEGF
induced Akt phosphorylation.
[0290] 2.2 AICAR Activation of AMPK Leads to PTEN Dependent
Dephosphorylation of Akt.
[0291] AICAR can mediate its function via both AMPK dependent and
independent pathways (34-37) and there have been many conflicting
reports on the relationship between AMPK and Akt phosphorylation
depending on cell types and experimental system (38-42). Once AICAR
enters a cell, it can be converted to either inosine or ZMP.
Inosine inhibits cells by raising the adenosine concentration,
which is independent of AMPK. By contrast, ZMP is activating the
AMPK pathway. AICAR is converted to ZMP by adenosine kinase (AK),
but this conversion is blocked by the AK inhibitor IODO (19,43). As
shown in FIG. 9, AICAR treatment of endothelial cells resulted in
AMPK and PTEN phosphorylation with similar time course, that was
followed by Akt dephosphorylation. Pretreatment with IODO,
suppressed and delayed the effects of AICAR on AMPK and PTEN
phosphorylation and Akt dephosphorylation. This indicates that AMPK
activation by AICAR is needed for the phosphorylation of PTEN and
dephosphorylation of Akt (FIG. 9A-D). In addition, PTEN knockdown
by siRNA abrogated the effects of AICAR on Akt dephosphyralation,
suggesting that its effects are PTEN dependent (FIG. 9E,F).
[0292] 2.3 AICAR Inhibits VEGF-Induced Albumin Endocytosis and
Leakage in HUVECs in an In Vitro Model and Suppresses VEGF-Induced
Phosphorylation of Caveolin-1 and c-Abl.
[0293] In addition to its role in angiogenesis, VEGF is also a
powerful stimulus of endocytosis and vascular leakage. Exposure of
HUVEC monolayer to VEGF results in increased albumin leakage (FIG.
10A) and endocytosis (FIG. 10B) that can be significantly
suppressed by pretreatment with AICAR (FIG. 10A,B). Increased VEGF
permeability is thought to be partially mediated by Caveolin-1
phosphorylation on Y14 (7,8). Caveolin-1 phosphorylation requires
c-Abl, at least under certain conditions such as oxidative stress
(15). As shown in FIG. 11A-C, AICAR significantly suppressed the
VEGF induced phosphorylation of caveolin-1 and c-Abl.
[0294] 2.4 AICAR Suppresses VEGF-Induced Caveolin-1, c-Abl and Akt
Phosphorylation Likely Via AMPK.
[0295] To determine if the effects of AICAR are mediated via AMPK,
several set of experiments were performed. AICAR administration
resulted in dose dependent AMPK phosphorylation in HUVEC cells
(FIG. 12A). To confirm AMPK phosphorylation was due to
intracellular AICAR, HUVEC cells were pretreated with Adenosine
transporter inhibitor DPY or with the AK inhibitor IODO (19,43).
Blocking AICAR receptors with DPY inhibited AMPK phosphorylation
(FIG. 12B). IODO inhibition of AICAR conversion to ZMP, the direct
activator of AMPK, significantly abolished the inhibitory effect of
AICAR on the phosphorylation of caveolin-1, c-Abl and Akt (FIG.
12D,E). These results suggest that AMPK activation is a key process
for the inhibitory effect of AICAR on VEGF induced phosphorylation
of caveolin-1, c-Abl and Akt phosphorylation.
[0296] 2.5 Both AMPK.alpha.1 and .alpha.2 Isoforms are Required for
AICAR Inhibition of VEGF Dependent Caveolin-1, c-Abl and Akt
Phosphorylation.
[0297] AMPK has two catalytic subunit isoforms (AMPK.alpha.1 and
.alpha.2) (16,17,26). To determine the role of each isoform, we
used siRNA. Knockdown of either isoform of AMPK.alpha. abolished
the inhibitory effect of AICAR on the VEGF-induced phosphorylation
of caveolin-1, c-Abl and Akt (FIG. 13). These results suggest that
both AMPK isoforms (.alpha.1 and .alpha.2) are required for AICAR
to inhibit VEGF dependent c-alb, Akt, and caveolin-1
phosphorylation.
[0298] 2.6 c-Abl is Required for VEGF Dependent Caveolin-1
Phosphorylation.
[0299] To determine the role of c-Abl in the VEGF-induced
phosphorylation of caveolin-1, we used a c-Abl inhibitor, imatinib
mesylate (44,45). As shown in FIG. 14 A-D, imatinib mesylate
inhibited the VEGF-induced phosphorylation of caveolin-1 and c-Abl
in a time and dose dependent manner, indicating that c-Abl is an
upstream kinase of caveolin-1 in HUVECs. We next investigated the
role of c-Abl in the inhibitory effect of AICAR on caveolin-1
phosphorylation by the knockdown of c-Abl with siRNA. Deletion of
c-Abl resulted in a significant decrease in caveolin-1
phosphorylation after VEGF exposure (FIG. 14 E-G). These results
indicate that VEGF induced caveolin-1 phosphorylation requires
c-abl.
[0300] 2.7 Prdx1 is Indispensable for the Inhibitory Effect of AMPK
Activator AICAR on the VEGF-Induced Phosphorylation of Caveolin-1
and c-Abl.
[0301] Prdx1, one of the antioxidant enzymes that exists in a
complex with c-Abl and plays a protective role in cells against
oxidative stress (46-49). Dissociation of this complex is thought
to lead to phosphorylation of c-Abl and subsequent Caveolin-1
phosphorylation by c-Abl. To investigate the role of this complex
on AICAR inhibition of VEGF induced Caveolin-1 phosphorylation, we
knocked down Prdx1 in HUVECs with siRNA. As shown in FIG. 15A-C,
knockdown of prdx1 resulted in increased phosphorylation of
caveolin-1 and c-Abl after VEGF exposure. Furthermore, lack of
prdx1 abolished the inhibitory effect of AICAR on the VEGF-induced
phosphorylation of caveolin-1 and c-Abl. These results indicate
that prdx1 is indispensable for the inhibitory effect of AMPK
activator AICAR on the VEGF-induced phosphorylation of caveolin-1
and c-Abl.
[0302] 2.8 AMPK Inhibits VEGF Dependent Caveolin-1 Phosphorylation
by Suppressing the Dissociation Between Prdx1 and c-Abl.
[0303] AICAR inhibited the VEGF induced dissociation between prdx1
and c-Abl (FIG. 15 D). This inhibition was abrogated by the AK
inhibitor IODO suggesting that the effects of AICAR were AMPK
mediated (FIGS. 15D,E). Supporting this conclusion, siRNA knockdown
of either AMPK.alpha.1 or .alpha.2 isoform decreased the ability of
AICAR to inhibit the VEGF induced dissociation between prdx1 and
c-Abl (FIGS. 15F,G). These results indicated that AMPK mediates the
AICAR suppression of VEGF induced prdx1/c-abl dissociation and
subsequent caveolin-1 phosphorylation.
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OTHER EMBODIMENTS
[0376] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
4119RNAArtificial Sequencealpha1 PRKAA1 siRNA 1ccauacccuu gaugaauua
19219RNAArtificial Sequencealpha2 PRKAA2 siRNA 2cgacuaagcc
caaaucuuu 19319RNAArtificial Sequencealpha1prime PRKAA1 siRNA
3gcccagaggu agauauaug 19419RNAArtificial Sequencealpha2prime PRKAA2
siRNA 4gagcauguac cuacguuau 19
* * * * *