U.S. patent application number 14/597390 was filed with the patent office on 2015-04-30 for modulation of estrogen receptor-related receptor gamma (err gamma) and uses therefor.
This patent application is currently assigned to Salk Institute for Biological Studies. The applicant listed for this patent is Salk Institute for Biological Studies. Invention is credited to Michael Downes, Ronald M. Evans, Vihang A. Narkar, Ruth T. Yu.
Application Number | 20150119315 14/597390 |
Document ID | / |
Family ID | 47219629 |
Filed Date | 2015-04-30 |
United States Patent
Application |
20150119315 |
Kind Code |
A1 |
Narkar; Vihang A. ; et
al. |
April 30, 2015 |
MODULATION OF ESTROGEN RECEPTOR-RELATED RECEPTOR GAMMA (ERR GAMMA)
AND USES THEREFOR
Abstract
This application provides methods of increasing vascularization,
muscle performance, muscle rehabilitation, and/or mitochondrial
activity in subjects in need thereof, by administering a
therapeutically effective amount of one or more agents that
increases ERR.gamma. activity to the subject. Such agents can
include one or more ERR.gamma. agonists. In some examples the
method does not require that the subject exercise, and as such, the
subject may be sedentary (such as bedridden or in a
wheelchair).
Inventors: |
Narkar; Vihang A.; (Houston,
TX) ; Downes; Michael; (San Diego, CA) ; Yu;
Ruth T.; (La Jolla, CA) ; Evans; Ronald M.;
(La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Salk Institute for Biological Studies |
La Jolla |
CA |
US |
|
|
Assignee: |
Salk Institute for Biological
Studies
La Jolla
CA
|
Family ID: |
47219629 |
Appl. No.: |
14/597390 |
Filed: |
January 15, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13410142 |
Mar 1, 2012 |
8962546 |
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14597390 |
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61447704 |
Mar 1, 2011 |
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Current U.S.
Class: |
514/1.1 ;
514/150; 514/438; 514/44R; 514/615 |
Current CPC
Class: |
A61K 38/177 20130101;
A61P 25/00 20180101; A61P 21/00 20180101; A61K 31/166 20130101;
A61K 31/655 20130101; A61K 48/0058 20130101; A61K 48/005 20130101;
A61P 9/00 20180101; A61K 48/0066 20130101; A61K 31/381 20130101;
A61P 13/12 20180101; A61K 38/1796 20130101; A61K 48/00
20130101 |
Class at
Publication: |
514/1.1 ;
514/44.R; 514/615; 514/438; 514/150 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 31/655 20060101 A61K031/655; A61K 31/381 20060101
A61K031/381; A61K 38/17 20060101 A61K038/17; A61K 31/166 20060101
A61K031/166 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under
AR053803-03 awarded by the National Institute of Arthritis and
Musculoskeletal and Skin Diseases; under HD027183 and DK057978
awarded by the National Institutes of Health, Department of Health
and Human Services; and under U19DK62434-01 awarded by the Nuclear
Receptor Signaling Atlas. The government has certain rights in the
invention.
Claims
1. A method of increasing mitochondrial activity, comprising:
administering a therapeutically effective amount of one or more
agents that increases ERR.gamma. activity to a mammal needing
increased mitochondrial activity; and not exercising the
mammal.
2. The method of claim 1, wherein the method further comprises:
selecting a mammal in need of increased mitochondrial activity or a
mammal at risk for developing a disorder that can benefit from
increased mitochondrial activity.
3. The method of claim 1, wherein increased mitochondrial activity
is needed in the mammal's muscle, brain or ear.
4. The method of claim 1, wherein the mammal has or is at risk for
muscle atrophy, muscle wasting, sarcopenia, hearing loss,
mitochondrial encephalomyopathy, lactic acidosis, Leber's
hereditary optic neuropathy (LHON), or stroke-like episode syndrome
(MELAS).
5. The method of claim 1, wherein the one or more agents that
increases ERR.gamma. activity comprises: a nucleic acid molecule
encoding ERR.gamma.; one or more ERR.gamma. agonists; an ERR.gamma.
protein; or combinations thereof.
6. The method of claim 1, wherein the ERR.gamma. agonist is:
##STR00003## or combinations thereof.
7. The method of claim 1, wherein the one or more agents that
increases ERR.gamma. activity is: ##STR00004##
8. The method of claim 1, wherein the one or more agents that
increases ERR.gamma. activity is: ##STR00005## wherein R is H
(DY162), p-CH.sub.3 (DY163), 2-Cl, 3-CF.sub.3 (DY165), p-CF.sub.3
(DY168), p-OCH.sub.3 (DY169), 3-NO.sub.2, 4CF.sub.3 (DY170),
2,3-O.sub.2CH.sub.3 (DY174), or m-CH.sub.3 (DY159), ##STR00006##
wherein X is S and R is 5-CH.sub.3 (DY166), 5-CH.sub.2CH.sub.3
(DY164), or 5-NO.sub.2 (DY167); wherein X is O and R is
4,5-CH.sub.3 (DY173) or CH.sub.2CH.sub.3 (DY175), or wherein X is
CH, and R is 2-Cl, 3-CF.sub.3, p-CF.sub.3; p-OCH.sub.3, 3-NO.sub.2,
4-CF.sub.3; or 2,3-O.sub.2CH.sub.3; ##STR00007## wherein R is H
(DY117) or R is Br (DY172), ##STR00008## wherein m is 0, 1 or 2; n
is 0, 1 or 2; R.sub.1 and R.sub.7 are independently selected from
1) H; 2) Halo; 3) OH; 4) (C=0).sub.a, O.sub.bC.sub.1-C.sub.4 alkyl,
wherein a is 0 or 1 and b is 0 or 1, wherein the alkyl can be
substituted by 0, 1 or more substituted groups independently
selected from H or C3C6 heterocyclyl; 5) (C=0),
O.sub.bC.sub.3-C.sub.6 cycloalkyl, wherein a is 0 or 1 and b is 0
or 1; R2 is selected from: 1) H; 2) C.sub.1-C.sub.3 alkyl, wherein
the alkyl can be substituted by 0, 1 or more substituted groups
independently selected from H or C.sub.3C.sub.6 heterocyclyl; 3)
C.sub.3-C.sub.6 cycloalkyl; ##STR00009## or combinations
thereof.
9. The method of claim 3, wherein increased mitochondrial activity
is needed in the mammal's skeletal muscle, and the mammal has or is
at risk for muscle atrophy, sarcopenia, or muscle wasting.
10. The method of claim 3, wherein increased mitochondrial activity
is needed in the mammal's cardiac muscle, and the mammal has or is
at risk for a heart attack or ischemia in a cardiac muscle.
11. The method of claim 1, wherein the mammal cannot exercise or is
sedentary.
12. The method of claim 1, wherein the mammal is a human.
13. The method of claim 1, wherein the administration comprises
parenteral, subcutaneous, intraperitoneal, intrapulmonary, or
intranasal administration.
14. A method of increasing mitochondrial activity by at least 25%,
comprising: administering a therapeutically effective amount of one
or more agents that increases ERR.gamma. activity to a mammal
needing increased mitochondrial activity; and not exercising the
mammal, wherein the increase of at least 25% as compared is
relative to an amount of mitochondrial activity in the absence of
administration of the one or more agents that increases ERR.gamma.
activity.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 13/410,142 filed Mar. 1, 2012, now U.S. Pat. No. ______, which
claims priority to U.S. Provisional Application No. 61/447,704
filed Mar. 1, 2011, herein incorporated by reference.
FIELD
[0003] This application relates to methods of increasing
vascularization, muscle performance, and/or mitochondrial activity
in subjects in need thereof, by administering a therapeutically
effective amount of one or more agents that increases ERR.gamma.
activity to the subject. In some examples the subject does not
receive additional exercise, and as such, the subject may be
sedentary (such as bedridden or in a wheelchair).
BACKGROUND
[0004] Tissue vascular supply is tightly coupled to its oxidative
capacity. This is especially evident in skeletal muscle beds, each
enriched in either oxidative slow-twitch or glycolytic fast-twitch
myofibers (Fluck and Hoppeler, 2003; Pette and Staron, 2000).
Slow-twitch muscles are characterized by high mitochondrial
content, fatigue resistant (type I) fibers and dense vascularity to
ensure a steady and prolonged supply of oxygen and nutrients (Annex
et al., 1998; Cherwek et al., 2000; Ripoll et al., 1979).
Fast-twitch (type II) muscles generally have lower oxidative
capacity, a reduced blood supply and are fatigue sensitive. How the
type I vs. the type II muscle vasculature is specified to match
oxidative capacity is unclear.
[0005] Previous studies established that nuclear receptors such as
PPAR.alpha., PPAR.delta. and ERR.alpha. along with co-regulators
PGC1.alpha., PGC1.beta. and Rip140 control diverse aspects of
aerobic respiration including fatty acid oxidation, oxidative
phosphorylation and mitochondrial biogenesis in skeletal muscle
(Arany et al., 2007; Huss et al., 2004; Lin et al., 2002; Minnich
et al., 2001; Muoio et al., 2002; Seth et al., 2007; Wang et al.,
2004). While signaling factors such as TGF.beta.1, platelet-derived
growth factor, fibroblast growth factor (FGF) 1 and 2, and vascular
endothelial growth factor (VEGF) are known to stimulate
angiogenesis (Carmeliet, 2000; Ferrara and Kerbel, 2005; Gustafsson
and Kraus, 2001), whether and how these factors orchestrate dense
vascularization of aerobic muscles is unclear. One possibility is
vascular arborization by co-activator PGC1.alpha. that is induced
by hypoxia and exercise (Arany et al., 2008). However, PGC1.alpha.
knockout mice are viable, still retain oxidative muscle, and have
normal vasculature (Arany et al., 2008; (Lin et al., 2004). Since
the intrinsic enrichment of blood flow to aerobic muscles in the
absence of exercise is unlikely to depend on PGC1.alpha. induction,
we speculate the existence of a novel regulatory angiogenic
pathway.
[0006] Estrogen receptor-related receptor .gamma. (ERR.gamma.),
like other members of the ERR subfamily, is a constitutively active
orphan nuclear receptor, though unlike ERR.alpha. and .beta., it is
more selectively expressed in metabolically active and highly
vascularized tissues such as heart, kidney, brain and skeletal
muscles (Giguere, 2008; Heard et al., 2000; Hong et al., 1999). In
vitro data indicate that ERR.gamma. activates genes such as PDK4
and MCAD that play a regulatory role in oxidative fat metabolism
(Huss et al., 2002; Zhang et al., 2006). Furthermore, a
comprehensive gene expression analysis identified ERR.gamma. as a
key regulator of multiple genes linked to both fatty acid oxidation
and mitochondrial biogenesis in cardiac muscles (Alaynick et al.,
2007; Dufour et al., 2007). Expression of ERR.gamma. is also
induced in variety of tumors with hyper-metabolic demands and
abundant vasculature (Ariazi et al., 2002; Cheung et al., 2005; Gao
et al., 2006).
SUMMARY
[0007] The ability of ERR.gamma. in controlling the intrinsic
angiogenic pathway in oxidative slow-twitch muscles is demonstrated
herein. It is shown herein that ERR.gamma. is exclusively and
abundantly expressed in oxidative (type I) slow-twitch muscles.
Transgenic expression of ERR.gamma. in fast-twitch type II muscle
triggered aerobic transformation, mitochondrial biogenesis, VEGF
induction and robust myofibrillar vascularization, all in the
absence of exercise. It was observed that these intrinsic effects
of ERR.gamma. do not depend on PGC1.alpha. induction, but rather
are linked to activation of the metabolic sensor AMPK. These
results reveal an exercise-independent ERR.gamma. pathway that
promotes and coordinates vascular supply and metabolic demand in
oxidative slow-twitch muscles.
[0008] Based on these observations, provided herein are methods for
increasing vascularization in a subject. For example, such methods
can include administering a therapeutically effective amount of one
or more agents that increases ERR.gamma. activity to a mammal
needing increased vascularization, such as vascularization in the
mammal's muscle (such as skeletal or cardiac muscle), brain,
kidney, or brown adipose tissue. In some examples, the method can
also include selecting a mammal in need of increased
vascularization or a mammal at risk for developing a disorder that
can benefit from increased vascularization.
[0009] Methods for muscle rehabilitation (such as increasing or
enhancing muscle performance) in a subject are provided. For
example, such methods can include administering a therapeutically
effective amount of one or more agents that increases ERR.gamma.
activity to a mammal needing muscle rehabilitation (such as
increased or enhanced muscle performance), such as a subject having
or at risk for muscle wasting. In some examples, the method can
also include selecting a mammal in need of muscle rehabilitation
(such as increased muscle performance) or a mammal at risk for
developing a disorder that can benefit from muscle rehabilitation
(such as increased muscle performance).
[0010] In addition, provided herein are methods for increasing
mitochondrial activity in a subject. For example, such methods can
include administering a therapeutically effective amount of one or
more agents that increases ERR.gamma. activity to a mammal needing
increased mitochondrial activity. In some examples, the method can
also include selecting a mammal in need of increased mitochondrial
activity or a mammal at risk for developing a disorder that can
benefit from increased mitochondrial activity.
[0011] In some examples, the methods do not include exercising the
mammal. As such, in some examples the subject is one who cannot
exercise or is sedentary, such as a person who is bedridden or
confined to a wheelchair.
[0012] The foregoing and other objects and features of the
disclosure will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-F show skeletal muscle ERR.gamma. expression. (A)
ERR.gamma. gene (lower panel) and/or protein (upper panel)
expression in quadriceps (QUADS), white gastrocnemius (WG), red
gastrocnemius (RG) and soleus (SOL) isolated from C57B1/6J mice
(N=4). (B) Representative images of .beta.-galactosidase stained
muscles. (C) Expression of transgene transcript (lower panel) and
protein (upper panel) in quadriceps of wild type (WT), founder TG
425 and 421. (D) Representative hindlimbs from WT and transgenic
mice. (E) Dissected hindlimb muscle beds [adductor (ADDT),
quadriceps, gastrocnemius (GASTROC) and soleus]. (F) Oxidative
biomarker expression in wild type and transgenic mice. Protein
expression levels of myoglobin and cytochrome c (cycs) in wild
type, TG 425 and TG 421 quadriceps (N=3) are shown. In (A) and (C)
data are presented as mean.+-.SD (N=4). See FIG. 1F.
[0014] FIGS. 2A-E show that ERR.gamma. promotes oxidative muscle
transformation. (A) Gene ontology classification of positively
regulated genes. Gene selection was based on p<0.05 on
Bonferroni's multiple comparison test for fold change (N=3). (B)
ERR.gamma. increases expression of oxidative metabolism (Ucp3,
Pdk4, Cycs, Cox5a, Lpl), oxidative muscle (Mhc1a, Mhc2a) but not
glycolytic muscle (Mhc2b) biomarker genes. Data are presented as
mean.+-.SD from N=6 samples. (C) ERR.gamma. increases protein
expression of myoglobin, cytochrome c and uncoupling protein 3
(N=3). (D) Representative images of SDH stained WT and transgenic
gastrocnemius cryo-sections. Similar results were obtained from N=4
mice. (E) OCAR/ECAR ratio representing a shift in cellular energy
production to oxidative phosphorylation. Data is presented as
mean.+-.SD. * represents statistically significant difference
between WT and transgenic mice or between WT and ERR.gamma.
over-expressing C2C12 cells (p<0.05, unpaired Student's
t-test).
[0015] FIGS. 2F-H show ERR.gamma. in cultured muscle cells. (F)
ERR.gamma. knockdown in primary myoblast. Primary myoblast were
prepared from soleus and red gastrocnemius and infected with either
control or ERR.gamma. siRNA. Expression of ERR.gamma. and oxidative
biomarkers (cycs, ucp3, Acscl1, Cox6a2, Ppara) was measured in
control (open bars) and ERR.gamma. (closed bars) knockdown primary
muscle cells. Data is presented as mean.+-.SD. (*) Indicates
statistically significant difference between control and ERR.gamma.
knockdown cells (p<0.05, unpaired Student's t-test). (G-H)
Mitochondrial bioenergetics in wild type and ERR.gamma.
over-expressing C2C12 cells. (G) Basal oxygen consumption rate
(OCR) representing mitochondrial respiration. (H) Basal
extracellular acidification rate (ECAR) representing glycolysis.
Data are presented as mean.+-.SD. (*) Indicates statistically
significant difference between the two groups. (p<0.05, unpaired
Student's t-test).
[0016] FIGS. 3A-I show that ERR.gamma. increases muscle
vascularization. (A) Increased PECAM 1 staining in transgenic
compared to WT gastrocnemius. (B) Increased alkaline phosphatase
staining in transgenic compared to WT tibialis muscles. (C)
Confocal images of microsphere perfused WT and transgenic
quadriceps. Similar results were obtained from N=4 experiments in
(A-C). (D-H) Expression of Vegfa-121, Vegfa-165, Vegfa-189, Vegfb
and Fgf1 transcript levels in WT and transgenic quadriceps. Data
are presented as mean.+-.SD from N=6 samples. (I) ERR.gamma.
increases VEGFa and FGF1 protein expression (N=4). * represents
significant difference between WT and transgenic mice (p<0.05,
unpaired Student's t-test).
[0017] FIG. 3J shows that ERR.gamma. activates Vegfa promoter.
Vegfa gene 5' of the transcriptional start site containing promoter
region was PCR cloned from mouse genomic DNA and sub-cloned
up-stream of luciferase gene in pGL3 vector. All three isoforms of
ERR (ERR.alpha., ERR.beta. and ERR.gamma.) transcriptionally
activated Vegfa promoter.
[0018] FIGS. 4A-E show paracrine stimulation of angiogenesis by
ERR.gamma.. (A) Tube formation in SVEC4-10 cells treated for 7-8 hr
with conditioned media from WT and ERR.gamma. over-expressing C2C12
myotubes. Similar results were obtained from 4-6 experiments. (B-D)
Expression of Vegfa isoforms in WT and ERR.gamma. over-expressing
C2C12 myotubes (N=6). (E) Vegfa concentrations (pg/ml) in
conditioned media from 2 day differentiated WT and ERR.gamma.
over-expressing C2C12 myotubes (N=3). Data in (B-E) are presented
as mean.+-.SD. * represents significant difference between WT and
transgenic mice (p<0.05, unpaired Student's t-test).
[0019] FIGS. 5A-E show the physiological effect of ERR.gamma.
over-expression. (A) Average oxygen consumption (N=6-7) and (B)
average RER (N=6-7) during the light and the dark cycle over a
period of 24 hr in WT and transgenic mice. (C) Running endurance as
a function of time and distance (N=6). (D) The ambulatory activity,
measured using CLMAC units, is comparable between the wild type and
the transgenic mice. (E) Average weight gain in wild type and
transgenic mice on high fat diet (N=6). Data are presented as
mean.+-.SEM in (A), (B), and (D) and as mean.+-.SD in (C) and (E).
(*) Indicates statistically significant difference between the two
groups. (p<0.05, unpaired Student's t-test).
[0020] FIGS. 6A-H show PGC1.alpha.-independent regulation by
ERR.gamma.. (A) Relative expression of Pgc1a, Erra and Errb genes
in WT and transgenic muscle (N=6). Data are presented as
mean.+-.SD. * represents significant difference between WT and
transgenic mice (p<0.05, unpaired Student's t-test). (B) Phospho
(upper panel) and total (lower panel) AMPK in soleus (SOL) and
quadriceps (QUAD) of WT and transgenic mice (N=3). (C)
Quantification of AMPK activation (phospho to total AMPK ratio) by
densitometric analysis, presented as fold of WT soleus (N=3). Data
is presented as mean.+-.SD. (D) Representative images of SDH
staining of muscle cryo-sections from vehicle and AICAR (500
mg/kg/day for 4 weeks) treated mice. Similar results were obtained
from N=3 mice. (E) Synchronization of metabolism and vasculature by
ERR.gamma. in aerobic muscle. (F) PGC1 a acetylation in the
skeletal muscle. Nuclear extracts were prepared from freshly
isolated quadriceps from wild type and ERRGO mice using a
commercially available kit according to the manufacturer's
instructions (Thermo Scientific* NE-PER* Nuclear and Cytoplasmic
Extraction Kit, Cat no. P1-78833). PGC1 it was immunoprecipitated
using anti-PGC1.alpha. antibody (Santacruz, Cat no. sc-13067) from
the nuclear extracts and acetylation levels detected using
anti-acetyl lysine antibody (Cell Signaling, Cat no. 9441S). Upper
panel. Representative blots of acetylated and total PGC1.alpha.
immunoprecipitated from wild type and ERRGO nuclear extracts. Lower
panel. Densitometric analysis (using Image J) presented as the
ratio of acetylated to total PGC1.alpha. in the wild type and ERRGO
muscles. There is no statistically significant difference between
the two groups. (G) Phospho-ACC levels in wild type and ERR.gamma.
transgenic muscle. ACC phosphorylation was measured in murine
quadriceps using an antibody that specifically detects phospho-ACC
(Cell Signaling, Cat no. 3661). The blot represents phospho-ACC
levels in wild type and ERR.gamma. transgenic quadriceps from N=3
samples in each group. (H) ATP levels in wild type and ERR.gamma.
over-expressing C2C12 cells. Absolute ATP level in C2C12 cells was
measured using the ATP Bioluminescent Assay Kit according to
manufacturer's instruction (Sigma, Cat No. FLAA-1KT). Briefly,
5.times.10.sup.4 cells were lysed with 100 .mu.l ATP releasing
reagent for 10 minutes and combined with 100 .mu.l water. The
standards (20, 10, 2, 1, 0.2, and 0.1 .mu.M) were made by mixing
100 .mu.l ATP releasing reagent with 100 .mu.l ATP solutions. Next,
100 .mu.l ATP assay solution was added to a 96-well black plate
with solid bottom and mixed with 100 .mu.l samples or standards.
Luminescence was measured using the EnVision plate reader (Perkin
Elmer) and absolute ATP levels were calculated. ATP levels in wild
type and ERR,/over-expressing 02012 cells are presented as
mean.+-.SD (nmol per 2.times.104 cells). * Represents statistically
significant difference between groups (p<0.05, unpaired
Student's t-test).
SEQUENCE LISTING
[0021] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand. In
the accompanying sequence listing:
[0022] SEQ ID NOS: 1 and 2 are the nucleic acid and corresponding
amino acid sequence of an exemplary ERR.gamma. sequence.
DETAILED DESCRIPTION
[0023] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which a disclosed invention
belongs. The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. "Comprising" means "including." Hence
"comprising A or B" means "including A" or "including B" or
"including A and B."
[0024] Suitable methods and materials for the practice and/or
testing of embodiments of the disclosure are described below. Such
methods and materials are illustrative only and are not intended to
be limiting. Other methods and materials similar or equivalent to
those described herein can be used. For example, conventional
methods well known in the art to which the disclosure pertains are
described in various general and more specific references,
including, for example, Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0025] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety for all purposes. All sequences associated with the
GenBank.RTM. Accession numbers mentioned herein are incorporated by
reference in their entirety as were present on Mar. 1, 2012.
Although exemplary GenBank.RTM. numbers are listed herein, the
disclosure is not limited to the use of these sequences. Many other
ERR.gamma. sequences are publicly available, and can thus be
readily used in the disclosed methods. In one example, an
ERR.gamma. sequence has at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, or at least
100% sequence identity to any of the GenBank.RTM. numbers are
listed herein.
[0026] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0027] Administration: The introduction of a composition, such as
an ERR.gamma. agonist, into a subject by a chosen route, for
example topically, orally, intravascularly such as intravenously,
intramuscularly, intraperitoneally, intranasally, intradermally,
transdermally, intrathecally, subcutaneously, via inhalation or via
suppository. Administration can be local or systemic, such as
intravenous or intramuscular. For example, if the chosen route is
intravenous, the composition is administered by introducing the
composition into a vein of the subject. In some examples an
ERR.gamma. agonist is administered to a subject at an effective
dose.
[0028] Estrogen receptor-related receptor .gamma. (ERR.gamma.):
(OMIM 602969) A constitutively active orphan nuclear receptor of
the ERR subfamily. Unlike ERR.gamma. and .beta., it is more
selectively expressed in metabolically active and highly
vascularized tissues such as heart, kidney, brain and skeletal
muscles.
[0029] ERR.gamma. sequences are publicly available. For example,
GenBank.RTM. Accession Nos. NM.sub.--001134285.1, AY388461,
AF058291.1 and NM.sub.--011935.2 disclose ERR.gamma. nucleic acids,
and GenBank.RTM. Accession Nos. NP.sub.--001127757.1, P62508.1,
AAQ93381.1, and NP.sub.--036065.1 disclose ERR.gamma. proteins. In
certain examples, ERR.gamma. has at least 80% sequence identity,
for example at least 85%, 90%, 95%, or 98% sequence identity to
such sequences (such as SEQ ID NO: 1 or 2), and retains ERR.gamma.
activity.
[0030] ERR.gamma. activity includes the ability to promote
vascularization (for example in skeletal muscle), increase
mitochondrial activity (such as mitochondrial respiration), promote
transformation of fast- to slow-twitch type muscle, promote muscle
rehabilitation, and/or enhance muscle performance.
[0031] Isolated: An "isolated" biological component (such as a
nucleic acid, protein or antibody) has been substantially
separated, produced apart from, or purified away from other
biological components in the cell of the organism in which the
component naturally occurs, such as, other chromosomal and
extrachromosomal DNA and RNA, and proteins. Nucleic acids and
proteins which have been "isolated" thus include nucleic acids and
proteins purified by standard purification methods. The term also
embraces nucleic acids and proteins prepared by recombinant
expression in a host cell as well as those chemically
synthesized.
[0032] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of an ERR.gamma. agonist or other agent that increases ERR.gamma.
activity.
[0033] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually include injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0034] Recombinant: A recombinant nucleic acid molecule or protein
is one that has a sequence that is not naturally occurring or has a
sequence that is made by an artificial combination of two otherwise
separated segments of sequence. This artificial combination can be
accomplished by methods known in the art, such as chemical
synthesis or by the artificial manipulation of isolated segments of
nucleic acids, e.g., by genetic engineering techniques. Cells that
express such molecules are referred to as recombinant or transgenic
cells.
[0035] Sequence identity: The similarity between amino acid or
nucleic acid sequences are expressed in terms of the similarity
between the sequences, otherwise referred to as sequence identity.
Sequence identity is frequently measured in terms of percentage
identity (or similarity or homology); the higher the percentage,
the more similar the two sequences are.
[0036] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and
Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and
Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989;
Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson
and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul
et al., Nature Genet. 6:119, 1994, presents a detailed
consideration of sequence alignment methods and homology
calculations.
[0037] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403, 1990) is available from several
sources, including the National Center for Biotechnology
Information (NCBI, Bethesda, Md.) and on the internet, for use in
connection with the sequence analysis programs blastp, blastn,
blastx, tblastn and tblastx. A description of how to determine
sequence identity using this program is available on the NCBI
website on the internet.
[0038] Variants of ERR.gamma. that retain ERR.gamma. activity are
encompassed by this disclosure typically characterized by
possession of at least about 75%, for example at least about 80%,
at least 85%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98% or at least 99% sequence identity counted over the full
length alignment with the amino acid or nucleic acid sequence of
interest, such as any of SEQ ID NOS: 1-2. Proteins with even
greater similarity to the reference sequences will show increasing
percentage identities when assessed by this method, such as at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%,
or at least 99% sequence identity. When less than the entire
sequence is being compared for sequence identity, homologs and
variants will typically possess at least 80% sequence identity over
short windows of 10-20 amino acids, and may possess sequence
identities of at least 85% or at least 90% or 95% depending on
their similarity to the reference sequence. Methods for determining
sequence identity over such short windows are available at the NCBI
website on the internet. One of skill in the art will appreciate
that these sequence identity ranges are provided for guidance only;
it is entirely possible that strongly significant homologs could be
obtained that fall outside of the ranges provided.
[0039] Subject: Living multi-cellular vertebrate organisms, a
category that includes human and non-human mammals.
[0040] Therapeutically effective amount: An amount of a
pharmaceutical preparation that alone, or together with a
pharmaceutically acceptable carrier or one or more additional
therapeutic agents, induces the desired response. A therapeutic
agent, such as an ERR.gamma. agonist, is administered in
therapeutically effective amounts. In some embodiments, a
therapeutically effective amount is the amount of one or more
agents that increase ERR.gamma. activity necessary to increase or
more of vascularization (for example in skeletal muscle),
mitochondrial activity (such as mitochondrial respiration),
transformation of fast- to slow-twitch type muscle, and/or muscle
performance (such as an increase of at least 20%, at least 50%, at
least 60%, at least 75%, at least 80%, or at least 95% as compared
to an absence of the ne or more agents that increase ERR.gamma.
activity). When administered to a subject, a dosage will generally
be used that will achieve target tissue concentrations that has
been shown to achieve a desired in vitro effect.
[0041] Effective amounts a therapeutic agent can be determined in
many different ways, such as assaying for an increase in
vascularization, mitochondrial activity transformation of fast- to
slow-twitch type muscle, and/or muscle performance or improvement
of physiological condition of a subject having or at risk for a
disease such as a mitochondrial disease, vascular disease (such as
cardiovascular disease, peripheral vascular disease, ischemia), or
muscular disease (such as atrophy or sarcopenia). Effective amounts
also can be determined through various in vitro, in vivo or in situ
assays.
[0042] Therapeutic agents can be administered in a single dose, or
in several doses, for example daily, during a course of treatment.
However, the effective amount of can be dependent on the source
applied, the subject being treated, the severity and type of the
condition being treated, and the manner of administration.
[0043] Treating a disease: "Treatment" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition after it has begun to develop, such a sign
or symptom of a mitochondrial disease, vascular disease (such as
cardiovascular disease, peripheral vascular disease, ischemia), or
muscular disease (such as atrophy or sarcopenia). Treatment can
also induce remission or cure of a condition, such as an ischemic
stroke, transient ischemia attacks (TIAs), muscle atrophy,
sarcopenia, MELAS, and hearing loss. Preventing a disease refers to
a therapeutic intervention to a subject who does not exhibit signs
of a disease or exhibits only early signs for the purpose of
decreasing the risk of developing pathology, such that the therapy
inhibits or delays the full development of a disease, such as
preventing development of a mitochondrial disease, vascular disease
(such as cardiovascular disease, peripheral vascular disease,
ischemia), or muscular disease (such as atrophy or sarcopenia).
Treatment and prevention of a disease does not require a total
absence of disease. For example, a decrease of at least 20% or at
least 50% can be sufficient. The beneficial effect can be
evidenced, for example, by a delayed onset of clinical symptoms of
the disease in a susceptible subject, a reduction in severity of
some or all clinical symptoms of the disease, a slower progression
of the disease, a reduction in the viral load, an improvement in
the overall health or well-being of the subject, or by other
parameters well known in the art that are specific to the
particular disease.
[0044] Upregulated or activation: When used in reference to the
expression of a nucleic acid molecule, such as an ERR.gamma. gene,
refers to any process which results in an increase in production of
a gene product. A gene product can be RNA (such as mRNA, rRNA,
tRNA, and structural RNA) or protein (such as an ERR.gamma.
protein). Therefore, gene upregulation or activation includes
processes that increase transcription of a gene or translation of
mRNA.
[0045] Examples of processes that increase transcription include
those that facilitate formation of a transcription initiation
complex, those that increase transcription initiation rate, those
that increase transcription elongation rate, those that increase
processivity of transcription and those that relieve
transcriptional repression (for example by blocking the binding of
a transcriptional repressor). Gene upregulation can include
inhibition of repression as well as stimulation of expression above
an existing level. Examples of processes that increase translation
include those that increase translational initiation, those that
increase translational elongation and those that increase mRNA
stability.
[0046] Gene upregulation includes any detectable increase in the
production of a gene product. In certain examples, production of a
gene product increases by at least 2-fold, for example at least
3-fold or at least 4-fold, as compared to a control (such an amount
of gene expression in an untreated cell, such as a cell not
contacted with an agent that increases ERR.gamma. activity).
[0047] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more selectable marker genes and other genetic
elements known in the art.
Methods of Enhancing Vascularization, Muscle Rehabilitation, and
Mitochondrial Activity
[0048] It is shown herein that nuclear receptor ERR.gamma. is
highly expressed in type I skeletal muscle. Type I muscle is
fatigue resistant, highly vascularized, aerobic, and slow-twitch.
When ERR.gamma. is transgenically expressed in anaerobic type II
muscles (ERRGO mice), dually induces metabolic and vascular
transformation in absence of exercise. ERRGO mice showed increased
expression of genes promoting fat metabolism, mitochondrial
respiration and type I fiber specification. Muscles in ERRGO mice
also display an activated angiogenic program marked by myofibrillar
induction and secretion of pro-angiogenic factors,
neo-vascularization and a 100% increase in running endurance. At a
functional level, these genetic changes impart high oxygen
consuming and exercising capacity as well as resistance to
diet-induced obesity to the ERRGO mice. Surprisingly, the induction
of type I muscle properties by ERR.gamma. does not involve
PGC1.alpha.. Instead, ERR.gamma. genetically activates the energy
sensor AMPK, in mediating the metabo-vascular changes in the ERRGO
mice. Therefore, ERR.gamma. represents a previously unrecognized
determinant that specifies intrinsic vascular and oxidative
metabolic features that distinguish type I from type II muscle.
[0049] Although skeletal muscle adapts to exercise by increasing
oxidative metabolism and vascular supply via induction of
transcriptional regulators such as PGC1.alpha. (Arany et al., 2008;
Baar et al., 2002; Huss et al., 2002; Pilegaard et al., 2003;
Russell et al., 2003; Russell et al., 2005), how type I fibers
achieve intrinsic vascularization even in absence of exercise is
poorly understood. It is shown herein that one such molecular
pathway involves nuclear receptor ERR.gamma., which is highly
expressed in oxidative slow-twitch muscles. Targeted expression of
ERR.gamma. to quadriceps and white gastrocnemius, where the
receptor is typically not expressed, morphologically endows these
muscles with dense vascular supply and numerous slow-twitch
characteristics.
[0050] Genome-wide expression analysis revealed that ERR.gamma.
acts by coordinately inducing gene networks promoting mitochondrial
biogenesis, oxidative transformation and angiogeneis. The
ERR.gamma. program includes mobilization and oxidation of fat
[e.g., Acadl, Acadm, Cpt1b, Cpt2, Lpl], electron transport [e.g.,
Atp5h, Cox6a2, Ndufab1, Ndufb2m Ndufv1, Sdhb], mitochondrial
biogenesis [e.g., Mfn1], and formation of energy efficient
slow-contractile muscle [e.g., Tnnc1, Tnni1, Tnnt1]. The observed
changes constituting transformation of the contractile apparatus to
a slow phenotype and increase in oxidative metabolic genes
reflected in profound increase in mitochondrial (SDH) staining
represents a fiber type switch. Notably, ERR.gamma. also induces
key transcriptional inducers of oxidative metabolism including
Esrrb, Ppara, Ppard and Ppargc1b (Table 4) (Lin et al., 2002;
Minnich et al., 2001; Muoio et al., 2002; Wang et al., 2004).
Therefore, ERR.gamma. may be an upstream genetic switch that
determines metabolic fate by presiding over the expression of
multiple aerobic regulators.
[0051] Without wishing to be bound by a particular model, it is
proposed that the vascular program triggered by myocellular
ERR.gamma. activates a transcriptional program that directs
secretion of paracrine signals into skeletal muscle
microenvironment to induce angiogenesis. This model is strongly
supported by the observation herein that conditioned media from
ERR.gamma. over-expressing C2C12 myotubules induces endothelial
cell tube formation in culture. Indeed, ERR.gamma.
transcriptionally induced all isoforms of angiokine Vegfa in C2C12
myotubes, resulting in increased Vegfa secretion into the media.
Vegfa is a key regulator of angiogenesis critical for guiding
endothelial cells to their targets (Grunewald et al., 2006;
Springer et al., 1998). Furthermore, ERR.gamma. stimulates the
Vegfa promoter containing putative ERR binding sites that is known
to transcribe all Vegfa isoforms (Arany et al., 2008). Vegfa mRNA
and protein expression is also induced in ERRGO muscle. These
findings collectively indicate direct transcriptional activation of
angiogenic genes by ERR.gamma.. However, the angiogenic effects of
ERR.gamma. cannot be solely attributed to Vegfa induction and
secretion. For example ERR.gamma. additionally activates the
expression of Fgf1 and Cxcl12, known to regulate endothelial cell
proliferation and migration (Forough et al., 2006; Gupta et al.,
1998; Partridge et al., 2000; Shao et al., 2008; Zheng et al.,
2007), along with ephrin B2 proposed to recruit mural cells that
are required for vessel maturation (Foo et al., 2006).
Additionally, up-regulated factors such as Notch4 as well as SOX17
are transcriptional regulators of vasculogenesis (Hainaud et al.,
2006; Leong et al., 2002; Matsui et al., 2006). In this aspect,
ERR.gamma. seems to serve a function similar to HIF1.alpha., a
regulator of angiogenesis during hypoxia (Pajusola et al., 2005).
Interestingly, it was recently demonstrated that ERRs might
physically interact with HIF1.alpha. in regulating its
transcriptional activity (Ao et al., 2008). HIF1.alpha. mRNA
levels--a marker for chronic hypoxia--did not change in ERRGO
compared to wild type muscles indicating an absence of hypoxia or
its involvement in the vascular effects of ERR.gamma. (Hoppeler and
Vogt, 2001a, b). Furthermore, HIF1.alpha. is known to negatively
regulate oxidative metabolism (Mason et al., 2004; Mason et al.,
2007) and is therefore unlikely to contribute to
ERR.gamma.-mediated remodeling of skeletal muscles.
[0052] ERRGO mice exhibited increased oxygen consumption, decreased
respiratory exchange ratio, high running endurance and resistance
to diet-induced weight gain. These changes are physiological
hallmarks of increased aerobic capacity in mice, and are a direct
consequence of engineering highly oxidative and vascularized muscle
by ERR.gamma.. While similar remodeling of skeletal muscle and
aerobic physiology are triggered by exercise, the data herein
demonstrate that generation of a fully functional "endurance
vasculature" is not exercise dependent (Bloor, 2005; Egginton,
2008; Gavin et al., 2007; Gustafsson and Kraus, 2001; Jensen et
al., 2004; Waters et al., 2004).
[0053] A surprising finding was lack of change in the expression of
PGC1.alpha., a known and inducible regulator of aerobic muscles, in
the ERR.gamma.-transformed muscle. One alternative possibility is
post-translational activation of PGC1.alpha. without change in its
expression (Jager et al., 2007; Puigserver et al., 2001; Rodgers et
al., 2005). De-acetylation of PGC1.alpha. is critical for its
activation in the skeletal muscle (Canto et al., 2010;
Gerhart-Hines et al., 2007; Lagouge et al., 2006). However,
ERR.gamma. over-expression did not lead to de-acetylation of
PGC1.alpha., which remained comparably acetylated in both the wild
type and ERRGO muscles. The lack of post-translational activation
of the co-factor in ERRGO mice is further underscored by a previous
report that non-genomic activation of PGC1.alpha. typically leads
to its transcriptional induction, which we did not observe in these
studies (Jager et al., 2007). Along the same lines, it was recently
shown that both PGC1.alpha. and .beta. are dispensable for fiber
type specification in the skeletal muscle (Zechner et al., 2010).
In contrast, an alternative aerobic master regulator, AMPK, was
found to be activated by ERR.gamma. in the skeletal muscles. AMPK
is typically activated by exercise (Fujii et al., 2000; Winder and
Hardie, 1996; Wojtaszewski et al., 2000) and is essential for
exercise-mediated switch to aerobic myofibers in the skeletal
muscle (Rockl et al., 2007). Indeed, transgenic activation of AMPK
in the skeletal muscle increases the proportions of oxidative
myofibers in absence of any exercise (Rockl et al., 2007). It is
shown herein that chemical activation of AMPK by AICAR triggers
aerobic transformation of type II muscle. However, AMPK alone is
unlikely to mediate all the ERR.gamma. effects, and contribution by
additional metabolic regulators (e.g., calcineurin, SIRT1, etc.) in
ERRGO mice cannot be ruled out. This is possible because, unlike
ERR.gamma., AMPK activation apparently does not lead to a complete
transformation to a type I phenotype, but to a more intermediate
type IIa and IIx oxidative myofibers (Rockl et al., 2007). In this
context, it is peculiar that AMPK was naturally and selectively
active in soleus (pre-dominantly type I myofibers) compared to
quadriceps (pre-dominantly type II myofibers). Previous studies
have suggested AMPK activity to be similar between soleus and EDL
(also pre-dominantly made up of type II myofibers) (Dzamko et al.,
2008; Jensen et al., 2007; Jorgensen et al., 2004). Speculatively,
this discrepancy may have technical attributes or may even be
linked to possible differences in recruitment of EDL and quadriceps
for postural activity that might affect basal AMPK activation.
Nevertheless, the results herein demonstrate that in the context of
over-expression, ERR.gamma. is sufficient to initiate both
metabolic and vascular pathways to drive aerobic remodeling of
sedentary muscle independent of PGC1.alpha. by recruiting
alternative regulators such as AMPK (see FIG. 6E).
[0054] Multiple diseases including obesity and diabetes are
commonly linked to deregulation of both oxidative metabolism and
vascularity. A shared therapeutic approach to these conditions
includes exercise that activates a plethora of transcriptional
pathways to increase aerobic metabolism and vascularization to
ultimately enhance performance (Bloor, 2005; Egginton, 2008; Gavin
et al., 2007; Gustafsson and Kraus, 2001; Jensen et al., 2004;
Waters et al., 2004). These findings indicate that regulation of
ERR.gamma. activity can be used to simultaneously regulate
oxidative capacity and vascularity. High expression levels of this
receptor in tissues most prone to metabolic and vascular diseases
(e.g., heart, skeletal muscle, brain and kidney) further
potentiates its value as a pharmacologic target (Ariazi et al.,
2002; Cheung et al., 2005; Gao et al., 2006; Giguere, 2008; Heard
et al., 2000; Hong et al., 1999). In summary, it is shown herein
that ERR.gamma. controls mitochondrial function and metabolism,
together with angiogenesis that anatomically synchronizes vascular
arborization to oxidative metabolism.
[0055] Based on this observation, provided herein are methods of
increasing vascularization, mitochondrial activity, muscle
rehabilitation and/or muscle endurance by increasing ERR.gamma.
activity, for example by use of ERR.gamma. nucleic acids, proteins,
agonists, or combinations thereof. Such methods can be used to
treat or prevent a disorder associated with defects in
vascularization or mitochondrial activity, as well as disorders
that may result from being sedentary or being unable to exercise.
The agent(s) which enhance ERR.gamma. activity can be administered
by any suitable means, including parenteral, subcutaneous,
intraperitoneal, intrapulmonary, and intranasal. Parenteral
infusions include intramuscular, intravenous, intracerebral,
intraarterial, intraperitoneal, or subcutaneous administration. In
some embodiments, the dosing is given by injections, such as
intravenous or intramuscular injections, depending in part on
whether the administration is brief or chronic.
[0056] In some examples, the methods do not include providing
additional exercise to the subject being treated with the agents
that increase ERR.gamma. activity. For example, the subject may
undergo some moderate activity during such treatment (such as that
needed to perform menial tasks), the subject does not undergo any
strenuous activity (such as running, biking, swimming, or walking
for more than 5 or 10 minutes at a time). In some examples, the
subject treated is one who cannot exercise, such as one confined to
a bed or wheelchair. In another example, the subject treated is one
who is sedentary, such as one with no or irregular physical
activity, for example one who sits or remains inactive for most of
the day with little or no exercise.
[0057] In one example, methods are provided for increasing
vascularization. Such methods can include administering a
therapeutically effective amount of one or more agents that
increase ERR.gamma. activity to a mammal needing increased
vascularization. In some examples, the method increases
vascularization in the desired tissue by at least 25%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, or at least 100%, as compared to an amount of
vascularization in the absence of administration of the one or more
agents that increases ERR.gamma. activity. Methods of measuring
vascularization are provided herein and are known in the art, and
can include measuring expression of one or more VEGF molecules,
detection of vasculature (for example using angiography), and the
like. In some examples, the method also includes selecting a mammal
in need of increased vascularization or a mammal at risk for
developing a disorder that can benefit from increased
vascularization. In one example, vascularization is needed or
occurs in the mammal's muscle, brain, kidney, or brown adipose
tissue. Thus, the agents that increase ERR.gamma. activity can be
used for the prophylaxis or treatment of a mammal, for instance, a
human subject who has been diagnosed with a vascular disorder, such
as ischemia or peripheral vascular disease (PVD), or a person at
risk for developing a vascular disorder, such as one who is
sedentary or is unable to exercise.
[0058] In some embodiments, the agents that increase ERR.gamma.
activity is administered to treat or prevent the development of a
vascular disorder in skeletal muscle. For example, the subject may
have or be at risk for muscle atrophy, sarcopenia, or peripheral
vascular disease (such as peripheral arterial disease, PAD). In
some embodiments, the agents that increase ERR.gamma. activity is
administered to treat or prevent the development of a vascular
disorder in cardiac muscle. For example, the subject may have or be
at risk for a heart attack or ischemia in a cardiac muscle. In some
embodiments, the agents that increase ERR.gamma. activity is
administered to treat or prevent the development of a vascular
disorder in the brain. For example, the subject may have or be at
risk for a cerebrovascular disease, such as ischemic stroke,
migraines, transient ischemic attacks (TIAs), dementia and the
like. In some embodiments, the agents that increase ERR.gamma.
activity is administered to treat or prevent the development of a
vascular disorder in the kidney. For example, the subject may have
or be at risk for a kidney failure. In some embodiments, the agents
that increase ERR.gamma. activity is administered to treat or
prevent the development of a vascular disorder in brown adipose
tissue. For example, the subject may have or be at risk for obesity
(such as diet-induced obesity). One skilled in the art will
appreciate that complete elimination of symptoms associated with
the vascular disorder is not required for the method to be
effective, as long as at least some symptoms are eased.
[0059] In one example, methods are provided for increasing
mitochondrial activity. Such methods can include administering a
therapeutically effective amount of one or more agents that
increase ERR.gamma. activity to a mammal needing increased
mitochondrial activity. In some examples, the method increases
mitochondrial activity by at least 25%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, or at least
100%, as compared to an amount of mitochondrial activity in the
absence of administration of the one or more agents that increases
ERR.gamma. activity. Methods of measuring mitochondrial activity
are provided herein and are known in the art, and can include
measuring mitochondrial respiration, expression of mitochondrial
genes, and the like. In some examples, the method also includes
selecting a mammal in need of increased mitochondrial activity or a
mammal at risk for developing a disorder that can benefit from
increased mitochondrial activity. In one example, increased
mitochondrial activity is needed or occurs in the mammal's muscle
(e.g., skeletal or cardiac), brain, or ear. Thus, the agents that
increase ERR.gamma. activity can be used for the prophylaxis or
treatment of a mammal, for instance, a human subject who has been
diagnosed with a mitochondrial disorder, such as mitochondrial
myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms
(MELAS), or a person at risk for developing a mitochondrial
disorder, such as one who is sedentary or is unable to
exercise.
[0060] In some embodiments, the agents that increase ERR.gamma.
activity is administered to treat or prevent the development of a
mitochondrial disorder in skeletal muscle. For example, the subject
may have or be at risk for muscle atrophy, sarcopenia, or muscle
wasting. In some embodiments, the agents that increase ERR.gamma.
activity is administered to treat or prevent the development of
hearing loss, Leber's hereditary optic neuropathy (LHON), or
mitochondrial encephalomyopathy, lactic acidosis, and stroke-like
episode syndrome (MELAS). One skilled in the art will appreciate
that complete elimination of symptoms associated with the
mitochondrial disorder is not required for the method to be
effective, as long as at least some symptoms are eased.
[0061] In one example, methods are provided for muscle
rehabilitation (such as increasing muscle performance). Such
methods can include administering a therapeutically effective
amount of one or more agents that increase ERR.gamma. activity to a
mammal needing muscle rehabilitation (such as increased muscle
performance). In some examples, the method increases muscle
rehabilitation or performance by at least 25%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
or at least 100%, as compared to an amount of muscle performance in
the absence of administration of the one or more agents that
increases ERR.gamma. activity. In some examples, the method
increases aerobic transformation of a type II muscle by at least
25%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, or at least 100%, as compared to an amount
of aerobic transformation of a type II muscle (e.g., quadriceps or
white gastrocnemius) in the absence of administration of the one or
more agents that increases ERR.gamma. activity. In some examples,
the method increases an amount of type I fibers in a muscle by at
least 25%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, or at least 100%, as compared to an
amount of type I fibers in a muscle in the absence of
administration of the one or more agents that increases ERR.gamma.
activity. Methods of measuring muscle performance are provided
herein and are known in the art, and can include measuring the
strength and endurance of the muscle, testing creatine kinase
levels in the blood, electromyography (measuring electrical
activity in muscles), elastography, and the like. In some examples,
the method also includes selecting a mammal in need of muscle
rehabilitation (such as increased muscle performance) or a mammal
at risk for developing a disorder that can benefit from muscle
rehabilitation (such as increased muscle performance). In one
example, the muscle rehabilitated is skeletal muscle or cardiac
muscle. Thus, the agents that increase ERR.gamma. activity can be
used for the prophylaxis or treatment of a mammal, for instance, a
human subject who has been diagnosed with a muscle performance
disorder, muscle atrophy or sarcopenia, or a person at risk for
developing a muscle performance disorder, such as one who is
sedentary or is unable to exercise.
[0062] Thus, increasing ERR.gamma. activity, for example without
the addition of exercise, can result in one or more of the
following in skeletal or cardiac muscle: increased oxidative
metabolism (such as one or more of aerobic transformation of
fast-twitch muscles, increased mitrochondrial biogenesis and
respiration, increased lipid metabolism, increased
fatigue-resistant type I fibers, increase running endurance),
increased vascularization (such as one or more of increased
synthesis and release of pro-angiogenic factors by myotubes,
increased vascularization of skeletal muscle (e.g.,
hyper-vascularization). Thus, increasing ERR.gamma. activity can
enhance muscle performance, such as performance of type II muscles,
such as quadriceps and white gastrocnemius. In one example,
increasing ERR.gamma. activity increases expression of or more of
myoglobin, cytochrome c and UCP3, and has increased oxidative
myofibers, relative to untreated muscle. In some examples such
increases are an at least 1.5-fold or an at least 2-fold
increase.
Increasing ERR.gamma. Activity
[0063] The present disclosure provides methods and pharmaceutical
compositions for increasing vascularization, mitochondrial
activity, and/or muscle performance by increasing ERR.gamma.
activity and thereby treating or preventing disorders associated
with decreased vascularization, muscle performance, or
mitochondrial activity. ERR.gamma. activity may be increased by
increasing the amount of ERR.gamma. protein being produced or by
enhancing the activity of ERR.gamma. protein. This can be achieved,
for example, by administering a nucleotide sequence encoding for an
ERR.gamma. protein, an agent which enhances ERR.gamma. expression,
a substantially purified ERR.gamma. protein, or an ERR.gamma.
agonist. An ERR.gamma. agonist includes compounds which increase
the ERR.gamma. activity in a cell or tissue.
[0064] Administration of ERR.gamma. Proteins
[0065] In one example, ERR.gamma. activity is increased by
administering to the subject an ERR.gamma. protein, such as a
pharmaceutical composition containing such a protein. ERR.gamma.
protein sequences are known. For example, GenBank.RTM. Accession
Nos. NP.sub.--001127757.1, P62508.1, AAQ93381.1, and
NP.sub.--036065.1 disclose exemplary ERR.gamma. protein sequences.
However, one skilled in the art will appreciate that variations of
such proteins can also retain ERR.gamma. activity. For example such
variants may include one or more deletions, substitutions, or
additions (or combinations thereof), such as 1-50 of such changes
(such as 1-40, 1-30, 1-20, or 1-10 of such changes). In certain
examples, ERR.gamma. has at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98% or at least 99% sequence
identity to such sequences (such as SEQ ID NO: 2), and retains
ERR.gamma. activity. In some examples, changes are not made to the
ERR.gamma. ligand binding domain (LBD). In some examples, residues
Asp328, Arg316 and/or Asp275 are not changed.
[0066] One of skill will realize that variants of ERR.gamma.
proteins can be used, such as a variant containing conservative
amino acid substitutions. Such conservative variants will retain
critical amino acid residues necessary for ERR.gamma. activity, and
will retain the charge characteristics of the residues in order to
preserve the low pI and low toxicity of the molecules. Amino acid
substitutions (such as at most one, at most two, at most three, at
most four, at most five, or at most 10 amino acid substitutions,
such as 1 to 10 or 1 to 5 conservative substitutions) can be made
in an ERR.gamma. protein sequence to increase yield. Conservative
amino acid substitution tables providing functionally similar amino
acids are well known to one of ordinary skill in the art. The
following six groups are examples of amino acids that are
considered to be conservative substitutions for one another: [0067]
1) Alanine (A), Serine (S), Threonine (T); [0068] 2) Aspartic acid
(D), Glutamic acid (E); [0069] 3) Asparagine (N), Glutamine (Q);
[0070] 4) Arginine (R), Lysine (K); [0071] 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); and [0072] 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0073] An ERR.gamma. protein can be derivatized or linked to
another molecule (such as another peptide or protein). For example,
the ERR.gamma. protein can be functionally linked (by chemical
coupling, genetic fusion, noncovalent association or otherwise) to
one or more other molecular entities, such as an antibody, a
detection agent, or a pharmaceutical agent.
[0074] Methods of making proteins are routine in the art, for
example by recombinant molecular biology methods or by chemical
peptide synthesis. In one example, an ERR.gamma. protein is
expressed in a cell from a vector encoding the protein. In some
examples, the expression vector encoding ERR.gamma. also encodes a
selectable marker. In some examples, the sequence encoding
ERR.gamma. also encodes a purification tag sequence (such as a
His-tag, .beta.-globin-tag or glutathione S-transferase- (GST) tag)
at the N- or C-terminus of ERR.gamma., to assist in purification of
the protein.
[0075] For example, expression of nucleic acids encoding ERR.gamma.
proteins can be achieved by operably linking the ERR.gamma. DNA or
cDNA to a promoter (which is either constitutive or inducible),
followed by incorporation into an expression cassette. The promoter
can be any promoter, including a cytomegalovirus promoter and a
human T cell lymphotrophic virus promoter (HTLV)-1. Optionally, an
enhancer, such as a cytomegalovirus enhancer, is included in the
construct. The cassettes can be suitable for replication and
integration in either prokaryotes (such as E. coli) or eukaryotes
(such as yeast or a mammalian cell). Typical expression cassettes
contain specific sequences useful for regulation of the expression
of the DNA encoding the protein. For example, the expression
cassettes can include appropriate promoters, enhancers,
transcription and translation terminators, initiation sequences, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signal for introns, sequences for the maintenance of the
correct reading frame of that gene to permit proper translation of
mRNA, and stop codons. The vector can encode a selectable marker,
such as a marker encoding drug resistance (for example, ampicillin
or tetracycline resistance).
[0076] To obtain high level expression of ERR.gamma., expression
cassettes can include a strong promoter to direct transcription, a
ribosome binding site for translational initiation (internal
ribosomal binding sequences), and a transcription/translation
terminator. Exemplary control sequences include the T7, trp, lac,
tac, trc, or lambda promoters, the control region of fd coat
protein, a ribosome binding site, and can include a transcription
termination signal. For eukaryotic cells, the control sequences can
include a promoter and/or an enhancer derived from, for example, an
immunoglobulin gene, HTLV, SV40, polyoma, adenovirus, retrovirus,
baculovirus, simian virus, promoters derived from the promoter for
3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,
the promoter of the yeast alpha-mating factors or cytomegalovirus,
and a polyadenylation sequence, and can further include splice
donor and/or acceptor sequences (for example, CMV and/or HTLV
splice acceptor and donor sequences). The cassettes can be
transferred into the chosen host cell by well-known methods such as
transformation or electroporation for E. coli and calcium phosphate
treatment, electroporation or lipofection for mammalian cells.
Cells transformed by the cassettes can be selected by resistance to
antibiotics conferred by genes contained in the cassettes, such as
the amp, gpt, neo and hyg genes.
[0077] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors may be used.
Eukaryotic cells can also be cotransformed with polynucleotide
sequences encoding EER.gamma., and a second foreign DNA molecule
encoding a selectable phenotype, such as the herpes simplex
thymidine kinase gene. Another method is to use a eukaryotic viral
vector, such as simian virus 40 (SV40), retrovirus, adenovirus,
adeno-associated virus, Herpes virus, or bovine papilloma virus, to
transiently infect or transform eukaryotic cells and express the
protein (see for example, Eukaryotic Viral Vectors, Cold Spring
Harbor Laboratory, Gluzman ed., 1982). One can readily use an
expression system, such as plasmids and vectors, to produce
proteins in cells including higher eukaryotic cells such as the
COS, CHO, HeLa, fibroblast cell lines, lymphoblast cell lines, and
myeloma cell lines.
[0078] Once expressed, the recombinant ERR.gamma. protein can be
purified according to standard procedures of the art, including
ammonium sulfate precipitation, affinity columns, column
chromatography, and the like (see, generally, R. Scopes, PROTEIN
PURIFICATION, Springer-Verlag, N.Y., 1982). The recovered
ERR.gamma. protein need not be 100% pure. Once purified, partially
or to homogeneity as desired, the ERR.gamma. protein can be used
therapeutically.
[0079] Modifications can be made to a nucleic acid encoding
ERR.gamma. without diminishing its biological activity. Some
modifications can be made to facilitate the cloning, expression, or
incorporation of ERR.gamma. into a fusion protein. Such
modifications are well known and include, for example, termination
codons, a methionine added at the amino terminus to provide an
initiation, site, additional amino acids placed on either terminus
to create conveniently located restriction sites, or additional
amino acids (such as poly His) to aid in purification steps.
[0080] In one example, ERR.gamma. protein is synthesized by
condensation of the amino and carboxyl termini of shorter
fragments. Methods of forming peptide bonds by activation of a
carboxyl terminal end (such as by the use of the coupling reagent
N,N'-dicylohexylcarbodimide) are well known.
[0081] Expression of ERR.gamma. in a Subject
[0082] In one example, ERR.gamma. activity is increased by
administering to the subject a nucleic acid molecule encoding an
ERR.gamma. protein. ERR.gamma. coding sequences are known. For
example, GenBank.RTM. Accession Nos. NM.sub.--001134285.1,
AY388461, AF058291.1 and NM.sub.--011935.2 disclose exemplary
ERR.gamma. nucleic acid sequences. However, one skilled in the art
will appreciate that variations of such sequences can also encode a
protein with ERR.gamma. activity. For example such variants may
include encode a protein with one or more deletions, substitutions,
or additions (or combinations thereof), such as 1-50 of such
changes (such as 1-40, 1-30, 1-20, or 1-10 of such changes). In
certain examples, an ERR.gamma. coding sequence has at least 80%,
at least 85%, at least 90%, at least 95%, at least 97%, at least
98% or at least 99% sequence identity to such sequences (such as
SEQ ID NO: 1), and encodes a protein having ERR.gamma. activity.
One of skill in the art can readily use the genetic code to
construct a variety of functionally equivalent nucleic acids, such
as nucleic acids which differ in sequence but which encode the same
ERR.gamma. protein sequence.
[0083] Nucleic acid sequences encoding a ERR.gamma. protein can be
prepared by any suitable method including, for example, cloning of
appropriate sequences or by direct chemical synthesis by methods
such as the phosphotriester method of Narang et al., Meth. Enzymol.
68:90-99, 1979; the phosphodiester method of Brown et al., Meth.
Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of
Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase
phosphoramidite triester method described by Beaucage &
Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using
an automated synthesizer as described in, for example,
Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984;
and, the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis produces a single stranded oligonucleotide. This can be
converted into double stranded DNA by hybridization with a
complementary sequence or by polymerization with a DNA polymerase
using the single strand as a template. One of skill will recognize
that longer sequences may be obtained by the ligation of shorter
sequences.
[0084] Exemplary ERR.gamma. nucleic acids can be prepared by
routine cloning techniques. Examples of appropriate cloning and
sequencing techniques, and instructions sufficient to direct
persons of skill through many cloning exercises are found in
Sambrook et al., supra, Berger and Kimmel (eds.), supra, and
Ausubel, supra. Product information from manufacturers of
biological reagents and experimental equipment also provide useful
information. Such manufacturers include the SIGMA Chemical Company
(Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia
Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo
Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen
(Carlsbad, Calif.), and Applied Biosystems (Foster City, Calif.),
as well as many other commercial sources. Nucleic acids can also be
prepared by amplification methods. Amplification methods include
but are not limited to polymerase chain reaction (PCR), ligase
chain reaction (LCR), transcription-based amplification system
(TAS), and the self-sustained sequence replication system (3SR). A
wide variety of cloning methods, host cells, and in vitro
amplification methodologies are well known.
[0085] In some examples, it may only be necessary to introduce the
ERR.gamma. genetic or protein elements into certain cells or
tissues. For example, introducing ERR.gamma. into only the muscle,
such as skeletal or cardiac muscle (or even a particular muscle),
may be sufficient. However, in some instances, it may be more
therapeutically effective and simple to treat all of the patient's
cells, or more broadly disseminate the ERR.gamma. nucleic acid or
protein, for example by intravascular administration.
[0086] Nucleic acids encoding ERR.gamma. can be introduced into the
cells of a subject using routine methods, such as by using
recombinant viruses (e.g., viral vectors) or by using naked DNA or
DNA complexes (non-viral methods). Thus, in some embodiments, a
method of increasing ERR.gamma. activity in persons suffering from,
or at risk for, a vascular disease, mitochondrial disease, and/or
muscle performance disease, is achieved by introducing a nucleic
acid molecule coding for ERR.gamma. into the person. A general
strategy for transferring genes into donor cells is disclosed in
U.S. Pat. No. 5,529,774. The nucleic acid encoding ERR.gamma. can
be administered to the subject by any method which allows the
recombinant nucleic acid to reach the appropriate cells. Exemplary
methods include injection, infusion, deposition, implantation, and
topical administration. Injections can be intradermal,
intramuscular, iv, or subcutaneous.
[0087] In one example, an ERR.gamma. coding sequence is introduced
into a subject in a non-infectious form, such as naked DNA or
liposome encapsulated DNA. Such molecules can be introduced by
injection (such as intramuscular, iv, ip, pneumatic injection, or a
gene gun), or other routine methods (such as oral or nasal). In one
example, ERR.gamma. coding sequence is part of a lipoplex,
dendrimer, or inorganic nanoparticle to assist in its delivery.
[0088] In one example, viral vectors are used. Generally, such
methods include cloning an ERR.gamma. coding sequence into a viral
expression vector, and that vector is then introduced into the
subject to be treated. The virus infects the cells, and produces
the ERR.gamma. protein sequence in vivo, where it has its desired
therapeutic effect. The nucleic acid sequence encoding ERR.gamma.
can be placed under the control of a suitable promoter. Suitable
promoters which may be employed include, but are not limited to,
the gene's native promoter; retroviral LTR promoter; adenoviral
promoters, such as the adenoviral major late promoter; the
cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV)
promoter; inducible promoters, such as the MMTV promoter; the
metallothionein promoter; heat shock promoters; the albumin
promoter; the histone promoter; the .beta.-actin promoter; TK
promoters; B19 parvovirus promoters; and the ApoAI promoter.
[0089] Exemplary viral vectors include, but are not limited to: pox
viruses, recombinant vacciniavirus, retroviruses (such as
lentivirus), replication-deficient adenovirus strains,
adeno-associated virus, herpes simplex virus, or poliovirus.
[0090] Adenoviral vectors may include essentially the complete
adenoviral genome. Alternatively, the adenoviral vector may be a
modified adenoviral vector in which at least a portion of the
adenoviral genome has been deleted. In one embodiment, the vector
includes an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral
encapsidation signal; a DNA sequence encoding a therapeutic agent
such as EDA1-II, dl or DL; and a promoter for expressing the DNA
sequence encoding a therapeutic agent. The vector is free of at
least the majority of adenoviral E1 and E3 DNA sequences, but is
not necessarily free of all of the E2 and E4 DNA sequences, and DNA
sequences encoding adenoviral proteins transcribed by the
adenoviral major late promoter. Such a vector may be constructed
according to standard techniques, using a shuttle plasmid which
contains, beginning at the 5' end, an adenoviral 5' ITR, an
adenoviral encapsidation signal, and an E1a enhancer sequence; a
promoter (which may be an adenoviral promoter or a foreign
promoter); a tripartite leader sequence, a multiple cloning site
(which may be as herein described); a poly A signal; and a DNA
segment which corresponds to a segment of the adenoviral genome.
The DNA segment serves as a substrate for homologous recombination
with a modified or mutated adenovirus, and may encompass, for
example, a segment of the adenovirus 5' genome no longer than from
base 3329 to base 6246. The plasmid may also include a selectable
marker and an origin of replication. The origin of replication may
be a bacterial origin of replication. A desired DNA sequence
encoding a therapeutic agent may be inserted into the multiple
cloning site of the plasmid. The plasmid may be used to produce an
adenoviral vector by homologous recombination with a modified or
mutated adenovirus in which at least the majority of the E1 and E3
adenoviral DNA sequences have been deleted. Homologous
recombination may be effected through co-transfection of the
plasmid vector and the modified adenovirus into a helper cell line,
such as 293 cells, by CaPO4 precipitation. The homologous
recombination produces a recombinant adenoviral vector which
includes DNA sequences derived from the shuttle plasmid between the
Not I site and the homologous recombination fragment, and DNA
derived from the E1 and E3 deleted adenovirus between the
homologous recombination fragment and the 3' ITR.
[0091] In one embodiment, the viral vector is a retroviral vector.
Examples of retroviral vectors which may be employed include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, and vectors derived from retroviruses such as Rous Sarcoma
Virus, Harvey Sarcoma Virus, avian leukosis virus, human
immunodeficiency virus, lentivirus, myeloproliferative sarcoma
virus, and mammary tumor virus. The vector can be a replication
defective retrovirus particle. Retroviral vectors are useful as
agents to effect retroviral-mediated gene transfer into eukaryotic
cells. Retroviral vectors are generally constructed such that the
majority of sequences coding for the structural genes of the virus
are deleted and replaced by the gene(s) of interest. Most often,
the structural genes (e.g., gag, pol, and env), are removed from
the retroviral backbone using genetic engineering techniques known
in the art. An ERR.gamma. coding sequence can be incorporated into
a proviral backbone using routine methods. In the most
straightforward constructions, the structural genes of the
retrovirus are replaced by a ERR.gamma. gene which then is
transcribed under the control of the viral regulatory sequences
within the long terminal repeat (LTR). Retroviral vectors have also
been constructed which can introduce more than one gene into target
cells. Usually, in such vectors one gene is under the regulatory
control of the viral LTR, while the second gene is expressed either
off a spliced message or is under the regulation of its own,
internal promoter. Alternatively, two genes may be expressed from a
single promoter by the use of an Internal Ribosome Entry Site.
[0092] In one example, the viral vector is an adeno-associated
virus (AAV). Gene therapy vectors using AAV can infect both
dividing and non-dividing cells and persist in an extrachromosomal
state without integrating into the genome of the host cell. In some
examples, the rep and cap are removed from the DNA of the AAV. The
ERR.gamma. coding sequence together with a promoter to drive
transcription is inserted between the inverted terminal repeats
(ITR) that aid in concatamer formation in the nucleus after the
single-stranded vector DNA is converted by host cell DNA polymerase
complexes into double-stranded DNA.
[0093] The viral particles are administered in an amount effective
to produce a therapeutic effect in a host. The exact dosage of
viral particles to be administered is dependent upon a variety of
factors, including the age, weight, and sex of the patient to be
treated, and the nature and extent of the disease or disorder to be
treated. The viral particles may be administered as part of a
preparation having a titer of viral particles of at least
1.times.10.sup.5 pfu/ml, at least 1.times.10.sup.6 pfu/ml, at least
1.times.10.sup.7 pfu/ml, at least 1.times.10.sup.8 pfu/ml, at least
1.times.10.sup.9 pfu/ml, or at least 1.times.10.sup.10 pfu/ml, and
in some examples not exceeding 2.times.10.sup.11 pfu/ml. The viral
particles can be administered in combination with a
pharmaceutically acceptable carrier, for example in a volume up to
10 ml. The pharmaceutically acceptable carrier may be, for example,
a liquid carrier such as a saline solution, protamine sulfate or
Polybrene.
[0094] Agonists
[0095] An ERR.gamma. agonist is an agent that induces or increases
ERR.gamma. activity or expression. Agonists of ERR.gamma. are
commercially available, and can be generated using routine methods.
In some examples, the agonist is an agonist of ERR.gamma., but not
ERR.alpha. or ERR.beta.. In some examples, the agonist is an
agonist of ERR.gamma., as well as of ERR.alpha. and/or
ERR.beta..
[0096] ERR.gamma. agonists are known in the art, and additional
ERR.gamma. agonists can be identified using known methods (e.g.,
see Zuercher et al., 2005, J. Med. Chem. 48(9):3107-9; Coward et
al. 2001, Proc Natl Acad Sci USA. 8(15):8880-4; and Zhou et al.,
1998, Mol. Endocrin. 12:1594-1604).
[0097] For example, phenolic acyl hydrazones GSK4716 (e.g., Santa
Cruz Catalog #sc-203986) and GSK9089 (also known as DY131, see for
example, U.S. Pat. No. 7,544,838)
(N-[(E)-[4-(diethylamino)phenyl]methylideneamino]-4-hydroxybenzamide;
e.g., Tocris Bioscience Catalog #2266 or Santa Cruz Catalog #
sc203571) are agonists of ERR.beta. and ERR.gamma..
##STR00001##
[0098] Kim et al. (J. Comb. Chem. 11:928-37, 2009) disclose a
screening assay for agonists of ERR.gamma. derived from GSK4716.
Such a screening method can also be used to identify other agonists
of ERR.gamma.. E6 was discovered as being selective for ERR.gamma.
but not ERR.alpha. and .beta..
##STR00002##
[0099] U.S. Pat. Nos. 7,544,838 and 8,044,241 also provide
ERR.gamma. agonists that can be used with the disclosed methods,
such as DY131. In addition, DY159, DY162, DY163 and DY164 were also
observed to activate ERR.gamma. (and ERR.alpha. and .beta., for
example in the presence of PGC-1a.
[0100] US Patent Application Publication Nos. 2011/0218196 and
2009/0281191 also provide ERR.gamma. agonists that can be used with
the disclosed methods.
Subjects
[0101] Exemplary subjects that can benefit from the disclose
therapies include human and veterinary mammalian subjects, such as
cats, dogs, horses, rodents, and the like. In one example, the
subject treated has, or is at risk for developing, a vascular
disease, mitochondrial disease, and/or muscle performance disease.
Thus, such therapies can be used to prevent or treat the disease
provided herein. In some examples the patient is at risk for such
diseases due to smoking, alcoholism, diabetes (e.g., diabetes
mellitus), obesity, head trauma, hypertension, stroke, heart
attack, dyslipidemia, atherosclerosis, sedentary lifestyle,
inability to exercise, and the like, and thus such patients can be
treated using the methods provided herein.
[0102] In one example, the subject has or is at risk to develop a
vascular disease that can be treated or prevented by increased
blood flow. In one example, the vascular disease is a
cerebrovascular disease, such as a vascular disease of the brain.
Exemplary diseases that can be treated or prevented by increased
blood flow in or to the brain include migraines, dementia, and
ischemia, such as ischemia resulting from or due to ischemic
stroke, transient ischemic attacks (TIAs), and carotid stenosis. In
some examples such patients are diabetic, smokers, have
hypertension, sedentary, or have suffered head trauma.
[0103] In one example, the vascular disease is a cardiovascular
disease, such as a vascular disease of the heart or blood vessels.
Exemplary diseases that can be treated or prevented by increased
blood flow in the heart or vessels include ischemic heart disease,
coronary heart disease, and cardiomyopathy. In some examples such
patients are diabetic, smokers, have hypertension, have suffered a
heart attack, or are sedentary.
[0104] In one example, the vascular disease is a vascular disease
of the kidney. Exemplary diseases that can be treated or prevented
by increased blood flow in the kidney include kidney failure. In
some examples such patients are diabetic, smokers, have
hypertension, or are sedentary.
[0105] In one example, the vascular disease is a vascular disease
of the brown adipose tissue. Exemplary diseases that can be treated
or prevented by increased blood flow in the brown adipose tissue
include obesity. In some examples such patients are diabetic,
smokers, have hypertension, or are sedentary.
[0106] In one example, the vascular disease is a vascular disease
of the skeletal muscle. Exemplary diseases that can be treated or
prevented by increased blood flow in the skeletal muscle include
peripheral vascular or artery disease, muscle atrophy, muscle
wasting and sarcopenia. In some examples such patients are
diabetic, smokers, have hypertension, or are sedentary.
[0107] In one example, the subject has or is at risk to develop a
mitochondrial disease that can be treated or prevented by increased
mitochondrial activity. Exemplary mitochondrial diseases that can
be treated or prevented by increased mitochondrial activity include
muscle disease (such as muscle atrophy, muscle wasting and
sarcopenia), diabetes mellitus and deafness (DAD), hearing loss,
Leber's hereditary optic neuropathy (LHON), Leigh syndrome,
neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP),
myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonic
epilepsy with ragged red fibers (MERRF) progressive myoclonic
epilepsy, mitochondrial myopathy, encephalomyopathy, lactic
acidosis, stroke-like symptoms (MELAS), and mitochondrial
neurogastrointestinal encephalomyopathy (MNGIE). In some examples
such patients are sedentary.
[0108] In one example, the subject has or is at risk to develop a
skeletal or cardiac muscle disease that can be treated or prevented
by increased muscle performance. Exemplary muscle diseases that can
be treated or prevented by muscle rehabilitation, such as increased
muscle performance, include muscle disease (such as muscle atrophy,
muscle wasting and sarcopenia, and heart disease). In some examples
such patients are diabetic, smokers, have hypertension, or are
sedentary.
[0109] Muscle atrophy, or disuse atrophy, is a decrease in the mass
of the muscle; it can be a partial or complete wasting away of
muscle. When a muscle atrophies, this leads to muscle weakness.
Muscle atrophy can result from cancer, AIDS, congestive heart
failure, COPD (chronic obstructive pulmonary disease), renal
failure, severe burns, Dejerine Sottas syndrome (HSMN Type III),
inactivity (e.g., when a cast is put on a limb), weightlessness
(e.g., due to spaceflight), extended bedrest (e.g., during a
prolonged illness), cachexia, liver failure, starvation, and
disuse. Thus, the disclosed methods can be used to treat or prevent
atrophy resulting from such conditions.
[0110] Sarcopenia refers to the process during aging, where there
is a gradual decrease in the ability to maintain skeletal muscle
function and mass.
[0111] In addition to the simple loss of muscle mass (atrophy), or
the age-related decrease in muscle function (sarcopenia), other
muscle diseases which may be caused by structural defects in the
muscle (muscular dystrophy), or by inflammatory reactions in the
body directed against muscle (the myopathies) can be treated using
the disclosed methods.
Administration of Agents that Increase ERR.gamma. Activity
[0112] Compositions that include one or more agents that increase
ERR.gamma. activity, such as ERR.gamma. nucleic acids, ERR.gamma.
proteins, and ERR.gamma. agonists that can be used to increase
vascularization, mitochondrial activity, and/or muscle performance,
are suited for the preparation of pharmaceutical compositions.
[0113] Pharmaceutical compositions that include one or more agents
that increase ERR.gamma. activity are provided. These
pharmaceutical compositions can be used in methods of
treatment/prevention of vascular, mitochondrial, and muscular
disorders, and can be formulated with an appropriate
physiologically acceptable solid or liquid carrier, depending upon
the particular mode of administration chosen. A variety of aqueous
carriers can be used, for example, buffered saline and the like.
These solutions are sterile and generally free of undesirable
matter. These compositions can be sterilized by conventional, well
known sterilization techniques. The compositions can contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
antibody in these formulations can vary widely, and will be
selected primarily based on fluid volumes, viscosities, body weight
and the like in accordance with the particular mode of
administration selected and the subject's needs. Compositions
including one or more agents that increase ERR.gamma. activity are
of use, for example, for the treatment of a vascular,
mitochondrial, or muscular disorders, such as those resulting from
inactivity.
[0114] The pharmaceutically acceptable carriers and excipients
useful in this disclosure, for either therapeutic or diagnostic
methods, are conventional. The one or more agents that increase
ERR.gamma. activity can be formulated for systemic or local (such
as inhalational) administration. In one example, the one or more
agents that increase ERR.gamma. activity is formulated for
parenteral administration, such as intravenous or intramuscular
administration. For instance, parenteral formulations usually
include injectable fluids that are pharmaceutically and
physiologically acceptable fluid vehicles such as water,
physiological saline, other balanced salt solutions, aqueous
dextrose, glycerol or the like. Excipients that can be included
are, for instance, other proteins, such as human serum albumin or
plasma preparations. If desired, the pharmaceutical composition to
be administered can also contain minor amounts of non-toxic
auxiliary substances, such as wetting or emulsifying agents,
preservatives, and pH buffering agents and the like, for example
sodium acetate or sorbitan monolaurate.
[0115] The compositions can be prepared in unit dosage forms for
administration to a subject. The dosage form of the pharmaceutical
composition will be determined by the mode of administration
chosen. For instance, in addition to injectable fluids, topical,
inhalation, oral and suppository formulations can be employed.
Topical preparations can include ointments, sprays and the like.
Inhalation preparations can be liquid (such as solutions or
suspensions) and include mists, sprays and the like. Oral
formulations can be liquid (for example, syrups, solutions or
suspensions), or solid (such as powders, pills, tablets, or
capsules). Suppository preparations can also be solid, gel, or in a
suspension form. For solid compositions, conventional non-toxic
solid carriers can include pharmaceutical grades of mannitol,
lactose, starch, or magnesium stearate. Actual methods of preparing
such dosage forms are known, or will be apparent, to those skilled
in the art.
[0116] The pharmaceutical compositions that include one or more
agents that increase ERR.gamma. activity can be formulated in unit
dosage form suitable for individual administration of precise
dosages. In addition, the pharmaceutical compositions may be
administered in a single dose or as in a multiple dose schedule. A
multiple dose schedule is one in which a primary course of
treatment may be with more than one separate dose, for instance
1-10 doses, followed by other doses given at subsequent time
intervals as needed to maintain or reinforce the action of the
compositions. Treatment can involve daily or multi-daily doses of
compound(s) over a period of a few days to months, or even years.
Thus, the dosage regime will also, at least in part, be determined
based on the particular needs of the subject to be treated, the
severity of the affliction, whether the therapeutic agent is
administered for preventive or therapeutic purposes, previous
prophylaxis and therapy, the subject's clinical history and
response to the therapeutic agent, and the manner of
administration, and can be left to the judgment of the prescribing
clinician. Within these bounds, the formulation to be administered
will contain a quantity of the active component(s) in amounts
effective to achieve the desired effect in the subject being
treated. A therapeutically effective amount of one or more agents
that increase ERR.gamma. activity is one that which provides either
subjective relief of a symptom(s) or an objectively identifiable
improvement as noted by the clinician or other qualified observer.
These compositions can be administered in conjunction with another
agent, such as angiogenic therapy (e.g., those that include a VEGF,
such as VEGF-A), either simultaneously or sequentially. The one or
more agents that increase ERR.gamma. activity also can be used or
administered as a mixture, for example in equal amounts, or
individually, provided in sequence, or administered all at
once.
[0117] Single or multiple administrations of the compositions can
be administered depending on the dosage and frequency as required
and tolerated by the subject. The composition should provide a
sufficient quantity of one or more agents that increase ERR.gamma.
activity to effectively treat the subject or inhibit the
development of the desired disease. The dosage can be administered
once but can be applied periodically until either a therapeutic
result is achieved or until side effects warrant discontinuation of
therapy. In one example, a dose of the one or more agents that
increase ERR.gamma. activity is infused for thirty minutes every
other day. In this example, about one to about ten doses can be
administered, such as three or six doses can be administered every
other day. In a further example, a continuous infusion is
administered for about five to about ten days. The subject can be
treated at regular intervals, such as monthly, until a desired
therapeutic result is achieved. Generally, the dose is sufficient
to treat or ameliorate symptoms or signs of a disease without
producing unacceptable toxicity to the patient.
[0118] In one specific, non-limiting example, a unit dosage for
intravenous or intramuscular administration of an ERR.gamma.
agonist includes at least 0.5 .mu.g agonist per dose, such as at
least 5 .mu.g agonist per dose, at least 50 .mu.g agonist per dose,
or at least 500 .mu.g agonist per dose. In some examples, doses are
administered three-times in one week.
[0119] In one specific, non-limiting example, an ERR.gamma. agonist
daily dosage is from about 0.01 milligram to about 500 milligram
per kilogram of animal body weight, for example given as a single
daily dose or in divided doses two to four times a day, or in
sustained release form. For most large mammals, the total daily
dosage is from about 0.01 milligrams to about 100 milligrams per
kilogram of body weight, such as from about 0.5 milligram to about
100 milligrams per kilogram of body weight, which can be
administered in divided doses 2 to 4 times a day in unit dosage
form containing for example from about 10 to about 100 mg of the
compound in sustained release form. In one example, the daily oral
dosage in humans is between 1 mg and 1 g, such as between 10 mg and
500 mg, 10 mg and 200 mg, such as 10 mg. The dosage regimen may be
adjusted within this range or even outside of this range to provide
the optimal therapeutic response. Oral administration of an
ERR.gamma. agonist can be carried out using tablets or capsules,
such as about 10 mg to about 500 mg of the ERR.gamma. agonist.
Exemplary doses in tablets include 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg,
1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, and 500 mg
of the ERR.gamma. agonist. Other oral forms can also have the same
dosages (e.g., capsules). In one example, a dose of an ERR.gamma.
agonist administered parenterally is at least 10 mg, such as 10 to
500 mg or 10 to 200 mg of the ERR.gamma. agonist.
[0120] In one specific, non-limiting example, a unit dosage for
oral administration (such as a table or capsule), or for oral
intravenous or intramuscular administration, of an ERR.gamma.
protein includes about 1 .mu.g to 1000 mg of ERR.gamma. protein per
dose, such as 1 .mu.g to 100 .mu.g ERR.gamma. protein per dose, 1
.mu.g to 500 .mu.g ERR.gamma. protein per dose, 1 .mu.g to 1 mg
ERR.gamma. protein per dose, 1 mg to 1000 mg ERR.gamma. protein per
dose, or 10 mg to 100 mg ERR.gamma. protein per dose. In some
examples, doses are administered at least three-times in one
week.
[0121] In one specific, non-limiting example, a unit dosage for
administration of an ERR.gamma. nucleic acid (such as injection,
gene gun, pneumatic injection, or topical) includes at least 10 ng,
at least 100 ng, at least 1 .mu.g, at least 10 .mu.g, at least 100
.mu.g, or at least 500 .mu.g nucleic acid per dose. Saline
injections can use amounts of DNA, such as from 10 .mu.g-1 mg,
whereas gene gun deliveries can require 100 to 1000 times less DNA
than intramuscular saline injection (such as 0.2 .mu.g-20 .mu.g).
These amounts can vary from species to species, with mice, for
example, requiring approximately 10 times less DNA than primates.
Saline injections may require more DNA because the DNA is delivered
to the extracellular spaces of the target tissue (e.g., muscle),
where it has to overcome physical barriers before it is taken up by
the cells, while gene gun deliveries bombard DNA directly into the
cells.
[0122] In one specific, non-limiting example, a unit dosage for
intravenous or intramuscular administration of a viral vector that
encodes ERR.gamma. includes at least 1.times.10.sup.8 viral
particles per dose, such as at least 1.times.10.sup.9 viral
particles per dose, at least 1.times.10.sup.10 viral particles per
dose, or at least 1.times.10.sup.11 viral particles per dose.
[0123] Actual methods for preparing administrable compositions will
be known or apparent to those skilled in the art and are described
in more detail in such publications as Remington's Pharmaceutical
Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).
[0124] Agents that increase ERR.gamma. activity (such as a
ERR.gamma. protein or agonist) can be provided in lyophilized form
and rehydrated with sterile water before administration, although
they are also provided in sterile solutions of known concentration.
The resulting solution can then added to an infusion bag containing
0.9% sodium chloride, USP, and can be administered in some examples
at a dosage of from 1 to 300 mg/kg of body weight. Considerable
experience is available in the art in the administration of
proteins or nucleic acids. Such molecules can be administered by
slow infusion, rather than in an intravenous push or bolus. In one
example, a higher loading dose is administered, with subsequent,
maintenance doses being administered at a lower level.
[0125] The agents that increase ERR.gamma. can be administered to
humans or other mammal using routine modes of administration, such
as topically, orally, intravascularly such as intravenously,
intramuscularly, intraperitoneally, intranasally, intradermally,
intrathecally, subcutaneously, intracraneally, via inhalation or
via suppository. The particular mode of administration and the
dosage regimen will be selected by the attending clinician, taking
into account the particulars of the case (for example the subject,
the disease, the disease state involved, and whether the treatment
is prophylactic).
[0126] Controlled release parenteral formulations of agents that
increase ERR.gamma. activity can be made as implants, oily
injections, or as particulate systems. For a broad overview of
protein delivery systems (see Banga, A. J., Therapeutic Peptides
and Proteins: Formulation, Processing, and Delivery Systems,
Technomic Publishing Company, Inc., Lancaster, Pa., 1995).
Particulate systems include microspheres, microparticles,
microcapsules, nanocapsules, nanospheres, and nanoparticles.
Microcapsules contain the therapeutic protein as a central core. In
microspheres the therapeutic is dispersed throughout the particle.
Particles, microspheres, and microcapsules smaller than about 1
.mu.m are generally referred to as nanoparticles, nanospheres, and
nanocapsules, respectively. Capillaries have a diameter of
approximately 5 .mu.m so that only nanoparticles are administered
intravenously. Microparticles are typically around 100 .mu.m in
diameter and are administered subcutaneously or intramuscularly
(see Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed.,
Marcel Dekker, Inc., New York, N.Y., pp. 219-342, 1994; Tice &
Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed.,
Marcel Dekker, Inc. New York, N.Y., pp. 315-339, 1992).
[0127] Polymers can be used for ion-controlled release. Various
degradable and nondegradable polymeric matrices for use in
controlled drug delivery are known in the art (Langer, R., Accounts
Chem. Res. 26:537, 1993). For example, the block copolymer,
polaxamer 407 exists as a viscous yet mobile liquid at low
temperatures but forms a semisolid gel at body temperature. It has
shown to be an effective vehicle for formulation and sustained
delivery of recombinant interleukin-2 and urease (Johnston et al.,
Pharm. Res. 9:425, 1992; and Pec et al., J. Parent. Sci. Tech.
44:58, 1990). Alternatively, hydroxyapatite has been used as a
microcarrier for controlled release of proteins (Ijntema et al.,
Int. J. Pharm. 112:215, 1994). In yet another aspect, liposomes are
used for controlled release as well as drug targeting of the
lipid-capsulated drug (Betageri, et al., Liposome Drug Delivery
Systems, Technomic Publishing Co., Inc., Lancaster, Pa., 1993).
Numerous additional systems for controlled delivery of therapeutic
proteins are known (see, for example, U.S. Pat. Nos. 5,055,303,
5,188,837, 4,235,871, 4,501,728, 4,837,028 4,957,735 and 5,019,369,
5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505;
5,506,206, 5,271,961; 5,254,342 and 5,534,496).
[0128] Site-specific administration of the agents that increase
ERR.gamma. activity can be used, for instance by applying the agent
to a region of the body in need of treatment, such as the brain,
particular muscle, or kidney. In some embodiments, sustained
release of the pharmaceutical preparation that includes a
therapeutically effective amount of the one or more agents that
increase ERR.gamma. activity may be beneficial.
[0129] The present disclosure also includes combinations of one or
more agents that increase ERR.gamma. activity with one or more
other agents useful in the treatment of a vascular, mitochondria,
or muscular disorder. For example, the compounds of this disclosure
can be administered in combination with effective doses of other
angiogenic agents, such as vascular endothelial growth factor
(VEGF), or fibroblast growth factor (FGF). The term "administration
in combination" or "co-administration" refers to both concurrent
and sequential administration of the active agents.
[0130] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
Example 1
Materials and Methods
[0131] This example describes the materials and methods used for
Examples 2-8.
[0132] Animals.
[0133] Mouse ERR.gamma. cDNA was placed downstream to the human
.alpha.-skeletal actin promoter and upstream of the SV40
intron/poly (A) sequence. The purified transgene was injected into
C57BL/6J.times.CBA F1 zygotes. Two transgenic founders (TG 425 and
421) were obtained that were backcrossed for 5 generations with
C57BL/6J. All experiments used age (2-3 months) and sex (male)
matched transgenic and wild type (WT) littermates. Mice were
maintained on a normal chow diet. ERR.gamma.+/-mice and tissue
.beta.-galactosidase staining has been described previously
(Alaynick et al., 2007).
[0134] Drug Treatment.
[0135] Male C57B1/6J mice (8 weeks old) were intra-peritoneally
injected with vehicle or AICAR (500 mg/kg/day), as previously
described (Narkar et al., 2008).
[0136] Gene and Protein Expression Analysis.
[0137] RNA was extracted using the Trizol extraction method from
quadriceps or soleus isolated from WT and transgenic mice.
Additionally, protein lysates were prepared from quadriceps and
analyzed by western blotting with myoglobin (Dako), CYCS
(Santacruz), UCP3 (Affinity Bioreagents), phospho-AMPK alpha (Cell
Signaling, Cat no #2535) and total-AMPK alpha (Cell Signaling, Cat
no #2532) antibodies. The AMPK antibodies detect both the alpha 1
and 2 catalytic subunits of AMPK (Narkar et al., 2008).
[0138] Microarray Analysis.
[0139] Global gene expression analysis was performed in quadriceps
from WT and transgenic mice, as previously described (Narkar et
al., 2008).
[0140] Fluorescence Micro-Angiography.
[0141] Blood vessel mapping was performed as previously described
(Johnson et al., 2004; Springer et al., 2000). Briefly, a red
fluorescent microsphere (0.1 .mu.M) suspension was
intra-ventricularly perfused (10 ml, 1 ml/min) followed by
euthanasia and tissue collection. Longitudinal cryo-sections (10
.mu.M) of frozen gastrocnemius were processed and subjected to
confocal microscopy to image skeletal muscle vasculature.
[0142] Cell Culture, In Vitro Angiogenesis and Vegfa ELISA.
[0143] C2C12 myoblasts were grown in 20% FBS-DMEM and
differentiated in 2% horse serum-DMEM [with
penicillin/streptomycin]. Conditioned media from two day
differentiated WT and ERR/over-expressing C2C12 myotubules were
used in the in vitro angiogenesis assay. Murine endothelial
SVEC4-10 cells were cultured and maintained in DMEM containing 10%
fetal bovine serum and penicillin/streptomycin. On the day of the
experiment 4.times.10.sup.5 cells/500 .mu.l/well were plated in
matrigel-coated 12-well plates. The cells were immediately treated
for 7 hr with C2C12 cell conditioned media (2500), followed by
evaluation of tube formation. Vegfa concentration in the
conditioned media was measured using commercial Elisa kit according
manufacturer's instructions [Research & Diagnostics].
[0144] Oxymetery and Treadmill Assays.
[0145] Oxygen consumption, respiratory exchange ratio and
ambulatory activity were measured in 3 month old, WT and transgenic
male mice (N=6-7/group) of comparable weight using Comprehensive
Lab Animal Monitoring System to obtain oxymetric measurements
(Columbus Instruments). These mice were first acclimated in the
monitoring system for 1 day, followed by data collection for 24 hr
to include a 12 hr light and dark cycle. For each animal, the
average of all the data points within the light or dark phase was
used as a representative value of the respective cycle. Diurnal
differences between the light and dark cycles were detectable in
all animals, validating the method of data collection.
[0146] Endurance was determined in WT and transgenic (N=6
mice/group), as previously described (Narkar et al., 2008). The
treadmill endurance test was performed as follows. WT and
transgenic mice were acclimated to treadmill running (8 meters/min
for 15 min) every other day for 1 week before the test. For the
endurance testing, the mice were run on a treadmill at 5.degree.
inclination as the speed was gradually increased to 14 meters/min.
After reaching 14 m/min, mice were run to exhaustion at constant
speed. Endurance was measured as the function of time and distance
ran.
[0147] Succinate Dehydrogenase (SDH) Staining.
[0148] SDH staining was performed on 6 .mu.M cryo-sections of
gastrocnemius. Briefly, WT and transgenic sections were incubated
at 37.degree. C. for 10 min in substrate buffer [0.2M Phosphate
buffer containing sodium succinate (250 mg/10 ml) and NBT (10 mg/10
ml)]. Following incubation, sections were washed three times with
water following by two washes each with increasing and decreasing
concentrations of acetone (30%, 60%, 90%). Finally, the sections
were washed three times with water and mounted in an aqueous
mounting media.
[0149] Immunohistochemistry.
[0150] Gastrocnemius muscles isolated from WT and transgenic mice
were equilibrated in 30% sucrose (in PBS) for 2-3 hr and frozen in
OCT. Cryo-sections (10[1M) were fixed (4% paraformaldehyde-PBS),
permeabilized (0.3% Triton X-PBS) and blocked (normal goat
serum-PBS) before antibody treatment. Further, the sections were
incubated overnight at 4.degree. C. with anti-PECAM 1 antibody
(1:25 in PBS, SEROTEC), washed three times with PBS, incubated with
anti-rat secondary antibody (1:250, ALEXA FLOR 344), washed three
times with PBS and mounted in VECTASHIELD. For negative controls,
primary antibody was replaced with normal goat serum-PBS for
overnight incubation.
[0151] Alkaline Phosphatase (AP) Staining.
[0152] For AP staining, 10 .mu.M muscle sections were fixed in
ice-cold acetone (5 min, -20.degree. C.), incubated in
Tris-buffered Naphthol AS-MX phosphate/N,N Dimethylformamide
solution (30 min, 37.degree. C.), rinsed with distilled water
(3.times.2 min) and mounted with aqueous media.
[0153] Data Analysis.
[0154] Data was analyzed using either one way ANOVA with an
appropriate post hoc test, or unpaired student's t-test, as
indicated.
[0155] The global gene expression data has been deposited in the
NCBI Gene Expression Omnibus under the GEO series accession number
(pending).
Example 2
Skeletal Muscle ERR.gamma. Expression
[0156] This example describes methods used to examine ERR.gamma.
expression in skeletal muscle.
[0157] Because skeletal muscle is a functionally heterogeneous
tissue containing both aerobic slow-twitch and glycolytic
fast-twitch muscles, ERR.gamma. expression was evaluated in the
context of different myofibrillar beds. The ERR.gamma. transcript
was highly expressed in oxidative muscles such as soleus and red
gastrocnemius, with minimal expression in glycolytic quadriceps and
white gastrocnemius (FIG. 1A, lower panel). ERR.gamma. protein is
undetectable in quadriceps, but highly expressed in soleus (FIG.
1A, upper panel).
[0158] Viable ERR.gamma.+/-mice are available in which a
.beta.-galactosidase protein-coding region without the promoter was
introduced in-frame with the initiation site of the Esrrg gene
(Alaynick et al., 2007) such that the enzyme mimics the expression
of endogenous ERR.gamma.. .beta.-Galactosidase staining of
different muscle beds from ERR.gamma.+/-adult mice confirmed that
the receptor is highly expressed in oxidative (e.g., soleus and red
gastrocnemius) compared to the minimal levels in glycolytic muscles
(e.g., quadriceps, white gastrocnemius) (FIG. 1B).
Example 3
Transgenic Muscle-Specific ERR.gamma. Over-Expression
[0159] This example describes methods used to demonstrate the role
of ERR.gamma. in oxidative and slow-twitch muscle biology.
[0160] Transgenic mice were generated that selectively expressed
ERR.gamma. in skeletal muscles under the control of the human
alpha-skeletal actin promoter (Muscat and Kedes, 1987; Wang et al.,
2004). Two ERR.gamma. over-expressing (ERRGO) transgenic lines were
obtained (TG 421 and 425) showing both transcript (lower panel) and
protein (upper panel) in fast-twitch quadriceps (FIG. 1C). Gross
anatomical analysis of hindlimb muscles (FIG. 1D) and dissection of
individual muscle beds (FIG. 1E) revealed enhanced red coloration
(characteristic of oxidative fibers) in transgenic compared to wild
type muscle. Importantly, slow-twitch (soleus) muscle, already high
in ERR.gamma. expression, was not affected (FIG. 1E), presumably
because it is already fully oxidative. In addition, oxidative
biomarkers myoglobin and cytochrome c were induced in the
quadriceps of both the transgenic lines compared to wild type mice
(FIG. 1F). TG 421 was used in later experiments due to slightly
higher biomarker expression in this progeny.
Example 4
Fast to Slow-Twitch Transformation of Skeletal Muscle by
ERR.gamma.
[0161] This example describes methods used to determine the
transcriptional effect of ERR.gamma. by measuring muscle gene
expression in quadriceps from wild type and ERRGO mice.
[0162] In gene array analysis, it was observed that ERR.gamma.
regulated 1123 genes in skeletal muscles, of which 623 genes were
induced. Gene ontology-based classification of these genes is
presented in FIG. 2A. The majority of the up-regulated genes belong
to either mitochondrial biology (90) or oxidative metabolism (43)
encoding various components of fatty acid oxidation pathway as well
as the oxidative respiratory chain reflective of aerobic adaptation
(Table 1).
TABLE-US-00001 TABLE 1 Global gene expression was compared between
wild type and ERR.gamma. transgenic quadriceps. The positively
regulated genes were subjected to gene ontology classification. The
genes linked to mitochondrial respiration and/or fatty acid
oxidation are described below (N = 3, each pooled from 3 mice, p
< 0.05, Bonferroni's multiple comparison test). Locus Fold
Description 1300010F03Rik 2.135 RIKEN cDNA 1300010F03 gene
1700020C11Rik 2.604 RIKEN cDNA 1700020C11 gene 1700034H14Rik 1.742
RIKEN cDNA 1700034H14 gene Acaa1a 2.169 acetyl-Coenzyme A
acyltransferase 1A Acaa2 3.473 acetyl-Coenzyme A acyltransferase 2
(mitochondrial 3- oxoacyl-Coenzyme A thiolase) Acadl 2.093
acyl-Coenzyme A dehydrogenase, long-chain Acadm 1.931 acyl-Coenzyme
A dehydrogenase, medium chain Acads 1.713 acyl-Coenzyme A
dehydrogenase, short chain Acadvl 2.2 acyl-Coenzyme A
dehydrogenase, very long chain Acat1 1.943 acetyl-Coenzyme A
acetyltransferase 1 Acot1 2.031 acyl-CoA thioesterase 1 Acot11
2.356 acyl-CoA thioesterase 11 Acot2 3.018 acyl-CoA thioesterase 2
Acot7 1.952 acyl-CoA thioesterase 7 Acsl1 2.564 acyl-CoA synthetase
long-chain family member 1 Adh1 1.843 alcohol dehydrogenase 1
(class I) Ak3l1 7.733 adenylate kinase 3 alpha-like 1 Akap1 2.569 A
kinase (PRKA) anchor protein 1 Aldh2 2.484 aldehyde dehydrogenase
2, mitochondrial Atad3a 1.78 ATPase family, AAA domain containing
3A Atp5h 1.685 ATP synthase, H+ transporting, mitochondrial F0
complex, subunit d Bcat2 1.793 branched chain aminotransferase 2,
mitochondrial Bdh1 2.899 3-hydroxybutyrate dehydrogenase, type 1
Cabc1 5.199 chaperone, ABC1 activity of bc1 complex like (S. pombe)
Cat 1.919 catalase Cds2 1.854 CDP-diacylglycerol synthase
(phosphatidate cytidylyltransferase) 2 Chkb 2.021 choline kinase
beta Cox15 1.727 COX15 homolog, cytochrome c oxidase assembly
protein (yeast) Cox6a2 1.643 cytochrome c oxidase, subunit VI a,
polypeptide 2 Cpt1b 1.698 carnitine palmitoyltransferase 1b, muscle
Cpt2 1.815 carnitine palmitoyltransferase 2 Ctsb 2.539 cathepsin B
D10Jhu81e 1.709 DNA segment, Chr 10, Johns Hopkins University 81
expressed Dci 2.037 dodecenoyl-Coenzyme A delta isomerase (3,2
trans- enoyl-Coenyme A isomerase) Decr1 2.745 2,4-dienoyl CoA
reductase 1, mitochondrial Dhrs4 2.103 dehydrogenase/reductase (SDR
family) member 4 Dlat 2.027 dihydrolipoamide S-acetyltransferase
(E2 component of pyruvate dehydrogenase complex) Ech1 2.297 enoyl
coenzyme A hydratase 1, peroxisomal Etfb 1.695 electron
transferring flavoprotein, beta polypeptide Etfdh 1.962 electron
transferring flavoprotein, dehydrogenase Fabp3 3.658 fatty acid
binding protein 3, muscle and heart Fdft1 2.287 farnesyl
diphosphate farnesyl transferase 1 Gcdh 2.246 glutaryl-Coenzyme A
dehydrogenase Gfm1 2.099 G elongation factor, mitochondrial 1
Ggtla1 1.767 gamma-glutamyltransferase-like activity 1 Glrx5 2.036
glutaredoxin 5 homolog (S. cerevisiae) Glud1 1.812 glutamate
dehydrogenase 1 Got2 2.262 glutamate oxaloacetate transaminase 2,
mitochondrial Hadh 1.969 hydroxyacyl-Coenzyme A dehydrogenase Hadha
2.415 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl- Coenzyme A
thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha
subunit Hadhb 1.775 hydroxyacyl-Coenzyme A
dehydrogenase/3-ketoacyl- Coenzyme A thiolase/enoyl-Coenzyme A
hydratase (trifunctional protein), beta subunit Hba-a1 4.779
hemoglobin alpha, adult chain 1 Herc2 1.786 hect (homologous to the
E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain
(RLD) 2 Hibadh 1.685 3-hydroxyisobutyrate dehydrogenase Hsdl2 2.283
hydroxysteroid dehydrogenase like 2 Hspa9 1.673 heat shock protein
9 Idh3b 1.749 isocitrate dehydrogenase 3 (NAD+) beta Ivd 1.843
isovaleryl coenzyme A dehydrogenase Ldhd 2.18 lactate dehydrogenase
D Lpl 1.873 lipoprotein lipase Me3 1.657 malic enzyme 3,
NADP(+)-dependent, mitochondrial Mfn1 1.855 mitofusin 1 Mlycd 1.761
malonyl-CoA decarboxylase Mrm1 1.868 mitochondrial rRNA
methyltransferase 1 homolog (S. cerevisiae) Mrpl14 2.465
mitochondrial ribosomal protein L14 Mrpl19 1.875 mitochondrial
ribosomal protein L19 Mrpl3 1.744 mitochondrial ribosomal protein
L3 Mrpl9 1.734 mitochondrial ribosomal protein L9 Msrb2 2.412
methionine sulfoxide reductase B2 Mterfd3 2.284 MTERF domain
containing 3 Mtx2 1.738 metaxin 2 Mut 1.666 methylmalonyl-Coenzyme
A mutase Ndufab1 1.645 NADH dehydrogenase (ubiquinone) 1,
alpha/beta subcomplex, 1 Ndufb2 1.734 NADH dehydrogenase
(ubiquinone) 1 beta subcomplex, 2 Ndufs8 1.895 NADH dehydrogenase
(ubiquinone) Fe--S protein 8 Ndufv1 1.648 NADH dehydrogenase
(ubiquinone) flavoprotein 1 Nnt 4.809 nicotinamide nucleotide
transhydrogenase Nrip1 1.821 nuclear receptor interacting protein 1
Nudt8 2.662 nudix (nucleoside diphosphate linked moiety X)-type
motif 8 Osbpl1a 2.225 oxysterol binding protein-like 1A Pdk4 2.692
pyruvate dehydrogenase kinase, isoenzyme 4 Phca 2.679
phytoceramidase, alkaline Pisd 1.679 phosphatidylserine
decarboxylase Pitpnc1 2.322 phosphatidylinositol transfer protein,
cytoplasmic 1 Pla2g4b 4.406 phospholipase A2, group IVB (cytosolic)
Plcb4 2.431 phospholipase C, beta 4 Plcd1 2.009 phospholipase C,
delta 1 Ppara 2.444 peroxisome proliferator activated receptor
alpha Ppif 1.989 peptidylprolyl isomerase F (cyclophilin F) Ppm1k
1.759 protein phosphatase 1K (PP2C domain containing) Prdx5 1.655
peroxiredoxin 5 Prdx6 1.643 peroxiredoxin 6 Qk 1.875 quaking
Rtn4ip1 2.032 reticulon 4 interacting protein 1 Sdhb 1.699
succinate dehydrogenase complex, subunit B, iron sulfur (Ip) Sfxn5
1.992 sideroflexin 5 Slc25a20 2.989 solute carrier family 25
(mitochondrial carnitine/acylcarnitine translocase), member 20
Slc25a22 2.49 solute carrier family 25 (mitochondrial carrier,
glutamate), member 22 Slc25a4 3.435 solute carrier family 25
(mitochondrial carrier, adenine nucleotide translocator), member 4
Slc27a1 1.769 solute carrier family 27 (fatty acid transporter),
member 1 Slc40a1 3.279 solute carrier family 40 (iron-regulated
transporter), member 1 Sod2 1.766 superoxide dismutase 2,
mitochondrial Sorl1 2.367 sortilin-related receptor, LDLR class A
repeats- containing Tfam 1.754 transcription factor A,
mitochondrial Timm44 1.683 translocase of inner mitochondrial
membrane 44 Tomm22 2.072 translocase of outer mitochondrial
membrane 22 homolog (yeast) Txn2 2.021 thioredoxin 2 Ucp3 2.838
uncoupling protein 3 (mitochondrial, proton carrier) Ung 3.577
uracil DNA glycosylase Uqcrq 2.108 ubiquinol-cytochrome c
reductase, complex III subunit VII
[0163] Furthermore, contractile genes, especially ones associated
with slow myofibers, were also activated raising the possibility of
fast-to-slow transformation linked to the metabolic switch (Table
2).
TABLE-US-00002 TABLE 2 Contractile genes induced by ERR.gamma. in
quadriceps of the transgenic mice (N = 3, each pooled from 3 mice,
p < 0.05, Bonferroni's multiple comparison test). Locus Fold
Description Abra 2.915 actin-binding Rho activating protein Actn2
4.281 actinin alpha 2 Ankrd2 9.885 ankyrin repeat domain 2 (stretch
responsive muscle) Csrp3 8.534 cysteine and glycine-rich protein 3
Kcnj8 1.824 potassium inwardly-rectifying channel, subfamily J,
member 8 Myh2 5.84 myosin, heavy polypeptide 2, skeletal muscle,
adult Myoz2 3.67 myozenin 2 Nrap 1.66 nebulin-related anchoring
protein Spna2 1.804 spectrin alpha 2 Tnnc1 3.256 troponin C,
cardiac/slow skeletal Tnni1 4.827 troponin I, skeletal, slow 1
Tnnt1 15 troponin T1, skeletal, slow Tpm3 3.49 tropomyosin 3,
gamma
[0164] Key biomarker genes associated with oxidative metabolism
[Ucp3, Pdk4, Cycs, Cox5a, Lpl] and oxidative myofibers [Mhc Ia, Mhc
IIa], but not glycolytic myofibers [Mhc IIb] were induced by
ERR.gamma. in quadriceps of transgenic mice (FIG. 2B). Conversely,
many of the biomarker genes [Ucp3, Cycs, Acscl1, Cox6a2, Ppara]
were found to be down-regulated by siRNA-mediated ERR.gamma.
knockdown in primary cultured myotubes (FIG. 2F) isolated from
oxidative muscles (soleus and red gastrocnemius). Moreover, the
oxidative changes were confirmed at the protein level as
exemplified by increased expression of myoglobin, cytochrome c and
UCP3 in transgenic relative to wild type muscle (FIG. 2C).
Furthermore, staining of gastrocnemius cryo-sections for defining
oxidative mitochondrial enzyme SDH activity revealed an increase in
oxidative myofibers in ERRGO compared to wild type mice (FIG. 2D),
which was confirmed by electron microscopy.
[0165] To access the metabolic effects of ERR.gamma. at the
cellular level, the mitochondrial bioenergetics were measured in
wild type and ERR.gamma. over-expressing C2C12 cells using an
extracellular flux analyzer. Specifically, the oxygen consumption
rate (OCR) (an indicator of mitochondrial respiration) along with
the extracellular acidification rate (ECAR) (a measure of
glycolysis) was determined in these cells (FIGS. 2G and H).
ERR.gamma. expression significantly induced mitochondrial
respiration (OCR), reduced cellular glycolysis (ECAR) resulting in
an 85% shift in the cellular energy production ratio towards
oxidative phosphorylation (FIG. 2E).
[0166] These results show that ERR.gamma. promotes an overt
conversion of glycolytic fast-twitch muscles, such as quadriceps,
to an oxidative slow-twitch phenotype.
Example 5
ERR.gamma. Promotes Skeletal Muscle Vascularization
[0167] Intrinsic vascularization of slow-twitch myofibers enables a
baseline of exercise independent fatigue resistance. This example
provides results demonstrating that ERR.gamma., by virtue of its
restricted expression to type I fibers can, in addition to
promoting oxidative metabolism, simultaneously induce vessel
formation to match the increased oxidative demand.
[0168] Muscle cryo-sections were stained for PECAM 1 (CD31), an
endothelial cell marker that is routinely used to detect
angiogenesis and changes in tissue vasculature. Transgenic muscles
showed increased PECAM 1 (FIG. 3A) staining compared to wild type.
Similarly, transgenic muscle cryo-sections showed an increase in
alkaline phosphatase staining, an alternative marker for tissue
endothelium (FIG. 3B). These findings point indicated induction of
angiogenesis and muscle vascularization by ERR.gamma..
[0169] To show that ERR.gamma. supports formation of functional
non-leaky blood vessels micro-angiography following
intra-ventricular perfusion of a fluorescent microspheres (0.1
.mu.M) was used. The impermeability of the microspheres allows
their vascular retention, enabling confocal angiographic "vascular
mapping" of intact and mature blood vessels. Examination of
perfused microspheres in wild type and transgenic gastrocnemius
revealed an increase in muscle vascularity by ERR.gamma. (FIG. 3C)
showing that ERR.gamma. dually promotes oxidative fiber
specification and neo-vascularization.
Example 6
Paracrine Regulation of Muscle Vascularization of ERR.gamma.
[0170] This example provides results showing how ERR.gamma.
expressed in myofibers can regulate proximal vascular
development.
[0171] Gene expression studies (FIG. 2A and Table 3) revealed
increased expression of 25 angiogenic genes, including vascular
endothelial growth factor A (Vegfa) in ERRGO quadriceps. Real time
PCR confirmed induction of two Vegfa isoforms (165 & 189) along
with Vegfb and Fgf1 in transgenic muscles (FIGS. 3D-H). Moreover,
ERR.gamma. as well as ERR.alpha. & ERR.beta. increased the
transcription of a Vegfa promoter-driven luciferase reporter in AD
293 cells (FIG. 3J). In addition, the protein levels of Vegfa and
Fgf1 were increased in the quadriceps of the transgenic mice (FIG.
3H), indicating that muscle ERR.gamma. activates paracrine networks
that are released into the microenvironment to promote
neo-vascularization.
TABLE-US-00003 TABLE 3 Angiogenic genes up-regulated in the
quadriceps of ERR.gamma. transgenic mice as compared to wild type
mice (N = 3, each pooled from 3 mice, p < 0.05, Bonferroni's
multiple comparison test). Locus Fold Description Cdh5 1.755
cadherin 5 Crhr2 2.072 corticotropin releasing hormone receptor 2
Cxcl12 2.05 chemokine (C-X-C motif) ligand 12 Efnb2 2.16 ephrin B2
Egfl7 1.958 EGF-like domain 7 Epas1 1.867 endothelial PAS domain
protein 1 Fgf1 4.123 fibroblast growth factor 1 Flt1 1.85 FMS-like
tyrosine kinase 1 Gja1 1.704 gap junction membrane channel protein
alpha 1 Kdr 1.718 kinase insert domain protein receptor Notch4
2.254 Notch gene homolog 4 (Drosophila) Nrp1 1.816 neuropilin 1
Pdgfrb 1.895 platelet derived growth factor receptor, beta
polypeptide Plcd1 2.009 phospholipase C, delta 1 Qk 1.875 quaking
Rhob 1.702 ras homolog gene family, member B Sox17 1.98 SRY-box
containing gene 17 Vegfa 2.505 vascular endothelial growth factor A
Vegfb 2.341 vascular endothelial growth factor B Vezf1 1.958
vascular endothelial zinc finger 1
[0172] As shown in Table 4, ERR.gamma. also induces key
transcriptional inducers of oxidative metabolism including Esrrb,
Ppara, Ppard and Ppargc1b.
TABLE-US-00004 TABLE 4 Transcriptional regulators are targets of
ERR' in the quadriceps of transgenic mice (N = 3, each pooled from
3 mice, p < 0.05, Bonferroni's multiple comparison test). LOCUS
DESCRIPTION FOLD Esrrb estrogen related receptor, beta 3 Ppara
peroxisome proliferator activated receptor alpha 2.444 Ppard
peroxisome proliferator activator receptor delta 2.065 Ppargc1b
peroxisome proliferative activated receptor, gamma, 1.988
coactivator 1 beta
[0173] To directly test whether ERR.gamma. triggers paracrine
angiogenesis an SVEC4-10 (murine endothelial cells) tube formation
assay was employed. It was reasoned that conditioned media from
ERR.gamma. over-expressing muscle cells would contain the
appropriate signals to induce tube formation in endothelial cells.
Indeed, treatment of SVEC4-10 cells with conditioned media from
ERR.gamma. over-expressing C2C12 myotubes stimulated tube formation
in 7-8 hr (FIG. 4A). To confirm that the conditioned media contains
angiogenic signals, the gene expression in cells and protein levels
in the media (by ELISA) of a representative angiokine, Vegfa was
examined. Over-expression of ERR.gamma. in C2C12 myotubes increased
expression of Vegfa-121, 165 and 189 genes (FIGS. 4B-D) and
increases total Vegfa secretion (by 4-fold) in the media (FIG. 4E).
These results demonstrate that ERR.gamma. can induce angiogenic
factors such as myocellular Vegfa to increase angiogenesis in a
paracrine fashion.
Example 7
Physiological Effects of ERR.gamma. Remodeled Muscle
[0174] Aerobic exercise-induced remodeling of skeletal muscles
depends on both an increase in oxidative capacity and new blood
vessel formation; changes that are a critical part of the
physiologic adaptation to training (Bloor, 2005; Egginton, 2008;
Gavin et al., 2007; Gustafsson and Kraus, 2001; Jensen et al.,
2004; Waters et al., 2004). This example describes results showing
the ability of ERR.gamma. to promote physiological re-modeling.
[0175] First, in metabolic cage oxymetric studies, the transgenic
mice exhibited an increase in oxygen consumption (during both the
light and dark cycles) in concert with the observed increased
oxidative metabolism and blood supply to skeletal muscles (FIG.
5A). Second, the ERGGO mice have a lower Respiratory Exchange Ratio
(RER) compared to the wild type mice indicative of a tendency to
preferentially oxidize fat over carbohydrate in the transgenic
skeletal muscles (FIG. 5B). The ambulatory activities of wild type
and transgenic mice were comparable, and therefore unlikely to
contribute to changes in oxymetric parameters (FIG. 5D).
[0176] Based on these combined changes the ability of ERRGO mice to
acquire enhanced running endurance was determined. ERR.gamma.
transgenic mice were able to run longer and further compared to the
wild type littermates (FIG. 5C). Finally, the ERRGO mice were
subjected to a high fat-high carbohydrate diet to establish whether
the induction of endurance muscle and oxidative RER affected global
metabolic balance. As expected ERRGO mice gained 35% less weight
than wild type controls on a high fat diet (FIG. 5E).
[0177] These findings demonstrate that targeting of ERR.gamma.
increases oxidative metabolism and blood supply to skeletal muscle
leading to increased oxygen consumption, better endurance and
resistance to weight gain.
Example 8
PGC1.alpha.-Independent Regulation of Aerobic Muscle by
ERR.gamma.
[0178] PGC1.alpha. is induced by hypoxia and exercise to promote
HIF1.alpha.-independent vascularization of type II muscle (Arany et
al., 2008) and further activated by post-translational
modifications such as deacetylation (Jager et al., 2007; Puigserver
et al., 2001; Rodgers et al., 2005). This example describes results
showing that the ERR.gamma.-induced changes in the muscle were due
to the induction and/or activation of PGC1.alpha..
[0179] The levels of PGC1.alpha. mRNA, protein and acetylation
remained unchanged in the ERR.gamma.-transformed skeletal muscle
(FIGS. 6A and 6F). Interestingly, of the two additional ERR
isoforms that can mediate PGC1.alpha. signaling, ERR.beta. but not
ERR.alpha., was also significantly induced in transgenic muscle
(Mootha et al., 2004; Schreiber et al., 2003; Huss et al.,
2002).
[0180] To determine if AMPK can ERR.gamma. control metabolism, VEGF
induction and vasculature remodeling in ERRGO mice in absence of
enhanced PGC1.alpha. signaling, the following methods were used.
AMPK is an alternative aerobic master-regulator, having a role in
metabolic (Fujii et al., 2008; Fujii et al., 2007) and vascular
adaptation (Zwetsloot et al., 2008). While AMPK is normally induced
by exercise or hypoxia, it was surprisingly found to be
constitutively activated in ERRGO muscle (FIGS. 6B and C). The AMPK
activation was further validated by measuring phospho-ACC levels
(an AMPK target and a bio-marker of AMPK activity), which was found
to be higher in the transgenic compared to the wild type muscles
(FIG. 6G). ATP consumption is critical to AMPK activation as AMP
stimulates and ATP inhibits the enzyme (Xiao et al., 2007). Indeed,
it was observed that ATP levels were lower in ERR.gamma.
over-expressing compared to control C2C12 muscle cells, providing a
biochemical basis for the observed AMPK activation (FIG. 6H). (Note
that cultured muscle cells were used for measuring ATP levels
because ERR.gamma. over-expression promotes both angiogenic gene
expression as well as oxidative respiration in a fashion similar to
transgenic muscle). Interestingly, in wild type mice, AMPK was more
active in predominantly oxidative slow-twitch compared to
predominantly glycolytic fast-twitch muscle, in resting state
(FIGS. 6B and C). Indeed, a synthetic activator AICAR, at a dose
(500 mg/kg/day) previously shown to stimulate AMPK in anaerobic
muscle and improve aerobic performance (Narkar et al., 2008), was
able to direct aspects of skeletal muscle transformation in a
fashion similar to ERR.gamma. (FIG. 6D).
[0181] These observations indicate a convergence between ERR.gamma.
and AMPK pathways that comprise an exercise-independent mechanism
to direct intrinsic vascularization and oxidative metabolism in
type I muscle, as depicted in FIG. 6E.
REFERENCES
[0182] Alaynick et al., (2007). ERRgamma directs and maintains the
transition to oxidative metabolism in the postnatal heart. Cell
Metab 6, 13-24. [0183] Annex et al., (1998). Induction and
maintenance of increased VEGF protein by chronic motor nerve
stimulation in skeletal muscle. Am J Physiol 274, H860-867. [0184]
Ao et al., (2008). Involvement of estrogen-related receptors in
transcriptional response to hypoxia and growth of solid tumors.
Proc Natl Acad Sci USA 105, 7821-7826. [0185] Arany et al., (2008).
HIF-independent regulation of VEGF and angiogenesis by the
transcriptional coactivator PGC-1alpha. Nature 451, 1008-1012.
[0186] Arany et al., (2007). The transcriptional coactivator
PGC-1beta drives the formation of oxidative type IIX fibers in
skeletal muscle. Cell Metab 5, 35-46. [0187] Ariazi et al., (2002).
Estrogen-related receptor alpha and estrogen-related receptor gamma
associate with unfavorable and favorable biomarkers, respectively,
in human breast cancer. Cancer Res 62, 6510-6518. [0188] Baar et
al., (2002). Adaptations of skeletal muscle to exercise: rapid
increase in the transcriptional coactivator PGC-1. FASEB J 16,
1879-1886. [0189] Bloor, C. M. (2005). Angiogenesis during exercise
and training. Angiogenesis 8, 263-271. [0190] Canto et al., (2010).
Interdependence of AMPK and SIRT1 for metabolic adaptation to
fasting and exercise in skeletal muscle. Cell Metab 11, 213-219.
[0191] Carmeliet, P. (2000). Mechanisms of angiogenesis and
arteriogenesis. Nat Med 6, 389-395. [0192] Cherwek et al., (2000).
Fiber type-specific differential expression of angiogenic factors
in response to chronic hindlimb ischemia. Am J Physiol Heart Circ
Physiol 279, H932-938. [0193] Cheung et al., (2005). Expression and
functional study of estrogen receptor-related receptors in human
prostatic cells and tissues. J Clin Endocrinol Metab 90, 1830-1844.
[0194] Dufour et al., (2007). Genome-wide orchestration of cardiac
functions by the orphan nuclear receptors ERRalpha and gamma. Cell
Metab 5, 345-356. [0195] Dzamko et al., (2008). AMPK-independent
pathways regulate skeletal muscle fatty acid oxidation. J Physiol
586, 5819-5831. [0196] Egginton, S. (2008). Invited review:
activity-induced angiogenesis. Pflugers Arch. [0197] Ferrara, N.,
and Kerbel, R. S. (2005). Angiogenesis as a therapeutic target.
Nature 438, 967-974. [0198] Fluck, M., and Hoppeler, H. (2003).
Molecular basis of skeletal muscle plasticity--from gene to form
and function. Rev Physiol Biochem Pharmacol 146, 159-216. [0199]
Foo et al., (2006). Ephrin-B2 controls cell motility and adhesion
during blood-vessel-wall assembly. Cell 124, 161-173. [0200]
Forough et al., (2006). Transcription factor Ets-1 regulates
fibroblast growth factor-1-mediated angiogenesis in vivo: role of
Ets-1 in the regulation of the PI3K/AKT/MMP-1 pathway. J Vasc Res
43, 327-337. [0201] Fujii et al., (2000). Exercise induces
isoform-specific increase in 5'AMP-activated protein kinase
activity in human skeletal muscle. Biochem Biophys Res Commun 273,
1150-1155. [0202] Fujii et al., (2008). Ablation of AMP-activated
protein kinase alpha2 activity exacerbates insulin resistance
induced by high-fat feeding of mice. Diabetes 57, 2958-2966. [0203]
Fujii et al., (2007). Role of AMP-activated protein kinase in
exercise capacity, whole body glucose homeostasis, and glucose
transport in skeletal muscle-insight from analysis of a transgenic
mouse model. Diabetes Res Clin Pract 77 Suppl 1, S92-98. [0204] Gao
et al., (2006). Expression of estrogen receptor-related receptor
isoforms and clinical significance in endometrial adenocarcinoma.
Int J Gynecol Cancer 16, 827-833. [0205] Gavin et al., (2007). No
difference in the skeletal muscle angiogenic response to aerobic
exercise training between young and aged men. J Physiol 585,
231-239. [0206] Gerhart-Hines et al., (2007). Metabolic control of
muscle mitochondrial function and fatty acid oxidation through
SIRT1/PGC-1alpha. EMBO J 26, 1913-1923. [0207] Giguere, V. (2008).
Transcriptional control of energy homeostasis by the
estrogen-related receptors. Endocr Rev 29, 677-696. [0208]
Grunewald et al., (2006). VEGF-induced adult neovascularization:
recruitment, retention, and role of accessory cells. Cell 124,
175-189. [0209] Gupta et al., (1998). Chemokine receptors in human
endothelial cells. Functional expression of CXCR4 and its
transcriptional regulation by inflammatory cytokines. J Biol Chem
273, 4282-4287. [0210] Gustafsson, T., and Kraus, W. E. (2001).
Exercise-induced angiogenesis-related growth and transcription
factors in skeletal muscle, and their modification in muscle
pathology. Front Biosci 6, D75-89. [0211] Hainaud et al., (2006).
The role of the vascular endothelial growth factor-Delta-like 4
ligand/Notch4-ephrin B2 cascade in tumor vessel remodeling and
endothelial cell functions. Cancer Res 66, 8501-8510. [0212] Heard
et al., (2000). Human ERRgamma, a third member of the estrogen
receptor-related receptor (ERR) subfamily of orphan nuclear
receptors: tissue-specific isoforms are expressed during
development and in the adult. Mol Endocrinol 14, 382-392. [0213]
Hong, H., Yang, L., and Stallcup, M. R. (1999). Hormone-independent
transcriptional activation and coactivator binding by novel orphan
nuclear receptor ERR3. J Biol Chem 274, 22618-22626. [0214]
Hoppeler, H., and Vogt, M. (2001a). Hypoxia training for sea-level
performance. Training high-living low. Adv Exp Med Biol 502, 61-73.
[0215] Hoppeler, H., and Vogt, M. (2001b). Muscle tissue
adaptations to hypoxia. J Exp Biol 204, 3133-3139. [0216] Huss et
al., (2002). Peroxisome proliferator-activated receptor
coactivator-1alpha (PGC-1alpha) coactivates the cardiac-enriched
nuclear receptors estrogen-related receptor-alpha and -gamma.
Identification of novel leucine-rich interaction motif within
PGC-1alpha. J Biol Chem 277, 40265-40274. [0217] Huss et al.,
(2004). Estrogen-related receptor alpha directs peroxisome
proliferator-activated receptor alpha signaling in the
transcriptional control of energy metabolism in cardiac and
skeletal muscle. Mol Cell Biol 24, 9079-9091. [0218] Jager et al.,
(2007). AMP-activated protein kinase (AMPK) action in skeletal
muscle via direct phosphorylation of PGC-1alpha. Proc Natl Acad Sci
USA 104, 12017-12022. [0219] Jensen et al., (2004). Effect of high
intensity training on capillarization and presence of angiogenic
factors in human skeletal muscle. J Physiol 557, 571-582. [0220]
Jensen et al., (2007). Possible CaMKK-dependent regulation of AMPK
phosphorylation and glucose uptake at the onset of mild tetanic
skeletal muscle contraction. Am J Physiol Endocrinol Metab 292,
E1308-1317. [0221] Johnson et al., (2004). Matrix
metalloproteinase-9 is required for adequate angiogenic
revascularization of ischemic tissues: potential role in capillary
branching. Circ Res 94, 262-268. [0222] Jorgensen et al., (2004).
Knockout of the alpha2 but not alpha1 5'-AMP-activated protein
kinase isoform abolishes
5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranosidebut not
contraction-induced glucose uptake in skeletal muscle. J Biol Chem
279, 1070-1079. [0223] Lagouge et al., (2006). Resveratrol improves
mitochondrial function and protects against metabolic disease by
activating SIRT1 and PGC-1alpha. Cell 127, 1109-1122. [0224] Leon
et al., (2002). Activated Notch4 inhibits angiogenesis: role of
beta 1-integrin activation. Mol Cell Biol 22, 2830-2841. [0225] Lin
et al., (2002). Transcriptional co-activator PGC-1 alpha drives the
formation of slow-twitch muscle fibres. Nature 418, 797-801. [0226]
Lin et al., (2004). Defects in adaptive energy metabolism with
CNS-linked hyperactivity in PGC-1alpha null mice. Cell 119,
121-135. [0227] Mason et al., (2004). Loss of skeletal muscle
HIF-1alpha results in altered exercise endurance. PLoS Biol 2,
e288. [0228] Mason et al., (2007). HIF-1alpha in endurance
training: suppression of oxidative metabolism. Am J Physiol Regul
Integr Comp Physiol 293, R2059-2069. [0229] Matsui et al., (2006).
Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in
mice. J Cell Sci 119, 3513-3526. [0230] Minnich, A., Tian, N.,
Byan, L., and Bilder, G. (2001). A potent PPARalpha agonist
stimulates mitochondrial fatty acid beta-oxidation in liver and
skeletal muscle. Am J Physiol Endocrinol Metab 280, E270-279.
[0231] Mootha et al., (2004). Erralpha and Gabpa/b specify
PGC-1alpha-dependent oxidative phosphorylation gene expression that
is altered in diabetic muscle. Proc Natl Acad Sci USA 101,
6570-6575. [0232] Muoio et al., (2002). Peroxisome
proliferator-activated receptor-alpha regulates fatty acid
utilization in primary human skeletal muscle cells. Diabetes 51,
901-909. [0233] Muscat, G. E., and Kedes, L. (1987). Multiple
5'-flanking regions of the human alpha-skeletal actin gene
synergistically modulate muscle-specific expression. Mol Cell Biol
7, 4089-4099. [0234] Narkar et al., (2008). AMPK and PPARdelta
agonists are exercise mimetics. Cell 134, 405-415. [0235] Pajusola
et al., (2005). Stabilized HIF-1alpha is superior to VEGF for
angiogenesis in skeletal muscle via adeno-associated virus gene
transfer. Faseb J 19, 1365-1367. [0236] Partridge et al., (2000).
Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated
endothelial cell migration. J Cell Biochem 78, 487-499. [0237]
Pette, D., and Staron, R. S. (2000). Myosin isoforms, muscle fiber
types, and transitions. Microsc Res Tech 50, 500-509. [0238]
Pilegaard et al., (2003). Exercise induces transient
transcriptional activation of the PGC-1alpha gene in human skeletal
muscle. J Physiol 546, 851-858. Puigserver et al., (2001). Cytokine
stimulation of energy expenditure through p38 MAP kinase activation
of PPARgamma coactivator-1. Mol Cell 8, 971-982. [0239] Ripoll et
al., (1979). Changes in the capillarity of skeletal muscle in the
growing rat. Pflugers Arch 380, 153-158. [0240] Rockl (2007).
Skeletal muscle adaptation to exercise training: AMP-activated
protein kinase mediates muscle fiber type shift. Diabetes 56,
2062-2069. [0241] Rodgers et al., (2005). Nutrient control of
glucose homeostasis through a complex of PGC-1alpha and SIRT1.
Nature 434, 113-118. [0242] Russell et al., (2003). Endurance
training in humans leads to fiber type-specific increases in levels
of peroxisome proliferator-activated receptor-gamma coactivator-1
and peroxisome proliferator-activated receptor-alpha in skeletal
muscle. Diabetes 52, 2874-2881. [0243] Russell et al., (2005).
Regulation of metabolic transcriptional co-activators and
transcription factors with acute exercise. FASEB J 19, 986-988.
[0244] Schreiber et al., (2003). The transcriptional coactivator
PGC-1 regulates the expression and activity of the orphan nuclear
receptor estrogen-related receptor alpha (ERRalpha). J Biol Chem
278, 9013-9018. [0245] Seth et al., (2007). The transcriptional
corepressor RIP140 regulates oxidative metabolism in skeletal
muscle. Cell Metab 6, 236-245. [0246] Shao et al., (2008). Statin
and stromal cell-derived factor-1 additively promote angiogenesis
by enhancement of progenitor cells incorporation into new vessels.
Stem Cells 26, 1376-1384. [0247] Springer et al., (1998). VEGF gene
delivery to muscle: potential role for vasculogenesis in adults.
Mol Cell 2, 549-558. [0248] Springer et al., (2000). Angiogenesis
monitored by perfusion with a space-filling microbead suspension.
Mol Ther 1, 82-87. [0249] Wang, Y. X., Zhang, C. L., Yu, R. T.,
Cho, H. K., Nelson, M. C., Bayuga-Ocampo, C. R., Ham, J., Kang, H.,
and Evans, R. M. (2004). Regulation of muscle fiber type and
running endurance by PPARdelta. PLoS Biol 2, e294. [0250] Waters et
al., (2004). Voluntary running induces fiber type-specific
angiogenesis in mouse skeletal muscle. Am J Physiol Cell Physiol
287, C1342-1348. [0251] Winder, W. W., and Hardie, D. G. (1996).
Inactivation of acetyl-CoA carboxylase and activation of
AMP-activated protein kinase in muscle during exercise. Am J
Physiol 270, E299-304. [0252] Wojtaszewski et al., (2000).
Isoform-specific and exercise intensity-dependent activation of
5'-AMP-activated protein kinase in human skeletal muscle. J Physiol
528 Pt 1, 221-226. [0253] Xiao et al., (2007). Structural basis for
AMP binding to mammalian AMP-activated protein kinase. Nature 449,
496-500. [0254] Zechner, et al., (2010). Total skeletal muscle
PGC-1 deficiency uncouples mitochondrial derangements from fiber
type determination and insulin sensitivity. Cell Metab 12, 633-642.
[0255] Zhang et al., (2006). Estrogen-related receptors stimulate
pyruvate dehydrogenase kinase isoform 4 gene expression. J Biol
Chem 281, 39897-39906. [0256] Zheng et al., H. (2007). Migration of
endothelial progenitor cells mediated by stromal cell-derived
factor-1alpha/CXCR4 via PI3K/Akt/eNOS signal transduction pathway.
J Cardiovasc Pharmacol 50, 274-280. [0257] Zwetsloot et al.,
(2008). AMPK regulates basal skeletal muscle capillarization and
VEGF expression, but is not necessary for the angiogenic response
to exercise. J Physiol 586, 6021-6035.
[0258] In view of the many possible embodiments to which the
principles of the disclosure may be applied, it should be
recognized that the illustrated embodiments are only examples of
the disclosure and should not be taken as limiting the scope of the
invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
Sequence CWU 1
1
213245DNAHomo sapiensCDS(488)..(1795) 1tatccacaca cagcatcgga
atattgctag ctaactcaac aaatgtgcaa atcaggggac 60tgttgtgtgt gtaccgattc
atgtctagac tgtttttatt ggtgaagtag gaactgcctc 120atcagtcatg
ggatcatagt gtcacagatg gaaaagcaac tatattagtc taaatatttg
180attctgcagt tgcatgcacc aaattcagtg aggttagatg ttaaatcatc
ttgttggctt 240tgggctgaat ttgatctaag agacaaaagt ctcaaacaac
agactactta ctgccaccac 300atctcgattc aaagaatagt tttcacatgt
tcgtggtgtg gaaaggactt tctgtttctc 360actaatttct tcagctatac
caagagtggt gttgtctttg aacaggaagg acagcaaaaa 420taaacataac
agttttttca taagatacac agctgcatta cacatcccaa aattgcttct 480ctgcaga
atg tca aac aaa gat cga cac att gat tcc agc tgt tcg tcc 529 Met Ser
Asn Lys Asp Arg His Ile Asp Ser Ser Cys Ser Ser 1 5 10 ttc atc aag
acg gaa cct tcc agc cca gcc tcc ctg acg gac agc gtc 577Phe Ile Lys
Thr Glu Pro Ser Ser Pro Ala Ser Leu Thr Asp Ser Val 15 20 25 30 aac
cac cac agc cct ggt ggc tct tca gac gcc agt ggg agc tac agt 625Asn
His His Ser Pro Gly Gly Ser Ser Asp Ala Ser Gly Ser Tyr Ser 35 40
45 tca acc atg aat ggc cat cag aac gga ctt gac tcg cca cct ctc tac
673Ser Thr Met Asn Gly His Gln Asn Gly Leu Asp Ser Pro Pro Leu Tyr
50 55 60 cct tct gct cct atc ctg gga ggt agt ggg cct gtc agg aaa
ctg tat 721Pro Ser Ala Pro Ile Leu Gly Gly Ser Gly Pro Val Arg Lys
Leu Tyr 65 70 75 gat gac tgc tcc agc acc att gtt gaa gat ccc cag
acc aag tgt gaa 769Asp Asp Cys Ser Ser Thr Ile Val Glu Asp Pro Gln
Thr Lys Cys Glu 80 85 90 tac atg ctc aac tcg atg ccc aag aga ctg
tgt tta gtg tgt ggt gac 817Tyr Met Leu Asn Ser Met Pro Lys Arg Leu
Cys Leu Val Cys Gly Asp 95 100 105 110 atc gct tct ggg tac cac tat
ggg gta gca tca tgt gaa gcc tgc aag 865Ile Ala Ser Gly Tyr His Tyr
Gly Val Ala Ser Cys Glu Ala Cys Lys 115 120 125 gca ttc ttc aag agg
aca att caa ggc aat ata gaa tac agc tgc cct 913Ala Phe Phe Lys Arg
Thr Ile Gln Gly Asn Ile Glu Tyr Ser Cys Pro 130 135 140 gcc acg aat
gaa tgt gaa atc aca aag cgc aga cgt aaa tcc tgc cag 961Ala Thr Asn
Glu Cys Glu Ile Thr Lys Arg Arg Arg Lys Ser Cys Gln 145 150 155 gct
tgc cgc ttc atg aag tgt tta aaa gtg ggc atg ctg aaa gaa ggg 1009Ala
Cys Arg Phe Met Lys Cys Leu Lys Val Gly Met Leu Lys Glu Gly 160 165
170 gtg cgt ctt gac aga gta cgt gga ggt cgg cag aag tac aag cgc agg
1057Val Arg Leu Asp Arg Val Arg Gly Gly Arg Gln Lys Tyr Lys Arg Arg
175 180 185 190 ata gat gcg gag aac agc cca tac ctg aac cct cag ctg
gtt cag cca 1105Ile Asp Ala Glu Asn Ser Pro Tyr Leu Asn Pro Gln Leu
Val Gln Pro 195 200 205 gcc aaa aag cca tat aac aag att gtc tca cat
ttg ttg gtg gct gaa 1153Ala Lys Lys Pro Tyr Asn Lys Ile Val Ser His
Leu Leu Val Ala Glu 210 215 220 ccg gag aag atc tat gcc atg cct gac
cct act gtc ccc gac agt gac 1201Pro Glu Lys Ile Tyr Ala Met Pro Asp
Pro Thr Val Pro Asp Ser Asp 225 230 235 atc aaa gcc ctc act aca ctg
tgt gac ttg gcc gac cga gag ttg gtg 1249Ile Lys Ala Leu Thr Thr Leu
Cys Asp Leu Ala Asp Arg Glu Leu Val 240 245 250 gtt atc att gga tgg
gcg aag cat att cca ggc ttc tcc acg ctg tcc 1297Val Ile Ile Gly Trp
Ala Lys His Ile Pro Gly Phe Ser Thr Leu Ser 255 260 265 270 ctg gcg
gac cag atg agc ctt ctg cag agt gct tgg atg gaa att ttg 1345Leu Ala
Asp Gln Met Ser Leu Leu Gln Ser Ala Trp Met Glu Ile Leu 275 280 285
atc ctt ggt gtc gta tac cgg tct ctt tca ttt gag gat gaa ctt gtc
1393Ile Leu Gly Val Val Tyr Arg Ser Leu Ser Phe Glu Asp Glu Leu Val
290 295 300 tat gca gac gat tat ata atg gac gaa gac cag tcc aaa tta
gca ggc 1441Tyr Ala Asp Asp Tyr Ile Met Asp Glu Asp Gln Ser Lys Leu
Ala Gly 305 310 315 ctt ctt gat cta aat aat gct atc ctg cag ctg gta
aag aaa tac aag 1489Leu Leu Asp Leu Asn Asn Ala Ile Leu Gln Leu Val
Lys Lys Tyr Lys 320 325 330 agc atg aag ctg gaa aaa gaa gaa ttt gtc
acc ctc aaa gct ata gct 1537Ser Met Lys Leu Glu Lys Glu Glu Phe Val
Thr Leu Lys Ala Ile Ala 335 340 345 350 ctt gct aat tca gac tcc atg
cac ata gaa gat gtt gaa gcc gtt cag 1585Leu Ala Asn Ser Asp Ser Met
His Ile Glu Asp Val Glu Ala Val Gln 355 360 365 aag ctt cag gat gtc
tta cat gaa gcg ctg cag gat tat gaa gct ggc 1633Lys Leu Gln Asp Val
Leu His Glu Ala Leu Gln Asp Tyr Glu Ala Gly 370 375 380 cag cac atg
gaa gac cct cgt cga gct ggc aag atg ctg atg aca ctg 1681Gln His Met
Glu Asp Pro Arg Arg Ala Gly Lys Met Leu Met Thr Leu 385 390 395 cca
ctc ctg agg cag acc tct acc aag gcc gtg cag cat ttc tac aac 1729Pro
Leu Leu Arg Gln Thr Ser Thr Lys Ala Val Gln His Phe Tyr Asn 400 405
410 atc aaa cta gaa ggc aaa gtc cca atg cac aaa ctt ttt ttg gaa atg
1777Ile Lys Leu Glu Gly Lys Val Pro Met His Lys Leu Phe Leu Glu Met
415 420 425 430 ttg gag gcc aag gtc tga ctaaaagctc cctgggcctt
cccatccttc 1825Leu Glu Ala Lys Val 435 atgttgaaaa agggaaaata
aacccaagag tgatgtcgaa gaaacttaga gtttagttaa 1885caacatcaaa
aatcaacaga ctgcactgat aatttagcag caagactatg aagcagcttt
1945cagattcctc cataggttcc tgatgagttc tttctacttt ctccatcatc
ttctttcctc 2005tttcttccca catttctctt tctctttatt ttttctcctt
ttcttctttc acctccctta 2065tttctttgct tctttcattc ctagttccca
ttctccttta ttttcttccc gtctgcctgc 2125cttctttctt ttctttacct
actctcattc ctctcttttc tcatccttcc ccttttttct 2185aaatttgaaa
tagctttagt ttaaaaaaaa aaatcctccc ttcccccttt cctttccctt
2245tctttccttt ttccctttcc ttttcccttt cctttccttt cctcttgacc
ttctttccat 2305ctttcttttt cttccttctg ctgctgaact tttaaaagag
gtctctaact gaagagagat 2365ggaagccagc cctgccaaag gatggagatc
cataatatgg atgccagtga acttattgtg 2425aaccataccg tccccaatga
ctaaggaatc aaagagagag aaccaacgtt cctaaaagta 2485cagtgcaaca
tatacaaatt gactgagtgc agtattagat ttcatgggag cagcctctaa
2545ttagacaact taagcaacgt tgcatcggct gcttcttatc attgcttttc
catctagatc 2605agttacagcc atttgattcc ttaattgttt tttcaagtct
tccaggtatt tgttagttta 2665gctactatgt aactttttca gggaatagtt
taagctttat tcattcatgc aatactaaag 2725agaaataaga atactgcaat
tttgtgctgg ctttgaacaa ttacgaacaa taatgaagga 2785caaatgaatc
ctgaaggaag atttttaaaa atgttttgtt tcttcttaca aatggagatt
2845tttttgtacc agctttacca cttttcagcc atttattaat atgggaattt
aacttactca 2905agcaatagtt gaagggaagg tgcatattat cacggatgca
atttatgttg tgtgccagtc 2965tggtcccaaa catcaatttc ttaacatgag
ctccagttta cctaaatgtt cactgacaca 3025aaggatgaga ttacacctac
agtgactctg agtagtcaca tatataagca ctgcacatga 3085gatatagatc
cgtagaattg tcaggagtgc acctctctac ttgggaggta caattgccat
3145atgatttcta gctgccatgg tggttaggaa tgtgatactg cctgtttgca
aagtcacaga 3205ccttgcctca gaaggagctg tgagccagta ttcatttaag
32452435PRTHomo sapiens 2Met Ser Asn Lys Asp Arg His Ile Asp Ser
Ser Cys Ser Ser Phe Ile 1 5 10 15 Lys Thr Glu Pro Ser Ser Pro Ala
Ser Leu Thr Asp Ser Val Asn His 20 25 30 His Ser Pro Gly Gly Ser
Ser Asp Ala Ser Gly Ser Tyr Ser Ser Thr 35 40 45 Met Asn Gly His
Gln Asn Gly Leu Asp Ser Pro Pro Leu Tyr Pro Ser 50 55 60 Ala Pro
Ile Leu Gly Gly Ser Gly Pro Val Arg Lys Leu Tyr Asp Asp 65 70 75 80
Cys Ser Ser Thr Ile Val Glu Asp Pro Gln Thr Lys Cys Glu Tyr Met 85
90 95 Leu Asn Ser Met Pro Lys Arg Leu Cys Leu Val Cys Gly Asp Ile
Ala 100 105 110 Ser Gly Tyr His Tyr Gly Val Ala Ser Cys Glu Ala Cys
Lys Ala Phe 115 120 125 Phe Lys Arg Thr Ile Gln Gly Asn Ile Glu Tyr
Ser Cys Pro Ala Thr 130 135 140 Asn Glu Cys Glu Ile Thr Lys Arg Arg
Arg Lys Ser Cys Gln Ala Cys 145 150 155 160 Arg Phe Met Lys Cys Leu
Lys Val Gly Met Leu Lys Glu Gly Val Arg 165 170 175 Leu Asp Arg Val
Arg Gly Gly Arg Gln Lys Tyr Lys Arg Arg Ile Asp 180 185 190 Ala Glu
Asn Ser Pro Tyr Leu Asn Pro Gln Leu Val Gln Pro Ala Lys 195 200 205
Lys Pro Tyr Asn Lys Ile Val Ser His Leu Leu Val Ala Glu Pro Glu 210
215 220 Lys Ile Tyr Ala Met Pro Asp Pro Thr Val Pro Asp Ser Asp Ile
Lys 225 230 235 240 Ala Leu Thr Thr Leu Cys Asp Leu Ala Asp Arg Glu
Leu Val Val Ile 245 250 255 Ile Gly Trp Ala Lys His Ile Pro Gly Phe
Ser Thr Leu Ser Leu Ala 260 265 270 Asp Gln Met Ser Leu Leu Gln Ser
Ala Trp Met Glu Ile Leu Ile Leu 275 280 285 Gly Val Val Tyr Arg Ser
Leu Ser Phe Glu Asp Glu Leu Val Tyr Ala 290 295 300 Asp Asp Tyr Ile
Met Asp Glu Asp Gln Ser Lys Leu Ala Gly Leu Leu 305 310 315 320 Asp
Leu Asn Asn Ala Ile Leu Gln Leu Val Lys Lys Tyr Lys Ser Met 325 330
335 Lys Leu Glu Lys Glu Glu Phe Val Thr Leu Lys Ala Ile Ala Leu Ala
340 345 350 Asn Ser Asp Ser Met His Ile Glu Asp Val Glu Ala Val Gln
Lys Leu 355 360 365 Gln Asp Val Leu His Glu Ala Leu Gln Asp Tyr Glu
Ala Gly Gln His 370 375 380 Met Glu Asp Pro Arg Arg Ala Gly Lys Met
Leu Met Thr Leu Pro Leu 385 390 395 400 Leu Arg Gln Thr Ser Thr Lys
Ala Val Gln His Phe Tyr Asn Ile Lys 405 410 415 Leu Glu Gly Lys Val
Pro Met His Lys Leu Phe Leu Glu Met Leu Glu 420 425 430 Ala Lys Val
435
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