U.S. patent application number 16/917074 was filed with the patent office on 2021-03-18 for method to enhance tissue regeneration.
This patent application is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. The applicant listed for this patent is CHILDREN'S MEDICAL CENTER CORPORATION, THE GENERAL HOSPITAL CORPORATION. Invention is credited to Wolfram GOESSLING, Trista E. NORTH, Leonard I. ZON.
Application Number | 20210077505 16/917074 |
Document ID | / |
Family ID | 1000005237013 |
Filed Date | 2021-03-18 |
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United States Patent
Application |
20210077505 |
Kind Code |
A1 |
ZON; Leonard I. ; et
al. |
March 18, 2021 |
METHOD TO ENHANCE TISSUE REGENERATION
Abstract
The present invention provides for compositions and methods for
modulating tissue growth using tissue growth modulators, which are
agents that either enhance or inhibit tissue growth as desired by a
particular indication by modulating the PG or Wnt signaling
pathways, or employing modulators of both PG and Wnt signaling
pathways for a synergistic effect or highly selective effect.
Inventors: |
ZON; Leonard I.; (Wellesley,
MA) ; NORTH; Trista E.; (Newton Center, MA) ;
GOESSLING; Wolfram; (Chestnut Hill, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S MEDICAL CENTER CORPORATION
THE GENERAL HOSPITAL CORPORATION |
Boston
Boston |
MA
MA |
US
US |
|
|
Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION
Boston
MA
THE GENERAL HOSPITAL CORPORATION
Boston
MA
|
Family ID: |
1000005237013 |
Appl. No.: |
16/917074 |
Filed: |
June 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15177365 |
Jun 9, 2016 |
10736906 |
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16917074 |
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12445986 |
Apr 17, 2009 |
9402852 |
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PCT/US2007/082093 |
Oct 22, 2007 |
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15177365 |
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60853351 |
Oct 20, 2006 |
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60853202 |
Oct 20, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/557 20130101;
A61K 31/5575 20130101; A61K 33/14 20130101; A61K 31/41 20130101;
A61K 31/5585 20130101; A61K 31/404 20130101; A61K 31/558 20130101;
A61K 31/655 20130101; A61K 31/201 20130101 |
International
Class: |
A61K 31/5575 20060101
A61K031/5575; A61K 31/557 20060101 A61K031/557; A61K 31/201
20060101 A61K031/201; A61K 31/404 20060101 A61K031/404; A61K 31/41
20060101 A61K031/41; A61K 31/558 20060101 A61K031/558; A61K 31/5585
20060101 A61K031/5585; A61K 31/655 20060101 A61K031/655; A61K 33/14
20060101 A61K033/14 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No. CA103846-02 and DK071940 awarded by the National Institutes of
Health. The government has certain rights in this invention.
Claims
1-3. (canceled)
4. A pharmaceutical composition comprising at least one
prostaglandin signaling pathway activator and a wnt pathway
activator, wherein the at least one prostaglandin signaling pathway
activator is a PGE.sub.2 receptor agonist.
5. The composition of claim 4, wherein the at least one PGE.sub.2
receptor agonist comprises at least one prostaglandin signaling
pathway activator selected from the group consisting of: PGE2,
PGI.sub.2, 16-phenyl tetranor PGE.sub.2, 16,16-dimethyl PGE.sub.2,
19(R)-hydroxy PGE.sub.2, 16,16-dimethyl PGE.sub.2
p-(p-acetamidobenzamido) phenyl ester,
9-deoxy-9-methylene-16,16-dimethyl PGE.sub.2, PGE.sub.2 methyl
ester, Butaprost, 15(S)-15-methyl PGE.sub.2, 15(R)-15-methyl
PGE.sub.2, 20-hydroxy PGE.sub.2, 11-deoxy-16,16-dimethyl PGE.sub.2,
9-deoxy-9-methylene PGE.sub.2, PGE serinol amide, and
Sulprostone.
6. The composition of claim 4, wherein the wnt pathway activator is
selected from the group consisting of a GSK-3 inhibitor and a
soluble Wnt ligand.
7. The composition of claim 5, wherein the wnt pathway activator is
selected from the group consisting of a GSK-3 inhibitor and a
soluble Wnt ligand.
8. The composition of claim 6, wherein the GSK-3 inhibitor is LiCl
or 6-bromoindirubin-3'-oxime (BIO).
9. The composition of claim 7, wherein the GSK-3 inhibitor is LiCl
or 6-bromoindirubin-3'-oxime (BIO).
10. The composition of claim 6, wherein the soluble Wnt ligand is
Wnt2b, Wnt3, or Wnt8.
11. The composition of claim 7, wherein the soluble Wnt ligand is
Wnt2b, Wnt3, or Wnt8.
12. A pharmaceutical composition comprising at least one
prostaglandin signaling pathway activator or a downstream mediator
of the prostaglandin signaling pathway, and a wnt pathway
activator; wherein the at least one prostaglandin signaling pathway
activator is a PGE.sub.2 receptor agonist; wherein the downstream
mediator of the prostaglandin signaling pathway is selected from
the group consisting of: Forskolin, 8-bromo-cAMP,
Sp-5,6,-DCI-cBiMPS, Bapta-AM, Fendiline, Nicardipine, Nifedipine,
Pimozide, Strophanthidin, and Lanatoside; and wherein the wnt
pathway activator is selected from the group consisting of a GSK-3
inhibitor and a soluble Wnt ligand.
13. The pharmaceutical composition of claim 12, wherein the
PGE.sub.2 receptor agonist comprises at least one prostaglandin
signaling pathway activator selected from the group consisting of:
PGE.sub.2, PGI.sub.2, 16-phenyl tetranor PGE.sub.2, 16,16-dimethyl
PGE.sub.2, 19(R)-hydroxy PGE.sub.2, 16,16-dimethyl PGE.sub.2
p-(p-acetamidobenzamido) phenyl ester,
9-deoxy-9-methylene-16,16-dimethyl PGE.sub.2, PGE.sub.2 methyl
ester, Butaprost, 15(S)-15-methyl PGE.sub.2, 15(R)-15-methyl
PGE.sub.2, 20-hydroxy PGE.sub.2, 11-deoxy-16,16-dimethyl PGE.sub.2,
9-deoxy-9-methylene PGE.sub.2, PGE serinol amide, and
Sulprostone.
14. A method for promoting tissue growth or regeneration,
comprising: contacting a tissue with at least one prostaglandin
signaling pathway activator or a downstream mediator of the
prostaglandin signaling pathway, and a wnt pathway activator;
wherein the at least one prostaglandin signaling pathway activator
is a PGE.sub.2 receptor agonist; wherein the downstream mediator of
the prostaglandin signaling pathway is selected from the group
consisting of: Forskolin, 8-bromo-cAMP, Sp-5,6,-DCI-cBiMPS,
Bapta-AM, Fendiline, Nicardipine, Nifedipine, Pimozide,
Strophanthidin, and Lanatoside; and wherein the wnt pathway
activator is selected from the group consisting of a GSK-3
inhibitor and a soluble Wnt ligand.
15. A method for promoting tissue growth or regeneration,
comprising: contacting a tissue the tissue with a prostaglandin
signaling pathway activator, and a wnt pathway activator; wherein
the prostaglandin signaling pathway activator is a PGE.sub.2
receptor agonist; and wherein the wnt pathway activator is selected
from the group consisting of a GSK-3 inhibitor and a soluble Wnt
ligand.
16. The method of claim 15, wherein the PGE.sub.2 receptor agonist
is selected from the group consisting of: PGE.sub.2, PGI.sub.2,
16-phenyl tetranor PGE.sub.2, 16,16-dimethyl PGE.sub.2,
19(R)-hydroxy PGE.sub.2, 16,16-dimethyl PGE.sub.2
p-(p-acetamidobenzamido) phenyl ester,
0-deoxy-9-methylene-16,16-dimethyl PGE.sub.2, PGE.sub.2 methyl
ester, Butraprost, 15(S)-15-methyl PGE.sub.2, 15(R)-15-methyl
PGE.sub.2, 20-hydroxy PGE.sub.2, 11-deoxy-16,16-dimethyl PGE.sub.2,
9-deoxy-9-methylene PGE.sub.2, PGE.sub.2 serinol amide, and
Sulprostone.
17. The method of claim 14, wherein the prostaglandin signaling
pathway activator is selected from the group consisting of:
PGE.sub.2 and 16,16-dimethyl PGE.sub.2.
18. The method of claim 14, wherein the GSK-3 inhibitor is LiCl or
6-bromoindirubin-3'-oxime (BIO).
19. The method of claim 15, wherein the GSK-3 inhibitor is LiCl or
6-bromoindirubin-3'-oxime (BIO).
20. The method of claim 14, wherein the soluble Wnt ligand is
Wnt2b, Wnt3, or Wnt8.
21. The method of claim 15, wherein the soluble Wnt ligand is
Wnt2b, Wnt3, or Wnt8.
22. The method of claim 14, wherein the tissue is selected from the
group consisting of liver tissue, myocardial tissue, bone tissue,
and skin tissue.
23. The method of claim 14, wherein the tissue is a non-cancerous
tissue.
24. The method of claim 14, wherein the contact is ex vivo or in
vivo.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/177,365 filed Jun. 9, 2016, which is a
divisional application of U.S. patent application Ser. No.
12/445,986 filed Apr. 17, 2009, which is now U.S. Pat. No.
9,402,852 issued Aug. 2, 2016, which a 35 U.S.C. .sctn. 371
National Phase Entry of the International Application No.
PCT/US2007/082093 filed on Oct. 22, 2007, which designates the
United States, and claims the benefit under 35 U.S.C. .sctn. 119(e)
of U.S. Provisional Patent applications No. 60/853,351, entitled
Method to Modulate Hematopoietic Stem Cell Growth, filed on Oct.
20, 2006 and No. 60/853,202, entitled Method to Enhance Tissue
Regeneration, filed on Oct. 20, 2006; and also claims the benefit
of WO/2007/112084 A2, entitled Method to Modulate Hematopoietic
Stem Cell Growth, filed Apr. 26, 2007, each by Leonard I. Zon,
Trista E. North and Wolfram Goessling, which applications are
incorporated herein by reference in their entirety.
FIELD OF THE DISCLOSURE
[0003] The present embodiments provide for modulators that either
enhance or inhibit tissue development or regeneration in vitro, in
vivo, and ex vivo. More specifically, for example, modulators that
interact with the prostaglandin or wnt signaling pathways may be
used to enhance tissue response to regeneration in organs such as
liver, hematopoietic stem cells, skin, vessels, and other organs
capable of regeneration.
BACKGROUND
[0004] Regenerative medicine holds extraordinary potential for the
development of therapies which may change the future for those
suffering from organ loss due to accident, defect, or disease.
Understanding developmental signaling pathways may unlock the
promise not only of tissue regeneration, but of cancer
inhibition.
[0005] For example, in the development of liver tissue, the
undifferentiated endodermal germ layer is patterned to form liver,
intestine, pancreas, and accessory organs by the action of a
variety of signaling pathways. The plasticity of endodermal
progenitors at early stages of development, and the mechanisms
regulating endodermal cell fate and subsequent organ growth are
poorly understood. The liver remains capable of repair and
regeneration in the adult, thus further elucidation of the pathways
regulating liver development may clarify mechanisms of tissue
homeostasis and regeneration. As the progression of disease states
involves the reaction of primitive cell programs of proliferation
and differentiation, a better understanding of tissue organogenesis
may provide targets for pharmaceutical intervention to, for
example, inhibit carcinogenesis or, conversely, enhance tissue
regeneration.
SUMMARY
[0006] The compositions and methods of the present embodiments
provide for tissue growth modulators, which are agents that either
enhance tissue development and growth or inhibit tissue development
as desired by a particular indication. These modulators act by
stimulating or suppressing signaling pathways important for tissue
growth or regeneration.
[0007] For example, wnt signaling may be manipulated to enhance
tissue regeneration, particularly liver regeneration, blood
repopulation, vessel growth, and wound healing. Activators of the
wnt signaling pathway can be used to enhance these processes in
both development and regeneration, such a modulator may be a
synthetic or soluble wnt ligand, an inhibitor of .beta.-catenin
destruction, or a transcriptional co-activator.
[0008] Prostaglandin signaling interacts with wnt signaling, and
thus may be used to alter wnt activity to modulate development and
tissue regeneration. A modulator of the present invention may be a
compound that alters prostaglandin signaling or its downstream
effectors, and may be used to modify wnt signaling in organ growth
and regeneration processes. For example, effectors downstream of
the prostaglandin receptor activation such as cyclic AMP,
PI3-kinase and protein kinase A can be directly manipulated to
exert effects on the wnt signaling pathway.
[0009] Modulators of the prostaglandin pathway may also be used as
a mechanism to regulate wnt activity, thereby allowing "fine
tuning" of growth and regeneration signals. For example, activation
of wnt signaling can enhance tissue growth, indomethacin can be
used to slow or stop this effect one the desired result has been
achieved.
[0010] Additionally, modulators of the prostaglandin and wnt
pathways may be used synergistically to increase/enhance total wnt
activity, while avoiding toxicities of using either high dose or
repeated dosing of compound/method to directly activate the wnt
pathway. The present invention has confirmed each of these
principles in both fish embryos and adults, as well as in adult
mammals.
[0011] Modulators of the wnt or prostaglandin signaling pathway may
be used to enhance liver regeneration after toxic injury, such as
acetaminophen poisoning, after surgical resection of tumors or
diseased liver tissue, or after resection of a healthy part of the
liver for organ donation. These modulators may be administered in a
systemic fashion or by direct application to the liver, such as
infusion into the portal vein. Furthermore, prostaglandin modifiers
may be used ex vivo and in vitro to enhance liver stem cell and
heptocyte growth in culture in preparation for hepatocyte
transplants, or in bioartifical liver assist devices for patients
with fulminant hepatic failure.
[0012] Further more, the modulation of wnt signaling either
directly or via manipulation of the prostaglandin pathway could be
used in other tissues to enhance organ repair and regeneration,
specifically in hematopoietic stem cell growth and homeostasis, in
wound healing and repair, in vessel growth and regeneration, and in
the repair and regeneration of other organs, such as heart and
nervous system.
[0013] In general, the compounds of the present embodiments can be
applied systemically to the patient, in a targeted fashion to the
organ in question, or ex vivo to cells or organ tissue.
[0014] Manipulation of the prostaglandin pathway can be
accomplished by pharmaceutical measure, such as the targeted
administration of activators or inhibitors of various components of
the prostaglandin pathway. Alternatively, genes can be targeted
through viruses or other devices to the organ of interest to modify
the regulation of the prostaglandin pathway.
[0015] One embodiment provides a method for promoting tissue cell
growth in a subject, comprising administering at least one
modulator and a pharmaceutically acceptable carrier.
[0016] For example, modulators found to enhance tissue development
and adult tissue homeostasis include dimethyl-prostaglandin E2
(dmPGE2) and agents that stimulate the PGE2 pathway.
[0017] In another embodiment, the tissue development modulator
increases growth by modifying the Wnt pathway. Modulators that
enhances tissue growth, such as liver regeneration, hematopoietic
stem cell recovery, wound healing, or other organ tissue repair, by
directly modifying the wnt pathway include, for example, BIO or
LiCl, or other compounds that modulate the wnt pathway at any level
or the wnt signaling cascade.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1E collectively show that elevated
wnt/.beta.-catenin signaling affects liver size. FIG. 1A compares
the size of livers developing in wild-type (normal) and APC+/-
mutant zebrafish. FIG. 1B compares the number of GFP positive
hepatocytes in APC+/-/LFABP: GFP crosses as determined by FACS
analysis. FIG. 1C shows the increase in hepatocyte number in
histological sections of APC+/- mutants as compared to control
fish. FIG. 1D reflects immunohistochemical analysis of
.beta.-catenin at 96 hours post-fertilization (hpf), showing an
increase in both cytoplasmic and nuclear staining in APC+/- mutants
as compared to wild-type. FIG. 1E illustrates that BrdU
incorporation in corresponding liver sections was significantly
up-regulated in APC+/- embryos. *=statistically significant
difference
[0019] FIGS. 2A-2B collectively show that increased wnt activity
accelerates liver regeneration. FIG. 2A illustrates the resection
margins in adult zebrafish subjected to partial hepatectomy. FIG.
2B graphically depicts the results of morphometric analysis of
inferior liver lobe regeneration, demonstrating that wnt activation
provides a regenerative advantage over wild-type fish, while wnt
inhibition diminishes liver regrowth.
[0020] FIG. 3. Wnt-mediated acceleration of liver regeneration is
evolutionarily conserved. APC heterozygosity in APCMin/+ mice
mediates a growth advantage during liver regeneration following 2/3
partial hepatectomy. *=statistically significant difference
[0021] FIG. 4. A diagram of the wnt signaling pathway, showing
potential sites of interaction with prostaglandin signaling.
[0022] FIGS. 5A-5D collectively show the effect of prostaglandin
modulation on wnt activity in the developing zebrafish. FIG. 5A
illustrates the experimental design of determining wnt activity in
TOP:dGFP fish . FIG. 5B demonstrates increased wnt activity in the
brain following PGE2 administration, and diminished wnt activity
after indomethacin exposure. FIGS. 5C and 5D demonstrate similar
effects in the developing liver and gut, respectively.
[0023] FIGS. 6A-6B collectively show the effect of prostaglandin
modulation, cAMP activation and wnt activity on endoderm and liver
development. FIG. 6A shows the effects of indomethacin and
forskolin, a cAMP activator, in wild-type and APC+/- zebrafish on
the endodermal progenitor cell population, demonstrating that
downstream mediators of prostaglandin signaling can have similar
effects compared to prostaglandins themselves. FIG. 6B shows the
effects of PGE2 and indomethacin on liver morphology.
[0024] FIGS. 7A-7B collectively show that modulators of the
prostaglandin signaling pathway alters the effects of wnt activity
on target gene expression. FIG. 7A illustrates one approach to
testing prostaglandin pathway modulators using a zebrafish model.
FIG. 7B shows the effects of prostaglandin modulators on the
expression of wnt target and endodermal genes in wild-type, wnt8,
and dkk fish as measured by quantitative PCR..
[0025] FIGS. 8A-8C collectively show that prostaglandin signaling
modifies liver regeneration. FIGS. 8A and 8B illustrate approaches
to testing prostaglandin pathway modulators in a zebrafish model,
by either measuring liver size during regeneration or by analyzing
the expression of genes involved in this process. FIG. 8C reveals
that inhibition of prostaglandin synthesis decreases wnt target
gene expression during liver regeneration.
[0026] FIG. 9A and FIG. 9B Prostaglandin modulation and wnt
activity affect liver regeneration. These figures illustrate how
both prostaglandin and wnt activation as well as treatment with
components influencing these pathways and downstream targets can
affect liver regeneration in zebrafish.
[0027] FIGS. 10A-10C collectively show the effect of prostaglandin
inhibition on liver tumor growth. FIG. 10A and FIG. 10B illustrate
approaches to testing prostaglandin pathway modulators in zebrafish
carcinogenesis. FIG. 10B depicts the model for testing whether the
inhibition of prostaglandin synthesis prevents liver tumor
formation in APC+/- zebrafish. FIG. 10C demonstrates the decrease
in tumor incidence by inhibition of prostaglandin synthesis in this
model.
[0028] FIGS. 11A-11B collectively show the effect of simultaneous
modulation of wnt and prostaglandin signaling pathway on mouse bone
marrow transplantation. This figure illustrates how wnt activation
by BIO enhances early spleen colony formation following bone marrow
transplantation. Indomethacin blocks this effect.
[0029] FIG. 12. Effect of prostaglandin modulation and wnt
activation on wound healing in zebrafish. Skin wounds following
partial hepatectomy heal faster and to a greater extent with PGE2
as well as in APC+/- fish. Wound healing is severely inhibited
after administration of indomethacin.
DETAILED DESCRIPTION
[0030] Unless otherwise defined herein, scientific and technical
terms used in connection with the present application shall have
the meanings that are commonly understood by those of ordinary
skill in the art. Further, unless otherwise required by context,
singular terms shall include pluralities and plural terms shall
include the singular.
[0031] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0032] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.1%.
[0033] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0034] Developmental signaling pathways hold keys to unlocking the
promise of adult tissue regeneration and inhibiting carcinogenesis.
The present invention provides insights into the regulation of
embryonic and adult hematopoietic stem cell growth. The present
invention also provides insights into the regulation of embryonic
and adult liver growth, including growth following liver
resection.
[0035] An embodiment of the present invention provides for
manipulation of the genetic interaction between PGE2 and
wnt/.beta.-catenin signaling, which regulates developmental
specification of stem cells and regeneration. Briefly,
prostaglandin (PG) E2 is required for the formation and function of
hematopoietic stem cells (HSCs) in vivo, yet its mechanism of
action in these cells is not completely understood. North et al.,
447(7147) Nature 1007-11 (2007). Clinical observations in patients
with APC mutations, a central regulator of the wnt/.beta.-catenin
pathway (Cruz-Correa et al., 122(3) Gastroenterology 641-45
(2002)), and in vitro data (Castellone et al., 310 Science 1504-10
(2005)), suggest that the prostaglandin and wnt/.beta.-catenin
signaling pathways interact. Wnt signaling positively regulates the
homeostatic function of adult HSCs (Reya et al., 423 Nature 409-14
(2003)), but its role in HSC formation has not been examined. In
order to demonstrate a direct interaction of PGE2 and the wnt
signaling pathway in vivo, TOP:dGFP wnt reporter zebrafish embryos
were exposed to a stabilized derivative of PGE2, dmPGE2 (10 .mu.M),
and indomethacin (10 .mu.M), a non-selective inhibitor of
cyclooxygenases (cox). In situ hybridization for GFP revealed a
striking increase in wnt activity throughout the embryo following
dmPGE2 exposure (99 observed/111 scored), particularly in the
region of the aorta-gonad mesonephros (AGM), where definitive HSCs
are formed (12.+-.3.4 vs. 3.+-.1.8 cells). Indomethacin treatment
abolished wnt activity in the AGM and markedly decreased GFP
expression globally (72/87). These results were confirmed by qPCR
analysis for GFP, revealing a 2-fold induction of wnt activity in
whole embryo extracts following dmPGE2 exposure and indicating the
direct influence of PGE2 on wnt signaling activity.
[0036] The functional consequences of the PGE2/wnt interaction in
HSC formation during embryonic development were analyzed by
examining the expression of the HSC markers runxl and c-myb. Heat
shock induction of a canonical wnt ligand, wnt8, at ten somites led
to enhanced HSC formation at 36 hours post fertilization (hpf;
47/54). When wnt8 induction was followed by exposure to
indomethacin (10 .mu.M, 16-36 hpf) HSC formation was reduced to or
below wild-type levels (43/46). These results indicate that PGE2
activity is required for the effects of wnt activation on HSC
development.
[0037] Inducible negative regulators of wnt activity were used in
combination with dmPGE2 treatment to functionally localize the
interaction between the PG and wnt pathways. Dkk1 antagonizes the
wnt pathway at the level of membrane binding and the initiation of
the wnt signaling cascade. Dkk induction in hs:dkk1 transgenic
embryos inhibited HSC development (34/49). Exposure to exogenous
dmPGE2 rescued the dkk1-mediated effect on HSC formation (28/51, 10
.mu.M, 16-36 hpf). Axin is a central component of the
.beta.-catenin destruction complex and thus a negative regulator of
the wnt signaling cascade in the cytosolic compartment. When
induced at ten somites, axin severely inhibited HSC formation
(47/52). Furthermore, this effect could not be overcome by dmPGE2
treatment. Similarly, a dominant-negative form of the
.beta.-catenin transcriptional co-activator TCF abolished HSC
formation (60/62), and no rescue was affected via dmPGE2 exposure.
These results indicate that the PG and wnt pathways interact at the
level of the .beta.-catenin destruction complex to regulate
definitive HSC formation in the embryo.
[0038] The wnt pathway can actively enhance HSC proliferation
through signaling from the HSC niche as well as within HSCs
themselves. PGE2 regulates HSC formation at the level of the
vascular niche as well as within the HSCs. North et al., 2007. In
order to identify transcriptional programs regulated by the wnt
signaling pathway, genes involved in HSC development were analyzed
by qPCR. The expression of stem cell markers runx1 and cmyb were
significantly enhanced following wnt8 induction, corresponding to
the in situ hybridization expression data. Conversely, a
significant reduction in runxl and cmyb was seen in response to
dkk1 induction. The general vascular marker flk1, as well as the
aorta specific vessel marker ephB2 were each similarly increased in
response to wnt8 and diminished following dkk1 induction. These
effects on the vascular niche and the HSCs developing therein could
be modified by the addition of the appropriate prostaglandin
pathway modifier: indomethacin to wnt8, and dmPGE2 to dkk1,
respectively. These data suggest that the interaction of PGE2 and
wnt, at least in part, affects HSC formation through regulation of
the developmental potential level of the hematopoietic niche.
Furthermore, analysis of the wnt target gene cyclin D1 by in situ
hybridization in the AGM and qPCR indicated that the wnt/PGE2
interaction was also activated in the HSCs themselves, likely
influencing their proliferation and self-renewal.
[0039] It has been hypothesized that wnt activation regulates HSC
self-renewal and repopulation. Wnt activation is also thought to be
involved in carcinogenesis, however, making the concept of direct
enhancement of wnt signaling problematic. Whether regulation of wnt
activity by prostaglandin can effectively modulate HSC homeostasis
in the adult, was determined by observing hematopoietic recovery
following irradiation in the zebrafish. Previously, it was shown
that the PGE2 treatment significantly enhanced regeneration and
that the effects on stem and progenitor numbers could be readily
detected by ten days post irradiation; lack of PGE2 suppressed stem
and progenitor cell proliferation. Wnt activity in the kidney
marrow is 2-fold increased following irradiation as analyzed by
FACS in TOP:dGFP reporter fish. Furthermore, wnt8 induction from 24
hours-36 hours post irradiation produced a 2.5-fold increase in the
stem and progenitor cell population by ten days post irradiation.
This effect was significantly diminished following the inhibition
of cox by indomethacin. The conservation of the interaction in
vertebrate species was shown using a murine model of constitutive
wnt activation. APCMin/+ mice have elevated .beta.-catenin levels
due to loss of APC function in the destruction complex. These mice
exhibit normal differential blood counts at baseline compared to
sibling controls. Following 5-FU chemical injury, bone marrow
recovery was enhanced in APCMin/+ mice compared to controls.
Indomethacin (1 mg/kg every 48 hrs) markedly diminished the
proliferative advantage in the APCMin/+ mice. These data confirm
that PG and wnt interact to regulate vertebrate hematopoietic
homeostasis.
[0040] To assess whether prostaglandin levels regulate wnt activity
in HSCs, murine transplantation assays were performed utilizing
purified HSCs. FACS-isolated cKit+Scal+Lineage-(KSL) bone marrow
cells were transplanted into lethally irradiated recipients. The
recipient mice were treated with the GSK inhibitor BIO (0.05
mg/kg), indomethacin (1 mg/kg and 2.5 mg/kg), or a combination of
both to affect both wnt activity and PG levels. CFU-S12 showed a
significant 2-fold increase in response to BIO treatment (p=0.03),
however, concurrent administration of indomethacin diminished CFU-S
number back to baseline levels. These results confirm that PGE2 and
wnt directly interact in HSCs.
[0041] To determine whether the interaction of PGE2 and
wnt/.beta.-catenin signaling is a conserved regulator of stem and
progenitor cell populations in other tissues, endodermal and
hepatic progenitor cells were examined during zebrafish
development. Zebrafish embryos exposed to indomethacin exhibited
decreased foxA3 expression, a marker for endodermal progenitor
cells (67/71). In particular, the developing liver bud was markedly
decreased, resulting in a smaller liver at 72 h.p.f. (51/56), as
detected by expression of liver fatty acid binding protein (lfabp).
Addition of dmPGE2 resulted in an expanded foxA3 population with an
increased liver anlage (75/83) and increased liver size (88/92),
revealing a novel role for PGE2 signaling in endoderm development.
This finding is supported by the detection of various components of
PGE2 signaling in foxA3 positive cells.
[0042] Wnt signaling has been shown to be required for endoderm and
liver formation. The APC mutant zebrafish to model constitutive
activation of the wnt signaling pathway, was used to characterize
the effects of the wnt/PG interaction in foxA3+endodermal
progenitors at 48 hpf. APC+/- embryos have enhanced foxA3
expression and and an increased liver bud (88/93) as well as
increased liver size (68/75) compared to wild-type siblings.
Indomethacin caused a decrease in foxA3 positive progenitor cells
(33/39) and liver size (61/67) in APC+/- embryos, comparable to
untreated wild-type controls, while dmPGE2 enhanced both endodermal
progenitors (47/54) and liver size (75/81) excessively. Use of the
heat-shock inducible transgenic lines confirmed that as in HSCs the
interaction of the PG and wnt pathways during endodermal
development occurs at the level of the destruction complex. qPCR
revealed that both markers of endodermal (foxA3) and hepatic
progenitors (hhex) are regulated, suggesting that the interaction
of PG and wnt is involved at different progenitor populations
during endoderm development. Insulin expression is not affected by
modulation of the PG pathway, indicating that the PG/wnt regulation
is not a general regulator of different endodermal lineages. Both
cyclinD1 and cmyc were co-regulated by wnt and prostaglandin during
development which may indicate that PGE2 exerts its effects on stem
cells through enhanced cell cycling and proliferation.
[0043] The continued importance of the PG/wnt pathways in adult
liver homeostasis was demonstrated clearly using a liver resection
model in the zebrafish. Following 1/3 partial hepatectomy, the
zebrafish liver regenerates within five days to seven days. This
process is accelerated in APC+/- fish. Treatment with indomethacin
from 6 hours to 18 hours post resection significantly diminished
the regenerative index in both wild-type and APC mutant fish.
Immunohistochemistry for .beta.-catenin reveals enhanced nuclear
staining in APC+/- fish post resection. Indomethacin caused a
decrease in overall .beta.-catenin levels, however, and the absence
of nuclear .beta.-catenin in both wild-type and APC+/- fish.
[0044] The mechanism by which PGE2 affects .beta.-catenin levels
and to demonstrate a conserved role of this interaction in
mammalian liver regeneration were further elucidated by performing
partial hepatectomies in wild-type and APCMin/+ mice. Here, APC
mutation caused increased total and nuclear .beta.-catenin levels,
particularly in the periportal region. Exposure to indomethacin
(2.5 mg/kg bid sq) decreased .beta.-catenin levels significantly in
both genotypes. Cell culture studies have suggested that PGE2 may
enhance b-catenin levels by phosphorylation and inactivation of
GSK3b through activation of adenylyl cyclase and protein kinase A
(PKA); IHC for P-GSK3b (at serine 9) revealed decreased amounts
after indomethacin exposure in both wild-type and APC+/- mice.
These findings were confirmed by western blot. .beta.-catenin may
cause increased cell proliferation through its target cyclin D1.
Cyclin D1 levels and resultant cell proliferation as measured by
BrdU incorporation were increased in APCMin/+ mice and markedly
diminished after indomethacin exposure.
[0045] The functional interaction of signaling processes in the
zebrafish, down stream from PG, was explored by increasing camp
production using forskolin and inhibiting PKA with H89. In both
HSCs and endodermal progenitors, forskolin exposure had similarly
enhancing effects as dmPGE2. Forskolin was able to rescue the
inhibitory effects of indomethacin in both wild-type and wnt8
transgenic fish. Inhibition of PKA by H89 reduced the increased HSC
formation induced by dmPGE2. Furthermore, the rescue of dkk effects
by dmPGE2 was eliminated by H89. These data suggest that PGE2 acts
through activation of camp and PKA and subsequent inactivation of
GSK3b to enhance .beta.-catenin levels in various stem and
progenitor populations
[0046] Another embodiment of the present invention provides for the
role of wnt/.beta.-catenin signaling in the processes of liver
development and growth. Briefly, embryos heterozygous for the
adenomatous polyposis coli gene (APC+/-), a critical regulator of
wnt signaling, developed enlarged livers. Conversely, APC-/-
embryos failed to specify liver. Elevated wnt signaling and
increased intracellular .beta.-catenin mediated both APC liver
phenotypes. Using transgenic zebrafish that expressed inducible
activators and repressors of wnt/.beta.-catenin signaling, the
requirement for wnt during embryogenesis was shown to be biphasic:
suppression of wnt signaling was required following gastrulation
for hepatic cell fate specification; conversely, activation of wnt
signaling was necessary for normal liver growth. Liver resections
were preformed in both zebrafish and mice to assess the functional
requirement of wnt signaling in hepatic regeneration. Intriguingly,
APC heterozygotes demonstrated accelerated liver regeneration,
while inhibition of wnt signaling severely diminished regrowth. The
present invention reveals an evolutionary conserved role for
wnt/.beta.-catenin signaling in endodermal organ specification,
hepatocyte growth and liver regeneration, which has implications
for regenerative medicine.
[0047] Another embodiment of the present invention provides for the
role of the prostaglandin signaling pathway as a potent modifier of
liver growth. Incubation of zebrafish embryos with cox1-, cox2-, or
dual-specific inhibitors (e.g., indomthacin) caused a marked
reduction in liver size by 72 hours post-fertilization compared to
controls. Conversely, exposure to dimethyl-prosaglandin E2 (dmPGE2)
enhanced liver development. Morpholino knock-down of either cox1 or
cox2 similarly inhibited growth, but such growth was rescued fully
by exposure to exogenous dmPGE2. Adult zebrafish subjected to
partial hepatectomy and exposed to indomethacin showed
significantly reduced liver regrowth compared to controls. Cox
inhibition also prohibited wound healing. In contrast, exposure to
dmPGE2 following resection led to enhanced liver regrowth with
noted increases in liver vascularity compared to untreated fish.
More rapid would healing was also observed in dmPGE2-treated fish.
Similar studies in zebrafish demonstrated that dmPGE2 could enhance
kidney marrow repopulation following injury. Hence, regulation of
the prostaglandin pathway may impact repair/regrowth in a variety
of tissue types such as cardiac, bone and wounded tissue.
[0048] The undifferentiated endodermal germ layer is patterned to
form liver, intestine, pancreas, and accessory organs by the action
of signaling pathways. Cui et al., 180 Dev. Biol. 22-34 (1996);
Zaret, 3 Nat. Rev. Genet. 499-512 (2002). Wnt signaling through its
main transcriptional mediator .beta.-catenin plays an important
role in controlling tissue patterning, cell fate decisions, and
proliferation in many embryonic contexts, including the development
and differentiation of organs. Clevers, 127 Cell 365-69 (2006). In
the absence of Wnt signaling, .beta.-catenin is phosphorylated by
the action of a destructive complex of Axin, APC, and Glycogen
Synthase Kinase (GSK) 3.beta., and targeted for degradation.
Binding of Wnt ligand to surface receptors allows .beta.-catenin to
accumulate in the cytoplasm and translocate to the nucleu, where is
modulates gene expression.
[0049] Genetic mutations in several components of the
Wnt/.beta.-catenin signaling pathway are frequently detected in
gastrointestinal neoplasia. Most prominently, patients carrying
mutations in the APC gene develop colon cancer at a very young age.
Kinzler et al., 251 Sci. 1366-70 (1991). Children with APC
mutations are 1000-times more likely to develop hepatoblastoma, an
embroyonal form of liver cancer. Hirschman et al., 147 J. Pediatr.
263-66 (2005). Mutations in .beta.-catenin as well as AXIN-1 and -2
are found in hepatocellular carcinoma (HCC) (Taniguchi et al., 21
Oncogene 4863-71 (2002). Based on the prevalence of defects in Wnt
pathway components found in both primitive and differentiated
hepatic neoplasms, it is likely that .beta.-catenin signaling
regulates several aspects of liver development.
[0050] The liver is derived from anterior endodermal progenitor
cells during embryogenesis. Following convergence of the endodermal
progenitors to the midline, the proliferation and specification of
the endodermal rod is initiated. In the zebrafish embryo,
endodermal progenitors fated to become liver can be identified at
22 hours to 24 hours post fertilization (hpf) as a thickening in
the anterior endoderm. Field et al., 253 Dev. Bio. 279-90 (2003).
As the endoderm develops further, the liver primordial appears as a
prominent bud extending to the left from the midline over the yolk
sac. Between 28hpf and 30hpf, the transcription of liver-specific
genes is initiated within cells specified to become liver. The
liver is fully developed by 48hpf and expresses mature
liver-specific genes such as liver fatty acid binding protein
(LFABP). Her et al., 538 FEBS Lett 125-33 (2003). Hepatic growth
continues as the zebrafish liver expands anteriorly and leftward.
The mechanisms by which liver specification, budding, and growth in
the vertebrate embryo are initiated and controlled appear to be
highly conserved across vertebrate species.
[0051] The requirement for Wnt signaling in the development of the
endoderm was described initially in C. elegans, and is
evolutionarily conserved. Lin et al., 83 Cell 599-609 (1995). An
analysis of the role of Wnt/.beta.-catenin signaling in vertebrate
endodermal development was slowed by early embryonic lethality of
mice with homozygous deletion of .beta.-catenin. Haegel et al., 121
Devel. 3529-37 (1995). APCMin homozygous mutant mice are also
embryonic lethal, although heterozygotes are viable and develop
neoplasia as adults. Su et al., (1992). In Xenopus, wnt is required
during gastrulation for endodermal patterning. Heasman et al.,
(2000). Through inducible inactivation of .beta.-catenin,
Wnt/.beta.-catenin signaling was shown to be required for the
development of the intestine and formation of intestinal
architecture. Ireland et a., (2004). Additionally, Wnt-dependent
regulation of intestinal crypt anatomy is maintained in the adult.
Pinto et al., (2003). Recent studies on Wnt signaling in liver
development have produced seemingly contradictory findings.
Emerging data in Xenopus suggest that repression of wnt in early
endodermal progenitor cells is needed to allow liver specification
to occur. In contrast, the zebrafish wnt2b mutant, prometheus,
reveals a requirement for mesodermally derived Wnt signals in
regulating liver growth. Ober et al., (2006). Homozygous prometheus
mutants are viable and eventually develop a liver. This suggests
that liver progenitors can be correctly specified in the absence of
wnt2b, but that lack of mesodermal wnt2 signaling impairs the
initial wave of liver growth. It is unknown whether other wnt
factors compensate for the lack of wnt2b in liver specification and
development, or if wnt is not required for later phases of
hepatocyte proliferation.
[0052] Zebrafish possessing mutation in APC were identified
previously through TILLING (targeting induced local lesions in
genomes). Hurlstone et al., 425 Nature 633-37 (2003). Although
APC+/- mutants dies by 96hpf, APC+/- fish are viable and have
increased susceptibility for the development of spontaneous
gastronintestinal neoplasia. Haramis et al., (2006). Liver tumors
arising in APC+/- mutant zebrafish resemble hepatoblastomas,
implying the APC mutation leads to a defect in wnt-regulated
differentiation of hepatic progenitors.
[0053] Utilizing APC mutant and transgenic zebrafish expressing
inducible activator and repressors of wnt/.beta.-catenin signaling,
the present invention provides for the characterization of the
temporal requirement of wnt/.beta.-catenin signaling during
embryonic development and in mediating tissue homeostasis in the
adult. There is a differential effect of APC loss on liver
development, mediated by changing temporal requirements of wnt
signaling during hepatogenesis. Wnt activation influences
endodermal progenitor fate decisions resulting in increased liver
and intestinal development at the expense of pancreatic tissue
formation. Through the creation of a zebrafish model for partial
hepatectomy, the present invention demonstrates a requirement for
wnt during liver regeneration in vivo. Additionally, the present
invention shows that the role of elevated wnt signaling in
enhancing the regenerative process is conserved in zebrafish and
mice. These data demonstrate that Wnt/.beta.-catenin signaling is
required and highly regulated during several aspects of liver
development and maintains a central role in organ homeostasis.
Hence, the present invention provides for methodology for the
transient upregulation of Wnt signaling, which may serve as an
attractive mechanism to enhance liver regeneration in mammals and
humans.
[0054] The differential effects of APC loss on liver organogenesis
was elucidated by crossing APC+/- zebrafish into a LFABP:GFP
fluorescent reporter line, and assessing liver development by
fluorescence microscopy. By 72hpf, APC+/- embryos showed a dramatic
increase in liver size (265/297) compared to wild-type siblings. In
contrast, no LFABP expression could be detected in homozygous
APC-/-mutant embryos (134/134) at any stage of development. To
ensure that the observed phenotypic changes in liver development
were not simply due to variation in the expression of LFABP, in
situ hybridization for sterol carrier protein and transferring were
performed, yielding similar results.
[0055] Flow cytometry analysis of GFP+cells in progeny of APC+/-:
LFABP:GFP incross revealed a three-fold increase in hepatocyte
number in APC+/- embryos, and confirmed the absence of GFP+
hepatocytes in APC+/- mutants. Hepatocyte nuclei counts in
corresponding histological sections corroborated the differential
effects of APC loss on liver development; ACP-/- embryos showed a
complete absence of hepatocytes by histological analysis, while
APC+/- embryos exhibited a significant increase compared to
wild-type. No change in the overall cellular morphology was
observed between wild-type and APC+/- samples.
[0056] As APC co-regulates the availability of .beta.-catenin in
the nucleus, the cellular content and localization of
.beta.-catenin within hepatocytes in wild-type and APC+/- embryos
was examined at 72hpf by immunohistochemistry (IHC). Wild-type
livers exhibited primarily membrane-bound 13-catenin. In the livers
of APC+/- embryos, .beta.-catenin staining was markedly increased,
with 4-fold enhanced cytoplasmic and 5-fold increased nuclear
staining. Wnt/.beta.-catenin signaling is known to mediatre effects
on the cell cycle, cellular proliferation and apoptosis in a
variety of tissues. Alonso & Fuchs, 17 Genes Devel. 1189-1200
(2003); Pinto et al., 17 Genes Devel. 1709-13 (2003); Reya et al.,
243 Nature 409-14 (2003). To determine whether the increase in
total hepatocyte number in the APC+/- embryos was due to increased
proliferative activity, BrdU incorporation was examined at 72hpf. A
significant increase in the percentage of BrdU positive cells per
liver was seen in APC+/- embryos compared to wild-type. Similar
results were seen with PCNA staining.
[0057] Although the absence of liver development precluded an
assessment of .beta.-catenin distribution in hepatocytes of
embryos, the adjacent endodermal tissue exhibited intense staining
for .beta.-catenin along the entire length of the gastrointestinal
tract and BrdU incorporation was seen in nearly every cell.
Aberrant wnt signaling due to APC loss in the intestine and
developing brain leads to a block in differentiation and eventually
apoptosis. Chenn & Walsh, 297 Sci. 365-69 (2002); Sansom et
al., 18 Genes Devel. 1385-90 (2004). To further assess why the
APC+/- embryos fail to develop hepatocytes despite the presence of
abundant wnt signaling, TUNEL staining was evaluated in
histological sections of APC+/- embryos. Hugh levels of
TUNEL-positive apoptotic cells were found along the entire length
of endoderm, including the area where liver differentiation failed
to occur. Capsase activity, a marker of apoptosis, was twice as
high in APC-/- embryos as wild-type. These data suggest that the
lack of liver in the APC+/- mutants is due to death of endodermal
progenitors.
[0058] Increased levels of .beta.-catenin are responsible for the
differential liver phenotypes in APC mutants. An intriguing finding
was that progressive loss of APC did not have a linear effect on
liver size. To demonstrates that .beta.-catenin caused both the
enlarged liver in the APC-/- embryos and failure of liver
specification in APC+/- mutants, .beta.-catenin levels were reduced
by a morpholino antisense oligonucleotide (MO) strategy. MOs
against the start sight of zebrafish .beta.-catenin (Lyman
Gingerich et al., 286 Devel. Bio. 427-39 (2005)) were injected into
the progeny of an APC+/- incross at the one-cell stage. By using a
low concentration of the MO (40 .mu.M), injected embryos were able
to successfully progress through gastrulation, and no effects on
overall gross morphology were noted compared to control MO-injected
embryos.
[0059] Targeted knockdown of .beta.-catenin led to a dramatic shift
in the districbution of the liver phenotypes. The majority of
embryos (74%) displayed a normal liver, and subsequent genotyping
revealed that this population included both wild-type and APC+/-
embryos. Some APC+/- embryos (15%) still exhibited an enlarged
liver, likely reflecting an insufficient functional knock-down of
.beta.-catenin caused by the low MO dosing. Of APC+/- embryos
injected with MO that survived until 72hpf, 43% now had evidence of
LFABP expression; these embryos still exhibited severe
developmental defects, however, and were not viable beyond 120hpf.
These data suggest that .beta.-catenin levels alone are sufficient
to cause both liver phenotypes, indicating that the
wnt/.beta.-catenin pathway as mediator of these effects. This
conclusion is supported by the fact that knockdown of canonical
wnt2b, wnt3, and wnt8 resulted in reduced liver size.
[0060] Wnt/.beta.-catenin signaling effects the endodermal
progenitor population. Inducible transgenic zebrafish expressing
activators or repressors of wnt signaling were employed to
determine at which stage of embryonic development wnt signaling
effects endodermal differentiation and liver size. hs:wnt-GFP fish
express the wnt ligand wnt8 under the control of a
heat-shock-inducible promoter, while hs:dkk-GFP and hs:dnTCF-GFP
allow inhibition of wnt/.beta.-catenin signaling either at the
level of the frizzled receptor or the nuclear transcript or the
nuclear transcription complex, respectively. Global induction of
wnt8 prior to the tail bud stage of development (10hpf) caused
severe disruption of gastrulation and overall embryonic patterning
resulting in growth arrest or death by 24hpf. Between the 1-somite
and 5-somite stages, wnt activation caused significant cardiac
edema, reduced body length and absence of liver formation in
embryos surviving until 72hpf, reminiscent of APC-/- mutants.
Heat-shock induction of wnt8 from 10-18 somites resulted in
markedly enlarged livers compared to heat-shocked wild-type
controls. In addition to the overall increase in organ size, 50% of
livers heat-shocked at 10 somites failed to segregate entirely from
the endodermal rod, resulting in increased liver-specific gene
expression at the midline, and a posterior extension of liver
cells; this phenotype was confirmed by confocal microscopy and
histological sectioning. Transient wnt activation at time points
between 24-hpf and 36-hpf produced moderate effect on liver size at
72hpf. Similarly, the effect of wnt inhibition on liver development
caused by the induction of dkk or dnTCF was most significant from
10-18 somites, and more modest later in embryonic maturation.
Global inhibition of wnt/.beta.-catenin signaling prior to the 5
somite stage resulted in early embryonic lethality.
[0061] The expression of liver-specific transcripts such as LFABP
begins at .about.44hpf, and the segregation of the endodermal tubes
into a region fated to become liver is thought to be established by
22hpf. The results obtained in the heat-shock assays suggested that
the wnt-mediated effects on liver development originated prior to
the formation of the mature organ, at or slightly before the stage
at which the fate of endodermal progenitors is determined. To
investigate the effect of wnt activation on the endodermal
progenitor cell population, the expression of the pan-endodermal
marker foxA3 was analyzed after heat-shock induction of wnt8. A
significant increase in the size of the liver bud was observed at
48hpf following heat activation at 10 somites; a decrease in the
size of the pancreatic anlage was also seen, while the effects of
later wnt8 induction (after 24hpf) were less notable. wnt8
activation also had a dose-dependent effect on endodermal
progentors: compared to controls, heat-shock for 5 minutes, 20
minutes, and 60 minutes at the 18-somite stage resulted in
progressive enlargement of the liver but at the expense of
pancreatic tissue.
[0062] .beta.-catenin activation in APC mutants resulted in altered
endodermal fate. The heat-shock experiments suggested that the
wnt-mediated enhancement of hepatocyte number occurs at the level
of endodermal progenitor cells. To evaluate the effects of
progressive APC loss on endodermal progenitors, expression in
progeny of an APC+/- incross was examined at 48hpf. Compared to
wild-type, APC+/- embryos exhibited increased liver and decreased
pancreatic buds. The phenotype was confirmed in vivo by confocal
microscopy of APC; gut:GFP incrosses, and by FACS analysis at
48hpf. The APC+/- embryos failed to exhibit clear patterns of
endodermal organization at 48hpf and had reduced numbers of
gut:GFP+progenitors by FACS analysis, implying that neither
definitive organ organ nor endodermal progenitor cell expansion
occurred.
[0063] As all populations of endodermal progenitors appeared to be
affected by APC loss or wnt activation, mature endodermal organs
were examined for consequences of this early alteration in
development. Insulin and trypsin expression, indicative of
endocrine and exocrine pancreas differentiation, respectively, were
decreased in APC+/- embryos at 72hpf. In APC-/- mutants, trypsin
expression was virtually undetectable; insulin expression, although
reduced, could still be observed. The effect of APC loss on
differentiated intestine as marked by expression of intestinal
fatty acid binding protein (IFABP) was similar to the liver: APC+/-
embryos had increased IFABP staining compared to wild-type, while
APC-/- embryos failed to express IFABP. Induction of wnt8 at 10
somites had similar effects on each endodermal organ but resulted
in more disorganized patterning, especially of the pancreas. These
data demonstrate that nascent wnt/.beta.-catenin signaling
regulates endodermal development prior to organ specification, and
that this effect mediates a shift in the differentiation of
endodermal progenitors into liver at the expense of pancreatic
tissue. In addition, excess wnt/.beta.-catenin activation at
tailbud and early somite stages leads to a failure of endodermal
specification and proliferation that results in elevated endodermal
cell death and the inability to develop mature endodermal
organs.
[0064] Wnt/.beta.-catenin signaling enhances hepatocyte growth. To
determine if wnt/.beta.-catenin signaling also mediates an effect
of the growth of differentiated hepatocytes, wnt8 expression was
induced at 48hpf. This resulted in a 2-fold increase in liver size
both by confocal microscopy of LFABP:GFP fish and in GFP+ cells by
FACS at 72hpf. As specified hepatic progenitor cells begin to
proliferate the liver expands dramatically in size: between 72hpf
and 120hpf the number of liver cells increases 2-fold to 3-fold in
wild-type embryos. By FACS analysis, wnt8-induced embryos still
possess increased number of GFP+ hepatocytes at 120hpf, although
this difference is no longer readily apparent by gross examination
of LFABP expression. Heat-shock induced inhibition of wnt signaling
at 48hpf demonstrated that wnt was required for optimal liver
growth; both dkk and dnTCF embryos had reduced liver size compared
to controls by in situ hybridization at 72hpf. These data
demonstrate that wnt signaling continues to be important in the
proliferation of differentiated hepatocytes.
[0065] Wnt/.beta.-catenin signaling is activated and required
during liver regeration. The vertebrate liver is a dynamic organ
that can repair limited damage throughout its lifetime. A model of
liver regeneration in the zebrafish was developed in order to
evaluate the role of wnt/.beta.-catenin signaling in the
maintenance of liver homeostasis in the adult. Adult zebrafish have
a trilobar liver; after 1/3 partial hepatectomy by removal of the
inferior lobe, >95%of wild-type zebrafish recover immediately
and their liver regenerates entirely within seven days. In
wnt/.beta.-catenin reporter fish (TOP:dGFP), GFP fluorescence could
be observed at the liver resection margin at 24 hours post
resection (hpr), indicating activation of the wnt signaling pathway
during the early stages of liver regeneration. This correlated with
increased nuclear .beta.-catenin in regenerating livers compared to
sham-operated controls.
[0066] To determine whether excess wnt activation provides a
regenerative advantage, wnt8 expression was induced by heat-shock
from 6hpr-18hpr. This treatment caused notable acceleration in
liver growth compared to wild-type controls at 3hpr. Similarly, the
APC+/- mutants displayed enhanced regenerative capacity compared to
controls. Use of dnTCF transgenics revealed that both liver
regeneration and wound healing were severely impaired and
demonstrated that wnt/.beta.-catenin signaling was required for
liver regeneration in zebrafish. Histological analysis confirmed
these findings at all stages of regeneration. Nuclear and cytosolic
.beta.-catenin levels as well as PCNA staining were increased in
zebrafish with elevated wnt signaling and enhanced regeneration.
These experiments highlight the persistent important of
wnt/.beta.-catenin signaling in liever homeostasis and growth
throughout the lifetime of the organism.
[0067] Importantly, elevated .beta.-catenin signaling can enhance
mammalian liver regeneration. To test that prediction that
increased levels of wnt/.beta.-catenin signaling could confer a
conserved regenerative advantage following partial hepatectomy in
mammals, liver resections were preformed in APCMin/+and wild-type
mice. After standard 2/3 partial hepatectomy, an assessment of the
liver weight:body weight ratios revealed as increased regenerative
capacity in the APCMin/+ mice compared with controls which was most
notable during the early stages of hepatic regrowth. In APCMin/+
mice, .beta.-catenin levels were increased at baseline, primarily
located around the portal tracts, and increased significantly
during the early phases of liver regeneration. Data from the
APCMin/+ mice demonstrates that wnt activation enhances the
kinetics of liver regeneration, and additionally suggests that
pharmacological manipulation of wnt/.beta.-catenin signaling would
accelerate hepatic regeneration following injury.
[0068] Indication of the molecular mechanisms controlling liver
development both sheds light on the biological basis of hepatic
tumor formation and provides targets for therapeutic manipulation.
As defects in wnt/.beta.-catenin signaling are prevalent in both
primitive and differentiated hepatic neoplasms, the present
invention provides for role of wnt signaling in regulating both
liver specification and growth. Through analysis of transgenic
zebrafish expressing activators and repressors of wnt signaling, as
well as mutant zebrafish with dysregulated .beta.-catenin activity,
the present invention provides for wnt/.beta.-catenin regulation
required for several aspects of liver development and adult tissue
homeostasis.
[0069] Progressive loss of APC did not have a linear effect on
liver size during embryonic development. Loss of APC led to
increased cytoplasmic and nuclear .beta.-catenin accumulation.
Although in the APC+/- embryos this resulted in higher hepatocyte
numbers, the complete absence of .beta.-catenin regulation in the
APC-/- mutants caused increased apoptosis and a failure to develop
differentiated endodermal organs. The use of heat-shock inducible
transgenic fish as reported herein demonstrates that the functional
requirements of wnt signaling in endodermal progenitors vary during
embryonic development. Surprisingly, although excess
wnt/.beta.-catenin signaling in early somatogenesis (.about.1
somite to 5 somites) could inhibit appropriate liver development,
wnt8 activation at 10 somites led to increased liver. Enhanced
progenitor proliferation mediated by elevated wnt/.beta.-catenin
signaling (10 somites to 24hpf) can expand the progenitor pool
rapidly and exponentially, resulting in the large differences in
liever size and cell number observed in wnt8 and APC+/- embryos.
After the differentiated liver has formed, elevated wnt signaling
(48hpf) again enhances in hepatocyte proliferation and overall
organ growth. Together, these data indicate several stage-dependent
requirements for regulation of wnt signaling for the proper
specification and development of the liver.
[0070] The observations support a biphasic wave model of the
effects of wnt/.beta.-catenin signaling on endodermal progenitors
and specified hepatocytes. Additionally, this reconciles
discrepancies in the reports of the effects of wnt/.beta.-catenin
signaling on endodermal development, specifically in the liver.
During early somitogenesis, high levels of wnt signaling are
detrimental to liver specification and development. The APC-/-
mutant embryos illustrate the need for some repression of
.beta.-catenin signaling for appropriate endodermal progenitors.
After endodermal fate is assigned, wnt is required to initiate
progenitor cell expansion and organ growth as demonstrated herein.
Elevated .beta.-catenin signaling in the APC+/- embryos confers a
growth advantage at this stage that is reflected by the increased
number of proliferating hepatoblasts and the subsequent enhacement
of liver cell number.
[0071] A provocative finding presented herein was that
wnt/.beta.-catenin signaling can alter developmental fate of
unspecified endodermal progenitors. The effect of
wnt/.beta.-catenin induction on the subsequent differentiation of
endodermal organs was striking in the heat-shock inducible wnt8
embryos. Wnt/.beta.-catenin signaling altered both the longitudinal
axial zone of liver specification and shifter the distribution of
progenitors to liver-specific cell fates. Most notably, excess wnt
signaling appeared to be particularly unfavorable to the
development of the pancreas. The zone of endodermal cells competent
to respond to liver-modulated signals may expand, truncating the
region normally available for pancreas development. Alternatively,
if bipotential hepato-pancreatic progenitors exist, biased pressure
to differentiate into liver cell fates would effectively diminish
the number of progenitors available to produce the pancreas. In
embryo explant analysis, ventral foregut endoderm was shown to
activate pancreatic gene programs in the absence of liver induction
signals, such as fibroblast growth factor, suggesting that
bipotential cells exist. Deutsch et al., 128 Devel. 871-81 (2001).
The nature of these populations of multipotent progenitors may be
manipulated therapeutically. Although hepatic progenitors capable
of differentiating into both hepatocyts and cholangiocytes have
been described (Strick-Marchand et al., 101 P.N.A.S. USA 8360-65
(2004)), no examination of the plasticity of the progenitors with
respect to pancreatic differentiation has been completed.
[0072] The present invention introduces the development of partial
hepatectomy as a novel technique in the zebrafish that allows for
regeneration studies in the liver. The zebrafish liver regenerates
to its original size within seven days, comparable with the
kinetics of murine liver regrowth. The size of the zebrafish and
the complexity associated with resection of an unencapsulated organ
preclude an exact quantitative analysis of liver regrowth based on
liver/body weight ratios, but the development of the en-bloc
dissection analysis as detailed herein, as well as thorough
histological characterization at several stages during regeneration
allow accurate and detailed analysis. For example, using the
wnt-reporter fish, the present work demonstrates in vivo that wnt
signaling is activated within the first 24 hours at the resection
margins.
[0073] Additionally, the present invention provides the first
example that the activation of wnt/.beta.-catenin signaling can
enhance the rate of liver regeneration. Analysis of wnt8 transgenic
and APC+/- fish revealed that increased levels of nuclear
.beta.-catenin led to enhanced cell proliferation and accelerated
liver regrowth. Furthermore, the regeneratice advantage is
evolutionarily conserved following hepatectomy in APCMin/+ mice.
(Murine liver resections were performed as described in Green &
Puder, 16 J. Investigational Surgery 99-102 (2003). Thus, the
present invention provides for the manipulation of the
wnt/.beta.-catenin pathway as a way to enhance liver regeneration
in patients, either after liver resection or during recovery from
acute liver failure induced by toxins such as acetaminophen.
[0074] As shown here, wnt/.beta.-catenin signaling effects several
endodermal and liver cell populations during liver development, and
this program is re-activated to regulate liver regeneration.
Wnt/.beta.-catenin signaling has a well-documented involvement in a
variety of forms of hepatic neoplasia: hepatoblastomas show
frequent mutation of APC, and cholangiocarcinomas and HCC
demonstrate alterations in .beta.-catenin, AXIN, and GSK-3.beta..
This suggests that just as wnt/.beta.-catenin signaling can
regulate hepatic cell fate at several stages during liver
development, wnt/.beta.-catenin signaling can contribute to
carcinogenesis within several populations of hepatic cell types.
Understanding the development effects of wnt/.beta.-catenin
signaling on these cell populations can uncover mechanisms of
carcinogenesis and how it can be inhibited in each cell type. The
zebrafish model provides a unique opportunity for the
identification of novel therapeutics to modulate wnt signaling. A
chemical genetic screen for modifiers of wnt-mediated regulation of
call growth during embryogenesis is currently underway; compounds
identified by this method may be further evaluated for conserved
function during adult zebrafish liver regeneration and in
modulation of carcinogenesis in APC+/- fish as well as in
chemically-induced zebrafish models of liver cancer. Thorough
analysis of the function of wnt/.beta.-catenin signaling in liver
development, hepatocyte proliferation and in carcinogenesis in
zebrafish, as a large-scale chemical screening, enhances the
ability to more effectively diagnose and treat cancer.
[0075] Another embodiment of the present invention provides for
compositions and methods that modulate vertebrate tissue growth or
regeneration via the prostaglandin signaling pathway. For example,
prostaglandin E2 enhances vertebrate tissue regeneration. A
chemical screen in zebrafish identified the prostaglandin signaling
pathway as a potent modifier of liver growth during embryonic
development. Incubation of embryos with cox1 -, cox2-, or
dual-specific inhibitors caused a marked reduction in liver size by
72hpf compared to wild-type controls, while exposure to
dimethylprostaglandin E2 (dmPGE2) enhanced liver development.
Morpholino knock-down of either cox1 or cox2 similarly inhibited
liver growth, and was fully rescued by exposure to exogenous
dmPGE2. As many molecular pathways controlling embryonic
development mediate tissue homeostasis in the adult, the effects of
prostaglandin signaling during liver regeneration were examined. A
novel methodology was devised to consistently resect 1/3 of the
liver of live zebrafish; following administration of tricaine
anesthetic, a small incision was made in the abdomen, just
posterior to the heart, and the inferior lobe of the tri-lobular
liver was removed using microdissection scissors. Fish were revived
and allowed to heal in fish water.
[0076] To test the requirement of functional prostaglandin
signaling during regeneration, fish were exposed to the
dual-specific cox inhibitor, indomethacin, from hour 6 to hour 18
following partial hepatectomy. Indomethacin significantly reduced
liver re-growth at day 1 and day 3 compared to controls, and failed
to regenerate fully by day 5 post-resection. Additionally,
indomethacin exposure resulted in altered architecture of the liver
both immediately in the region of the resection margin and
throughout the un-injured portions for the organ. Cox inhibition
also prohibited wound healing at the incision site.
[0077] Exposure to dmPGE2 following resection led to enhanced liver
regrowth with noted increases in liver vascularity compared to
untreated fish. This enhancement was seen as early as day 1
post-resection and led to a faster complete regeneration of the
organ. In addition, a more rapid wound healing of the connective
tissue was observed. Regulation of prostaglandin E2 levels may
function to repair/regrow a variety of tissue types, such as
cardiac, bone, and wound repair.
[0078] Additionally, the prostaglandin pathway interacts with wnt
signaling: dmPGE2, an activator of the prostaglandin pathway, was
found to enhance wnt signaling in the developing brain, liver, and
gut, while indomethicin resulted in the virtual absence of wnt
signaling. Furthermore, cox inhibition could mitigate the
growth-promoting effects of wnt activation on liver development and
liver regeneration. Results suggest that the prostaglandin pathway
is directly affecting the transcription activity of .beta.-catenin,
the central mediator of the wnt pathway. Similarly, wnt signaling
can also modulate formation and recovery of hematopoietic stem
cells, as described in WO 07/112084. As with the liver,
wnt-mediated enhancement of HSC number can be blocked by inhibition
of prostaglandin signaling. This suggests that the interaction of
the wnt and prostaglandin pathways is conserved in growth and
repair of a number of tissues. The wnt signaling pathway is a
potentially attractive target for therapeutic manipulation:
activation of the pathway enhances tissue growth and regeneration
after injury, conversely inhibition might be important in cancer
therapy. Wnt inhibitors discovered to date, however, have not yet
been fully developed for clinical use, perhaps due to toxicity or
side-effects. Using prostaglandins or prostaglandin inhibitors to
regulate wnt signaling provide an alternative, balanced approach:
wnt activation could provide a benefit in the acute repair phase
after injury, while inhibition of prostaglandins might serve to
prevent unwanted effects of wnt signaling.
[0079] Tissue growth modulators of the present invention that
affect the prostaglandin pathway such that tissue growth is
inhibited include Indomethacin, NS398, SC560, Celecoxib, Sulindac,
Fenbufen, Aspirin, Naproxen, Ibuprofen, AH6809 (EP1/2 antag),
andAH23848 (EP4 antag).
[0080] Tissue growth modulators that affect the prostaglandin
pathway such that tissue growth is enhanced include dmPGE2, PGE2,
PGI2, Linoleic Acid, 13(s)-HODE, LY171883, ONO-259, Cayl0397,
Eicosatrienoic Acid, Epoxyeicosatrienoic Acid, and Arachidonic
Acid.
[0081] Tissue growth modulators that affect the wnt pathway such
that growth is inhibited include Kenpaullone (HDAC effect, not
GSK3b), Valproic Acid, (HDAC effect, not GSK3b), and Soluble
dkk.
[0082] Conversely, tissue growth modulators that affect the wnt
pathway such that growth is enhanced include BIO, LiCl, and Soluble
wnt ligand.
[0083] Additionally, tissue growth modulators that affect
cAMP/PI3K/AKT second messenger modifiers--acting downstream of
initial prostaglandin signaling such that tissue growth is
inhibited include, H89, PD98059, KT5720, U0126, LY294002 and
Wortmannin.
[0084] Tissue growth modulators that affect cAMP/PI3K/AKT second
messenger modifiers--acting downstream of initial prostaglandin
signaling such that tissue growth is enhanced include Forskolin,
8-bromo-cAMP, and Sp-5,6,-DCI-cBiMPS.
[0085] Other tissue growth modulators that may act downstream of
initial prostaglandin signaling include Ca2+ second messenger
modifiers. Those that inhibit tissue growth include BayK 8644 and
Thioridazine. Those that are considered growth enhancers include
Bapta-AM, Fendiline, Nicardipine, Nifedipine, Pimozide,
Strophanthidin, and Lanatoside.
[0086] The NO/Angiotensin signaling modifiers pathways can interact
with prostaglandin and wnt signaling according to the present
invention. Those that inhibit tissue growth include L-NAME,
Enalapril, Captopril, AcSDKP, Losartan, Telimasartan, Histamine,
Ambroxol, Chrysin, Cycloheximide, Methylene Blue, Epinephrine,
Dexamethazone, Proadifen, Benzyl isothiocyanate, and Ephedrine.
[0087] NO/Angiotensin modifiers that can interact with
prostaglandin and wnt signaling to enhance tissue growth include
L-Arg, Sodium Nitroprusside, Sodium Vanadate, and Bradykinin.
[0088] Early experimental evidence suggests that liver growth may
be inhibited by tissue growth modulators such as Norethindrone,
3-estradiol, Beta-Carotene, and BMS189453. Conversely, liver growth
was enhanced by Flurandrenolide, All-trans retinoic acid, Vitamin
D, and Retinol.
[0089] Prostaglandin E2 (PGE2), a product of cyclooxygenase (COX),
exerts functions by binding to four G protein-coupled receptors
(EP1-EP4). Thus, tissue growth modulators of the present invention
include PGE2 receptor agonists and PGE2 receptor antagonists.
[0090] EP4-selective agonists include ONO-AE1-734 (methyl-7-[(1R,
2R, 3R)-3-hydroxy-2-[(E)-(3
S)-3-hydroxy-4-(m-methoxymethylphenyl)-1-butenyl]-5-oxocyclopenth1]-5-thi-
aheptanoate), ONO-AE1-437, ONO-AE1-329, ONO-4819 (each from Ono
Pharma. Co., Osaka, Japan), APS-999 Na (Toray Indus., Inc., Tokyo,
Japan), AGN205203, an analog from the 8-azapiperidinone series of
EP4 agonists (Allergan, Inc., Irvine, Calif.), L-902,688 (Merck
Frosst Canada, Ltd.), 1,6-disubstituted piperidin-2-one,
3,4-disubstituted 1,3-oxazinan-2-one, 3,4-disubstituted
1,3-thiazinan-2-one and 4,5-disubstituted morpholin-3-one
derivatives, see U.S. Pat. No. 7,053,085 (Merck & Co. Inc,
Rahway, N.J.).
[0091] Conversely, EP4-selective antagonists include ONO-AE3-208
(4-{4-Cyano-2[2-(4-fluoronaphthalen-1-yl) propionylamino] phenyl}
butyric acid) (Ono Pharma. Co., Osaka, Japan), CJ-023,423
(N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo [4,5-c] pyridin-1-yl)
phenyl]ethyl}amino) carbonyl]-4-methylbenzenesulfonamide) (Pfizer),
BGC20-1531 (BTC Intl, Ltd.), AH23848,
((4Z)-7-[(rel-1S,2S,5R)-5-((1,1'-Biphenyl-4-yl)
methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid
hemicalcium salt hydrate), AH22921 ([1.alpha.(Z),2.beta..alpha.,
5.alpha.]-(.+-.)-7-[5-[[(1,1'-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-
-oxocyclopentyl]-5-heptenoic acid) (GlaxoSmithKline), L-161,982
(N-[[4'[[3-Butyl-1,5-dihydro-5-oxo-1-[2-(trifluoromethyl)
phenyl]-4H-1,2,4-triazol-4-yl]methyl][1,1'-biphenyl]-2-yl]sulfonyl]-3-met-
hyl-2-thiophenecarboxamide) (Merck Frosst Ltd., Canada).
[0092] EP2-selective agonists include ONO-AE1-259, ONO-8815Ly,
ONO-8815,
(L-lysine(Z)-7-[(1R,2R,3R,5R)-5-chloro-3-hydroxy-2[(E)-(S)-4-(1-ethylcycl-
obutyl)-4-hydroxy-l-butenyl] cyclopentyl]-5-heptenoate) (Ono
Pharma. Co., Osaka, Japan), AH13205 (trans-2-[4-(1-hydroxyhexyl)
phenyl]-5-oxocyclopentane-heptanoic acid) (GlaxoSmithKline).
[0093] Several prostaglandin derivatives exhibit relative potency
to increase liver growth as shown on the following chart:
TABLE-US-00001 Prostaglandin Derivatives ( indicates relative
potency to increase liver growth): PGE2 PGI2 16-phenyl tetranor
PGE2 16,16-dimethyl PGE2 19(R)-hydroxy PGE2 16,16-dimethyl PGE2
p-(p-acetamidobenzamido) phenyl ester
9-deoxy-9-methylene-16,16-dimethyl PGE2 PGE2 methyl ester Butaprost
15(S)-15-methyl PGE2 15(R)-15-methyl PGE2 20-hydroxy PGE2
11-deoxy-16,16-dimethyl PGE2 9-deoxy-9-methylene PGE2 9-keto
Fluprostenol PGE2 serinol amide Sulprostone 17-phenyl trinor PGE2
8-iso-15-keto PGE2 8-iso PGE2 isopropyl ester toxic 5-trans
PGE2
[0094] Other example molecules involved in the wnt pathway, which
that may serve as modulators encompassed within the scope of the
present invention, have been reported. See, e.g., Barker &
Clever, 5 Nature Rev. Drug Discovery 997-1014 (2007); Janssens et
al., 24 Investigational New Drugs 263-80 (2006). It should be
noted, however, that there have been significant adverse side
effects reported with attempts to directly suppress wnt signaling
in cancer patients. Barker & Clever, 2007. Hence, as the
present invention suggests, indirect modulation of the wnt
signaling pathway via the prostaglandin pathway may prove more
efficacious in therapeutic development.
[0095] The tissue growth modulators within the scope of the present
invention may be identified in a variety of ways, such as the
zebrafish genetic system. The zebrafish (Danio rerio) is an
excellent genetic system for the study of vertebrate development
and diseases. See e.g., Hsia & Zon, 33(9) Exp. Hematol. 1007-14
(2005); de Jong & Zon; 39 Ann. Rev. Genet. 481-501 (2005);
Paffett-Lugassy & Zon, 105 Meth. Mol. Med. 171-98 (2005);
Haffner & Nusslein-Volhard, 40 Int'l J. Devel. Biol. 221-27
(1996). The embryo developing externally is transparent and organs
can be easily visualized. Zebrafish and mammals share many of the
same gene programs in development. When zebrafish mate, they give
rise to large numbers (100-200 weekly) of transparent embryos. Many
embryos can be placed in a relatively small space, and there is a
short generation time (about 3 months). Large-scale screens have
generated more than 2000 genetic mutants with specific defects that
affect virtually every aspect of embryogenesis. Driever et al., 123
Devel. 37-46 (1996); Eisen, 87 Cell 969-77 (1996). Many of the
blood mutants have been useful in describing key events in
hematopoeisis. Dooley & Zon, 10 Curr. Op. Genet. Devel. 252-56
(2000). Zebrafish have been used to perform whole organism-based
small molecule screens because large numbers of the embryos can be
arrayed into microtiter plates containing compounds from a chemical
library. For example, Peterson and colleagues tested 1,100
compounds for developmental defects. Peterson et al., 97 P.N.A.S.
USA 12965-69 (2000). From this screen, about 2% of the compounds
were lethal, and 1% caused a specific phenotype. For example, one
compound suppressed formation of inner ear structures called
otoliths, but caused no other defects.
[0096] It is also possible to screen for chemical suppressors of
mutant phenotypes. Peterson et al., 22 Nat. Biotech. 595-99 (2004);
Stern et al., 1 Nat. Chem. Biol. 366-70 (2005). In one such screen,
chemicals were found to rescue the gridlock mutant, a model of
congenital coarctation of the aorta. Peterson et al., 2004. The
mechanism of this rescue involved the induction of VEGF which
corrected the angiogenesis defect. These data demonstrate that
highly potent and specific compounds can be identified using
zebrafish.
[0097] Further regarding zebrafish, a high-density genetic map has
been constructed that includes microsatellite markers, genes, and
expressed sequence tags (ESTs). Knapuk et al., 18 Nat. Genet.
338-43 (1998); Shimoda et al., 58 Genomic 219-32 (1999); Kelly et
al., 10 Genome Res. 558-67 (2000); Woods et al., 20 Genome Res.
1903-14 (2000). A full-length cDNA project has also been undertaken
as an extension to the zebrafish EST project. A dense RH map has
been constructed and integrated with data for the genome sequencing
project at the Sanger Center. An important web resource supported
by the NIH is the zebrafish information network (ZFIN), a focal
point for the community. A stock center and supportive laboratory
called the Zebrafish International Resource Center (ZIRC) also
greatly helps the field. The Sanger Center is sequencing the
zebrafish genome.
[0098] Using the techniques described herein, wild-type and
transgenic zebrafish may be exposed to numerous compounds to assess
the effect of the compound as modulators of the prostaglandin
and/or wnt/.beta.-catenin signaling pathways. For example, test
compounds can be administered to transgenic fish harboring an
exogenous construct containing the expression sequence of a
reporter protein. By comparing the expression of the reporter
protein in fish exposed to a test compound to those that are not
exposed, the effect of the compound on the modulation of the
prostaglandin signaling pathway may be determined. Similarly,
comparing the expression of the reporter protein in fish exposed to
a test compound to negative controls, the effect of the compound on
the modulation of the wnt/.beta.-catenin signaling pathway can be
assessed. Test compounds can act as either inhibitors or activators
of the reporter gene. Importantly, modulators of the individual
pathways may then be combined and contacted with reporter fish and
the expression of the reporter protein compared with the
appropriate positive and negative controls. In this manner,
modulators that are useful as drugs for treating conditions
associated with wnt/.beta.-catenin signaling pathway which may be
affected by modulators of the prostaglandin pathway, as described
herein, are identified.
[0099] The modulators of the present invention include those that
directly modulate the wnt signaling pathway; modulate the
prostaglandin pathway to effect modulation of the wnt signaling
pathway; or modulate the downstream effects of prostaglandin to
modulate wnt. Additionally, these modulators may be combined to
"fine tune" the signal. For example, a wnt signaling activator may
be employed until the desired enhancement is noted, followed by a
prostaglandin inhibitor to limit the effects of the wnt activator.
Or, for example, a low-dose wnt activator could be combined with a
low-dose prostaglandin activator to avoid toxicity. Thus, the
interactions of the modulators of the wnt and prostaglandin
signaling pathways may be used in any direction or in any
combination to elicit a desired response while limiting toxicity or
exuberant growth. The modulators may be used simultaneously or
sequentially.
[0100] Patients may benefit from the present invention in several
ways: for example, patients undergoing liver resection surgery may
regain their hepatic function faster, decreasing complications and
hospitalization. Conceivably, patients receiving a liver transplant
may have a higher rate of organ survival. As applied to other
aspects of organ and tissue regeneration, for example, enhanced
recovery in the wound healing process, after myocardial infarction,
and after bone fracture may be positively impacted. Additionally,
for example, subjects suffering from traumatic injury, drug
toxicity, poisoning (e.g., Amanita ingestion), industrial toxins,
surgery, liver donation, cancer, skin grafts, burns, etc., may have
the modulators of the present invention added to their treatment
regimen. The present growth modulators may be useful on any tissue
capable of regeneration, repair, or regrowth, including
hematopoietic stem cells, liver, skin, or vessels.
[0101] Direct ex vivo administration of modulators may enable
significant in vivo tissue development or regenerations, such that
even smaller amounts of tissue can then be enough in
transplantation. Origin of such tissue is not limited.
Alternatively, a tissue source sample, such as skin, may be
harvested and then stored immediately in the presence of a
modulator, such as PGE2, and initially incubated (prior to
implantation) in the presence of the modulator before introduction
into a subject.
[0102] Additionally, one or more modulator might be used to enhance
the function of the tissue source. For example, modulating the
wnt/PGE2 pathway may hasten a graft's ability to assume its
physiological role and consequently lead to a decrease in the time
during which the subject has insufficient tissue (for example liver
tissue) thus reducing complication risks. Additionally, a modulator
could be administered to a living donor after removal of the tissue
to speed healing.
[0103] The tissue growth modulators may be used in vivo to enhance
tissue growth and ex vivo to increase tissue growth. This is
accomplished by administering one or more of the compounds to a
subject or to the resected tissue. For example, in reconstruction
of the uterus, excised tissue may be treated with tissue growth
modulator in the context of a biocompatible scaffold (see e.g.,
U.S. Pat. No. 7,04,057, "Tissue engineered uterus" issued to Atala
et al.) to provide a enhance autologous tissue growth before
implantation.
[0104] Various kits and collection devices are known for the
collection, processing, and storage of source cells are known in
the art. The modulators of the present invention may be introduced
to the cells in the collection, processing, and/or storage. Thus,
not being limited to any particular collection, treatment, or
storage protocols, an embodiment of the present invention provides
for the addition of a modulator, such as, for example, wnt
activators, PGE2 or dmPGE2, or their analogs, cAMP activators,
etc., to a tissue sample. This may be done at collection time, or
at the time of preparation for storage, or upon thawing and before
implantation.
[0105] The method of the invention thus provides the following
benefits: (1) Allows transplantation to proceed in patients who
would not otherwise be considered as candidates because of the
unacceptably high risk of failed engraftment or failure of primary
graft function; (2) Reduces the size of the donor tissue required
to generate a minimum acceptable harvest; (3) Reduces the incidence
of primary and secondary failure of engraftment by increasing the
tissue sample available for transplantation; and (4) Reduces the
time required for primary engraftment by enhancing the growth of
the implanted tissue.
[0106] The modulators of the invention, e.g., modulators that
inhibit tissue growth, may also be of use in treating subjects
suffering from hyperproliferative disorders including those of the
hematopoietic system or other cancers. In particular, modulators
may be useful in therapies to treat liver disease.
[0107] The modulators of the present invention also include
derivatives of such modulators. Derivatives, as used herein,
include a chemically modified compound wherein the modification is
considered routine by the ordinary skilled chemist, such as
additional chemical moieties (e.g., an ester or an amide of an
acid, protecting groups, such as a benzyl group for an alcohol or
thiol, and tert-butoxycarbonyl group for an amine). Derivatives
also include radioactively labeled modulators, conjugates of
modulators (e.g., biotin or avidin, with enzymes such as
horseradish peroxidase and the like, with bioluminescent agents,
chemoluminescent agents or fluorescent agents). Additionally,
moieties may be added to the modulator or a portion thereof to
increase half-life in vivo. Derivatives, as used herein, also
encompasses analogs, such as a compound that comprises a chemically
modified form of a specific compound or class thereof, and that
maintains the pharmaceutical and/or pharmacological activities
characteristic of said compound or class, are also encompassed in
the present invention. Derivatives, as used herein, also
encompasses prodrugs of the modulators, which are known to enhance
numerous desirable qualities of pharmaceuticals (e.g., solubility,
bioavailability, manufacturing, etc.).
[0108] The compounds or agents of the present invention can be
contained in pharmaceutically acceptable formulations. Such a
pharmaceutically acceptable formulation may include a
pharmaceutically acceptable carrier(s) and/or excipient(s). As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and anti fungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. For example, the carrier can be
suitable for injection into the cerebrospinal fluid. Excipients
include pharmaceutically acceptable stabilizers. The present
invention pertains to any pharmaceutically acceptable formulations,
including synthetic or natural polymers in the form of
macromolecular complexes, nanocapsules, microspheres, or beads, and
lipid-based formulations including oil-in-water emulsions,
micelles, mixed micelles, synthetic membrane vesicles, and resealed
erythrocytes.
[0109] When the agents or compounds are delivered to a patient,
they can be administered by any suitable route, including, for
example, orally (e.g., in capsules, suspensions or tablets) or by
parenteral administration. Parenteral administration can include,
for example, intramuscular, intravenous, including direct
administration into the portal vein, including direct
administration into the portal vein, intraarticular, intraarterial,
intrathecal, subcutaneous, or intraperitoneal administration. The
agent can also be administered orally, transdermally, topically, by
inhalation (e.g., intrabronchial, intranasal, oral inhalation or
intranasal drops) or rectally. Administration can be local or
systemic as indicated. Agents can also be delivered using viral
vectors, which are well known to those skilled in the art.
[0110] Both local and systemic administration are contemplated by
the invention. Desirable features of local administration include
achieving effective local concentrations of the active compound as
well as avoiding adverse side effects from systemic administration
of the active compound. In a preferred embodiment, the antagonist
is administered locally. Localized delivery techniques are
described in, for example, 51 J. Biomed. Mat. Res. 96-106 (2000);
100(2) J. Control Release 211-19 (2004); 103(3) J. Control Release
541-63 (2005); 15(3) Vet. Clin. North Am. Equine Pract. 603-22
(1999); 1(1) Semin. Interv. Cardiol. 17-23 (1996)
[0111] The pharmaceutically acceptable formulations can be
suspended in aqueous vehicles and introduced through conventional
hypodermic needles or using infusion pumps.
[0112] The amount of agent administered to the individual will
depend on the characteristics of the individual, such as general
health, age, sex, body weight and tolerance to drugs as well as the
degree, severity and type of rejection. The skilled artisan will be
able to determine appropriate dosages depending on these and other
factors.
[0113] Several embodiments will now be described further by
non-limiting examples.
EXAMPLES
Example 1
Techniques Associated with the Zebrafish Model
[0114] Zebrafish husbandry: Zebrafish were maintained according to
IACUC protocols. The LFABP:GFP (a gift from G.M. Her & J.L. Wu,
Nat'l Cheng Kung Univ., Taiwan), gut:GFP, hs:wnt8-GFP,
hs:dnTCF-GFP, and hs:dkk-GFP transgenic lines were used. Dorsky et
al., 2002: Her et al., 538 FEBS Lett 125-133 (2003); Lewis et al.,
131 Devel. 1299-1308 (2004); Ober et al., 120 Mech. Devel. 5-18
(2003); Stoick-Cooper et al., 134 Devel. 479-89 (2007); Weidingder
et al., 15 Curr. 489-500 (2005). Genotyping for APC mutants was
performed as described. Hurlstone et al., 2003.
[0115] Heat shock activation/repression of wnt signaling: Embryonic
heat-shock experiments were conducted at 38.degree. C. for a
duration of 20 minutes unless otherwise noted. Genotype was
determined by the presence of GFP fluorescence at three hours post
heat-induction, non-fluorescence (wild-type) siblings were used as
controls.
[0116] Morpholino knockdown: MO (Gene Tools, LLC, Philomath, Oreg.)
direcetd against zebrafish .beta.-catenin, wnt2b, wnt3, wnt5, wnt8,
and wnt11 (Buckles et al., 121 Mech. Devel. 437-47 (2004); Lekven
et al., 1 Cell Devel. 103-14 (2001); Lele et al., 30 Genesis 190-94
(2001); Lyman Gingerich et al., 2004; Ober et al., 442 Nature
688-91 (2006)), or mismatched controls were injected into zebrafish
embryos at the one-cell stage at a concentration of 40 .mu.M.
[0117] In situ hybridization: Paraformaldehyde (PFA)-fixed embryos
were processed for in situ hybridization using standard zebrafish
protocols such as those found on the internet at, for example,
ZFIN: The Zebrafish Model Organism Database (hosted by the Univ.
Oregon, Eugene, Oreg.). The following RNA probes were used to
detect alterations in endodermal and liver development: GFP, LFABP,
sterol carrier protein, transferrin, foxA3, insulin, trypsin, and
IFABP. Changes in expression compared to wild-type controls are
reported as the # altered/# scored per genotype; a minimum of three
independent experiments were conducted per analysis.
[0118] Immunohistochemistry: Embryos, adults, and en bloc abdominal
sections were fixed with PFA, paraffin embedded, and cut in 40
.mu.m serial step-sections for histological analysis.
Hematoxylin/eosin staining was performed on alternate sections
using standard techniques. Antibodies to .beta.-catenin (1:100) (BD
610154, BD Transduction Laboratories.TM., San Jose, Calif.), TUNEL
(Chemicon/Millipore, Billerica, Mass.), BrdU (1:2000) (clone BU-33,
B2531, Sigma-Aldrich, St. Louis, Mo.) and PCNA (1:80) (Clone PC10,
NA03, Calbiochem/EMD Chemicals, Inc., San Diego, Calif.) were
visualized by DAB and counterstained with hematoxylin or methylene
green.
[0119] Caspase Assay: Single embryos were manually dissociated in
lysis buffer and centrifuged. The supernatant (100 ml) was used for
the Caspase-Glo.RTM. 3/7 Assay System according to the manufacturer
protocol (Promega Corp., Madison, Wis.). DNA isolated from the cell
pellet was used to confirm APC genotype.
[0120] Confocal Microscopy: GFP transgenic zebrafish embryos were
embedded in 1% low-melting point agarose containing 0.4 mg/m1
Tricaine-S in glass-bottom culture dishes for visualization through
a Zeiss LSM Meta confocal microscope (Carl Zeiss MicroImaging,
Inc., Thornwood, N.Y.).
[0121] Flow Cytometry Analysis: Individual embryos were manually
dissociated in 0.9% PBS and examined for GFP fluorescence and
forward scatter. Genotyping for APC was performed by PCR on excess
cells following FACS anaysis.
[0122] Liver Resection: Folling administration of tricaine
anesthetic, 1/3 partial hepaectomy of the adult zebrafish liver was
performed under brightfield imaging on a dissection microscope. An
incision was made using microdissection scissors posterior to the
heart on the left lateral portion of the abdomen. Forceps were then
used to resect the entire length of the inferior lobe.
Example 2
Zebrafish Tumor Model
[0123] Although zebrafish are a valuable vertebrate model to study
carcinogenesis, noninvasive imaging remains challenging because
adult fish are not transparent. Tumors can be readily detected in
vivo, however, using high-resolution microscopic ultrasound. This
technique facilitates tissue perfusion calculations, cellular
aspirates, tumor progression analysis, and responses to treatment.
Ultrasound biomicroscopy allows longitudinal studies of tumor
development and real-time assessment of therapeutic effects in the
zebrafish model. The visualization techniques employed herein are
described by Goessling et al., 4(7) Nature Methods 551-53
(2007).
[0124] As shown in FIG. 10, zebrafish were exposed to DMBA at three
and four weeks of age. Tumor occurrence was monitored over about
five months. Tumors were identified by ultrasound biomicroscopy.
The cancerous fish were then treated with indomethacin for twelve
hours over night, every three days for one month, and the tumors
visualized as described. Goessling et al., 2007. In representative
fish, tumor size increased and architecture changed after three
treatments. After six treatments, the solid portion of the tumor
decreased in size, and an area of liquefaction appeared in the
posterior aspect of the abdomen, suggestive of tumor necrosis.
After ten treatments, the tumor had shrunk substantially, and
appeared diminished in size compared to the initial ultrasound.
After one month of drug treatment, the fish appeared healthy, but
were sacrificed to confirm the presence of cancer by histology.
These observations demonstrate the effect of prostaglandin
inhibition on liver tumor growth, and the successful chemotherapy
of fish.
[0125] In another experiment, detailed in FIG. 10B and FIG. 10C,
fish were treated every week for tumor prevention. Here, treatment
with the carcinogen led to liver tumors in wild-type fish, and to
twice as many tumors in APC+/- mutant fish. Weekly treatment with
indomethacin decreased tumor formation in both wild-type, but
especially in APC+/- fish.
* * * * *