NZ614203B2 - Treatment of disorders with altered vascular barrier function - Google Patents
Treatment of disorders with altered vascular barrier function Download PDFInfo
- Publication number
- NZ614203B2 NZ614203B2 NZ614203A NZ61420312A NZ614203B2 NZ 614203 B2 NZ614203 B2 NZ 614203B2 NZ 614203 A NZ614203 A NZ 614203A NZ 61420312 A NZ61420312 A NZ 61420312A NZ 614203 B2 NZ614203 B2 NZ 614203B2
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- NZ
- New Zealand
- Prior art keywords
- rasip1
- cell
- disorder
- barrier function
- modulator
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Abstract
Disclosed is a use of a RASIP1 modulator in the manufacture of a medicament for treating a disorder associated with altered vascular barrier function in a subject wherein the disorder is selected from the group consisting of sepsis, age-related macular degeneration (AMD), edema, ischemic stroke, hemorrhage, and hypertension. Also disclosed is a use of a RASIP1 agonist in the manufacture of a medicament for treating cancer or a proliferative retinopathy. orrhage, and hypertension. Also disclosed is a use of a RASIP1 agonist in the manufacture of a medicament for treating cancer or a proliferative retinopathy.
Description
TREATMENT OF DISORDERS WITH
ALTERED VASCULAR BARRIER FUNCTION
RELATED APPLICATIONS
This application claims the benefit under U.S. Provisional Application Serial No.
61/451,540 filed on 10 March 2011, which is incorporated by reference in entirety.
FIELD OF THE INVENTION
The present invention relates generally to use of agents that are useful for treatment
of conditions and disorders associated with altered vascular barrier function. Specifically,
the present invention relates to the use of modulators of Ras-Interacting Protein 1 (Rasip1).
BACKGROUND OF THE INVENTION
In murine embryos, onset of circulation (Ji et al., Circ. Res. 92:133-35, (2003))
coincides with active growth of the major vessels such as the dorsal aorta (Walls et al.,
PLoS One 3:e2853, (2008); Strilic et al., Dev. Cell 17:505-15, (2009)). Subsequently,
vigorous circulation ensues amid rapid vascular expansion via vasculogenesis,
angiogenesis, and remodeling – dynamic processes involving extensive cell movement and
positional exchange between cells (Carmeliet, Nat. Med. 6:389-95, (2000); Coultas et al.,
Nature 438:937-45, (2005); Jakobsson et al., Nat. Cell. Biol. 12:943-53, (2010)). These
concomitant events pose a unique challenge to the developing vasculature: nascent
endothelial cell-cell junctions must be stable enough to permit lumen formation, circulation
and to withstand increasing shear stress, yet be flexible enough to allow cell movement
during dynamic growth and remodeling. In addition, vascular permeability through
regulation of cell-cell junctions are tightly regulated, as increased permeability contributes
to pathologic conditions including hemorrhage, edema, ischemic stroke, inflammation, and
sepsis (Dejana et al., Dev. Cell 16:209-21, (2009); Spindler et al., Cardiovascular Research
87:243-53, (2010)). Although key components in endothelial cell-cell tight and adherens
junctions have been shown to critically influence vascular development and lumen
stabilization (Dejana, Nat. Rev. Mol. Cell. Biol. 5:261-70, (2004); Crosby et al., Blood
105:2771-76 (2005); Dejana et al. (2009; supra)), much remains to be learned regarding the
molecular ensemble that regulates endothelial cell-cell junctions in development.
A key regulator of cell-cell junction formation in both epithelial and endothelial
cells is the small G protein Rap1 (Kooistra et al., J. Cell Science 120:17-22, (2007)). In
many contexts, activation of protein kinase A (PKA) at the cell membrane promotes the
formation of cyclic AMP (cAMP) that binds to the guanine exchange factor (GEF) Epac1
and triggers exchange of GDP to GTP on Rap1, activating the protein and triggering a
cascade of molecular interactions leading to stabilization and linkage of cortical actin to
proteins of adherens and tight junctional complexes (Kooistra et al., FEBS Letters
579:4966-72, (2005)). Modulation of Rap1 activity affects endothelial barrier function
(Spindler et al. (2010; supra)). In this network, regulators of small G proteins, such as
EPAC1, also play critical roles (Pannekoek et al., Biochim. Biophys. Acta 1788:790-96,
(2009)). In the past several years, effectors of Ras and RAP1, such as MLLT4/AFADIN-6,
RADIL, and KRIT1, have been identified and shown to play important roles in mediating
cell-cell adhesion and migration (Boettner et al., Proc. Natl. Acad. Sci. USA 97:9064-69,
(2000); Glading et al., J. Cell Biol. 161:1163-77, (2007); Mitin et al., J. Biol. Chem.
279:22353-61, (2004); Smolen et al., Genes Dev. 21:2131-36, (2007); Xu et al., Dev. Biol.
329:269-79, (2009)). In addition, Rasip1 has been shown to bind overexpressed Ras and
Rap1 (Mitin et al., (2004; supra)) and knockdown of Rasip1 abolishes vessel formation in
Xenopus laevis (Mitin et al., (2004; supra)); Xu et al., (2009; supra)).
Despite the many advances in our understanding of the development and
maintenance of normal and pathological vasculature, there remains a need to identify targets
and develop means that can supplement or enhance the efficacy of existing therapies in this
area.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that
such documents, or such sources of information, in any jurisdiction, are prior art, or form part
of the common general knowledge in the art.
In the description in this specification reference may be made to subject matter that is
not within the scope of the claims of the current application. That subject matter should be
readily identifiable by a person skilled in the art and may assist in putting into practice the
invention as defined in the claims of this application.
SUMMARY OF THE INVENTION
The present invention relates to use of a RASIP1 modulator in the manufacture of a
medicament for treating a disorder associated with altered vascular barrier function in a
subject wherein the disorder is selected from the group consisting of sepsis, age-related
macular degeneration (AMD), edema, ischemic stroke, hemorrhage, and hypertension.
The invention also relates to use of a RASIP1 agonist in the manufacture of a
medicament for treating cancer or a proliferative retinopathy.
BRIEF DESCRIPTION
The present invention is based, at least in part, on the discovery that Ras-Interacting
Protein 1 (Rasip1) is essential to maintain endothelial junctional stability. Therefore,
targeting Rasip1 with agents that activate it, or the signaling cascade in which it lies, is
useful in the treatment of disorders with decreased vascular barrier function, including
sepsis, age-related macular degeneration (AMD), edema, and hemorrhage. Accordingly,
described are novel methods for treating such disorders using agents that activate Rasip1
activity. In addition, the invention is based, at least in part, on the discovery that Rasip1 is
required for the formation of stable vessels. Therefore, targeting Rasip1 with agents that
inhibit it, or the signaling cascade in which it lies, is useful in the treatment of disorders
where new vessel formation is required, including cancers and proliferative diabetic
retinopathy. Accordingly, described are novel methods for treating such disorders using
agents that inhibit Rasip1 activity.
Also described is a method of treating a disorder associated with altered vascular
barrier function in a subject comprising administering to the subject a RASIP1 modulator.
In some embodiments, the disorder is associated with reduced vascular barrier function and
wherein the RASIP1 modulator is a RASIP1 agonist, including where the disorder is, e.g.,
sepsis, age-related macular degeneration (AMD), edema, ischemic stroke or hemorrhage.
In some embodiments, the disorder is associated with increased vascular barrier function
and wherein the RASIP1 modulator is a RASIP1 antagonist, including where the disorder
is, e.g., hypertension.
Also described is a method of reducing or inhibiting vascular barrier function in a
subject in need thereof, comprising administering to the subject a RASIP1 agonist.
Also described is a method of increasing or enhancing vascular barrier function in a
subject in need thereof, comprising administering to the subject a RASIP1 antagonist.
Also described is a method of treating a disorder that requires new vessel formation
in a subject comprising administering to the subject a RASIP1 inhibitor, including where
the disorder is, e.g., cancer or a proliferative retinopathy, including diabetic retinopathy.
In some embodiments, the RASIP1 modulator is a small molecule. In some
embodiments where the RASIP1 modulator is an antagonist it is an antisense RNA, RNAi
or ribozyme.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Rasip1 knockout mice die in mid-gestation with vascular defects
(A) Brightfield image of heterozygous control (+/–) embryo at E9.0. (B) Brightfield
image of Rasip1 –/– embryo at E9.0, displaying smaller size, pericardial edema, and
hemorrhage. (C, D) Wholemount CD31 plus CD105 immunofluorescence (red) in Rasip1
+/– (C) and –/– (D) E8.5 embryos. Ventral view, rostral is to the left. (E, F) Wholemount
immunofluorescence of the trunk vasculature of Rasip1 +/– (E) and –/– (F) E9.0 embryos.
Lateral view, rostral to the left. Red: CD31+CD105, Green: RBC autofluorescence; Blue:
DAPI. Arrows: Dorsal aortae. Asterisks: Cardiac crescent. so: somite.
Figure 2: Axial vessel defects in Rasip1 knockout mice
Transverse sections of caudal dorsal aortae from Rasip1 +/– (A-D) and –/– (E-H)
embryos, at 1-2 somite stage (ss) (A, E), 3-6 ss (B, F), and 7-10 ss (C, D, G, H). Note that
sections in (C) and (D), as well as (G) and (H) are from adjacent sections, indicating
localized collapse of the aorta in Rasip1 –/– embryos at 7-10 ss. (I) Scatter plot of dorsal
aortae luminal areas from Rasip1 +/– (n=5) and –/– (n=5) embryos at 7-10 ss. The 20 μm
cutoff line (red) indicates functional capillary diameter. Rasip1 –/– aortae display a wider
variation of luminal areas. Each point represents an individual area measurement of an
aorta.
Figure 3: Disruption of rasip1 and rafadil expression in zebrafish causes aberrant EC-
EC association and vascular leakage
(A) Lateral view of control morpholino oligo (ctrl) injected Tg(kdrl:EGFP)s843
zebrafish embryos at 26 hours post fertilization (hpf). Rostral is to the left. (B) rasip1 and
rafadil morpholino oligos (MO) injected embryos at 26 hpf. Intersomitic vessels (ISVs) are
stunted and axial vessels are morphologically abnormal. (C) Lateral views of a 27 hpf
embryo showing the dorsal aorta (small bracket) and cells migrating ventrally to form the
posterior cardinal vein (large bracket). (D) Lateral view of a 27 hpf double morphant. Axial
positioning of angioblasts is normal (brackets), but vessel coalescence is aberrant and
numerous gaps appear (arrows). (E) Fluorescent angiography of a control 54 hpf embryo,
showing fluorescent microbeads (red) within vasculature (green). (F) Angiography of
rasip1 and rafadil MO embryo, showing leaked extravascular microbeads.
Figure 4: Loss of RASIP1 alters cell-cell connectivity
(A) Control (Ctrl) HUVEC angiogenic sprouting at 24 hours. (B) Sprouts formed by
HUVEC stably expressing RASIP1 short hairpin RNA. Note the increase in detached
cells/sprouts in RASIP1 knockdown (KD) sample. (C) Quantification of detached sprouts in
Ctrl and KD samples at 24 and 48 hours. A total of 40 beads per condition from two
experiments were quantified, represented as means +/- SEM. (* indicates P <0.0001,
determined by unpaired, Welch-corrected t-test). (D) Migration rate (μm/min) of Ctrl and
RASIP1 KD HUVEC in a two-dimensional (2D) wound healing assay. (E) Quantification of
cells transiently detaching from wavefront in 2D migration assay. 21-23 movies per
condition were quantified. Error bars are SEM. (F) Permeability increases in RASIP1 KD
HUVEC as normalized to control. Paracellular flux was measured using 40 kDa FITC-
dextran. Values are the mean of 4 independent replicate pairs. Error bars are SD. (**
indicates P <0.02 determined by paired t-test).
Figure 5: RASIP1 controls junctional refinement through GTP loading of RAP1
Ctrl (A-C) or RASIP1 KD (D-F) HUVECs were treated with EGTA followed by
cBiMPS, and stained with Phalloidin (A, D; green in C, F) and VE-cadherin (B, E; red in E,
F). Blue in E and F indicates nuclei (DAPI). (G) Staining of RASIP1 antibody on control
HUVEC. Diffuse cytoplasmic / perinuclear as well as junctional staining (arrows) is
observed. (H) Cytoplasmic and junctional signal from the RasIP1 pAb staining is markedly
reduced in RASIP1 KD HUVEC. (I) GTP loading of RAP1 in control and KD HUVEC.
GTP bound RAP1 is reduced in KD HUVEC as compared to control.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
Unless defined otherwise, technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd
ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For
purposes of the present invention, certain terms are defined below.
The term “comprising” as used in this specification means “consisting at least in part
of”. When interpreting each statement in this specification that includes the term
“comprising”, features other than that or those prefaced by the term may also be present.
Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.
As used herein, the terms “RASIP1” or “RASIP1 polypeptide” refer to a polypeptide
having the amino acid sequence of a RASIP1 polypeptide derived from nature, regardless of
its mode of preparation or species. Thus, such polypeptides can have the amino acid
sequence of naturally occurring RASIP1 from a human, a mouse, or any other species. A
full-length human RASIP1 amino acid sequence is:
MLSGERKEGGSPRFGKLHLPVGLWINSPRKQLAKLGRRWPSAASVKSSSSDTGSRSSEPLPPPPP
HVELRRVGAVKAAGGASGSRAKRISQLFRGSGTGTTGSSGAGGPGTPGGAQRWASEKKLPELAA
GVAPEPPLATRATAPPGVLKIFGAGLASGANYKSVLATARSTARELVAEALERYGLAGSPGGGPGE
SSCVDAFALCDALGRPAAAGVGSGEWRAEHLRVLGDSERPLLVQELWRARPGWARRFELRGREE
ARRLEQEAFGAADSEGTGAPSWRPQKNRSRAASGGAALASPGPGTGSGAPAGSGGKERSENLSL
RRSVSELSLQGRRRRQQERRQQALSMAPGAADAQIGTADPGDFDQLTQCLIQAPSNRPYFLLLQG
YQDAQDFVVYVMTREQHVFGRGGNSSGRGGSPAPYVDTFLNAPDILPRHCTVRAGPEHPAMVRP
SRGAPVTHNGCLLLREAELHPGDLLGLGEHFLFMYKDPRTGGSGPARPPWLPARPGATPPGPGWA
FSCRLCGRGLQERGEALAAYLDGREPVLRFRPREEEALLGEIVRAAAAGSGDLPPLGPATLLALCVQ
HSARELELGHLPRLLGRLARLIKEAVWEKIKEIGDRQPENHPEGVPEVPLTPEAVSVELRPLMLWMA
NTTELLSFVQEKVLEMEKEADQEDPQLCNDLELCDEAMALLDEVIMCTFQQSVYYLTKTLYSTLPAL
LDSNPFTAGAELPGPGAELGAMPPGLRPTLGVFQAALELTSQCELHPDLVSQTFGYLFFFSNASLLN
SLMERGQGRPFYQWSRAVQIRTNLDLVLDWLQGAGLGDIATEFFRKLSMAVNLLCVPRTSLLKAS
WSSLRTDHPTLTPAQLHHLLSHYQLGPGRGPPAAWDPPPAEREAVDTGDIFESFSSHPPLILPLGS
SRLRLTGPVTDDALHRELRRLRRLLWDLEQQELPANYRHGPPVATSP (SEQ ID NO: 1).
A full-length mouse RASIP1 amino acid sequence is:
MLSGERKEGGSPRFGKLHLPVGLWINSPRKQLAKLGRRWPSAASVKSSSSDTGSRSSEPLPPPPP
PPHVELRRVGAVKAAGGASGSRAKRISQLFRGSGAGGAGGPGTPGGAQRWASEKKLPELAAGVA
40 PEPPLPTRAAVPPGVLKIFASGLASGANYKSVLATERSTARELVAEALERYGLTGGRGAGDSGCVD
AYALCDALGRPAVGVGGGEWRAEHLRVLADAERPLLVQDLWRARPGWARRFELRGREEARRLEQ
EAFGAADADGTNAPSWRTQKNRSRAASGGAALASPGPGSGSGTPTGSGGKERSENLSLRRSVSE
LSLQGRRRRQQERRQQALSMAPGAADAQMVPTDPGDFDQLTQCLIQAPSNRPYFLLLQGYQDAQ
DFVVYVMTREQHVFGRGGPSSSRGGSPAPYVDTFLNAPDILPRHCTVRAGPEPPAMVRPSRGAPV
THNGCLLLREAELHPGDLLGLGEHFLFMYKDPRSGGSGPARPSWLPARPGAAPPGPGWAFSCRLC
GRGLQERGEALAAYLDGREPVLRFRPREEEALLGEIVRAAASGAGDLPPLGPATLLALCVQHSAREL
ELGHLPRLLGRLARLIKEAVWEKIKEIGDRQPENHPEGVPEVPLTPEAVSVELRPLILWMANTTELLS
FVQEKVLEMEKEADQEGLSSDPQLCNDLELCDEALALLDEVIMCTFQQSVYYLTKTLYSTLPALLDS
NPFTAGAELPGPGAELEAMPPGLRPTLGVFQAALELTSQCELHPDLVSQTFGYLFFFSNASLLNSLM
ERGQGRPFYQWSRAVQIRTNLDLVLDWLQGAGLGDIATEFFRKLSIAVNLLCVPRTSLLKASWSSL
RTDYPTLTPAQLHHLLSHYQLGPGRGPPPAWDPPPAERDAVDTGDIFESFSSHPPLILPLGSSRLRL
TGPVTDDALHRELRRLRRLLWDLEQQELPANHRHGPPVASTP (SEQ ID NO: 2).
Such RASIP1 polypeptides can be isolated from nature or can be produced by
recombinant and/or synthetic means.
“Isolated” in reference to a polypeptide means that it has been purified from an
natural source or has been prepared by recombinant or synthetic methods and purified. A
“purified” polypeptide is substantially free of other polypeptides or peptides. “Substantially
free” here means less than about 5%, preferably less than about 2%, more preferably less
than about 1%, even more preferably less than about 0.5%, most preferably less than about
0.1% contamination with other source proteins.
The term “agonist” is used in the broadest sense, and includes any molecule that
partially or fully activates a biological activity of a polypeptide. For example, an agonist of
RASIP1 would increase the ability of RASIP1 to influence GTP loading of Rap1, or to
increase cell-cell junctional stability, or to increase endothelial cell barrier function.
Methods for identifying agonists of a RASIP1 polypeptide may comprise contacting the
RASIP1 polypeptide with a candidate agonist molecule and measuring an appropriate
detectable change in one or more biological activities normally associated with the
polypeptide.
The term “antagonist” is used in the broadest sense, and includes any molecule that
partially or fully blocks, inhibits, or neutralizes a biological activity of a polypeptide. For
example, an antagonist of RASIP1 would partially or fully block, inhibit, or neutralize the
ability of RASIP1 to modulate RAP1-GTP loading, and to regulate stable endothelial cell-
cell connection. Suitable antagonist molecules specifically include antisense RNAs,
ribozymes, RNAi, small organic molecules, etc. Methods for identifying antagonists of a
RASIP1 polypeptide may comprise contacting the RASIP1 polypeptide with a candidate
antagonist molecule and measuring a detectable change in one or more biological activities
normally associated with the polypeptide.
The term “modulators” is used to refer to agonists and/or agonists collectively.
“Active” or “activity” for the purposes herein refers to form(s) of RASIP1 which
retain a biological and/or an immunological activity, wherein “biological” activity refers to
a biological function caused by RASIP1 other than the ability to induce the production of an
antibody and an “immunological” activity refers to the ability to induce the production of an
antibody against an antigenic epitope possessed by RASIP1. Principal biological activities
of RASIP1 are transduction or initiation of Rap1-induced signaling and refining cellular
junctions in the vasculature.
As used herein, “treatment” is an approach for obtaining beneficial or desired
clinical results. For purposes of this invention, beneficial or desired clinical results include,
but are not limited to, alleviation of symptoms, diminishment of extent of disease or
disorder, stabilized (i.e., not worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and remission (whether partial or
total), whether detectable or undetectable. “Treatment” is an intervention performed with
the intention of preventing the development or altering the pathology of a disorder.
Accordingly, “treatment” may refer to therapeutic treatment or prophylactic or preventative
measures. Those in need of treatment include those already with the disorder as well as
those in which the disorder is to be prevented. Specifically, the treatment may directly
prevent, slow down or otherwise decrease the pathology of cellular degeneration or damage,
such as the pathology of tumor cells in cancer treatment, or may render the cells more
susceptible to treatment by other therapeutic agents.
“Chronic” administration refers to administration of the agent(s) in a continuous
mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity)
for an extended period of time. “Intermittent” administration is treatment that is not
consecutively done without interruption, but rather is cyclic in nature.
A “disorder with altered vascular barrier function” is a disorder characterized by
increased or decreased vascular barrier function. Disorders with increased vascular barrier
function include, but are not limited to, hypertension. Disorders with decreased vascular
barrier function include, but are not limited to, sepsis, age-related macular degeneration
(AMD), edema, and hemorrhage.
A “disorder that requires new vessel formation” is characterized by dependency on
the formation of functional new vessels. Such disorders include, but are not limited to,
cancer and proliferative diabetic retinopathy.
The “pathology” of a disorder includes all phenomena that compromise the well-
being of the patient.
Administration “in combination with” one or more further therapeutic agents
includes simultaneous (concurrent) and consecutive administration in any order.
“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or
stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages
and concentrations employed. Often the physiologically acceptable carrier is an aqueous
pH buffered solution. Examples of physiologically acceptable carriers include buffers such
as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low
molecular weight (less than about 10 residues) polypeptide; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions
such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG),
and PLURONICS™.
A “small molecule” is defined herein to have a molecular weight below about 500
Daltons.
Methods for carrying out the invention
Preparation and identification of antagonists of RASIP1 activity
Screening assays for antagonist drug candidates are designed to identify compounds
that bind or complex with RASIP1 polypeptides, or otherwise interfere with their activity
and/or interaction with other cellular proteins.
Small molecules may have the ability to act as RASIP1 agonists or antagonists and
thus to be therapeutically useful. Such small molecules may include naturally occurring
small molecules, synthetic organic or inorganic compounds and peptides. However, small
molecules useful in the present invention are not limited to these forms. Extensive libraries
of small molecules are commercially available and a wide variety of assays are taught
herein or are well known in the art to screen these molecules for the desired activity.
In some embodiments, small molecule RASIP1 agonists or antagonists are identified
by their ability to activate or inhibit one or more of the biological activities of RASIP1.
Thus a candidate compound is contacted with RASIP1 and a biological activity of RASIP1
is then assessed. In one embodiment the ability of RASIP1 to modulate RAP1 GTP loading
is assessed. A compound is identified as an agonist where the biological activity of RASIP1
is stimulated and a compound is identified as an antagonist where the biological activity of
RASIP1 is inhibited.
Another potential RASIP1 antagonist is an antisense RNA or DNA construct
prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts
to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which methods are based on
binding of a polynucleotide to DNA or RNA. For example, the 5’ coding portion of the
polynucleotide sequence, which encodes the mature RASIP1 polypeptides herein, is used to
design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A
DNA oligonucleotide is designed to be complementary to a region of the gene involved in
transcription (triple helix - see, Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al.,
Science 241:456 (1988); Dervan et al., Science 251:1360 (1991)), thereby preventing
transcription and the production of RASIP1. A sequence “complementary” to a portion of
an RNA, as referred to herein, means a sequence having sufficient complementarity to be
able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded
antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex
helix formation may be assayed. The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid. Generally, the longer the
hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still
form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a
tolerable degree of mismatch by use of standard procedures to determine the melting point
of the hybridized complex. The antisense RNA oligonucleotide hybridizes to the mRNA in
vivo and blocks translation of the mRNA molecule into RASIP1 (antisense - Okano,
Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression (CRC Press: Boca Raton, FL, 1988).
The antisense oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or double-stranded. The
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide
may include other appended groups such as peptides (e.g., for targeting host cell receptors
in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al.,
Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre, et al., Proc. Natl. Acad. Sci.
U.S.A. 84:648-652 (1987); PCT Publication No. WO88/09810, published December 15,
1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published
April 25, 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al.,
BioTechniques 6:958-976 (1988)) or intercalating agents (see, e.g., Zon, Pharm. Res. 5:539-
549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g.,
a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-
triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety
which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil,
-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-
(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethylthiouridine, 5-
carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-
isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-
methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethylthiouracil, beta-D-
mannosylqueosine, 5’-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine, uraciloxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-methylthiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil
oxyacetic acid methylester, uraciloxyacetic acid (v), 5-methylthiouracil, 3-(3-amino-
3-Ncarboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar moiety
selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose,
and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least one
modified phosphate backbone selected from the group consisting of a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an anomeric
oligonucleotide. An anomeric oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual units, the strands run parallel to each
other (Gautier, et al., Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a 2’
methylribonucleotide (Inoue, et al., Nucl. Acids Res. 15:6131-6148 (1987)), or a chimeric
RNA-DNA analogue (Inoue, et al., FEBS Lett. 215:327-330 (1987)).
In some embodiments, the antagonists are inhibitory duplex RNAs, e.g. siRNA,
shRNA, etc.
Oligonucleotides useful in the invention may be synthesized by standard methods
known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially
available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein, et al. (Nucl. Acids Res.
16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled
pore glass polymer supports (Sarin, et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451
(1988)), etc.
The oligonucleotides described above can also be delivered to cells such that the
antisense RNA or DNA may be expressed in vivo to inhibit production of RASIP1. When
antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation
site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are
preferred.
Potential antagonists further include small molecules that bind to RASIP1, thereby
blocking its activity. Examples of small molecules include, but are not limited to, small
peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl
organic or inorganic compounds.
Additional potential antagonists are ribozymes, which are enzymatic RNA
molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-
specific hybridization to the complementary target RNA, followed by endonucleolytic
cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified
by known techniques. For further details see, e.g., Rossi, Current Biology 4:469-471
(1994), and PCT publication No. WO 97/33551 (published September 18, 1997).
While ribozymes that cleave mRNA at site specific recognition sequences can be
used to destroy target gene mRNAs, the use of hammerhead ribozymes is preferred.
Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions which
form complementary base pairs with the target mRNA. The sole requirement is that the
target mRNA have the following sequence of two bases: 5’-UG-3’. The construction and
production of hammerhead ribozymes is well known in the art and is described more fully
in Myers, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH
Publishers, New York (1995), (see especially Figure 4, page 833) and in Haseloff and
Gerlach, Nature, 334:585-591 (1988), which is incorporated herein by reference in its
entirety.
Preferably the ribozyme is engineered so that the cleavage recognition site is located
near the 5’ end of the target gene mRNA, i.e., to increase efficiency and minimize the
intracellular accumulation of non-functional mRNA transcripts.
The ribozymes useful in of the present invention also include RNA
endoribonucleases (hereinafter “Cech-type ribozymes”) such as the one which occurs
naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which
has been extensively described by Thomas Cech and collaborators (Zaug, et al., Science,
224:574-578 (1984); Zaug and Cech, Science, 231:470-475 (1986); Zaug, et al., Nature,
324:429-433 (1986); published International patent application No. WO 88/04300 by
University Patents Inc.; Been and Cech, Cell, 47:207-216 (1986)). The Cech-type
ribozymes have an eight base pair active site that hybridizes to a target RNA sequence
whereafter cleavage of the target RNA takes place. The invention encompasses the use of
those Cech-type ribozymes that target eight base-pair active site sequences that are present
in the target gene.
As in the antisense approach, the ribozymes can be composed of modified
oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to
cells that express the target gene in vivo. A preferred method of delivery involves using a
DNA construct “encoding” the ribozyme under the control of a strong constitutive pol III or
pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme
to destroy endogenous target gene messages and inhibit translation. Because ribozymes,
unlike antisense molecules, are catalytic, a lower intracellular concentration is required for
efficiency.
Nucleic acid molecules in triple-helix formation used to inhibit transcription should
be single-stranded and composed of deoxynucleotides. The base composition of these
oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on
one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551,
supra.
Administration Protocols, Schedules, Doses, and Formulations
The RASIP1 agonists and antagonists are pharmaceutically useful as a prophylactic
and therapeutic agent for various disorders and diseases as set forth above.
Therapeutic compositions of the agonists or antagonists are prepared for storage by
mixing the desired molecule having the appropriate degree of purity with optional
pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical
Sciences, 16th edition, Osol, A. ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients
at the dosages and concentrations employed, and include buffers such as phosphate, citrate,
and other organic acids; antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol;
and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins,
such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN ,
PLURONICS or polyethylene glycol (PEG).
Additional examples of such carriers include ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium
hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal
silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and
polyethylene glycol. Carriers for topical or gel-based forms of antagonist include
polysaccharides such as sodium carboxymethylcellulose or methylcellulose,
polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,
polyethylene glycol, and wood wax alcohols. For all administrations, conventional depot
forms are suitably used. Such forms include, for example, microcapsules, nano-capsules,
liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained-release
preparations. RASIP1 antagonists will typically be formulated in such vehicles at a
concentration of about 0.1 mg/ml to 100 mg/ml.
Another formulation comprises incorporating RASIP1 agonists or antagonists into
formed articles. Such articles can be used in modulating endothelial cell growth and
angiogenesis. In addition, tumor invasion and metastasis may be modulated with these
articles.
RASIP1 agonists or antagonists to be used for in vivo administration must be sterile.
This is readily accomplished by filtration through sterile filtration membranes, prior to or
following lyophilization and reconstitution. If in lyophilized form, RASIP1 agonists or
antagonists is typically formulated in combination with other ingredients for reconstitution
with an appropriate diluent at the time for use. An example of a liquid formulation of
RASIP1 agonists or antagonists is a sterile, clear, colorless unpreserved solution filled in a
single-dose vial for subcutaneous injection. Preserved pharmaceutical compositions
suitable for repeated use may contain, for example, depending mainly on the indication and
type of polypeptide:
RASIP1 agonist or antagonist;
a buffer capable of maintaining the pH in a range of maximum stability of the
polypeptide or other molecule in solution, preferably about 4-8;
a detergent/surfactant primarily to stabilize the polypeptide or molecule against
agitation-induced aggregation;
an isotonifier;
a preservative selected from the group of phenol, benzyl alcohol and a
benzethonium halide, e.g., chloride; and
water.
If the detergent employed is non-ionic, it may, for example, be polysorbates (e.g.,
TM TM TM
POLYSORBATE (TWEEN ) 20, 80, etc.) or poloxamers (e.g., POLOXAMER 188).
The use of non-ionic surfactants permits the formulation to be exposed to shear surface
stresses without causing denaturation of the polypeptide. Further, such surfactant-
containing formulations may be employed in aerosol devices such as those used in a
pulmonary dosing, and needleless jet injector guns (see, e.g., EP 257,956).
An isotonifier may be present to ensure isotonicity of a liquid composition of
RASIP1 agonists or antagonists, and includes polyhydric sugar alcohols, preferably
trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and
mannitol. These sugar alcohols can be used alone or in combination. Alternatively, sodium
chloride or other appropriate inorganic salts may be used to render the solutions isotonic.
The buffer may, for example, be an acetate, citrate, succinate, or phosphate buffer
depending on the pH desired. The pH of one type of liquid formulation useful in of this
invention is buffered in the range of about 4 to 8, preferably about physiological pH.
The preservatives phenol, benzyl alcohol and benzethonium halides, e.g., chloride,
are known antimicrobial agents that may be employed.
Therapeutic polypeptide compositions described herein generally are placed into a
container having a sterile access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle. The formulations may be
administered as repeated intravenous (i.v.), subcutaneous (s.c.), or intramuscular (i.m.)
injections, or as aerosol formulations suitable for intranasal or intrapulmonary delivery (for
intrapulmonary delivery see, e.g., EP 257,956). The formulations are preferably
administered as intravitreal (IVT) or subconjuctival delivery.
Therapeutic polypeptides can also be administered in the form of sustained-released
preparations. Suitable examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the protein, which matrices are in the
form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release
matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as
described by Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981) and Langer, Chem.
Tech. 12:98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,
Biopolymers 22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer et al.,
supra), degradable lactic acid-glycolic acid copolymers such as the Lupron Depot®
(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-D-(-)hydroxybutyric acid (EP 133,988).
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable
release of molecules for over 100 days, certain hydrogels release proteins for shorter time
periods. When encapsulated proteins remain in the body for a long time, they may denature
or aggregate as a result of exposure to moisture at 37ºC, resulting in a loss of biological
activity and possible changes in immunogenicity. Rational strategies can be devised for
protein stabilization depending on the mechanism involved. For example, if the aggregation
mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing
from acidic solutions, controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
Sustained-release RASIP1 agonists or antagonists compositions also include
liposomally entrapped antagonists. Such liposomes are prepared by methods known per se:
DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et
al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP
143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent Nos. 4,485,045
and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800
Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. %
cholesterol, the selected proportion being adjusted for the optimal therapy.
The therapeutically effective dose of a RASIP1 agonist or antagonist will, of course,
vary depending on such factors as the pathological condition to be treated (including
prevention), the method of administration, the type of compound being used for treatment,
any co-therapy involved, the patient's age, weight, general medical condition, medical
history, etc., and its determination is well within the skill of a practicing physician.
Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of
administration as required to obtain the maximal therapeutic effect.
With the above guidelines, the effective dose generally is within the range of from
about 0.001 to about 1.0 mg/kg, more preferably about 0.01-1.0 mg/kg, most preferably
about 0.01-0.1 mg/kg.
The route of agonist or antagonist administration is in accord with known methods,
e.g., by injection or infusion by intravenous, intramuscular, intracerebral, intraperitoneal,
intracerobrospinal, subcutaneous, intraocular (including intravitreal), intraarticular,
intrasynovial, intrathecal, oral, topical, or inhalation routes, or by sustained-release systems
as noted.
Examples of pharmacologically acceptable salts of molecules that form salts and are
useful hereunder include alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth
metal salts (e.g., calcium salt, magnesium salt), ammonium salts, organic base salts (e.g.,
pyridine salt, triethylamine salt), inorganic acid salts (e.g., hydrochloride, sulfate, nitrate),
and salts of organic acid (e.g., acetate, oxalate, p-toluenesulfonate).
The following Examples are offered for illustrative purposes only, and are not
intended to limit the scope of the present invention in any way.
The disclosures of all patent and literature references cited in the present
specification are hereby incorporated by reference in their entirety.
EXAMPLES
Commercially available reagents referred to in the Examples were used according to
manufacturer's instructions unless otherwise indicated. All references cited herein are
hereby incorporated by reference.
EXAMPLE 1. Rasip1–/– mice exhibit abnormal cardiovascular development
In a bioinformatics screen for genes whose expression is enriched in the vasculature,
we identified RASIP1 as being highly expressed in endothelial cells (EC), and we
confirmed vascular selective expression by in situ hybridization in mouse embryos. To
investigate the in vivo role of Rasip1, we generated a conventional knockout targeting exon
3 of the mouse Rasip1 locus, predicted to create a truncated protein of approximately 40
amino acids. Briefly, a BAC-based targeted vector was designed with loxP sites flanking
exon 3 of the mouse Rasip1 locus. This construct was introduced into C57BL/6 ES cells
and recombination events screened by PCR and sequencing. To generate a conventional
knockout, the targeted ES cells were infected with adenovirus encoding Cre recombinase to
delete exon 3. Two founder lines backcrossed and maintained on a pure C57BL/6
background were selected for analysis and yielded identical phenotypes. Genotyping was
performed by PCR using the RED Extract-N-Amp kit (Sigma).
No homozygous mutant offspring were obtained from heterozygous parents, so we
examined Rasip1 mutant embryos. In contrast to wildtype and heterozygous littermates at
embryonic day (E) 9.0, Rasip1–/– animals were slightly smaller in size, pale, and displayed
multifocal hemorrhage and pericardial edema, indicative of defects in the cardiovascular
system (Fig. 1A, B). Yolk sacs of Rasip1–/– embryos were pale and exhibited abnormal
vascular morphology (data not shown). At E10.5, Rasip1–/– mutant embryos were
markedly smaller than control littermates with exacerbated edema and hemorrhage. Rasip1–
/– embryos were not detected past E12.5, and no overt morphological defects were seen at
stages earlier than E8.75. We confirmed loss of full-length RASIP1 protein in null embryos
at E9.0 using a rabbit polyclonal antibody directed against the extreme C-terminus of
RASIP1. No full-length protein corresponding to the predicted molecular weight was
observed in Rasip1–/– whole embryo lysates. Taken together, these data demonstrated that
targeted disruption of mouse Rasip1 results in abnormal cardiovascular development and
mid-gestational lethality.
We next analyzed the vasculature in Rasip1 knockout mice in more detail. Whole
mount embryos at 7-10 somite stage (ss) stained with EC markers revealed that the paired
dorsal aortae (DA) of Rasip1–/– embryos were assembled in the appropriate lateral
positions (Fig 1C, 1D). However, the width of the DA was irregular along the rostral-caudal
axis with the appearance of poorly formed or refined cell-cell contacts. By 18 ss, the
cardinal veins (CV) and DA appeared to either collapse or dilate in Rasip1–/– embryos,
with accompanying hemorrhage. ECs were also disorganized or appear dispersed (Figure
1E,F). Red blood cells (RBC) appeared to collect within the remnants of vessels, or were
found in extravascular space.
Formation of the murine DA initiates when clusters of ECs elongate into cords,
accompanied by extracellular lumen formation, defined as a space larger than 5 µm between
ECs (Strilic et al., Dev. Cell 17:505-15 (2009)). This process is largely complete by 6 ss,
although the diameter of the DA continues to enlarge, and angiogenic sprouting off the
vessels occurs (Strilic et al., supra (2009)). To rigorously determine whether vascular
lumen formed in Rasip1–/– embryos, we analyzed transverse sections of DA from mutant
and littermate control embryos at 1-2 ss, 3-6 ss, and 7-10 ss. At 1-2 ss, small spaces (slits)
were detected between EC clusters in both Rasip1+/– and –/– embryos (Figure 2), which
generally had a lumenal cross-sectional area around 20 µm . By 3-6 ss, DA had developed
a luminal space greater than 20 µm regardless of genotype, although the extent of this
space was considerably more variable in Rasip1–/– embryos (Figure 2, data not shown).
From 7-10 ss, Rasip1–/– embryos showed extensive variation in the size of the DA luminal
space along the rostral-caudal axis, even between adjacent sections that are 20 µm apart,
with pronounced indications of vascular collapse in one section adjacent to another with
seemingly normal lumen (Fig 2G,H). This phenomenon was also observed when examining
contralaterally paired DA, and persisted through later stages of embryogenesis. We
conclude that loss of Rasip1 does not preclude initial establishment of vascular lumen, but
leads to a slight delay in lumenal expansion, followed by localized dilation or collapse of
the major axial vessels. Further, the mutant vasculature appears to be partially functional,
allowing circulation of primitive erythrocytes for a period prior to the onset of hemorrhage.
EXAMPLE 2. Investigating the role of Rasip1 in zebrafish
We next used zebrafish to examine the role of Rasip1 in vascular development at the
cellular level. Using the human RASIP1 Ras-associated and Forkhead-associated domains
as the query sequence, we identified two expressed sequence tag (EST) clones in the
Ensembl database (www.ensembl.org) as potential RASIP1 orthologs. EST clones with
partial sequences of rasip1 and rafadil (GenBank: BM03633, EB781618.1) were from
Open Biosystems. Additional cDNAs were cloned by 5’ and 3’ RACE with the SMART
RACE cDNA Amplification kit (Clontech) using KOD Hot Start DNA polymerase (EMD
Biosciences). Sequences of RACE clones were used to obtain full-length cDNAs by RT-
PCR using total RNA from 30 hours post-fertilization zebrafish embryos. rasip1 and rafadil
cDNAs were subcloned using TopoXL PCR cloning kit (Invitrogen) into pCS2+ for in vitro
synthesis of 5’ capped mRNA using the Message Machine Sp6 kit (Ambion).The ESTs
were fully sequenced and used to clone both full-length cDNAs. The first encodes a protein
with high sequence similarity to mouse and human Rasip1/RASIP1, which we infer to be
the zebrafish ortholog. The second gene bore similarity to both zebrafish rasip1 and RADIL,
a related member of the afadin-6 family (Smolen et al., Genes Dev. 21:2131-36 (2007)). We
named this gene rafadil (for Ras-Associated, Forkhead-Associated, DILute domain protein).
Phylogenetic analysis indicates that rafadil is a fish-specific gene, which likely arose
through an ancestral gene duplication event. Both rasip1 and rafadil are highly expressed in
the developing vasculature.
In zebrafish, development and lumenization of the major axial vessels is
independent of circulation (Isogai et al. 2003), and we sought to visualize vascular
s843
development using the established Tg(kdrl:EGFP) line (Beis et al., Development
132:4193-204 (2005)). Knockdown of the zebrafish rasip1 via morpholino oligonucleotide
s843
injection into Tg(kdrl:EGFP) embryos resulted in the formation of aberrant and leaky
intersomitic vessels (ISVs), whereas knockdown of rafadil had no overt effect. Combined
knockdown of both rasip1 and rafadil resulted in notable morphological alteration in the
axial vessels and stunted ISVs (Fig. 3 A, B). In a staged developmental series, we observed
normal formation of angioblast aggregates ventral to the notochord in control and double
morphant embryos at 22 ss, as previously reported (Parker et al., Nature 428:754-58 (2004);
Jin et al., Development 132:5199-209 (2005)). At 24 ss, a subset of angioblasts dissociate
from the midline aggregates and migrate/sprout ventrally (Herbert et al., Science 326:294-
98 (2009)), which subsequently coalesce into the PCV around 26 ss. Although angioblasts
in the morphants dissociated from the midline aggregates and migrated properly, they were
defective in coalesceing into the PCV, as gaps indicating aberrant cell-cell connections
appeared and persisted (Fig. 3C, D). These defects ultimately led to a dysfunctional, leaky
vasculature, as assessed by micro-angiography (Fig. 3E, F). Vascular defects were rescued
with injection of in vitro transcribed RNA encoding rasip1 and rafadil.
Taken together, our data in the zebrafish indicate that loss of rasip1/rafadil
expression leads to formation of unstable vasculature that leaks and collapses as a result of
compromised EC-EC coherence.
EXAMPLE 3. Investigating the role of Rasip1 in cultured human cells
To better understand the cellular changes underlying the vascular defects in Rasip1
mutant embryos we undertook a series of in vitro analyses of human umbilical vein
endothelial cells (HUVEC) lacking significant RASIP1 expression. Knockdown of RASIP1
protein with both siRNA and lentivirally delivered shRNA was confirmed by qPCR and
Western blot, and had no significant effect on the ability of HUVEC to proliferate, migrate,
or survive under stress. We perfomed a three-dimensional angiogenic sprouting assays as
described (Nakatsu et al., Microvasc. Res. 66:102-12 (2003)). DIC images of sprouts were
acquired on a Zeiss Observer Z.1, with a 10X Fluar objective, NA 0.5, using Slidebook
software (Intelligent Imaging Innovations). In the three-dimensional angiogenic sprouting
assay we observed loss of RASIP1 resulted in fragmentation of sprouts (Fig. 4A,B),
suggesting deficiencies in maintaining connections between cells. A “detachment ratio”,
measuring the number of fragmented sprouts in relation to the total number of sprouts,
showed a significant increase in RASIP1 knockdown HUVEC versus control (Fig. 4C). At
later time points (3-5 days), where control sprouts have established lumen, RASIP1
knockdown sprouts showed transient lumen formation, with an increased number of break
points over control HUVEC. Thus, as in mice and fish, our in vitro angiogenesis system
provides strong evidence of disrupted cell-cell contacts and transient and unstable lumen
formation resulting from loss of RASIP1 expression.
We set out to determine which of the following reasons account for the lack of cell-
cell cohesion in RASIP1 knockdown HUVEC: a change in migratory capacity of the cells,
alteration in adhesion to extracellular matrix (ECM), or a change in cell-cell junction
composition or stability. In contrast to a prior report, which utilized transformed MS1 cells
(Xu et al., Dev. Biol. 329:269-79 (2009)), we observed no significant difference in the
ability of control and RASIP1 knockdown HUVEC to migrate in a scratch wound assay
where confluent HUVEC monolayers in 24-well plates were scratched with pipette tips and
monitored for 24 hours in EGM-2 (Lonza) using an Essen Incucyte system (Essen
BioScience) (Fig. 4D). However, an increase in the number of cells that detached briefly
and reassembled with the migrating wavefront was seen (Fig. 4E). We did not detect a
significant difference in the ability of control and knockdown HUVEC to adhere either to
type I collagen or fibronectin (Parker et al., Nature 428:754-58 (2004)). We then used a
paracellular flux assay to measure junctional integrity (Zhao et al., J. Cell. Biol. 189:955-65
(2010)), and found that passage of 40 kDa FITC-dextran increased by approximately 50-
60% in RASIP1 knockdown HUVEC compared to control (Fig. 4F). Taken together, our
data indicate that loss of RASIP1 does not significantly impact the ability of ECs to migrate
or adhere to common ECM substrates, but instead impairs cell-cell connectivity
Next, we investigated whether knockdown of RASIP1 impacted the ability of tight
or adherens junctions to form. In steady-state, confluent, serum-starved HUVEC cultures,
loss of RASIP1 did not alter the localization or protein levels of tight junction (CLAUDIN-
, OCCLUDIN, ZO-1), adherens junction (α-CATENIN, β-CATENIN, p120-CATENIN,
VE-cadherinCADHERIN), focal adhesion (activated β1-INTEGRIN, FAK, PAXILIN,
vinculinVINCULIN) or actin cytoskeleton-related proteins (alpha-ACTININ, non-muscle
myosin IIA) in discontinuous junctions formed under this culture condition (Millan et al.,
BMC Biology 8:11 (2010)). To monitor the initiation and formation of new, continuous EC-
EC junctions, we used EGTA to disrupt calcium-dependent adherens junctions (Sakurai et
al., Molec. Biol. Cell. 17:966-76 (2006)) followed by treatment with Sp-5,6-diCl-cBiMPS,
an EPAC1-selective cAMP analog expected to activate the small GTPase RAP1, and thus
promote junctional re-assembly (Christensen et al., J. Biol. Chem. 278:35394-402 (2003)).
Under these conditions, junctions re-assembled into tight complexes 30-60 minutes after
exposure to cBiMPS, as determined by VE-CADHERIN, β-CATENIN, CLAUDIN-5 and
ZO-1 staining (Fig. 5C), with accompanying association of a thin belt of cortical actin that
closely paralleled the junctional markers (Fig. 5A). Remarkably, staining of cortical
ACTIN, VE-CADHERIN, β-CATENIN and ZO-1 was either irregular and/or discontinuous
in RASIP1 knockdown HUVEC in this assay (Fig. 5D-F). Numerous ‘spikes’ or short actin
filaments emerged perpendicular to cell-cell contact, and VE-cadherin and phalloidin
staining was irregular and jagged in the RASIP1 knockdown cells as opposed to the refined,
compact junctions formed in control cells (Fig. 5A-F). The lack of refinement of both tight
and adherens junction markers, as well as ACTIN, suggests that loss of RASIP1 affects a
process that coordinates the linkage of sub-membranous actin to junctional proteins.
The inability of Rasip1 knockdown cells to form continuous, refined junctions in a
model requiring RAP1 stimulation of barrier formation prompted us to investigate a direct
relationship between RASIP1, junctions, and RAP1. First, we examined RASIP1 localization
using our RASIP1 antibody. In the barrier reformation model, RASIP1 signal was
prominent at newly formed cell-cell junctions and overlapped with β-CATENIN staining,
indicating junctional or sub-membranous localization (Fig. 5G, data not shown). This signal
was not seen in RASIP1 knockdown HUVEC (Fig. 5H), confirming antibody specificity.
We then reinforced a functional link to EPAC1/ RAP1 by confirming our results with
cBiMPS using the Epac1-specific cAMP analog 8-pCPT-2’-O-Me-cAMP. Finally, given
the reported association between RASIP1 and RAP1A (Mitin et al., J. Biol. Chem.
279:22353-61, (2004)), we investigated whether GTP loading of RAP1 was compromised
in RASIP1 knockdown HUVEC. Significant levels of RAP1-GTP is seen in HUVEC treated
with cBiMPS, this level is markedly decreased in RASIP1 knockdown HUVEC. Thus, loss
of RASIP1 affects GTP loading of RAP1, which may explain the lack of refinement of
nascent junctions. As RASIP1 does not possess GAP or GEF domains, we speculate that it
may affect RAP1 function by controlling its localization, or accessibility of factors such as
EPAC1. We propose that the unrefined junctions observed in the barrier reformation assay
are a hallmark of the increased fragility of EC-EC junctions that result when the cells are
exposed to contractile or tensile forces. This underlying defect in junctional stability
explains the inability of Rasip1 mouse mutants and fish morphants to form stable lumen, as
affected nascent vessels are unlikely to constantly withstand increased tensional forces
brought about by vascular expansion, as well as to resist the hydrodynamic forces of
circulation.
Our work demonstrates an essential role for Rasip1 in vertebrate vascular
development, and shows that Rasip1 is critical for stabilizing new cell-cell junctions during
active vascular growth. These studies lay the groundwork for the investigation of the role of
Rasip1 in pathologic conditions affected by compromised vascular junctional integrity, and
we anticipate that activation of RASIP1, or the signaling cascade in which it lies, would
have protective effects in diseases with altered vascular barrier function, such as sepsis, age-
related macular degeneration (AMD), edema, and hemorrhage.
The foregoing written specification is considered to be sufficient to enable one
skilled in the art to practice the invention. However, various modifications of the invention
in addition to those shown and described herein will be apparent to those skilled in the art
from the foregoing description and fall within the scope of the appended claims.
Claims (9)
1. Use of a RASIP1 modulator in the manufacture of a medicament for treating a disorder associated with altered vascular barrier function in a subject wherein the disorder is selected from the group consisting of sepsis, age-related macular degeneration (AMD), edema, ischemic stroke, hemorrhage, and hypertension.
2. The use of claim 1, wherein the disorder is associated with reduced vascular barrier function and wherein the RASIP1 modulator is a RASIP1 agonist.
3. The use of claim 2, wherein the disorder is selected from the group consisting of sepsis, age-related macular degeneration (AMD), edema, ischemic stroke and hemorrhage.
4. The use of claim 1, wherein the disorder is associated with increased vascular barrier function and wherein the RASIP1 modulator is a RASIP1 antagonist.
5. The use of claim 4, wherein the disorder is hypertension.
6. The use of any one of claims 1-5, wherein the RASIP1 modulator is a small molecule.
7. The use of claim 6, wherein the modulator is compound with a molecular weight less than 500 daltons.
8. Use of a RASIP1 agonist in the manufacture of a medicament for treating cancer or a proliferative retinopathy.
9. A use as claimed in any one of claims 1-8, substantially as herein described and with or without reference to the accompanying drawings.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161451540P | 2011-03-10 | 2011-03-10 | |
US61/451,540 | 2011-03-10 | ||
PCT/US2012/028588 WO2012122515A1 (en) | 2011-03-10 | 2012-03-09 | Treatment of disorders with altered vascular barrier function |
Publications (2)
Publication Number | Publication Date |
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NZ614203A NZ614203A (en) | 2015-12-24 |
NZ614203B2 true NZ614203B2 (en) | 2016-03-30 |
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