Note: Descriptions are shown in the official language in which they were submitted.
CA 0222~460 1997-12-22
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TR~SCRIPTIONAL REGULATION OF GENES ENCODING VASCULAR
ENDOTHELIAL GROW~H FACTOR RECEPTORS
Backqround of the Invention
This invention relates to endothelial cell-
specific gene transcription and transcriptional
regulation by TNF-~.
Vascular endothelial growth factor (VEGF) is a
potent and specific endothelial cell mitogen (Connolly et
10 al., 1989, J. Clin. Invest. 84:1470-1478; Leung et al.,
1989, Science 246:1306-1309). Through interactions with
its receptors, Kinase-insert Domain-cont~; n ing
Receptor/fetal liver kinase-l (KDR/flk-l) and fltl, VEGF
plays critical roles in growth and maintenance of
15 vascular endothelial cells and in the development of new
blood vessels in physiologic and pathologic states
(Aiello et al., 1994, New Engl. J. Med. 331:1480-1487;
Shweiki et al., 1992, Nature 359:843-845; Berkman et al.,
1993, J. Clin. Invest. 91:153-159). The patterns of
20 embryonic expression of VEGF suggest that it is crucial
for differentiation of endothelial cells from
hemangioblasts and for development of blood vessels at
all stages of growth (Jakeman et al., 1993, Endocrinology
133:848-859; Breier et al., 1992, Development 114:521-
25 532). Among many potentially angiogenic factors, VEGF is
the only one with patterns of expression, secretion, and
activity that suggest a specific angiogenic function in
normal development (Klagsbrun et al., 1993, Current
Biology 3:699-702).
High-affinity receptors for VEGF are found only on
endothelial cells, and VEGF binding has been demonstrated
on macro- and microvascular endothelial cells and in
~ quiescent and proliferating endothelial cells (Jakeman et
al., 1993, Endocrinology 133:848-859; Jakeman et al.,
CA 0222~460 1997-12-22
W O 97/00957 ~ PCTrUS96/10725
1992, Clin. Invest. 89:244-253). The tyrosine kinases
KDR/flk-l and fltl have been identified as candidate VEGF
receptors by affinity cross-linking and
competition-binding assays (de Vries et al., 1992,
5 Science 255:989-991; Millauer et al., 1993, Cell 72:835-
846; Terman et al., 1992, Biochem. Biophys. Res. Commun.
187:1579-1586). These two receptor tyrosine kinases
contain seven similar extracellular immunoglobulin
do~; n~ and a conserved intracellular tyrosine kinase
10 domain interrupted by a kinase insert (de Vries et al.,
1992. Science 2S5:989-991; Matthews et al., 1991, Proc.
Natl. Acad. Sci. U.S.A 88:9026-9030; Terman et al., 1001,
Oncogene 6:1677-1683); they are expressed specifically by
endothelial cells in vivo (Millauer et al., 1993, Cell
15 72:835-846; Peters et al., 1993, Proc. Natl. Acad. Sci.
USA 90:8915-8919; Quinn et al., 1993, Proc. Natl. Acad.
Sci. USA 90:7533-7537; Yamaguchi et al., 1993,
Development 118:489498). In situ hybridization in the
developing mouse has demonstrated that KDR/flk-l is
20 expressed in endothelial cells at all stages of
development, as well as in the blood islands in which
endothelial cell precursors first appear (Millauer et
al., 1993, Cell 72:835-846). KDR/flk-1 is a marker for
endothelial cell precursors at their earliest stages of
25 development (Yamaguchi et al., 1993, Development 118:489-
498).
The vascular endothelium is critical for
physiologic responses including thrombosis and
thrombolysis, lymphocyte and macrophage homing,
30 modulation of the immune response, and regulation of
vascular tone. The endothelium is also intimately
involved in the pathogenesis of vascular diseases such as
atherosclerosis (Ross, 1993, Nature 362:801-809).
Although a number of genes expressed in the endothelium
35 have been characterized (Collins et al., 1991, J. Biol.
CA 02225460 1997-12-22
W O 97/00957 PCT~US96/107~5
Chem. 266:2466-2473; Iademarco et al., 1992, J. 8iol.
Chem. 267:16323-16329; Jahroudi et al., 1994, Mol. Cell.
~ Biol. 14:999-1008; Lee et al., l99o, J. Biol. Chem.
265:10446-10450), expression of these genes is either not
5 limited to vascular endothelium (e.g., the genes encoding
von Willebrand factor, endothelin-1, vascular cell
adhesion molecule-1), or is restricted to specific
subpopulations of endothelial cells (e.g., the gene for
endothelial-leukocyte adhesion molecule-1).
SummarY of the Invention
The invention features substantially pure DNA,
i.e., a promoter sequence, which regulates endothelial
cell-specific transcription of a polypeptide-encoding
sequence to which it is operably linked. The DNA of the
15 invention contains a sequence substantially identical to
nucleotides -225 to -164 of the KDR/flk-l promoter, i.e.,
5' TTGTTGCTCTGGGATGTTCTCTCCTGGGCGACTTGGGGCCCAGCGCAGTCCAGT
TGTGTGGG 3' (SEQ ID N0:1). By "substantially identical"
is meant at least 80% identical to a reference DNA
20 sequence, that is, up to 20% of the basepairs of the
reference DNA sequence can be replaced with an
alternative basepair (e.g., G-C replaced with A-T, T-A,
or C-G), provided that the transcription-promoting
activity of the altered sequence is the same or greater
25 than that of the reference sequence. The DNA may also
include a sequence substantially identical to nucleotides
-95 to -77 of the KDR/flk-1 promoter, i.e.,
5' GCTGGCCGCACGGGAGAGC 3' (SEQ ID NO:2), a sequence
substantially identical to nucleotides -95 to -60 of the
30 KDR/flk-l promoter, i.e.,
5' GCTGGCCGCACGGGAGAGCCCCTCCTCCGC
CCCGGC 3' (SEQ ID N0:3), a sequence substantially
identical to nucleotides +105 to +127 of the KDR/flk-1
promoter, i.e., 5' GGATATCCTCTCCTACCGGCAC 3' (SEQ ID
-
CA 0222~460 1997-12-22
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NO:4), or a combination thereof. Preferably, the 5' to
3' orientation of sequences is SEQ ID NO:l; SEQ ID NO:2
or SEQ ID NO:3; and SEQ ID NO:4. However, any
orientation of these sequences which promotes endothelial
5 cell-specific transcription is within the invention. The
DNA may include a nonspecific sequence between any two of
the defined sequences, and/or at either or both ends.
Preferably, this nonspecific (i.e., sequence other than
SEQ ID NO:1-4 will constitute no more than 80% of the
10 entire promoter sequence. Most preferably, it is
substantially identical to the sequence shown in Table 1
(SEQ ID N0:5) or Table 2 (SEQ ID NO:6).
A "substantially pure DNA," as used herein, refers
to a DNA which has been purified from the sequences which
15 flank it in a naturally occurring state, i.e., a DNA
fragment which has been removed from the sequences which
are normally adjacent to the fragment, e.g., the
se~l~nc~ adjacent to the fragment in the genome in which
it naturally occurs.
A substantially pure DNA containing a sequence
substantially identical to nucleotides -225 to +268 of
the KDR~flk-l promoter (SEQ ID N0:5; Table 1) or
nucleotides -225 to +127 of the KDR/flk-1 promoter (SEQ
ID NO:6; Table 2) and which regulates endothelial cell-
25 specific transcription of a polypeptide-encoding sequence
or antisense template to which it is operably linked is
also within the invention.
TAB~E 1: -225 to ~268
TTGTTGCTCTGGGATGTTCTCTCCTGGGCGACTTGGGGCCCAGCGCAGTCCAGTTGT
30 GTGGGGAAATGGGGAGATGTAAATGGGCTTGGGGAGCTGGAGATCCCCGCCGGGTAC
CCGGGTGAGGGGCGGGGCTGGCCGCACGGGAGAGCCCCTCCTCCGCCCCGGCCCCGC
CCCGCATGGCCCCGCCTCCGCGCTCTAGAGTTTCGGCTCCAGCTCCCACCCTGCACT
GAGTCCCGGGACCCCGGGAGAGCGGTCAGTGTGTGGTCGCTGCGTTTCCTCTGCCTG
CGCCGGGCATCACTTGCGCGCCGCAGAAAGTCCGTCTGGCAGCCTGGATATCCTCTC
35 CTACCGGCACCCGCAGACGCCCCTGCAGCCGCCGGTCGGCGCCCGGGCTCCCTAGCC
CTGTGCGCTCAACTGTCCTGCGCTGCGGGGTGCCGCGAGTTCCACCTCCGCGCCTCC
TTCTCTA~ACAGGCGCTGG~-A~AAA~-~ACCGGCTCCC (SEQ ID N0:5)
CA 0222~460 1997-12-22
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T ~ ~E 2: -22S to 1~27
TTGTTGCTCTGGGATGTTCTCTCCTGGGCGACTTGGGGCCCAGCGCAGTCCAGTTGT
GTGGGGAAATGGGGAGATGTAAATGGGCTTGGGGAGCTGGAGATCCCCGCCGGGTAC
CCGGGTGAGGGGCGGGGCTGGCCGCACGGGAGA~tCCCCTCCTCCGCCCCGGCCCCGC
5 CCCGCATGGCCCCGCCTCCGCGCTCTAGAGTTTCGGCTCCAGCTCCCACCCTGCACT
GAGTCCCGGGACCCCGGGAGAGCGGTCAGTGTGTGGTCGCTGCGTTTCCTCTGCCTG
CGCCGGGCATCACTTGCGCGCCG~ r-~ A AGTCCGTCTGGCAGCCTGGATATCCTCTC
CTACCGGCAC (SEQ ID NO:6)
The DNA of the invention may be operably linked
10 to, and functions to regulate endothelial cell-specific
transcription of, a sequence encoding a polypeptide that
is not KDR/flk-l. Examples of such polypeptides include
tiss~e plasminogen activator (tPA), p21 cell cycle
inhibitor, and nitric oxide synthase. By "operably
15 lil~ed" is meant able to promote transcription of an mRNA
corresponding to a polypeptide-encoding or antisense
template located downstream on the same DNA strand.
The invention also includes a vector cont~; n; ng
the DNA of the invention, a method of directing
20 endothelial cell-specific expression of a polypeptide by
introducing the vector into an endothelial cell, and an
endothelial cell containing the vector.
The vector of the invention can be used for gene
therapy, such as a method of inhibiting arteriosclerosis
25 in an ~n;r~l involving contacting an artery of the animal
with the vector of the invention which directs the
production of a polypeptide capable of reducing or
preventing the development of arteriosclerosis, e.g., a
polypeptide which reduces proliferation of smooth muscle
30 cells, e.g., interferon-y or atrial natriuretic
polypeptide.
The invention also includes compositions and
methods of carrying out antisense therapy. For example,
the invention includes a substantially pure DNA with a
35 sequence substantially identical to SEQ ID NO:l which
regulates endothelial cell-specific transcription of an
antisense template to which it is operably linked, e.g.,
CA 0222~460 1997-12-22
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an antisense template the transcription product of which
prevents translation of mRNA into an endothelial cell
polypeptide. By the term "antisense template" is meant a
DNA which is transcribed into an RNA which hybridizes to
5 mRNA. Preferably, the endothelial cell polypeptide is
KDR/flk-1. For example, the antisense RNA transcript
which binds to and thereby prevents or reduces
translation of an mRNA encoding KDR/flk-1, a protein
involved in angiogenesis, can be used to treat cancer by
10 contacting a tumor site in an animal with the DNA of the
invention to reduce or prevent angiogenesis at the tumor
site.
Translation of other endothelial cell polypeptides
may also be reduced or prevented in this manner. For
15 example, translation of cell cycle proteins, coagulation
factors, e.g., von Willebrand factor, and endothelial
cell adhesion factors, e.g., intercellular adhesion
molecule-1 (ICAM-l) or vascular cell adhesion molecule-1
~VCAM-1) may be re~llce~ or prevented.
The invention also features a method of measuring
the ability of a candidate compound to modulate TNF-~
downregulation VEGF receptor (e.g., KDR/flk-1 or fltl)
gene expression. In this method, a cell cont~; n; ng the
promoter of a VEGF receptor gene operably linked to a
25 reporter gene is cultured in the presence of TNF-~ and
the candidate compound. The level of expression of the
reporter gene is then determined as a measure of the
ability of the candidate compound to modulate TNF-~
downregulation of VEGF receptor gene expression.
Another method included in the invention involves
measuring the ability of a candidate compound to modulate
TNF-~ inhibition of VEGF-induced endothelial cell
proliferation. In this method, an endothelial cell is
cultured in the presence of TNF-~, VEGF, and the
35 candidate compound. The level of endothelial cell growth
CA 0222~460 1997-12-22
W O 97/00957 PCTAUS96/10725
is determined (e.g., by measurement of uptake Of [methyl-
[3H]thymidine) as a measure of the ability of the
candidate compound to modulate TNF-~ inhibition of VEGF-
induced endothelial cell proliferation.
The invention also features a method of inhibiting
angiogenesis in a patient involving a~; n; ~tering to the
patient a non-TNF-~ compound which activates the TNF-~
pathway of downregulating VEGF receptor (e.g., KDR/flk-1
or fltl) gene expression in an endothelial cell.
An additional method of the invention for
inhibiting angiogenesis in a patient involves
a~;n;stering to the patient a polypeptide which inhibits
VEGF receptor (e.g., KDR/flk-l or fltl) gene expression
in an endothelial cell by binding to the TNF-~-responsive
15 element in the promoter of the VEGF receptor gene.
The invention also features a method of enhancing
angiogenesis in a patient involving A~; n; ~tering to the
patient a non-TNF-~ compound which inhibits the TNF-
~pa~hway of downregulating VEGF receptor (e.g., KDR/flk-1
20 or fltl) gene in an endothelial cell.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, from the drawings, and
from the claims.
Detailed DescriPtion
The drawings are first described.
Drawinqs
Fig. lA is a diagram of the human KDR/flk-1
promoter. Restriction enzyme sites are indicated above
30 the nucleotide sequence, and nucleotide sequences -780 to
+487 (SEQ ID NO:7) are numbered on the left of the
nucleotide sequence. The transcription start site is
indicated by a curved arrow. Potential cis-acting
elements are underlined. The PstI sites which were used
CA 0222~460 1997-12-22
W O 97/00957 PCT~US96/10725
to generate the riboprobe are double underlined, and the
sequence corresponding to the oligonucleotide which was
used for primer extension is underlined with an arrow.
Fig. lB is a diagram of the murine KDR/flk-1
5 promoter. Restriction enzyme sites are indicated above
the nucleotide sequence. Nucleotide sequences -295 to
+205 (SEQ ID NO:8) are numbered and potential cis-acting
elements are indicated as in Fig. lA. An asterisk
indicates the 5' end of the cDNA.
Fig. 2A is a photograph of an electrophoretic gel
showing the results of a primer extension analysis of the
KDR/flk-1 transcription start site. The oligonucleotide
underlined with an arrow in Fig. lA was hybridized to 20
~g of total RNA from human umbilical vein endothelial
15 cells (H W EC) and HeLa cells or 3 ~g of polyA+ H W EC RNA
and yeast tRNA. Extension products were analyzed on an
8% polyacrylamide gel (lanes 1-4: Yeast tRNA; HeLa total
RNA; HUVEC total RNA; and HUVEC polyA+ RNA). A Sanger
sequencing reaction primed on a plasmid DNA template
(with the same oligonucleotide primer) was run next to
the primer extension analyses (lanes 5-8: G; A; T; C).
Fig. 2B is a diagram showing the strategy for
mapping the transcription start site of the KDR/flk-1
gene by ribonuclease protection.
Fig. 2C is a photograph of an electrophoretic gel
showing a ribonuclease protection analysis of the
KDR/flk-l transcription start site. Total RNA from H W EC
and HeLa cells or polyA+ H W EC RNA and yeast tRNA were
incubated with a 559-bp 32P-labeled riboprobe spAnn;ng the
30 ; ~;ate 5' region of the human KDR/flk-1 gene. The
~n~e~l ing products were digested with RNase. Protected
fragments were analyzed on a 4% polyacrylamide gel. The
size markers (bp) were prepared by radiolabeling ~X174 RF
DNA digested with HaeIII. Fig. 3A is a diagram
35 showing the location of 5' deletion sites in the
CA 0222~460 1997-12-22
W O 97/00957 PCT~US96/10725
_ g
KDR/flk-1 promoter. Location of deletion sites is shown
in relation to consensus sequences for known nuclear
proteins.
Fig. 3B is a bar graph showing the results of a
5 ~unctional analysis of the human KDR/fl~-l promoter by
transfection of luciferase reporter constructs cont~ining
serial 5' deletions into bovine aortic endothelial cells
(BAEC). All constructs were cotransfected with pSV~gal
to correct for transfection efficiency, and luciferase
10 activity was expressed as a percentage of pGL2 Control
(mean + SEM). Fig. 4A is a diagram showing the
location of 3' deletion sites in the KDR/flk-1 promoter.
Location of deletion sites is shown in relation to
consensus sequences for known nuclear proteins.
Fig. 4B is a bar graph showing the results of a
functional analysis of 3' deletions on KDR/flk-1 promoter
activity in BAEC. Luciferase activity is represented as
a percentage of pGL2 control.
Fig. 5 is a bar graph showing the effect of a GATA
20 site mutation on KDR/flk-l promoter activity. Mutation
of the GATA site at position +107 does not decrease the
ability of the KDR/flk-l promoter to direct
transcription. When transfected into BAEC, the plasmid
pGL2-225+268 directed luciferase expression cs _=~able
25 to that directed by pGL2 Control, which contains the SV40
promoter and enhancer. When three bp of the GATA motif
at +107 were mutated to create pGL2 GATA-MUT, there was
no significant difference in promoter activity.
Fig. 6A is a photograph of a Northern blot
30 analysis showing that KDR/flk-l RNA expression is
restricted to endothelial cells in culture. RNA was
extracted from cells in culture and analyzed by Northern
blotting using a human KDR/flk-l cDNA probe. The
following cell types were tested: HUVEC (human umbilical
35 vein endothelial cells), HASMC (human aortic smooth
-
CA 0222~460 1997-12-22
W O 97/00957 PCTAJS96110725
-- 10 --
muscle cells), HISMC (human intestinal smooth muscle
cells), fibroblasts (human cultured fibroblasts), RD
(human embryonal rhabdomyosarcoma cells) HeLa (human
epidermoid carcinoma cells), HepG2 (human hepatoma
5 cells), MCF7 (human breast adenocarcinoma cells), and
U937 (human histiocytic lymphoma cells).
Fig. 6B is a photograph of the same agarose gel
shown in Fig. 6A which was stA; ne~ with ethidium bromide
(to visualize ribosomal RNA) to show the amount of RNA
lO loaded in each lane.
, Fig. 7 is a bar graph showing the results of a
luciferase assay. High-level activity of the KDR/flk-1
promoter was found to be specific to endothelial cells.
The luciferase reporter construct pGL2-4kbf296 was
15 transfected into cells in culture, and transfection
efficiency was assessed by monitoring cotransfection with
pSV~gal. Results are corrected for transfection
efficiency and expressed as a percentage of pGL2 Control
activity for each cell type. The following cell types
20 were tested: BAEC, bovine aortic endothelial cells;
JEG-3, human choriocarcinoma cells; Saos-2, human
osteosarcoma cells; A7r5, rat fetal smooth muscle cells;
3T3, mouse fibroblasts; and HeLa, human epidermoid
carcinoma cells.
Fig. 8 is a bar graph showing the effect of TNF--~
on VEGF-induced proliferation of HUVEC, as measured by
uptake of methyl - [ 3H]thymidine.
Fig. 9A is a photograph of a Northern blot
analysis of a time course of TNF-a-induced downregulation
30 of KDR/flk-l mRNA expression in H WEC. From left to
right, the time points are: O, 1, 2, 3, 6, 12, and 24
hours, as indicated in Fig. 9B.
Fig. 9B is a bar graph of the results of the time
course of TNF-~-induced downregulation of KDR/flk-1 mRNA
35 expression shown in the photograph of Fig. 9A.
CA 0222~460 1997-12-22
W O 97/00957 PCT~US96/10725
Fig. lOA is a photograph o~ a Northern blot
analysis of a dose-response experiment of TNF-~-induced
downregulation of KDR/flk-1 mRNA expression in HUVEC.
From le~t to right, 1. o.1, 1, 1o, 50, and loo ng/ml TNF-
~ 5 ~ were used, as indicated in Fig. lOB.
Fig. lOB is a bar graph of the results of thedose-response experiment of TNF-~-induced downregulation
of KDR/flk-l mRNA expression in H W EC shown in the
photograph of Fig. lOA.
Fig. 11 is a graph showing the effect of
Actinomycin D (ACD) on the levels of }~DR/flk-1 RNA in
HUVEC.
Fig. 12. is a photograph of immunoprecipitation
analysis of KDR/flk-l protein in HUVEC treated with TNF-
~
15 for o, 12, and 24 hours.
Tsolation and Characterization of KDR/flk-1 Genomic
Clones
8creening of ~um~n an~ Mouse Genomic hibraries
A 567-bp human KDR/flk-1 cDNA fragment was
20 generated from HW EC total RNA by reverse-transcriptase
polymerase chain reaction (RT-PCR). This fragment was
radiolabeled with t~-32P]dCTP and used to screen a phage
library of human placenta genomic DNA in the vector
lFixII (Stratagene, La Jolla, CA). Likewise, a 451-bp
25 mouse KDR/flk-1 cDNA was generated by RT-PCR from mouse
lung total RNA and used to screen a phage library of
mouse placenta genomic DNA in the vector ADashII
(Stratagene). Hybridizing clones were isolated and
purified from each library, and phage DNA was prepared
30 according to standard procedures.
Cell Culture and mR~A Isolation
BAEC were isolated and cultured in Dulbecco's
modified Eagle's medium (JRH Biosciences, Lenexa, KS)
supplemented with 10% fetal calf serum (HyClone, Logan,
CA 0222~460 1997-12-22
W O 97/00957 - PCT~US96/10725
UT), 600 ~g of glutamine/ml, 100 units of penicillin/ml,
and 100 ~g of streptomycin/ml. Cells were passaged every
3-5 days and cells from passages 4-8 were used for
transfection experiments. Saos-2 human osteosarcoma
5 cells (ATCC HTB-85), HeLa human epidermoid carcinoma
cells (ATCC CRL-7923), HepG2 human hepatoma cells (ATCC
HB-8065), human fibroblasts (ATCC CRL-1634), U937 human
histiocytic lymphoma cells (ATCC CRL-7939), RD human
embryonal rhabdomyosarcoma cells (ATCC CCL-136), MCF7
10 human breast adenocarcinoma cells (ATCC HTB-22), JEG-3
human choriocarcinoma cells (ATCC HTB-36), A7r5 fetal rat
aortic smooth muscle cells (ATCC CRL-1444), and NIH 3T3
mouse fibroblasts (ATCC CRL-1658) were obtained from the
American Type Culture Collection. Primary-culture HUVEC
15 were obtained from Clonetics Corp. (San Diego, CA) and
were grown in EGM medium contAin;ng 2% fetal calf serum
(Clonetics). Primary-culture human aortic and intestinal
smooth muscle cells were also obtA;ne~ from Clonetics
Corp. All cells were cultured in conditions identical to
20 those for BAEC, with the exception that medium used for
smooth muscle cells was supplemented with 25 mM HEPES
(Sigma, St. Louis, M0) and that HUVEC were cultured in
EGN medium containing 2~ fetal calf serum.
Primary-culture cells were passaged every 4-6 days, and
25 cells from passages 3-5 were analyzed. Total RNA from
cells in culture was prepared by guanidinium
isothiocyanate extraction and centrifugation through
cesium chloride.
DNA 8eguencing
Restriction fragments derived from the human and
mouse KDR/flk-1 genomic phage clones were subcloned using
s~n~l~rd techn;ques into pSP72 (PL~J ?ga, Madison, WI) or
pBluescript II SK (Stratagene) and sequenced from
alkaline- denatured double-stranded plasmid templates by
35 the dideoxy chain termination method with SEQUENASE~ 2.0
CA 02225460 1997-12-22
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DNA polymerase (United States Biochemical, Cleveland,
OH). DNA was sequenced from both directions at least
twice, and both dGTP and dITP sequencing protocols were
used to resolve compression artifacts in the highly
5 GC-rich 5' flanking region of the human and mouse
KDR/flk-1 genes. Sequence analysis was performed with
the GCG software package (Genetics Computer Group,
Madison, WI).
Primer Exte~sion Analyqis
Primer extension analysis was performed according
to known methods, e.g., the method of Fen et al., 1993,
Biochemistry 32:7932-7938. A synthetic oligonucleotide
primer (5' CTGTCTA~-~-A~GGAGGCGCGGAGGTGGAACT 3'; SEQ ID
NO:9) complementary to the 5' end of the human KDR/~lk-1
15 cDNA (Fig. lA) was end-labeled with [y-32P]ATP and
hybridized to 20 ~g of each RNA sample, which was then
subjected to reverse transcription. Extension products
wexe analyzed by electrophoresis on an 8~ denaturing
polyacrylamide gel.
20 ~hon~cle~se Protection Ass~y
A 559-bp PstI-PstI fragment of the human KDR/flk-1
gene (Fig. 2B) was cloned in pSP72 as the template for in
vitro transcription of an ~-32P-labeled antisense RNA with
T7 RNA polymerase (Boehringer Mannheim, Indianapolis,
25 IN). Gel-purified riboprobe (5 x 105 cpm) was hybridized
with 20 ~g of total RNA or 3 ~g of polyA RNA plus 17 ~g
of yeast tRNA at 55~C for 16 hours in an annealing buffer
cont~;ning 20 mM Tris-HCl, pH 7.40, 400 mM NaCl, 1 mM
EDTA, and 0.1% sodium dodecyl sulfate in 75~ formamide.
30 After the RNA had been annealed, the unhybridized RNA was
digested for 45 minutes at room temperature with 200 U
RNAse Tl (Boehringer Mannheim) and 0.3 U RNAse A
(Boehringer Mannheim) in a buffer cont~;~; ng 10 mM
Tris-HCl, pH 7.50, 300 mM NaCl, 5 mM EDTA. The digestion
35 products were then treated with proteinase K, extracted
CA 0222~460 1997-12-22
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with phenol:chloroform, and analyzed by electrophoresis
on a 4% denaturing polyacrylamide gel.
Northcrn Analysis
Total RNA (10 ~g) from cells in culture was
5 fractionated on a 1.3% formaldehyde-agarose gel and
transferred to a nitrocellulose filter. The human
KDR/flk-l cDNA probe was labeled with 32p by random
priming, the labeled probe was then used to hybridize the
filter. The filter was then autoradiographed for 16
10 hours on Kodak XAR film at -80~C.
Plasmids
Plasmids pGL2 Basic and pGL2 Control contained the
firefly luciferase gene (Promega). pGL2 Basic had no
promoter, whereas pGL2 Control was driven by the SV40
15 promoter and enhancer. The plasmid pSV~GAL (Promega)
contained the ~-galactosidase gene driven by the SV40
promoter and ~nh~nC~.
Reporter constructs contA;n;ng fragments of the
human KDR/flk-l 5' flanking region were inserted into
20 pGL2 Basic and named according to the length of the
fragment (from the transcription start site) in the 5'
and 3' directions. For example, plasmid pGL2-4kb+296
contained a human KDR/flk-1 promoter fragment exten~ing
from approximately -4 kb 5' of the transcription start
25 site to position +296 inserted into pGL2 Basic. Plasmids
pGL2-4kb+296 and pGL2-900+296 were created by restriction
digestion of purified phage DNA by using 5' BamHI and
PvuII sites, respectively, and the 3' XhoI site at
position +296. Plasmids pGL2-716+268, pGL2-570+268,
30 pGL2-323+268, pGL2-225+268, pGL2-164+268, pGL2-37+268,
pGL2-225+127, pGL2-225+105, pGL2-225+56, and pGL2-225+5
were created from promoter fragments generated by PCR of
human KDR/flk-l phage DNA. Plasmids pGL2-116+268, pGL2-
95+268, pGL2-77+268, pGL2-60+268, and pGL2-12+268 were
35 created by digesting the promoter fragment contained in
CA 0222~460 1997-12-22
W O 97/00957 PCT~US96/1072
- 15 -
plasmid pGL2-164+268 from the 5' end with exonuclease III
(Pharmacia Biotech, Piscataway, NJ). Plasmid pGL2
GATA-MUT was identical to pGL2-225+268 except that bp
+108 to +110 were mutated in the plasmid pGL2 GATA-MUT.
; 5 All constructs were sequenced from the 5' and 3' ends to
confirm orientation and sequence.
Mu~agene3is
Site-directed mutagenesis of the atypical GATA
sequence located in the first exon of the human KDR/flk-1
10 5' flAnk;ng region was performed by PCR using to the
method of Higushi et al., 1988, Nucleic Acids Res.
1607351-7367. A DNA fragment cont~;n;ng human KDR/flk-1
bp -225 to +268 was used as a template. The sequence
TGGATATC was mutated to TGGTCGTC by using one set of
15 mismatched primers, 5' TCTGGCAGCCTGGTCGTCCTCTCCTA 3' (SEQ
ID NO:10) and 5'TAGGAGAGGACGACCAGGCTGCCAGA 3' (SEQ ID
NOo 11) t and one set of primers fl~nki~g both ends of the
template, 5' TGCCTCGA~ll~llGCTCTGGGATGTT 3' (SEQ ID
NO- 12) and 5' TGTAAGCTTGGGAGCCGGTTCTTTCTC 3'(SEQ ID
20 NO 13 ) ~ The sequence of the mutated PCR fragment was
confirmed by the dideoxy chain termination method.
Tr~nsfeotions
All cell types were transfected by the calcium
phosphate method known in the art with the exception of
25 A7r5 cells, which were transfected with DOTAP (Boehringer
MAnnheim) as instructed by the manufacturer. In all
cases, 20 ~g of the appropriate reporter construct was
transfected along with 2.5 ~g of pSV~gal to correct for
variability in transfection efficiency. Cell extracts
30 were prepared 48 hours after transfection by a detergent
lysis method (Promega). Luciferase activity was measured
in duplicate for all samples with an EG&G Autolumat 953
luminometer (Gaithersberg, MD) and the Promega Luciferase
Assay system. ~-Galactosidase activity was assayed using
CA 0222~460 1997-12-22
W O 97/00957 PCTAUS96/10725
known methods, e.g., Lee et al., 1990, J. Biol. Chem.
265:10446-10450.
The ratio of luciferase activity to
~-galactosidase activity in each sample served as a
5 measure of the normalized luciferase activity. The
normalized luciferase activity was divided by the
activity of pGL2 Control and expressed as relative
luciferase activity. Each construct was transfected at
least six times, and data for each construct are
10 presented as the mean + SEM. Relative luciferase
activity among constructs was compared by a factorial
analysis of variance followed by Fisher's least
significant difference test. Statistical significance
was accepted at p<0.05.
15 Isol~tion and Char~cterization of ~uman ~nd Murine
XDR/flk-1 Genomic Clones
Initial screening of a human placental phage
library with a human KDR/flk-1 cDNA probe yielded a
positive clone that was examined by restriction enzyme
20 DNA mapping, subcloning, and sequencing. The 780-bp
sequence of the promoter and first exon is shown in Fig.
lA. Likewise, a murine KDR/flk-1 cDNA probe was used to
screen a murine placental phage library, and one clone
was identified and characterized. The sequence of the
25 mouse KDR/flk-1 promoter is shown in Fig. lB.
Identific~tion of the Tran~cription ~tart Site of ~um~n
RDR/fl~-1
To identify the transcription start site of the
human KDR/flk-l gene, primer extension was performed with
30 a complementary oligonucleotide probe corresponding to bp
+212 to +243 (underlined with arrow, Fig. lA). Primer
extension was performed on total RNA from HWEC and HeLa
cells and on polyA RNA from HWEC. Gene transcription
was found to be initiated only in endothelial cells (Fig.
35 2A). A single transcription start site, corresponding to
CA 0222~460 1997-12-22
W O 97/00957 PCT~US96/10725
~ 17 ~
a nucleotide located 303 bp 5' of the site of translation
initiation, i.e., the methionine initiation codon, was
~ identified. This nucleotide was designated +1. The
transcription start site is highlighted in bold in the
5 sequence, CCCTGCACTGA (SEQ ID N0:14) (see Figs. lA and
2A). The 5'CA3' nucleotide pair at this position is the
most common site for transcription initiation.
To confirm the results of the primer extension
studies, a ribonuclease protection analysis was performed
10 using an antisense riboprobe generated from a 559-bp
genomic PstI-PstI fragment ext~n~; ng 5' from position
+146 (Fig. 2B; the PstI sites are double underlined in
Fig. lA). Incubation of this probe with HUVEC polyA RNA
and HUVEC total RNA, but not with total RNA from HeLa
15 cells, resulted in protection of a single fragment
corresponding in length to the distance between the 3'
PstI site and the transcription start site identified by
primer extension (Fig. 2C). Despite the absence of a
TATA consensus sequence, transcription of the human
20 KDR/flk-1 gene was found to begin from a single site
located 303 bp 5' of the translation initiation codon
(Fig. lA, curved arrow).
Identification of Cis-Acting 8e~n~Q
The 5' flanking sequence of the human KDR/flk-1
25 gene contains regions rich in G and C residues and lacks
TATA and CCAAT boxes near the transcription start site
(Fig. lA). Comparison of this 5' flAnking sequence with
sequences in the Transcription Factors Database revealed
a series of five Spl sites located between human
30 KDR/flk-1 nucleotides -124 and -39. There are two AP-2
consensus sites at positions -95 and -68 and two inverted
NF~B binding elements at -130 and -83 interspersed among
the Spl sites. Two atypical GATA consensus sequences
(both GGATAT) are present in the KDR/flk-1 promoter, one
35 at position -759 and the other at position +107 within
CA 0222~460 1997-12-22
W O 97/00957 PCTAJS96/10725
-- 18 --
the untranslated portion of the first exon. In addition,
multiple CANNTG elements are located in the promoter at
positions -591, -175, +71, and +184; CANNTG elements can
be bound by E-box binding proteins. The sequence
5 AAACCAAA, which is conserved among genes expressed
preferentially in keratinocytes, is present at human
KDR/flk-1 position -508.
The human and mouse KDR/flk-l promoters were
cnmE-~red to identify conserved consensus sequences for
10 nuclear proteins (Fig. lB). Elements conserved between
the two species include two Spl sites located at
positions -244 and -124 relative to the 5' end of the
reported mouse cDNA sequence, two AP-2 sites at positions
-168 and -148, a noninverted NFIcB site at position -153,
15 and the keratinocyte element AAACCAAA at position -195.
An atypical GATA element (GGATAA) is located in the
untranslated portion of the first exon of the mouse
promoter at position +18; an atypical GATA element
(GGATAT) is located similarly in the human promoter.
20 Also, a CANNTG sequence is present 12 bp 5' of the G- and
C--rich sequences of the promoter at mouse KDR/flk-1
position -257, a location analogous to that of the CANNTG
element at position -175 of the human promoter.
Conservation of these elements across species suggests
25 that these regulatory elements have functional
significance.
Deletion Analy~is of the Human RDR/flk--l Promoter
To identify DNA elements important for basal
expression of KDR/flk-1 in endothelial cells, a series of
30 luciferase reporter plasmids cont~;n;ng serial 5'
deletions through the promoter region was constructed
(Figs. 3A and 3B). These plasmid constructs in pGL2
Basic were cotransfected into BAEC with pSV~gal (to
correct for differences in transfection efficiency) and
35 the luciferase activity was normalized to that of the
CA 0222~460 1997-12-22
WO 97/009S7 PCTrUS96/10725
-- 19 --
pGL2 Control vector driven by the SV40 promoter/enhancer.
The activity of the longest human KDR/flk-1 genomic
- fragment, sp~nn; ng bp -4kb to +296, was similar to that
of the powerful SV40 promoter/enhancer and consistent
- 5 with the high level of KDR/flk-1 mRNA expression in
endothelial cells. Similar levels o~ activity were
produced in constructs cont~;n;ng as much as 15.5 kb of
5' flanking sequence. Serial 5' deletions from bp -4kb
to -225 caused no significant change in promoter
10 activity, implying that elements in this region are not
important for basal activity of the KDR/flk-1 promoter.
Deletion of sequences between bp -225 and -164
significantly reduced KDR/flk-1 promoter activity to 63%
of the activity of the full promoter fragment (p<0.05).
15 These data suggest the presence of positive regulatory
elements in this region. Deletion of bp from -95 to -77,
a sequence that contains one AP-2 site and one NF~B site,
resulted in a further significant decrease in activity to
20% that of pGL2-4kb+296 (p<0.05). Further deletion of
20 bp from -77 to -60, an area cont~;n;ng an overlapping
AP 2/Spl site, significantly reduced ICDR/flk-1 promoter
activity to less than 5% that of pGL2~4kb+296 (p<0.05).
Thus, 5' deletion analysis revealed that many positive
regulatory elements in the KDR/flk-l promoter are
25 necessary for high-level expression of the gene.
The deletion analyses described above indicate
that three sequences within the 5' flanking region of the
KDR/flk-l gene contain elements important for expression
in endothelial cells. Potential binding sites for Spl,
30 AP-2, NF~B, and E-box proteins located within these three
positive regulatory elements in the human KDR/flk-1 gene
are also present in the mouse 5' flanking sequence, thus
suggesting that they are functional binding domains.
AP-2 is a developmentally regulated trans-acting factor
(Mitchell et al., 1991, Genes & Dev. 5:105-119) without a
CA 0222~460 1997-12-22
W O 97/00957 PCTrUS96/10725
- 20 -
demonstrated role in endothelial cell gene regulation.
NFKB is thought to trans-activate the inducible
expression of vascular cell adhesion molecule-1 and
tissue factor in endothelial cells (Iademarco, 1992, J.
5 Biol. Chem. 267:16323-16329; Moll et al., 1995, J. Biol.
Chem. 270:3849-3857) and is known to be a mediator of
tissue-specific gene regulation (Lenardo et al., 1989,
Cell 58:227-229). Nuclear proteins that bind the E-box
motif include the basic helix-loop-helix family of
10 trans-acting factors. E-box binding proteins have not
been clearly associated with endothelial cell gene
expression, although m~h~rs of this family are critical
for proper maturation of many cell types, including
skeletal muscle and B lymphocytes (Buskin et al., 1989,
15 Mol. Cell. Bio. 9:2627-2640; Murre et al., 1989, Cell
58:537-544)-
To determine whether sequences in the first exonof human KDR/flk-l are important for basal expression, a
series of 3' deletion constructs from the vector pGL2-
20 225+268, which is the smallest construct that possessedfull promoter activity, was made (Figs. 4A and 4B).
Deletion of a fragment sp~nning bp +105 to +127 (SEQ ID
NO:4) caused a five-fold reduction in promoter activity
(p<0.05), indicating the pr~C~nce of a positive
25 regulatory element in this region.
The functional importance of the atypical GATA
site located between bp +105 and +127 of human KDR/flk-1
was also examined. Three bp of the GATA motif in the
fragment -225 to +268 were mutated to GTCG by PCR to
30 create the mutant, pGL2 GATA-MUT. Mutation of these bp
in the GATA motif eliminates GATA-2 binding activity in
the endothelin-1 gene promoter. In contrast, there was
no significant decrease in promoter activity in BAEC with
the pGL2 GATA-MUT construct containing the mutated
CA 0222~460 1997-12-22
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- 21 -
atypical GATA sequence compared to the native pGL2-
225+268 promoter construct, (p>0.05; Fig. 5).
Four zinc finger-containing transcription factors
in the GATA protein family bind to the consensus sequence
(A/T)GATA(A/G) and regulate cell type-specific gene
expression in many cell lineages (Orkin, 1992, Blood
80:575-581); among these, GATA-2 has been most closely
linked to endothelial cell gene expression. GATA-2
functions as an enhancer of endothelin-l gene expression
10 and acts to restrict expression of von Willebrand factor
to endothelial cells. Human KDR/flk-1 5' fl~nk;ng region
was found to have two potential GATA-binding se~lence~,
at positions -759 and +107. Loss of the element located
at position -759 had no effect on expression of KDR/flk-1
lS in endothelial cells. The potential GATA element at
position +107 is located in a region of the first exon
which has now been identified as a powerful positive
regulatory element (SEQ ID NO:4). Although this GATA
sequence (GGATAT) differs from the GATA-binding sequences
20 of endothelin-l and von Willebrand factor and from the
consensus GATA sequence (A/T)GATA(A/G), the data suggests
that it is the functional motif in the region between
+105 and +127 because the functional GATA site in the von
Willebrand factor gene is located similarly in the first
25 exon, and because a similar GATA element is found in the
first exon of the mouse KDR/flk-1 gene. Mutation of
three bp in this element (GATA to GTCG), which had been
observed to prevent trans-activation of the GATA
cis-acting element in the endothelin-1 promoter, was
30 found to have no significant effect on KDR/flk-1 promoter
activity (Fig. 5). Thus, the deletion analyses and
mutagenesis studies do not support a functional role for
the two GATA sequences in the human promoter in its
high-level activity in endothelial cells. These
35 observations suggest that transcription factors other
CA 0222~460 1997-12-22
O 97/00957 PCTAUS96/10725
- 22 -
than GATA proteins are necessary for expression of the
human KDR/flk-1 gene.
High-Level Expre Qion Induced by the RDR/flk-1 Promoter
Is ~pecific to En~othelial Cells
Although KDR/flk-l expression is restricted to
endothelial cells in ~ivo, it does not necessarily follow
that its expression would be limited to endothelial cells
in culture. To determine whether a tissue culture system
is suitable for studying cell-type specific regulation of
the KDR/flk-l gene, Northern analysis of RNA extracted
from various cells in culture was performed. KDR/flk-1
message was detected in HUVEC but not in primary-culture
cells (human aortic and intestinal smooth muscle cells
and fibroblasts) or human cell lines (RD, HeLa, HepG2,
MCF7, and U937) (see Figs. 6A and 6B). Similarly,
KDR/flk-l message was not detected by RT-PCR in HeLa,
A7r5, or 3T3 cells. Thus, expression of KDR/flk-1
message in tissue culture appears to be restricted to
endothelial cells, as it is in vivo.
To determine whether 5' flanking sequences of the
KDR/flk-l gene can confer endothelial cell-specific
expression in cultured cells, pGL2-4kb+296, which
contains over 4 kb of the human KDR/flk-1 5' fl~nk;ng
sequence and includes most of the untranslated portion of
the first exon, was transfected into a variety of cell
types in culture (Fig. 7). Reporter gene expression
driven by the pGL2-4kb+296 promoter fragment was similar
to that driven by the potent SV40 promoter/enhancer. In
JEG-3, Saos-2, A7r5, 3T3, and HeLa cells, however,
expression driven by the pGL2-4k~+296 promoter was
markedly lower, demonstrating that induction of
high-level expression by these promoter sequences is
specific to endothelial cells. A similar expression
pattern was observed using a reporter plasmid containing
15.5 kb of KDR/flk-1 5' flanking sequence.
CA 0222~460 1997-12-22
O 97/00957 PCTAUS96/10725
- 23 -
These data indicate that the activity of the
KDR/flk-1 promoter in endothelial cells is similar to
that of the potent SV40 promoter/enhancer and that this
high-level activity is specific to endothelial cells;
activity in other cell types is markedly ~iri ~i ~hed.
Low, but detectable, promoter activity was observed in
transient transfection assays of cell types that do not
express the KDR/flk-1 gene in vivo; it is possible that
other silencer elements outside of the 15.5 kb 5'
fl~nk; ~g region are nececs~ry to block promoter activity
completely in non-endothelial cells. Alternatively, the
context of the promoter in relation to normal chromatin
structure may be essential for precise regulation of the
gene. The results described above suggest that
tissue-specific regulation of KDR/flk-l involves a
complex interaction between known, widely distributed
nuclear factors and other, undefined elements.
TNF-~ Downrequlates KDR/flk-1 and fltl ExPression
CQll Culture and mRNA Isolation
Primary-culture HUVEC and HAEC were obtained from
Clonetics Corp. (San Diego, CA) and were grown in M199
medium supplemented with 20% fetal calf serum (Hyclone,
Logan, UT), 30 mg endothelial cell growth substance
(ECGS, Collaborative Biomedical, Bedford, MA), 25 mg
heparin, 600 ~g of glutamine/ml, 100 units of
penicillin/ml, and 100 ~g of streptomycin/ml, in gelatin-
coated tissue culture plates. Bovine aortic endothelial
cells (BAEC) were isolated and cultured in Dlllh~co~s
modified Eagle~s medium (JRH Biosciences, Lenexa, RS)
supplemented with 10~ fetal calf serum. Primary-culture
cells were passaged every 4-6 days and experiments were
performed on cells three to six passages from primary
culture. After the cells had grown to confluence, they
were placed in serum-deprived medium (Ml99 medium
CA 0222~460 l997-l2-22
WO 97/00957 PCT/US96/10725
-- 24 --
supplemented with 5% fetal calf serum without ECGS).
Recombinant human TNF-~ (Genzyme, Cambridge, MA) was
aliquoted and stored at -80~C until use. Total RNA from
cells in culture was prepared by guanidinium
5 isothiocyanate extraction and centrifugation through
cesium chloride (Sambrook et al., 1989, Mol~c~llAr
Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New
York).
10 Northern Analysis
RNA blots were hybridized as described (Li et al.,
1995, J. Biol. Chem. 270:308--312). Total RNA (10 ~g)
from cells in culture was fractionated on a 1. 396
formaldehyde-agarose gel and transferred to
15 nitrocellulose filters. cDNA probes were labeled with 32p
by random priming and used to hybridize to the filters.
Filters were then washed and subject to autoradiography
for 4-8 hours on Kodak XAR film at -80~C. Filters were
stripped of radioactive probe in a 50% formamide solution
20 at 80~C and rehybridized with an end-labeled 18S
ribosomal RNA oligonucleotide to correct for loading.
Filters were scanned and radioactivity was measured on a
PhosphorImager running the ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). To correct for differences in
25 RNA loading, the signal intensity for each RNA sample
hybridized to the cDNA probes was divided by that for
each sample hybridized to the 18S ribosomal RNA probe.
Plasmids
A 567-bp human KDR/flk-1 cDNA fragment was
30 generated from human umbilical vein endothelial cell
(HWEC) total RNA by the reverse-transcriptase polymerase
chain reaction (RT-PCR) (Sambrook et al., 1989 , Molecular
Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New
35 York), as previously described (Patterson et al., 1995,
CA 0222~460 1997-12-22
W O 97/00957 PCTAUS96/10725
J. Biol. Chem. 270:2311-23118). The human fltl cDNA
clone was generously provided by Dr. Timothy Quinn
~ (University of California, San Francisco).
Nuclear Run-on Analysis
Confluent HUVEC were treated with either vehicle
(control) or TNF-~ (1 ng/ml) for 18 hours. The cells
were subsequently lysed, and the nuclei were isolated, as
described in Perrella et al. (Perrella et al., 1994, J.
Biol. Chem. 269:14595-14600). Nuclear suspension (200
10 ~1) was ;ncllh~ted with 0.5 mM each of CTP, ATP, and GTP,
and with 20 ~Ci of 32P-labeled UTP (3000 Ci/mmol,
DuPont/NEN, Boston, MA). The samples were extracted with
phenol/chloroform, precipitated, and resuspended at equal
counts/minute/ml in hybridization buffer (15 x 106
15 counts/minute/ml). Denatured probes (1 ~g) dot-blotted
onto nitrocellulose filters were hybridized with the
samples at 40~C for 4 days in the presence of formamide.
cDNAs for the KDR/flk-1 and ~-actin genes were used as
probes. The filters were sc~nn~ and radioactivity was
20 measured on a PhosphorImager running ImageQuant software.
The amount of sample hybridizing to the KDR/flk-1 probe
was divided by that hybridizing to the ~-actin probe, and
the corrected density was reported as the percentage
change from the control.
25 Immunoprecipitation
HUVEC in confluent monolayers were serum-deprived
for 24 hours and treated with TNF-~ (1 ng/ml) or vehicle
for the indicated times. The cells were ;nsllh~ted with
35S-methionine (100 ~Ci/ml, DuPont/NEN) for two hours and
30 lysed in RIPA buffer at 4~C for ten minutes. After
s~;~entation of the insoluble fraction, the protein
extract was pre-cleaned with Protein A sepharose
(0.1 ~g/~l, Pharmacia Biotech, Piscataway, NJ) for one
hour at 4~C followed by centrifugation and collection of
35 the supernatant. Protein concentrations in the whole
CA 0222~460 1997-12-22
W O 97/00957 PCT~US96/10725
cell lysates were determined by a modified Lowry
procedure (DC protein assay; Bio-Rad, Melville, NY) and
were confirmed by SDS-polyacrylamide gel fractionation of
samples followed by Coomassie Blue staining. Protein
5 samples (500 ~g) were diluted to 1 ~g/~l with
immunoprecipitation buffer (50 mM Tris-Cl, pH 8.0, 150 mM
NaCl, 0.1% SDS, 1 NP40, 0.5% sodium deoxycholate, 2 mM
EDTA, 0.5 mM DTT, 0.02% sodium azide) plus 4 mg/ml BSA,
and rocked gently at 4~C for one hour. Specific antibody
10 was added to a concentration of 50 ~g/~l and the sample
was rocked at 4~C for 1.5 hours. Protein A sepharose (10
~g) was added and rocking was continued for 1.5 hours.
The antigen-antibody-Protein A sepharose conjugates were
removed by centrifugation and washed four times with
15 immunoprecipitation buffer. The conjugates were
denatured at 100~C for 5 minutes in Laemmli buffer and
size fractionated on a 7% SDS-polyacrylamide gel, which
was then vacuum-dried and subject to autoradiography.
Autoradiograms were scanned with a Howtek Sc~ ~ter 3+
(Hudson, NH) using Adobe Photoshop 3.0, and Scion Image
1.55 was used to quantitate the immunoprecipitated
protein.
t3~Thymid~ne Inoorporation
HUVEC grown to near confluence in gelatin-coated
25 24-well tissue culture plates were serum-deprived and
pretreated with vehicle or TNF-~ (1 ng/ml) for 12 hours
before addition of recombinant human VEGF (10 ng/ml,
Collaborative) or vehicle. Cells were treated with VEGF
for 24 hours and were labeled with methyl-[3H]thymidine
(DuPont/NEN) at 1 ~Ci/ml during the last three hours of
VEGF treatment. After labeling, the cells were washed
with phosphate-buffered saline, fixed in cold
10% trichloroacetic acid, and washed with 95% ethanol.
Incorporated [3H]thymidine was extracted in 0.2 M NaOH
35 and measured in a liguid scintillation counter. Values
CA 0222~460 1997-12-22
W O 97/00957 ~ PCTrUS96/10725
- 27 -
were expressed as the mean + SEM from 6 wells from two
separate experiments.
- ~t~ti~tic~l ~nalysis
When appropriate, data from image analyses and
[3H]thymidine incorporation were expressed as the mean
+ SEM. Statistical analysis of multiple treatment groups
was performed using a factorial analysis of variance
followed by Fisher's least significant difference test.
Statistical significance was accepted at p<0.05.
10 Bffect of ~NF-~ on VEGF-induced Endothelial Cell
Proliferation
TNF-a has previously been demonstrated to blunt
the mitogenic action of acidic and basic fibroblast
growth factors on bovine aortic endothelial cells in a
15 concentration-dependent manner (Frater-Schroder et al.,
1987, Proc. Natl. Acad, Sci. USA 84:5277-5281). To
determine whether TNF-~ also blocks the proliferative
effect of VEGF on human endothelial cells, t3H]thymidine
incorporation was measured as a marker for DNA synthesis
20 after stimulating HUVEC with human recombinant VEGF. In
serum-deprived H W EC, pretreatment with TNF-~ alone in
concentrations similar to those used by Frater-Schroder
et al. (Frater-Schroder et al., 1987, Proc. Natl. Acad,
Sci. USA 84:5277-5281) decreased [3H]thymidine
25 incorporation only slightly in comparison to HUVEC
pretreated with vehicle (Fig. 8). In comparison to
control cells, VEGF treatment alone potently enhanced
[3H]thymidine incorporation in HWEC by 2.3-fold, as has
been previously demonstrated (Connolly et al., 1989, J.
30 Clin. Invest. 84:1470-1478). However, pre-treatment of
H WEC with TNF-~ totally abolished the effect of VEGF on
DNA synthesis. These results demonstrate that TNF-
~blocks the proliferative response of H WEC to VEGF.
CA 0222~460 1997-12-22
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- 28 -
Downregulation of VEGF Receptor mRNA by TNF-~ in ~UVEC
The 5.7 kb KDR/flk-1 mRNA is constitutively
expressed in HUVEC (Patterson et al., 1995, J. Biol.
Chem. 270:2311-23118). The 7.0 kb fltl mRNA is also
5 abundantly expressed by H W EC in culture. Treatment of
HUVEC with
TNF-~ (10 ng/ml) resulted in a decrease in the mRNA for
both receptors (KDR/flk-1 and fltl) that was evident by 6
hours, and that reached 28% and 33% of 0-hour values,
10 respectively, for KDR/flk-1 and fltl after 24 hours of
treatment (Figs. 9A and 9B). That this effect was due to
TNF-~ alone, and not to serum deprivation, was
demonstrated by including a control sample which was
serum-deprived for 24 hours and treated with vehicle
15 alone; serum deprivation alone actually slightly induced
both KDR/flk-1 and fltl messages. To exclude the
possibility that the downregulation of these two
receptors was due to a generalized decrease in mRNA
production ;n~tlc~ by TNF-a, the same blots were
20 hybridized to a human heparin-binding epidermal growth
factor-like factor (HB-EGF) probe. Under these
conditions, TNF-~ induced a biphasic increase in HB-EGF
message, consistent with the results of previous
experiments (Yoshizumi et al., 1992, J. Biol. Chem.
25 267(14):9467-9469). To demonstrate that the effect of
TNF-~ was not specific to endothelial cells of venous
origin, identical experiments were performed with human
aortic endothelial cells (HAEC). A similar potent
decrease in the message for both receptors was noted in
30 HAEC. The message for KDR/flk-1 was also decreased by
TNF-~ in bovine aortic endothelial cells, demonstrating
that the effect of TNF-~ is not species-specific.
TNF-~ also decreased the messages for KDR/flk-1
and fltl in a dose-dependent fashion (Figs. lOA and lOB).
35 As little as 1 ng/ml TNF-~ inhibited the mRNA for both
CA 0222~460 1997-12-22
W O 97/00957 PCTAJS96/10725
receptors to near ~Y; ~l levels, with KDR/flk-1 being
slightly more sensitive than fltl to the effects of TNF-
~in H WEC. Thus, TNF-~ specifically downregulates the
mRNA for the VEGF receptors KDR/Elk--1 and fltl in a time
5 and dose-dependent fashion in human endothelial cells.
TNF-~ Decrea~ed the Rate of Transcription, But ~ad No
Effect on the ~alf-Life, of KDR/flk-1
To determine whether TNF-~ affected the steady-
state level of KDR/flk-1 mRNA by increasing its rate of
10 degradation, KDR/flk-1 mRNA was measured in the presence
of actinomycin D (ACD; 5 ~g/ml). The KDR/flk-1 mRNA
half-life was 1.9 hours in the absence of TNF-a and
increased slightly, to 2.6 hours, in the presence of TNF-
a (Fig. 11). In similar experiments, the mRNA half-life
15 of fltl was found not to be decreased by TNF-~ in HWEC.
Thus, the TNF-~-induced decrease in the level of KDR/flk-
1 and fltl mRNAs in HUVEC was not due to a decrease in
the stability of the mRNA.
Nuclear run-on experiments were performed to
20 determine the rate of KDR/flk-1 gene transcription in the
presence or absence of TNF-~, and to compare it with the
rate of transcription of the constitutively expressed ~-
actin gene. TNF-~ decreased the rate of KDR/flk-l gene
transcription (measured in PhosphorImager units) to 40~
25 of baseline, but had no effect on the transcription of ~-
actin. Thus, the TNF-~-induced decrease in KDR/flk-1
mRNA was due to a decrease in the rate of transcription
of the gene in H W EC and not to a change in the stability
of the mRNA.
30 The D__~ea~e in RDR/flk-1 mRNA by TNF-~ is Protein
Synthesis-Dependent
Whether the decrease in KDR/flk-1 mRNA required
protein synthesis was e~m; ned using the protein
synthesis inhibitor anisomycin. Concentrations of
35 anisomycin used (50 ~M) were five times higher than those
CA 0222~460 1997-12-22
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- 30 -
which inhibit protein synthesis in HUVEC by greater than
95%, as measured by [3H]leucine uptake. Anisomycin alone
had little effect on KDR/flk-l expression at this dose.
However, pretreatment with anisomycin significantly
5 blunted the effect of TNF-~ on KDR/flk-l expression (29%
vs. 65%, p<O.05), indicating that the effect of TNF-~ on
KDR/flk-1 in HUVEC was at least partly dependent on new
protein synthesis. These results did not vary with the
protein synthesis inhibitor used, as cycloheximide caused
10 an identical inhibition of the TNF-~ effect. Our studies
also show that the effect of TNF-~ on fltl expression is
similarly protein synthesis-dependent in H W EC.
TNF-~ Decreased New RDR/flk-l Protein Synthesis in HUVEC
Immunoprecipitation of 35S-labeled HUVEC lysates
15 was performed to demonstrate the production of
immunoreactive KDR/flk-l protein by HUVEC and to
determine whether the decrease in KDR/flk-l mRNA was
accompanied by a decrease in protein synthesis. A rabbit
anti-human KDR/flk-l antibody (Santa Cruz SC-xxx)
20 immunoprecipitated a single species with a molecular mass
of approximately 205 kDa, consistent with the size of
full length KDR/flk-l protein when expressed in, and
immunoprecipitated from, NIH 3T3 or COS7 cells (Quinn et
al., 1993, Proc. Natl. Acad. Sci. USA 90(16):7533-7537;
25 Millauer et al., 1994, Nature 367(6463):576-579). An
identically sized species was detected by an antibody
raised against a different KDR/flk-l epitope (Santa Cruz
SC-xxy), but not by an rabbit antibody raised in a
similar fashion to the transcription factor Spl,
30 demonstrating the specificity of this interaction.
Treatment of HUVEC with TNF-c~ for 12 hours increased 35S-
labeled KDR-flk-l protein levels slightly (<40%), but
reproducibly, raising the intriguing possibility that
TNF-~ also regulates KDR/flk-l at the translational or
35 post-translational level. After 24 hours of TNF-
~
CA 0222~460 1997-12-22
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- 31 -
treatment, 5S-labeled KDR/flk-1 protein levels were
decreased to 18% of control levels, confirming that the
decrease in KDR/flk-1 mRNA induced by TNF-~ is
a~o~r~n;ed by a similar decrease in KDR/flk-1 protein
5 expression (Fig. 12).
U e
The DNA of the invention promotes endothelial
cell-specific transcription of DNA sequences to which it
is operably linked. These promoter sequences are useful
10 to direct or prevent the expression of genes specifically
in endothelial cells. The invention provides the basis
for novel therapeutic approaches to vascular diseases
such as arteriosclerosis, as well as non-vascular
~ic~ c such as cancer, e.g., solid tumors, and
15 inflammatory diseases, e.g., rheumatoid arthritis and
diabetic retinopathy, as described in Examples 1 and 2
below.
The invention also provides methods for
identifying compounds which (1) modulate TNF-
~
20 downregulation VEGF receptor (e.g., KDR/flk-1 or fltl)
gene expression (Example 3, below), or (2) modulate TNF-
~inhibition of VEGF-induced endothelial cell proliferation
(Example 4). Compounds found to enhance TNF-
~downregulation of expression of a VEGF receptor gene or
25 enhance TNF-~ inhibition of VEGF-induced endothelial cell
proliferation can be used in methods to inhibit
angiogenesis, while compounds found to enhance TNF-
~downregulation of KDR/flk-1 or enhance TNF-~ inhibition
of VEGF-induced endothelial cell proliferation can be
30 used in methods to promote angiogenesis, for example, to
promote wound healing (e.g., healing of broken bones,
burns, diabetic ulcers, and traumatic or surgical wounds)
or to treat peripheral vascular disease, atherosclerosis,
cerebral vascular disease, hypoxic tissue damage (e.g.,
CA 0222~460 1997-12-22
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hypoxic damage to heart tissue), diabetic pathologies
such as chronic skin lesions, or coronary vascular
disease. These compounds can also be used to treat
patients who have, or have had, transient ischemic
attacks, vascular graft surgery, balloon angioplasty,
frostbite, gangrene, or poor circulation. As is
described in Example 5, identification of the cis-acting
sequences in the KDR/flk-1 gene required for
downregulation by TNF-~ provides the basis for additional
therapeutic methods for these conditions.
Exam~le 1: Gene Thera~y
The invention can be used for gene therapy
treatment of vascular diseases. The DNA of the invention
can be used alone or as part of a vector to express
heterologous genes, e.g., genes which encode proteins
other than KDR/flk-1, in cells of the blood vessel wall,
i.e., endothelial cells, for gene therapy of vascular
diseases such as arteriosclerosis. The DNA or vector
cont~;n;ng a sequence encoding a polypeptide of interest
is introduced into endothelial cells which in turn
produce the polypeptide of interest. For example,
sequences encoding t-PA (Pennica et al., 1982, Nature
301:214), p21 cell cycle inhibitor (El-Deiry et al.,
1993, Cell 75:817-823), or nitric oxide synthase (Bredt
et al., 1990, Nature 347:768-770) may be operably linked
to the endothelial cell-specific promoter sequences of
the invention and expressed in endothelial cells. For
example, thrombolytic agents can be expressed under the
control of the endothelial cell-specific promoter
sequences for expression by vascular endothelial cells in
blood vessels, e.g., vessels occluded by aberrant blood
clots. Other heterologous proteins, e.g., proteins which
inhibit smooth muscle cell proliferation, e.g.,
interferon-y and atrial natriuretic polypeptide, may be
CA 0222~460 1997-12-22
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- 33 -
specifically expressed in endothelial cells to ensure the
delivery of these therapeutic peptides to an
arteriosclerotic lesion or an area at risk of developing
an arteriosclerotic lesion, e.g., an injured blood
vessel.
The endothelial cell-specific promoter sequences
of the invention may also be used in gene therapy to
promote angiogenesis to treat diseases such as peripheral
vascular disease or coronary artery disease (Isner et
al., 1995, Circulation 91:2687-2692). For example, the
DNA of the invention can be operably linked to se~lt~oeC
encoding cellular growth factors which promote
angiogenesis, e.g., VEGF, acidic fibroblast growth
factor, or basic fibroblast growth factor.
According to the invention, the DNA of the
invention is located sufficiently close to the coding
sequence to be transcribed that it functions to direct
expression of the polypeptide in an endothelial cell.
For example, SEQ ID N0:1, 2, and 3 are preferably located
5~ to the transcription start site, and SEQ ID N0:4 is
located 3' of the transcription start site. However,
these sequences may be in any order relative to the
transcription start site provided that endothelial cell-
specific promoter activity is preserved.
Exam~le 2: Antisense Thera~y
The DNA of the invention may also be used in
methods of antisense therapy. Antisense therapy may be
carried out by At' ;niStering to an ~n; ~l, e.g., a human
patient, DNA containing the endothelial cell-specific
promoter sequences of the invention operably linked to a
DNA sequence, i.e., an antisense template, which is
transcribed into an antisense RNA. The antisense RNA may
a short (generally at least 10, preferably at least 14
nucleotides, and up to 100 or more nucleotides)
nucleotide sequence formulated to be complementary to a
CA 0222~460 1997-12-22
WO 97/00957 PCTAUS96/10725
portion of a specific mRNA sequence. The antisense
template is preferably located downstream from the
promoter sequences of the invention. A poly A tail is
typically located at the end of the antisense se~uence to
5 signal the end of the sequence. StAn~rd methods
relating to antisense technology have been described
(Melani et al., Cancer Res. 51:2897-2901, 1991).
Following transcription of the DNA sequence into
antisense RNA, the antisense RNA binds to its target mRNA
10 molecules within a cell, thereby inhibiting translation
of the mRNA and down-regulating expression of the protein
encoded by the mRNA. For example, an antisense sequence
complementary to a portion of or all of the KDR-flk-1
mRNA (Terman et al., 1991, Oncogene 6:1677-1683) would
15 inhibit the expression of KDR-flk-1, which in turn would
inhibit angiogenesis. Such antisense therapy may be used
to treat cancer, particularly to inhibit angiogenesis at
the site of a solid tumor, as well as other pathogenic
conditions which are caused by or exacerbated by
20 angiogenesis, e.g., inflammatory ~ic~ such as
rheumatoid arthritis, and diabetic retinopathy.
The expression of other endothelial cell proteins
may also be inhibited in a similar manner. For example,
the DNA of the invention can be operably linked to
25 antisense templates which are transcribed into antisense
RNA capable of inhibiting the expression of the following
endothelial cell proteins: cell cycle proteins (thereby
inhibiting endothelial cell proliferation, and therefore,
angiogenesis); coagulation factors such as von Willebrand
30 factor; and endothelial cell adhesion factors, such as
ICAM-1 and VCAM-1 (Bennett et al., 1994, J. Immunol.
152:3530-35~0).
For gene therapy or antisense therapy, the
claimed DNA may be introduced into target cells of an
35 ~n;~l ~ e.g., a patient, using standard vectors and/or
CA 0222~460 1997-12-22
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- 35 -
gene delivery systems. Suitable gene delivery systems
may include liposomes, receptor-mediated delivery
systems, naked DNA, and viral vectors such as herpes
viruses, retroviruses, adenoviruses, and adeno-associated
5 viruses, among others. Delivery of nucleic acids to a
specific site in the body for gene therapy or antisense
therapy may also be accomplished using a biolistic
delivery system, such as that described by Williams et
al., 1991, Proc. Natl. Acad. Sci. USA 88:2726-2729.
10 S~An~Ard methods for transfecting cells with isolated DNA
are well known to those skilled in the art of molecular
biology. Gene therapy and antisense therapy to prevent
or decrease the development of arteriosclerosis or
inhibit angiogenesis may be carried out by directly
15 administering the claimed DNA to a patient or by
transfecting endothelial cells with the claimed DNA ex
vivo and infusing the transfected cells into the patient.
DNA or transfected cells may be a~minictered in a
pharmaceutically acceptable carrier. Pharmaceutically
20 acceptable carriers are biologically compatible vehicles
which are suitable for a~in;stration to an animal, e.g.,
physiological saline. A therapeutically effective amount
is an amount of the DNA of the invention which is capable
of producing a medically desirable result in a treated
2S An;~l As is well known in the medical arts, dosages
for any one patient depends upon many factors, including
the patient's size, body surface area, age, the
particular compound to be A~il''; n;!:tered, sex, time and
route of ~;n;~tration~ general health, and other drugs
30 being aA~; n;~:tered concurrently. Dosages will vary, but
a preferred dosage for intravenous a~;n;~tration of DNA
is from approximately 106 to 1022 copies of the DNA
molecule. The compositions of the invention may be
administered locally or systemically. A~;n;stration
35 will generally be parenterally, e.g., intravenously; DNA
CA 0222~460 1997-12-22
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- 3 6 -
may also be administered directly to the target site,
e.g., by biolistic delivery to an internal or external
target site or by catheter to a site in an artery.
~mnle 3: Identification of ComPounds Which Modulate
5 TNF-~ Downrequlation of VEGF Rece~tor (KDR/flk-l or fltl)
Gene ExPression
As is discussed above, TNF-~ downregulates
expression of KDR/flk-1 and fltl genes, each of which
encodes a VEGF receptor. Thus, potentiating TNF-
~
10 downregulation of KDR/flk-l or fltl expression can be
useful in decreasing endothelial cell growth and,
therefore, in inhibiting processes such as angiogenesis.
Conversely, inhibiting TNF-~ downregulation of KDR/flk-1
or fltl expression can be useful in increasing
15 endothelial cell growth in order to promote angiogenesis,
as would be desirable in promoting wound healing or in
the treatment of peripheral vascular disease.
Modulation of endothelial cell growth can be
achieved by administering a compound which blocks or
20 ~nh~nce~ TNF-~-mediated inhibition of KDR/flk-1 or fltl
expression. Such a compound can be identified by methods
ranging from rational drug design to screening of random
compounds. The latter method is preferable, as a simple
and rapid assay for carrying out the method is available.
25 Small organic molecules are desirable candidate compounds
for this analysis, as frequently these molecules are
capable of passing through the plasma membrane so that
they can potentially modulate TNF-~ regulation of
KDR/flk-l or fltl gene expression within the cell.
The screening of small, membrane-permeable organic
molecules for the ability to modulate TNF-~
downregulation of KDR/flk-1 or fltl is carried out as
follows. Cells expressing KDR/flk-1 or fltl (e.g.,
HUVEC) are cultured in the presence of TNF-~ and the
35 candidate compound. (Cells cont~in;ng the KDR/flk-1 (or
CA 0222~460 1997-12-22
W O 97/00957 PCTrUS96/10725
- 37 -
fltl) promoter operably linked to a reporter gene may
also be used in this method, provided that the promoter
is active in the cells in the absence of TNF-~.) The
level of KDR/flk-1 (or fltl) expression (as measured by,
7 5 e.g., Northern blot analysis (see above), RNase
protection analysis, or other standard methods) in these
cells is compared to the level in cells cultured with
TNF-~, but without the candidate compound.
An increase in KDR/flk-l (or fltl) expression
10 indicates identification of a compound which blocks TNF-~
downregulation of KDR/flk-1 (or fltl) expression. As is
mentioned above, such a compound can be used in the
treatment of conditions in which enhancement of
endothelial cell growth or angiogenesis is desired, e.g.,
15 peripheral vascular disease, as well as for promoting
wound healing. One specific condition in which
angiogenesis is desired involves interruption of cardiac
blood flow. In such situations, TNF-~ may h; n~ the
natural angiogenic process which could ~o~ ol damage to
20 cardiac tissue.
A decrease in KDR/flk-1 (or fltl) expression
indicates identification of a compound which potentiates
TNF-~ downregulation of KDR/flk-1 expression. Such a
compound can be used to treat conditions in which
25 decreased endothelial cell growth or angiogenesis is
desired. For example, the growth of a tumor may be
inhibited by treatment with such a compound.
Compounds identified as having the desired effect
(i.e., enhancing or inhibiting TNF-~ downregulation of
30 KDR/flk-1 or fltl expression) can be tested further in
appropriate models of endothelial cell growth and
angiogenesis which are known to those skilled in the art.
The therapeutic compounds identified using the
method of the invention may be a~;n;~tered to a patient
35 by any appropriate method for the particular compound,
CA 0222~460 1997-12-22
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- 38 -
e.g., orally, intravenously, parenterally, transdermally,
transmucosally, or by surgery or implantation (e.g., with
the compound being in the form of a solid or semi-solid
biologically compatible and resorbable matrix) at or near
5 the site where the effect of the compound is desired.
For example, a salve or transdermal patch that can be
directly applied to the skin so that a sufficient
quantity of the compound is absorbed to increase
vascularization locally may be used. This method would
10 apply most generally to wounds on the skin. Salves
contA; n; ng the compound can be applied topically to
;n~llC~ new blood vessel formation locally, thereby
improving oxygenation of the area and hastening wound
healing. Therapeutic doses are determined specifically
15 for each compound, most administered within the range of
0.001 to 100.0 mg/kg body weight, or within a range that
is clinically determined to be appropriate by one skilled
in the art.
ExamPle 4: Identification of Com~ounds Which Modulate
20 the Effect of TNF-~ on VEGF-Induced E~ithelial Cell
Growth
As is discussed above, TNF-a inhibits VEGF-induced
proliferation of endothelial cells (see, e.g., Fig. 8 and
the corresponding text). Accordingly, compounds which
25 modulate the effect of TNF-~ on VEGF-induced endothelial
cell growth can be used to treat conditions associated
with endothelial cell growth, such as angiogenesis. Such
compounds can be identified using the methods described
above. For example, endothelial cells can be cultured
30 with VEGF and TNF-~ in the presence and absence of the
candidate compound in order to determine whether the
compound affects endothelial cell growth, which can be
measured, e.g., by monitoring uptake of [3H]thymidine.
As is discussed above, compounds found to have the
35 desired effect (i.e., enhancing or inhibiting TNF-
~
CA 0222~460 1997-12-22
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- 3 9 -
inhibition of VEGF-induced endothelial cell
proliferation) can be tested further in appropriate
models of endothelial cell growth and angiogenesis, which
are known to those skilled in the art. Compounds
- 5 identified using this method are a~ tered to patients
as is described above in Example 3. This method may also
be carried out without the addition of VEGF, in order to
identify compounds which modulate the effect of TNF-~ on
the growth of endothelial cells in the absence of VEGF.
10 Example 5: Identification of the Cis-Actinq Element in
the KDR/flk-1 Gene Reauired for TNF-~ Downrequlation
Identification of the cis-acting element in the
KDR/flk-l gene required for TNF-~-mediated downregulation
(the TNF-~-responsive element), as well as the trans-
15 acting factor which interacts with the TNF-~-responsive
element, will form the basis for the development of novel
therapeutics for modulating conditions associated with
endothelial cell growth, such as angiogenesis, vascular
disease, and wound healing.
Identification of the cis-acting elements of a
gene, as well as the corresponding trans-acting factors,
are carried out using st~n~rd methods in the art (see,
e.g., Sambrook et al., 1989, Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor
25 Laboratory Press, Cold Spring Harbor, New York; Ausubel
et al., eds., Current Protocols in Molecular Biology,
Wiley ~ Sons, New York, 1989). For example, as a
starting point, DNAse I hypersensitivity experiments can
be carried out in order to identify regions in the gene
30 which potentially bind regulatory factors.
Identification of the precise seguences of the cis-acting
element (e.g., the TNF-~ responsive element in the
KDR/flk-1 gene) can be carried out using st~n~rd
promoter deletion analysis. A construct including
35 KDR/flk-1 sequences that confer TNF-~ downregulation to a
-
CA 0222~460 1997-12-22
W O 97/00957 ~ PCTAUS96tlO725
- 4 O -
reporter gene to which the sequences are operably linked,
can be progressively deleted, by 5', 3', and/or nested
deletions, until the effect of TNF-~ on the expression of
the reporter gene in transfected cells is reduced.
5 Promoter deletion constructs, such as those described
above, can be used to begin this analysis. To confirm
the identification of the TNF-~-responsive element
identified in the deletion studies, point mutations can
be introduced into the element, using st~~~Ard methods,
10 in the context of the full promoter.
I The KDR/flk-1 TNF-~-responsive element can then be
used as a tool for identifying trans-acting factors which
bind to it, and thus are likely to be components of the
pathway of TNF-~ downregulation of KDR/flk-1. To
15 determine whether a protein binds to the element,
st~n~rd DNA footprinting and/or native gel-shift
analyses can be carried out. In order to identify the
trans-acting factor which binds to the TNF-~-responsive
element, the element can be used as an affinity reagent
20 in st~n~rd protein purification methods, or as a probe
for screening an expression library. Once the trans-
acting factor is identified, modulation of its binding to
the TNF-~-responsive element in the KDR/flk-1 gene can be
pursued, beginning with, for example, screening for
25 inhibitors of trans-acting factor binding. Enhancement
of TNF-~ downregulation of KDR/flk-1 expression in a
patient, and thus inhibition of angiogenesis, may be
achieved by administration of the trans-acting factor, or
the gene encoding it (e.g., in a vector for gene
30 therapy). In addition, if the active form of the trans-
acting factor is a dimer, do ;n~nt-negative mutants of
the trans-acting factor could be made in order to inhibit
its activity. Furthermore, upon identification of the
TNF-~-responsive element in the KDR/flk-l gene, and its
35 corresponding trans-acting factor, further components in
CA 0222~460 1997-12-22
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- 4 1 -
the TNF-~ pathway of KDR/flk-1 downregulation can be
identified. Modulation of the activities of these
~ components can then be pursued, in order to develop
additional drugs and methods for modulating endothelial
5 cell growth and angiogenesis. The methods described in
this example can also be carried out with the fltl gene.
Other Embodiments
In addition to antisense therapy for inhibition of
angiogenesis, expression of KDR/flk-1 in endothelial
10 cells can also be carried out by inhibiting the binding
of transcription factors, e.g., AP-2, SP-1 and NFKB, to
the cis-acting binding sites in the promoter sequences of
the invention. For example, transcription can be
inhibited using dominant negative mutants of
lS transcription factors, e.g., a dominant negative mutant
of AP-2 which binds to the AP-1 binding site but fails to
activate transcription. Alternatively, compounds which
downregulate production of transcription factors, e.g.,
retinoic acid or dexamethasone which downregulate
20 production of AP-2 and NF~B, can be A~;ni~tered to
inhibit angiogenesis by inhibiting expression of
KDR/flk-1.
CA 02225460 1997-12-22
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- 42 -
~:yu~ : LISTING
(1) ~.~NT'PAT. INFORMATION:
(L) APPLICANT: Pre~ident and Fellowff of Harvard College
(ii) TITLE OF lwv~:h-lON:TRANCCRTPTIONAL REGULATION OF GENES ENCODING
VASCULAR ~N~.~ .TAT GROWTH FACTOR R ~ORS
(iii) NUMBER OF S~QU~N~S: 16
( iv) Ct~RRTc.c:pc~NL~ c AnDRT.'.CS
'AJ AnDRrCCSF~: Fish & Richard~on P.C.
BJ STREET: 225 Franklin Street
C CITY: Bo~ton
,DI STATE: MA
El COuh.nY: USA
~I,F,l ZIP: 02110-2804
(v) CO:~u.~n RT~AnART.Tc FORM:
~A~l MEDIUM TYPE: Floppy di~k
BI C~ ~ul~n: IBM PC compatible
'C OPERATING SYSTEM: PC-DOS/MS-DOS
,D. SOFTWARE: PatentIn Relea~e ~1.0, Ver~ion ~1.30
(Vi) ~unK~h~ APPLICATION DATA:
(A) APPLICATION NU~BER: 08/573,692
(B) FILING DATE: DEC-18-1995
(C) CLASSIFICATION:
(vil) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/494,282
(B) FILING DATE: JUN-23-1995
(C) CLASSIFICATION:
(viii) A~..~Y/AGENT lN~-Ok~ATION:
(A) NAME: Fra~er, Jani~ K.
(B) REGISTRATION NUMBER: 34,819
(C) k~n~h~/DOCXET NUMBER: 05433/021001
(ix) TT~T.T~c~M~nJNIcATIoN lN~vRMATION:
(A) TELEPHONE: 617/542-5070
(B) TELEFAX: 617/542-8906
(C) TELEX: 200154
(2) INFORMATION FOR SEQ ID NO:l:
(i) ~yU~N~ ~ARA~TT~'RTSTICS:
'A' LENGTH: 62 ba~e pairs
~B TYPE: nucleic acid
,C, STRANnT~n-N~cs: both
l.DI TOPOLOGY: line~r
( ii ) MoT~TccuT~rc TYPE: DNA
CA 02225460 1997-12-22
W O 97/00957 PCTrUS96/107Z5
~ 43 ~
(xi) ~yu~ DT~'.~T~TPTION: SEQ ID NO:l:
TTGTTGCTCT GGGATGTTCT CTCCTGGGCG ACTTGGGGCC Q GCG QGTC CA~L~G 60
GG 62
- (2) INFORMATION FOR SEQ ID NO:2:
( i ) ~yU~N~ CHaRACTERISTICS:
'A'I LENGTH: l9 base pair~
~B TYPE: nucleic acid
,CI STR~NnT~nNEss: both
~D, TOPOLOGY: linear
(Li) MOLECULE TYPE: DNA
( Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:2:
GCTGGCCGCA CGGr~ GC l9
(2) INFORMATION FOR SEQ ID No:3:
( i ) ~Q~N~ CH~RACTERISTICS:
,'A' LENGTH: 36 base pairs
~B~ TYPE: nucleic acid
,C~ STRANDEDNESS: both
~DJ TOPOLOGY: linear
( ii ) M~T~CUT-T~' TYPE: DNA
(xi) ~:~u~ DESCRIPTION: SEQ ID NO:3:
G~GCCGCA CGGr-~-AGCC C~C~CCGC CCCGGC 36
(2) INFORMATION FOR SEQ ID NO:4:
( i ) ~yU~N~ CHARACTERISTICS:
,A~I LENGTH: 22 base pair~
IB TYPE: nucleic acid
,C~ STR~Nn~nNESS: both
~D~ TOPOLOGY: linear
( ii ) M~T~T~CUT~ TYPE: DNA
(xi) ~:~u~ DESCRIPTION: SEQ ID NO:4:
GGATATCCTC TCCTACCGGC AC 22
(2) INFORMATION FOR SEQ ID NO:5:
( i ) ~ ~:yU ~:N~: CHARACTERISTICS:
(A) LENGTH: 493 ba~e pair~
(B) TYPE: nucleic acid
(C) STR~NnT~'nNESS: both
CA 0222~460 l997-l2-22
W O 97/00957 PCTrUS96/10725
- 44 -
(D) TOPOLOGY: linear
( ii ) MrJT-~cur~ TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
TTGTTGCTCT GGGATGTTCT CTCCTGGGCG ACTTGGGGCC CAGCGCAGTC CAGL~ G 60
GGGAAATGGG GAGATGTA~A TGGGCTTGGG GAGCTGGAGA TCCCCGCCGG GTACCCGGGT 120
GAGGGGCGGG GCTGGCCGCA CGGGAGAGCC C~C~lCCGC CCCGGCCCCG CCCCGCATGG 180
CCCCGCCTCC GCGCTCTAGA GTTTCGGCTC CAGCTCCCAC CCTGCACTGA GTCCCGGGAC 240
CCCGGGAGAG CGGTCAGTGT ~ 'GCTG C~l.C~ GCCTGCGCCG GGCATCACTT 300
GCGCGCCGCA GA~AGTCCGT CTGGCAGCCT GGATATCCTC TCCTACCGGC ACCCGCAGAC 360
GCCCCTGCAG CCGCCGGTCG GCGCCCGGGC TCCCTAGCCC TGTGCGCTCA ACTGTCCTGC 420
GCTGCGGGGT GCCGCGAGTT CCACCTCCGC GC~-C~ CTAr-~r~r-GC GCTGGGAGAA 480
Ar-~rcGGcT CCC 493
(2) INFORMATION FOR SEQ ID NO:6:
(i) ~yu~N~ CHARACTERISTICS:
~A'I LENGTH: 352 base pairs
,BI TYPE: nucleic acid
,CJ ST~N~ :CS: both
,DI TOPOLOGY: linear
($i) MOLECULE TYPE: DNA
(xi) ~yu~..~ DESCRIPTION: SEQ ID NO:6:
TTGTTGCTCT GGGATGTTCT ~l~lGGGCG A~GGGCC CAGCGCAGTC CA~ G 60
GGGA~ATGGG GAGATGTA~A TGGGCTTGGG GAGCTGGAGA TCCCCGCCGG GTACCCGG~l 120
GAGGGGCGGG GCTGGCCGCA CGGr-~r-AGCC C~C~.CCGC CCCGGCCCCG CCCCGCATGG 180
CCCCGCCTCC GCGCTCTAGA GTTTCGGCTC CAGCTCCCAC CCTGCACTGA GTCCCGGGAC 240
CCCGGGAGAG CGGTCAGTGT GLGGlCGCTG C~l~l~l ~ GCCTGCGCCG GGCATCACTT 300
GCGCGCCGCA GA~AGTCCGT CTGGCAGCCT GGATATCCTC TCCTACCGGC AC 352
(2) lN~ukMATION FOR SEQ ID NO:7:
(i) ~yU~N~ CHARACTERISTICS:
,~A~I LENGTH: 1267 base pairs
IB~I TYPE: nucleic acid
,C, sT~Nn~nNEss: both
~D, TOPOLOGY: linear
( ii ) Mr~r~cur~ TYPE: DNA
CA 0222~460 l997-l2-22
O 97/009S7 PCTrUS96/10725
- 45 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7
C~l~llCCC CTGGGCCTAA GGATATCTTG GCTGGAAGCT CTGCTCTGAA AAGGGGCATG 60
GCCAaACTTT Q CTAGGGCT ~.~C~..GGG GAGCACGATG ~ArAAA~GCC TTCTTGGGGC 120
TAGGCAGGTC ACTT Q AACT TGGAGCCGCC AaATATTTTG Gr-AAATAr-CG GGAATGCTGG 180
CGAACTGGGC AAGTGCGTTT TCTGATTAAG AGrA~A~A TTCAGCTTTT TAAACTACAA 240
TTATACTGGC rAAArAAAAT ACCCTTATAC AAAAACCAAA ACTACTGG Q GGAGTCGCTG 300
CCAGCTTGCG ACCCGGCATA CTTGGCTGAG TATCCGCTTC ~CC~lGG CTGGAAACTG 360
ATGCAGATTC TCGGCCACTT CAGACGCGCG CGATGGCGAA GAGG~l~.~ CACTTTGACG 420
CGC~l~.GA GGGAGCGGTG ~ lCGCAG CGCTCCTGGT GATGCTCCCC AAA~CGGG 480
GACCGGCAAG CGATTAAATC TTGGAGTTGC T QGCGCCCG TTArCr-~r-TA ~llll~ ATTT 540
ArAr~A~AAA CAaAGTTGTT G~'-.aGGAT ~ C~I GGGCGACTTG GGGCCCAGCG 600
CAGTC Q GTT Gl~lGGGGAA ATGGGGAGAT GTAAATGGGC TTGGGGAGCT GGAGATCCCC 660
GCCGGGTACC CGGGTGAGGG GCGGGGCTGG CCGCACGGGA GAGCCC~.CC TCCGCCCCGG 720
CCCCGCCCCG CATGGCCCCG CCTCCGCGCT CTAGAGTTTC GGCTCCAGCT CCrACCCTGC 780
ACTGAGTCCC GGGACCCCGG GAGAGCGGTC A~.~.~lGGT CGCTGCGTTT CCTCTGCCTG 840
CGCCGGGCAT CACTTGCGCG CCG~r-AAAG lCCGl~lGGC AGCCTGGATA lC~~ ~lA 900
CCGG Q CCCG Q GACGCCCC TG Q GCCGCC GGTCGGCGCC CGGGCTCCCT AGCC~l~.GC 960
GCT QACTGT CCTGCGCTGC GGGGTGCCGC GAGTTCCACC TCCGCGCCTC ~l.~l~lAGA 1020
Q GGCGCTGG GArAAA~AAr, CGGCTCCCGA ~ll~GGCA TTTCGCCCGG CTCGAGGTGC 1080
AGGATGCAGA GCAAGGTGCT GCTGGCCGTC GCC~l~lGGC TCTGCGTGGA GACCCGGGCC 1140
GC~l l~lGG GTAAGGAGCC CA~l~l~GAG GAGGAAGGCA GACAGGTCGG GTGAGGGCGG 1200
AGAGGACCTG AaAGCCAGAT CTAACTCGGA ATCGTAGAGC TGGAGAGTTG r-~rAr-r-~rTT 1260
GA Q TTT 1267
(2) lNrOk~ATION FOR SEQ ID NO:8:
( i ) ~U~N~ CHARACTERISTICS:
lAj LENGTH: 500 base pair~
BI TYPE: nucleic acid
Cl STRANDEDNESS: both
D, TOPOLOGY: linear
( ii ) M~T~T~cuT~ TYPE DNA
( Xi ) ~yuh~ - ~ DESCRIPTION: SEQ ID NO:8:
CA 0222~460 1997-12-22
WO 97/00957 PCTrUS96/10725
- 46 -
ACTTCTACCA r-~A~rCr-Ar-C TGCGTCCAGA TTTGCTCTCA GATGCGACTT GCCGCCCGGC 60
ACAGTCCGGG GTAGTGGGGG AGTGGGCGTG Gr-~A~rcGGG A~Arcc~AAr, CTGGTATCCA 120
GTGGGGGGCG TGGCCGGACG CAGGGAGTCC crArCc~ cc CGGTAATGAC CCCGCCCCCA 180
TTCGCTAGTG TGTAGCCGGC G~ L~ ~C TGCCCTGAGT CCTr~Gr-~C Cr~r-~r-~r-T 240
AAG~ TCCTTAGATT CGGGGACCGC TACCCGGCAG GACTGAAAGC CCAGACTGTG 300
TCCCGCAGCC GGGATAACCT GGCTGACCCG ATTCCGCGGA CACCGCTGCA GCCGCGGCTG 360
GAGCCAGGGC GCCGGTGCCC CGCGCTCTCC CCGGTCTTGC GAAGGAGTCT GTGCCTGAG~ 420
AACTGGGCTC TGTGCCCAGG CGCGAGGTGC AGGATGGAGA GCAAGGCGCT GCTAGCTGTC 480
G~~ GGT TCTGCGTGGA 500
(2) INFORMATION FOR SEQ ID NO:9:
( i ) ~yU~N~ CHARACTERISTICS:
IA'I LENGTH: 32 base pair~
,BI TYPE: nucleic acid
C, STRANDEDNESS: both
,,D,, TOPOLOGY: linear
(iL) MnT ~CUT~ TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GAG AAGGAGGCGC GGAGGTGGAA CT 32
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
~A'l LENGTH: 26 ba~e pair~
~B TYPE: nucleic acid
~C, STRANDEDNESS: both
~D,, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) ~Qu~w~ DESCRIPTION: SEQ ID NO:l0:
TCTGGCAGCC ~G~lC~C~ CTCCTA 26
(2) INFORMATION FOR SEQ ID NO:ll:
(i) ~yu~:w~ CHARACTERISTICS:
,'AJ LENGTH: 26 base pair~
~B, TYPE: nucleic acid
C, STRANDEDNESS: both
,DJ TOPOLOGY: linear
( ii ) M~T.T~'CTJT.T~! TYPE: DNA
CA 02225460 1997-12-22
W O 97/00957 PCT~US96/10725
~ 47 -
tXi) ~U~N~ DESCRIPTION: SEQ ID NO:ll:
TAr~r-~r~r-GA cr-~rr~r~cT GCCAGA 26
(2) INFORMATION FOR SEQ ID NO:12:
- ( i ) ~yU~N~ CEARACTERISTICS:
,~AI LENGTH: 27 ba~e pairs
~Bl TYPE: nucleic acid
,C sT~ANnT2n~cs: both
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:12:
TGCCTCGAGT TGTTGCTCTG GGATGTT 27
(2) INFORMATION FOR SEQ ID NO:13:
(i) ~yU~:N~: CHARACTERISTICS:
~A'l LENGTH: 27 ba~e pair~
BJ TYPE: nucleic acid
C STRPNn~nNESS: both
~D,, TOPOLOGY: linear
( ii ) Mr~T~uT~ TYPE: DNA
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:13:
TGTAAGCTTG GGAGCCGGTT ~lC 27
(2) INFORMATION FOR SEQ ID NO:14:
( i ) ~yU~N~: CHARACTERISTICS:
~A'l LENGTH: 11 ba~e pair~
Bl TYPE: nucleic acid
,C, STRANn~nN~SS: both
lD) TOPOLOGY: linear
( ii ) M~T ~CTTT~ TYPE: DNA
(xi) ~yu~ DESCRIPTION: SEQ ID NO:14:
CCCTGCACTG A 11
(2) INFORMATION FOR SEQ ID NO:15:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 22 amino acid~
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02225460 1997-12-22
W O 97/00957 PCTAJS96/10725
- 4 8 -
( ii ) MnT.T'CUT.T~' TYPE: protein
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:15:
Met Gln Ser Ly~ Val Leu Leu Ala Val Ala Leu Trp Leu Cy~ Val Glu
1 5 10 15
~hr Arg Ala Ala Ser Val
~2) INFORMATIOW FOR SEQ ID NO:16:
(i) SEQUENCE CaARACTERISTICS:
(A) LENGTH: 16 amino acidR
(B) TYPE: amino acid
(D) TOPOLOGY: linear
( ii ) MOT~T~CUT ~ TYPE: protein
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:16:
~et Glu Ser Ly~ Ala Leu Leu Ala Val Ala Leu Trp Phe Cy~ Val Ly~
1 5 lO 15
Other embodiments are within the following claims.
What is claimed is:
