Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• TRANSCRIPTIONAL REGULATION OF GENES ENCODING VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS Background of the Invention This invention relates to endothelial cell- specific gene transcription and transcriptional regulation by TNF- ⁇ .
• VEGF Vascular endothelial growth factor
• KDR/flk-1 Kinase-insert Domain-containing Receptor/fetal liver kinase-1
• fltl fetal liver kinase-1
• VEGF plays critical roles in growth and maintenance of vascular endothelial cells and in the development of new blood vessels in physiologic and pathologic states (Aiello et al., 1994, New Engl. J. Med.
• VEGF vascular endothelial growth factor
• 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., 1992, Clin. Invest. 89:244-253).
• the tyrosine kinases KDR/flk-1 and fltl have been identified as candidate VEGF receptors by affinity cross-linking and competition-binding assays (de Vries et al., 1992, Science 255:989-991; Millauer et al., 1993, Cell 72:835- 846; Terman et al., 1992, Biochem. Biophys. Res. Commun. 187:1579-1586).
• KDR/flk-1 is 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 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, 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).
• vascular diseases such as atherosclerosis (Ross, 1993, Nature 362:801-809).
• expression of these genes is either not 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) .
• 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 invention contains a sequence substantially identical to nucleotides -225 to -164 of the KDR/flk-1 promoter, i.e., 5' TTGTTGCTCTGGGATGTTCTCTCCTGGGCGACTTGGGGCCCAGCGCAGTCCAGT TGTGGG 3' (SEQ ID NO:l).
• substantially identical is meant at least 80% identical to a reference DNA 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 than that of the reference sequence.
• an alternative basepair e.g., G-C replaced with A-T, T-A, or C-G
• 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 KDR/flk-1 promoter, i.e.,
• 5' GCTGGCCGCACGGGAGAGCCCCTCCTCCGC CCCGGC 3' (SEQ ID NO:3), a sequence substantially identical to nucleotides +105 to +127 of the KDR/flk-1 promoter, i.e., 5' GGATATCCTCTCCTACCGGCAC 3' (SEQ ID NO:4), or a combination thereof.
• 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.
• any orientation of these sequences which promotes endothelial 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.
• this nonspecific (i.e., sequence other than SEQ ID NO:1-4 will constitute no more than 80% of the entire promoter sequence. Most preferably, it is substantially identical to the sequence shown in Table 1 (SEQ ID NO: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 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 sequences 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-1 promoter (SEQ ID NO: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- specific transcription of a polypeptide-encoding sequence or antisense template to which it is operably linked is also within the invention.
• the DNA of the invention may be operably linked to, and functions to regulate endothelial cell-specific transcription of, a sequence encoding a polypeptide that is not KDR/flk-1.
• polypeptides include tissue plasminogen activator (tPA) , p21 cell cycle inhibitor, and nitric oxide synthase.
• tPA tissue plasminogen activator
• p21 cell cycle inhibitor p21 cell cycle inhibitor
• nitric oxide synthase nitric oxide synthase.
• operably linked 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 containing the DNA of the invention, a method of directing 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 in an animal 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 cells, e.g., interferon- ⁇ or atrial natriuretic polypeptide.
• the invention also includes compositions and methods of carrying out antisense therapy.
• the invention includes a substantially pure DNA with a 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.. an antisense template the transcription product of which prevents translation of mRNA into an endothelial cell polypeptide.
• antisense template is meant a DNA which is transcribed into an RNA which hybridizes to mRNA.
• the endothelial cell polypeptide is KDR/flk-1.
• 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 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.
• translation of cell cycle proteins, coagulation factors, e.g., von Willebrand factor, and endothelial cell adhesion factors, e.g., intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1) may be reduced or prevented.
• IAM-1 intercellular adhesion molecule-1
• VCAM-1 vascular cell adhesion molecule-1
• 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.
• TNF- ⁇ downregulation VEGF receptor e.g., KDR/flk-1 or fltl
• a cell containing the promoter of a VEGF receptor gene operably linked to a 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.
• an endothelial cell is cultured in the presence of TNF- ⁇ , VEGF, and the candidate compound.
• the level of endothelial cell growth is determined (e.g., by measurement of uptake of [methyl- [ 3 H]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 administering 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 administering to the patient a polypeptide which inhibits VEGF receptor (e.g., KDR/flk-1 or fltl) gene expression in an endothelial cell by binding to the TNF- ⁇ -responsive element in the promoter of the VEGF receptor gene.
• VEGF receptor e.g., KDR/flk-1 or fltl
• the invention also features a method of enhancing angiogenesis in a patient involving administering to the patient a non-TNF- ⁇ compound which inhibits the TNF- ⁇ pathway of downregulating VEGF receptor (e.g., KDR/flk-1 or fltl) gene in an endothelial cell.
• a non-TNF- ⁇ compound which inhibits the TNF- ⁇ pathway of downregulating VEGF receptor (e.g., KDR/flk-1 or fltl) gene in an endothelial cell.
• Fig. IA is a diagram of the human KDR/flk-1 promoter. Restriction enzyme sites are indicated above 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 Pstl sites which were used 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. IB is a diagram of the murine KDR/flk-1 promoter.
• 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. IA was hybridized to 20 ⁇ g of total RNA from human umbilical vein endothelial cells (HUVEC) and HeLa cells or 3 ⁇ g of polyA + HUVEC 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 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-1 transcription start site.
• Total RNA from HUVEC and HeLa cells or polyA + HUVEC RNA and yeast tRNA were incubated with a 559-bp 32 P-labeled riboprobe spanning the immediate 5' region of the human KDR/flk-1 gene.
• the annealing products were digested with RNase. Protected fragments were analyzed on a 4% polyacrylamide gel.
• Fig. 3A is a diagram showing the location of 5' deletion sites in the 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 functional analysis of the human KDR/flk-1 promoter by transfection of luciferase reporter constructs containing serial 5' deletions into bovine aortic endothelial cells (BAEC) . All constructs were cotransfected with pSV ⁇ gal to correct for transfection efficiency, and luciferase 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 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.
• the plasmid pGL2-225+268 directed luciferase expression comparable to that directed by pGL2 Control, which contains the SV40 promoter and enhancer.
• pGL2 Control which contains the SV40 promoter and enhancer.
• Fig. 6A is a photograph of a Northern blot 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.
• HUVEC human umbilical vein endothelial cells
• HASMC human aortic smooth 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 cells
• MCF7 human breast adenocarcinoma cells
• U937 human histiocytic lymphoma cells
• Fig. 6B is a photograph of the same agarose gel shown in Fig. 6A which was stained with ethidium bromide (to visualize ribosomal RNA) to show the amount of RNA loaded in each lane.
• Fig. 7 is a bar graph showing the results of a luciferase assay. High-level activity of the KDR/flk-l promoter was found to be specific to endothelial cells.
• the luciferase reporter construct pGL2-4kb+296 was 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.
• Fig. 8 is a bar graph showing the effect of TNF- ⁇ on VEGF-induced proliferation of HUVEC, as measured by uptake of -methyl-[ 3 H]thymidine.
• Fig. 9A is a photograph of a Northern blot analysis of a time course of TNF- ⁇ -induced downregulation of KDR/flk-l mRNA expression in HUVEC. From left to right, the time points are: 0, 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-l mRNA expression shown in the photograph of Fig. 9A.
• Fig. lOA is a photograph of a Northern blot analysis of a dose-response experiment of TNF- ⁇ -induced downregulation of KDR/flk-l mRNA expression in HUVEC. From left to right, 1. 0.1, 1, 10, 50, and 100 ng/ml TNF- ⁇ were used, as indicated in Fig. 10B.
• Fig. 10B is a bar graph of the results of the dose-response experiment of TNF- ⁇ -induced downregulation of KDR/flk-l mRNA expression in HUVEC shown in the photograph of Fig. 10A.
• Fig. 11 is a graph showing the effect of
• Actinomycin D on the levels of KDR/flk-l RNA in HUVEC.
• Fig. 12. is a photograph of immunoprecipitation analysis of KDR/flk-l protein in HUVEC treated with TNF- ⁇ for 0, 12, and 24 hours.
• a 567-bp human KDR/flk-l cDNA fragment was generated from HUVEC total RNA by reverse-transcriptase polymerase chain reaction (RT-PCR) . This fragment was radiolabeled with [ ⁇ - 32 P]dCTP and used to screen a phage library of human placenta genomic DNA in the vector ⁇ FixII (Stratagene, La Jolla, CA) . Likewise, a 451-bp mouse KDR/flk-l 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 ⁇ Dashll (Stratagene) . Hybridizing clones were isolated and purified from each library, and phage DNA was prepared according to standard procedures. Cell Culture and mRNA 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, 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.
• Dulbecco's modified Eagle's medium JRH Biosciences, Lenexa, KS
• 10% fetal calf serum HyClone, Logan, UT
• Saos-2 human osteosarcoma 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 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.
• HUVEC Primary-culture HUVEC were obtained from Clonetics Corp. (San Diego, CA) and were grown in EGM medium containing 2% fetal calf serum (Clonetics) .
• Primary-culture human aortic and intestinal smooth muscle cells were also obtained from Clonetics Corp. All cells were cultured in conditions identical to those for BAEC, with the exception that medium used for smooth muscle cells was supplemented with 25 mM HEPES (Sigma, St. Louis, MO) and that HUVEC were cultured in EGM medium containing 2% fetal calf serum.
• Primary-culture cells were passaged every 4-6 days, and cells from passages 3-5 were analyzed.
• RNA from cells in culture was prepared by guanidinium isothiocyanate extraction and centrifugation through cesium chloride.
• DNA Sequencing Restriction fragments derived from the human and mouse KDR/flk-l genomic phage clones were subcloned using standard techniques into pSP72 (Promega, Madison, WI) or pBluescript II SK (Stratagene) and sequenced from alkaline- denatured double-stranded plasmid templates by the dideoxy chain termination method with SEQUENASE® 2.0 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 GC-rich 5' flanking region of the human and mouse
• a 559-bp Pstl-PstI fragment of the human KDR/flk-l gene (Fig. 2B) was cloned in pSP72 as the template for in vitro transcription of an ⁇ - 32 P-labeled antisense RNA with T7 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) .
• Gel-purified riboprobe (5 x IO 5 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 containing 20 mM Tris-HCl, pH 7.40, 400 mM NaCl, 1 mM EDTA, and 0.1% sodium dodecyl sulfate in 75% formamide.
• RNAse Tl Boehringer Mannheim
• RNAse A Boehringer Mannheim
• the digestion products were then treated with proteinase K, extracted with phenol:chloroform, and analyzed by electrophoresis on a 4% denaturing polyacrylamide gel.
• RNA (10 ⁇ g) from cells in culture was fractionated on a 1.3% for aldehyde-agarose gel and transferred to a nitrocellulose filter.
• the human KDR/flk-l cDNA probe was labeled with 32 P by random priming, the labeled probe was then used to hybridize the filter.
• the filter was then autoradiographed for 16 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 promoter and enhancer.
• the plasmid pSV,9GAL contained the 3-galactosidase gene driven by the SV40 promoter and enhancer.
• Reporter constructs containing fragments of the human KDR/flk-l 5' flanking region were inserted into pGL2 Basic and named according to the length of the fragment (from the transcription start site) in the 5' and 3' directions.
• plasmid pGL2-4kb+296 contained a human KDR/flk-l promoter fragment extending from approximately -4 kb 5' of the transcription start 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 Pvull sites, respectively, and the 3' Xhol site at position +296.
• Plasmids pGL2-716+268, pGL2-570+268, 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 created by digesting the promoter fragment contained in 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. All constructs were sequenced from the 5' and 3' ends to confirm orientation and sequence. Mutagenesis
• Site-directed mutagenesis of the atypical GATA sequence located in the first exon of the human KDR/flk-l 5' flanking region was performed by PCR using to the method of Higushi et al. , 1988, Nucleic Acids Res. 16:7351-7367.
• a DNA fragment containing human KDR/flk-l bp -225 to +268 was used as a template.
• the sequence TGGATATC was mutated to TGGTCGTC by using one set of mismatched primers, 5' TCTGGCAGCCTGGTCGTCCTCTCCTA 3' (SEQ ID NO:10) and 5'TAGGAGAGGACGACCAGGCTGCCAGA 3' (SEQ ID NO:11), and one set of primers flanking both ends of the template, 5' TGCCTCGAGTTGTTGCTCTGGGATGTT 3' (SEQ ID NO:12) and 5' TGTAAGCTTGGGAGCCGGTTCTTTCTC 3' (SEQ ID NO:13).
• the sequence of the mutated PCR fragment was confirmed by the dideoxy chain termination method.
• the ratio of luciferase activity to 3-galactosidase activity in each sample served as a 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 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. Isolation and Characterization of Human and Murine KDR/flk-l Genomic clones
• primer extension was performed with a complementary oligonucleotide probe corresponding to bp +212 to +243 (underlined with arrow. Fig. IA) .
• Primer extension was performed on total RNA from HUVEC and HeLa cells and on polyA RNA from HUVEC.
• Gene transcription was found to be initiated only in endothelial cells (Fig. 2A) .
• the transcription start site is highlighted in bold in the sequence, CCCTGCACTGA (SEQ ID NO:14) (see Figs. IA and 2A) .
• the 5'CA3' nucleotide pair at this position is the most common site for transcription initiation.
• a ribonuclease protection analysis was performed using an antisense riboprobe generated from a 559-bp genomic Pstl-PstI fragment extending 5' from position +146 (Fig. 2B; the Pstl sites are double underlined in Fig. IA) .
• the 5' flanking sequence of the human KDR/flk-l gene contains regions rich in G and C residues and lacks TATA and CCAAT boxes near the transcription start site (Fig. IA) .
• Comparison of this 5' flanking sequence with sequences in the Transcription Factors Database revealed a series of five Spl sites located between human KDR/flk-l nucleotides -124 and -39. There are two AP-2 consensus sites at positions -95 and -68 and two inverted NFKB binding elements at -130 and -83 interspersed among the Spl sites.
• Two atypical GATA consensus sequences are present in the KDR/flk-l promoter, one at position -759 and the other at position +107 within the untranslated portion of the first exon.
• 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 AAACCAAA which is conserved among genes expressed preferentially in keratinocytes, is present at human KDR/flk-l position -508.
• the human and mouse KDR/flk-l promoters were compared to identify conserved consensus sequences for nuclear proteins (Fig. IB) .
• 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 NFKB site at position -153, 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.
• a CANNTG sequence is present 12 bp 5' of the G- and C-rich sequences of the promoter at mouse KDR/flk-l 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 that these regulatory elements have functional significance. Deletion Analysis of the Human KDR/flk-l Promoter
• luciferase reporter plasmids containing serial 5' deletions through the promoter region was constructed (Figs. 3A and 3B) .
• pGL2 Basic were cotransfected into BAEC with pSV ⁇ gal (to correct for differences in transfection efficiency) and the luciferase activity was normalized to that of the pGL2 Control vector driven by the SV40 promoter/enhancer.
• NFKB is thought to trans-activate the inducible expression of vascular cell adhesion molecule-1 and tissue factor in endothelial cells (Iademarco, 1992, J. 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 trans-acting factors.
• E-box binding proteins have not been clearly associated with endothelial cell gene expression, although members of this family are critical for proper maturation of many cell types, including skeletal muscle and B lymphocytes (Buskin et al., 1989, Mol. Cell. Bio. 9:2627-2640; Murre et al., 1989, Cell 58:537-544) .
• GATA-2 has been most closely linked to endothelial cell gene expression.
• GATA-2 functions as an enhancer of endothelin-1 gene expression and acts to restrict expression of von Willebrand factor to endothelial cells.
• Human KDR/flk-l 5' flanking region was found to have two potential GATA-binding sequences, at positions -759 and +107. Loss of the element located at position -759 had no effect on expression of KDR/flk-l 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).
• this GATA sequence differs from the GATA-binding sequences of endothelin-1 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 exon, and because a similar GATA element is found in the first exon of the mouse KDR/flk-l gene.
• KDR/flk-l expression is restricted to endothelial cells in vivo, it does not necessarily follow that its expression would be limited to endothelial cells in culture.
• 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-l 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) .
• KDR/flk-l message was not detected by RT-PCR in HeLa, A7r5, or 3T3 cells.
• expression of KDR/flk-l message in tissue culture appears to be restricted to endothelial cells, as it is in vivo.
• pGL2-4kb+296, which contains over 4 kb of the human KDR/flk-l 5' flanking 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.
• expression driven by the pGL2-4kb+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-l 5' flanking sequence.
• TNF- ⁇ Downrecrulates KDR/flk-l and fltl Expression Cell 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.
• fetal calf serum Hyclone, Logan, UT
• endothelial cell growth substance ECGS, Collaborative Biomedical, Bedford, MA
• Bovine aortic endothelial cells were isolated and cultured in Dulbecco's modified Eagle's medium (JRH Biosciences, Lenexa, KS) 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 (M199 medium supplemented with 5% fetal calf serum without ECGS) . Recombinant human TNF- ⁇ (Genzyme, Cambridge, MA) was aliquoted and stored at -80°C until use.
• Dulbecco's modified Eagle's medium JRH Biosciences, Lenexa, KS
• 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 (M199 medium supplemented with 5% fetal calf serum without
• RNA from cells in culture was prepared by guanidinium isothiocyanate extraction and centrifugation through cesium chloride (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) .
• 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 1.3% formaldehyde-agarose gel and transferred to nitrocellulose filters.
• cDNA probes were labeled with 32 P 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 at 80°C and rehybridized with an end-labeled 18S ribosomal RNA oligonucleotide to correct for loading.
• a 567-bp human KDR/flk-l cDNA fragment was generated from human umbilical vein endothelial cell
• HUVEC 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 York), as previously described (Patterson et al., 1995, J. Biol. Chem. 270:2311-23118).
• RT-PCR reverse-transcriptase polymerase chain reaction
• 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 ⁇ l was incubated with 0.5 mM each of CTP, ATP, and GTP, and with 20 ⁇ Ci of 32 P-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 IO 6 counts/minute/ml) .
• 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 incubated with 35 S-methionine (100 ⁇ Ci/ml, DuPont/NEN) for two hours and lysed in RIPA buffer at 4°C for ten minutes. After sedimentation 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 the supernatant.
• Protein concentrations in the whole 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 samples (500 ⁇ g) were diluted to 1 ⁇ g/ ⁇ l with immunoprecipitation buffer (50 mM Tris-CI, 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.
• immunoprecipitation buffer 50 mM Tris-CI, 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
• HUVEC grown to near confluence in gelatin-coated 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-[ 3 H]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 [ 3 H]thymidine was extracted in 0.2 M NaOH and measured in a liquid scintillation counter. Values were expressed as the mean + SEM from 6 wells from two separate experiments. Statistical analysis
• TNF- ⁇ has previously been demonstrated to blunt the mitogenic action of acidic and basic fibroblast growth factors on bovine aortic endothelial cells in a concentration-dependent manner (Frater-Schroder et al., 1987, Proc. Natl. Acad, Sci. USA 84:5277-5281) .
• [ 3 H]thymidine incorporation was measured as a marker for DNA synthesis after stimulating HUVEC with human recombinant VEGF.
• the 5.7 kb KDR/flk-l mRNA is constitutively expressed in HUVEC (Patterson et al., 1995, J. Biol. Chem. 270:2311-23118).
• the 7.0 kb fltl mRNA is also abundantly expressed by HUVEC in culture. Treatment of HUVEC with
• TNF- ⁇ (10 ng/ml) resulted in a decrease in the mRNA for both receptors (KDR/flk-l and fltl) that was evident by 6 hours, and that reached 28% and 33% of 0-hour values, respectively, for KDR/flk-l 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 alone; serum deprivation alone actually slightly induced both KDR/flk-l and fltl messages.
• TNF- ⁇ also decreased the messages for KDR/flk-l and fltl in a dose-dependent fashion (Figs. 10A and 10B) .
• TNF- ⁇ specifically downregulates the mRNA for the VEGF receptors KDR/flk-l and fltl in a time and dose-dependent fashion in human endothelial cells.
• TNF- ⁇ Decreased the Rate of Transcription, But Had No Effect on the Half-Life, of KDR/flk-l
• KDR/flk-l mRNA was measured in the presence of actinomycin D (ACD; 5 ⁇ g/ml) .
• ACD actinomycin D
• the KDR/flk-l mRNA half-life was 1.9 hours in the absence of TNF- ⁇ and increased slightly, to 2.6 hours, in the presence of TNF- ⁇ (Fig. 11) .
• the mRNA half-life of fltl was found not to be decreased by TNF- ⁇ in HUVEC.
• 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.
• TNF- ⁇ decreased the rate of KDR/flk-l gene transcription (measured in Phosphorimager units) to 40% of baseline, but had no effect on the transcription of ⁇ - actin.
• the TNF- ⁇ -induced decrease in KDR/flk-l mRNA was due to a decrease in the rate of transcription of the gene in HUVEC and not to a change in the stability of the mRNA.
• the Decrease in KDR/flk-l mRNA by TNF- ⁇ is Protein Synthesis-Dependent
• a rabbit anti-human KDR/flk-l antibody (Santa Cruz SC-xxx) 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; Millauer et al., 1994, Nature 367(6463) :576-579) .
• the DNA of the invention promotes endothelial cell-specific transcription of DNA sequences to which it is operably linked. These promoter sequences are useful 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 diseases such as cancer, e.g., solid tumors, and inflammatory diseases, e.g., rheumatoid arthritis and diabetic retinopathy, as described in Examples l and 2 below.
• the invention also provides methods for identifying compounds which (1) modulate TNF- ⁇ downregulation VEGF receptor (e.g., KDR/flk-l or fltl) gene expression (Example 3, below), or (2) modulate TNF- ⁇ inhibition of VEGF-induced endothelial cell proliferation (Example 4) .
• TNF- ⁇ downregulation VEGF receptor e.g., KDR/flk-l or fltl
• Compounds found to enhance TNF- ⁇ downregulation of expression of a VEGF receptor gene or 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-l or enhance TNF- ⁇ inhibition of VEGF-induced endothelial cell proliferation can be 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..
• hypoxic damage to heart tissue a progressive hypertension disease
• 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.
• identification of the cis-acting sequences in the KDR/flk-l gene required for downregulation by TNF- ⁇ provides the basis for additional therapeutic methods for these conditions.
• 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-l, in cells of the blood vessel wall, i.e., endothelial cells, for gene therapy of vascular diseases such as arteriosclerosis.
• heterologous genes e.g., genes which encode proteins other than KDR/flk-l
• endothelial cells e.g., endothelial cells
• the DNA or vector containing a sequence encoding a polypeptide of interest is introduced into endothelial cells which in turn produce the polypeptide of interest.
• 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.
• 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.
• heterologous proteins e.g., proteins which inhibit smooth muscle cell proliferation, e.g., interferon- ⁇ and atrial natriuretic polypeptide, may be 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).
• the DNA of the invention can be operably linked to sequences encoding cellular growth factors which promote angiogenesis, e.g., VEGF, acidic fibroblast growth factor, or basic fibroblast growth factor.
• 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.
• SEQ ID NO:l, 2, and 3 are preferably located 5' to the transcription start site, and SEQ ID NO:4 is located 3' of the transcription start site.
• these sequences may be in any order relative to the transcription start site provided that endothelial cell- specific promoter activity is preserved.
• the DNA of the invention may also be used in methods of antisense therapy.
• Antisense therapy may be carried out by administering to an animal, 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 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 sequence to signal the end of the sequence.
• Standard methods relating to antisense technology have been described (Melani et al., Cancer Res. 51:2897-2901, 1991).
• the antisense RNA binds to its target mRNA molecules within a cell, thereby inhibiting translation of the mRNA and down-regulating expression of the protein encoded by the mRNA.
• 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 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 angiogenesis, e.g., inflammatory diseases such as rheumatoid arthritis, and diabetic retinopathy.
• the expression of other endothelial cell proteins may also be inhibited in a similar manner.
• the DNA of the invention can be operably linked to 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 factor; and endothelial cell adhesion factors, such as ICAM-1 and VCAM-1 (Bennett et al., 1994, J. Immunol. 152:3530-3540) .
• the claimed DNA may be introduced into target cells of an animal, e.g., a patient, using standard vectors and/or 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 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.
• Standard 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 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 administered in a pharmaceutically acceptable carrier.
• Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration 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 animal.
• dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for intravenous administration of DNA is from approximately IO 6 to IO 22 copies of the DNA molecule.
• the compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA 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.
• Example 3 Identification of Compounds Which Modulate TNF- ⁇ Downregulation of VEGF Receptor (KDR/flk-l or fltl- Gene Expression
• TNF- ⁇ downregulates expression of KDR/flk-l and fltl genes, each of which encodes a VEGF receptor.
• potentiating TNF- ⁇ downregulation of KDR/flk-l or fltl expression can be useful in decreasing endothelial cell growth and, therefore, in inhibiting processes such as angiogenesis.
• inhibiting TNF- ⁇ downregulation of KDR/flk-l or fltl expression can be useful in increasing 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 enhances TNF- ⁇ -mediated inhibition of KDR/flk-l or fltl expression.
• a compound which blocks or enhances TNF- ⁇ -mediated inhibition of KDR/flk-l 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. 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.
• KDR/flk-l or fltl The screening of small, membrane-permeable organic molecules for the ability to modulate TNF- ⁇ downregulation of KDR/flk-l or fltl is carried out as follows.
• Cells expressing KDR/flk-l or fltl e.g., HUVEC are cultured in the presence of TNF- ⁇ and the candidate compound.
• Cells containing the KDR/flk-l (or 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-l (or fltl) expression is compared to the level in cells cultured with TNF- ⁇ , but without the candidate compound.
• KDR/flk-l (or fltl) expression indicates identification of a compound which blocks TNF- ⁇ downregulation of KDR/flk-l (or fltl) expression.
• a compound which blocks TNF- ⁇ downregulation of KDR/flk-l (or fltl) expression.
• angiogenesis is desired involves interruption of cardiac blood flow. In such situations, TNF- ⁇ may hinder the natural angiogenic process which could control damage to cardiac tissue.
• a decrease in KDR/flk-l (or fltl) expression indicates identification of a compound which potentiates TNF- ⁇ downregulation of KDR/flk-l expression.
• a compound can be used to treat conditions in which decreased endothelial cell growth or angiogenesis is desired. For example, the growth of a tumor may be inhibited by treatment with such a compound.
• the therapeutic compounds identified using the method of the invention may be administered to a patient by any appropriate method for the particular compound. 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 the site where the effect of the compound is desired.
• 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 apply most generally to wounds on the skin.
• Salves containing the compound can be applied topically to induce new blood vessel formation locally, thereby improving oxygenation of the area and hastening wound healing.
• Therapeutic doses are determined specifically 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 Compounds Which Modulate the Effect of TNF- ⁇ on VEGF-induced Epithelial Cell Growth
• TNF- ⁇ inhibits VEGF-induced proliferation of endothelial cells (see, e.g.. Fig. 8 and the corresponding text) .
• compounds which 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.
• endothelial cells can be cultured 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 [ 3 H]thymidine.
• TNF- ⁇ -responsive element the cis-acting element in the KDR/flk-l gene required for TNF- ⁇ -mediated downregulation (the TNF- ⁇ -responsive element) , as well as the trans- 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 precise sequences of the cis-acting element can be carried out using standard promoter deletion analysis.
• a construct including KDR/flk-l sequences that confer TNF- ⁇ downregulation to a 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.
• Promoter deletion constructs such as those described above, can be used to begin this analysis.
• point mutations can be introduced into the element, using standard methods, in the context of the full promoter.
• the KDR/flk-l 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-l.
• standard DNA footprinting and/or native gel-shift analyses can be carried out.
• the element can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library.
• the trans- acting factor modulation of its binding to the TNF- ⁇ -responsive element in the KDR/flk-l gene can be pursued, beginning with, for example, screening for inhibitors of trans-acting factor binding.
• Enhancement of TNF- ⁇ downregulation of KDR/flk-l 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 therapy) .
• the active form of the trans- acting factor is a dimer, dominant-negative mutants of the trans-acting factor could be made in order to inhibit its activity.
• TNF- ⁇ -responsive element in the KDR/flk-l gene, and its corresponding trans-acting factor
• further components in the TNF- ⁇ pathway of KDR/flk-l 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 cell growth and angiogenesis.
• the methods described in this example can also be carried out with the fltl gene.
• expression of KDR/flk-l in endothelial 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.
• transcription can be inhibited using dominant negative mutants of transcription factors, e.g., a dominant negative mutant of AP-2 which binds to the AP-l binding site but fails to activate transcription.
• compounds which downregulate production of transcription factors e.g., retinoic acid or dexamethasone which downregulate production of AP-2 and NFKB, can be administered to inhibit angiogenesis by inhibiting expression of KDR/flk-l.
• MOLECULE TYPE DNA
• MOLECULE TYPE DNA
• MOLECULE TYPE DNA

Applications Claiming Priority (5)

Publications (1)

Family

ID=27051386

Family Applications (1)

Country Status (5)

Families Citing this family (85)

Family Cites Families (3)

• 1996
  • 1996-06-21 JP JP9503975A patent/JPH11509088A/ja active Pending
  • 1996-06-21 EP EP96921746A patent/EP0833907A1/de not_active Ceased
  • 1996-06-21 WO PCT/US1996/010725 patent/WO1997000957A1/en not_active Application Discontinuation
  • 1996-06-21 AU AU62884/96A patent/AU723325B2/en not_active Ceased
  • 1996-06-21 CA CA002225460A patent/CA2225460A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events