EP1144606A2

EP1144606A2 - Human nucleic acid and protein sequences obtained from endothelial cells

Info

Publication number: EP1144606A2
Application number: EP00909340A
Authority: EP
Inventors: Karl-Heinz Thierauch; Jens Glienke; Bernd Hinzmann; Christian Pilarsky
Original assignee: Schering AG
Current assignee: Bayer Pharma AG
Priority date: 1999-03-09
Filing date: 2000-03-08
Publication date: 2001-10-17
Also published as: WO2000053734A3; AU3165500A; JP2002537832A; WO2000053734A2

Abstract

The invention relates to nucleic acid sequences - mRNA, cDNA, genome sequences - obtained from human endothelial cells and coding for gene products or parts thereof, as well as to their use. The invention also relates to the polypeptides obtained by means of said sequences and to their use.

Description

Human nucleic acid and protein sequences from EndoϊhelzeTlen

The invention relates to nucleic acid sequences -mRNA, cDNA, genomic sequences- from tissue of human endothelial cells, which code for gene products or parts thereof, and their use. The invention further relates to the polypeptides obtainable via the sequences and their use.

Angiogenesis is a process that can be observed in the adult in the cyclical processes of reproduction in women, in wound healing and in various pathological situations, such as. B. tumor growth, rheumatic diseases, endometriosis, in collateral formation in the heart and in the periphery, etc.

Persistent angiogenesis can be the cause of various diseases such as psoriasis, arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma, eye diseases such as diabetic retinopathy, neovascular glaucoma, renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombogenic microangiopathic syndrome, transplant rejections and glomerulopathy, fibrotic Diseases such as cirrhosis of the liver, mesangial cell proliferative diseases and atherosclerosis or lead to an exacerbation of these diseases.

If it were possible to induce or inhibit angiogenesis, several diseases could be treated fundamentally. To do this you would have to

Know genes or the nucleic acid sequences relevant for angiogenesis.

It is not yet known which genes or nucleic acid sequences or parts thereof are relevant to angiogenesis.

Nucleic acid sequences relevant to angiogenesis have now been found. These sequences have either not been described to date or are only known as rodent nucleic acid sequences, but without evidence of angiogenesis. Further sequences are described as human genes or parts thereof, but not with regard to possible angiogenesis-related properties.

To search for genes relevant to angiogenesis, endothelial cells were obtained from the foreskins of adults, which were cultivated in two ways:

a) on a rat tail collagen matrix in subconfluent density

and

b) on a gel made of extracellular matrix (Matrigel).

Under culture form a), the cells form the classic cobblestone-like monolayers.

Under culture form b), the cells form network-like structures with tubular structures.

The cell culture form a) represents an early state of angiogenesis with a predominantly proliferative phenotype.

The cell culture form b) represents a model for a later phase of angiogenesis, in which the differentiation of the endothelial cells leads to the formation of tubular structures. These structures are a prerequisite for blood flow that is separated from the tissue surface.

MRNA is isolated from both cell culture forms, transcribed into cDNA, and with a restriction endonuclease into fragments of the size from 200 to 1500 bp cut. The differentially occurring fragments of both states were amplified using a subtractive PCR technique. They were built into vectors and cloned. The clones were first sequenced and then their sequences were completed using bioinformatory techniques.

Using a quantitative PCR technique described in the literature (Pilarsky et al., 1998, see description of the experiment), it was first examined whether the genes are differentially expressed in the two culture states. The expression of the 23 kDalton protein (see.

Experiment description) used as an internal marker. In the differential expression, ratios of 2-7 times occurred.

The nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59 are found that play a role as candidate genes in angiogenesis.

The invention thus relates to nucleic acid sequences encoding a gene product or a part thereof, comprising

a) a nucleic acid sequence selected from the group of the nucleic acid sequences Seq ID Nos. 1 to Seq. ID No. 59

b) an allelic variation of the nucleic acid sequences mentioned under a)

or

c) a nucleic acid sequence which is complementary to the nucleic acid sequences mentioned under a) or b). The invention further relates to nucleic acid sequences according to one of the

Sequences Seq ID Nos. 1 to Seq. ID No. 59 or a complementary or allelic variant thereof and the nucleic acid sequences thereof, one

Have 90% to 95% homology to a human nucleic acid sequence.

The invention also relates to the nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59, which are expressed in increased endothelial cell tissue

The invention further relates to nucleic acid sequences comprising a part of the above-mentioned nucleic acid sequences, in such a sufficient size that they can be combined with the sequences Seq. ID No. 1 to Seq. ID No. 59 hybridize.

The nucleic acid sequences according to the invention generally have a length of at least 50 to 3000 bp, preferably a length of at least 150 to 2800 bp, particularly preferably a length of 150 to 2600 bp.

With the sequences Seq. ID No. 1 to Seq. ID No. 59, expression cassettes can also be constructed in accordance with current process practice, with at least one of the nucleic acid sequences according to the invention together with at least one control or regulatory sequence known to the person skilled in the art, such as, for example, on the cassette. B. a suitable promoter is combined. The sequences according to the invention can be inserted in sense or antisense orientation.

A large number of expression cassettes or vectors and promoters are known in the literature which can be used.

Expression cassettes or vectors are to be understood: 1. bacterial, such as. B., phagescript, pBs, φX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),

2. eukaryotic, such as B. pWLneo, pSV2cat, pOG44, pXT1, pSG

(Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).

Suitable control or regulatory sequence means suitable promoters. Two preferred vectors are the pKK232-8 and the PCM7 vector. Specifically, the following promoters are meant: lad, lacZ, T3, 17, gpt, lambda PR, trc, CMV, HSV thymidine kinase, SV40, LTRs from retrovirus and mouse metallothionein-I.

The DNA sequences on the expression cassette can encode a fusion protein which comprises a known protein and a biologically active polypeptide fragment.

The expression cassettes are also the subject of the present invention.

The nucleic acid sequences according to the invention can also be used to produce full-length genes. The available genes are also the subject of the present invention.

The invention also relates to the use of the nucleic acid sequences according to the invention and the gene fragments obtainable from the use.

The nucleic acid sequences according to the invention can be brought into host lines with suitable vectors, in which the heterologous part contains the genetic information contained on the nucleic acid sequences which is expressed. The host cells containing the nucleic acid sequences are also the subject of the present invention.

Suitable host cells are e.g. B. prokaryotic cell systems such as E. coli or eukaryotic cell systems such as animal or human cells or yeasts.

The nucleic acid sequences according to the invention can be used in sense or antisense form.

The polypeptides or their fragments are produced by cultivating the host cells in accordance with common cultivation methods and then isolating and purifying the peptides or fragments, likewise by means of conventional methods. The invention further relates to nucleic acid sequences which encode at least a partial sequence of a biologically active polypeptide.

Furthermore, the present invention relates to partial polypeptide sequences, so-called ORF (open reading frame) peptides, which are expressed by the inventive partial sequences.

The invention further relates to the polypeptide sequences which have at least 80% homology, in particular 90% homology to the polypeptides.

The invention also relates to antibodies which are directed against a polypeptide or a fragment which are derived from the nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59 can be encoded.

Antibodies are to be understood in particular as monoclonal antibodies.

The polypeptides encoded by the nucleic acid sequences according to the invention can also be used as a tool for finding active substances angiogenic diseases can be used, which is also the subject of the present invention.

The present invention also relates to the use of the nucleic acid sequences according to the sequences Seq. ID No. 1 to Seq. ID No. 59 for the expression of polypeptides that can be used as tools for finding active substances against angiogenic diseases.

The invention also relates to the use of the nucleic acid sequences Seq according to the invention. ID No. 1 to Seq. ID No. 59 expressed polypeptides as drugs in gene therapy for the treatment of angiogenic diseases, or for the manufacture of a drug for the treatment of angiogenic diseases.

The nucleic acids according to the invention or the proteins expressed via these nucleic acids can thus be used either alone or in formulation as a medicament for the treatment of psoriasis, arthritis, such as rheumatoid arthritis, hemangioma, angiofribroma, eye diseases, such as diabetic retinopathy, neovascular glaucoma, kidney diseases, such as glomerulonephritis, diabetic Nephropathy, malignant nephrosclerosis, thrombic microangiopatic syndromes, transplant rejections and glomerulopathy, fibrotic diseases such as cirrhosis of the liver, mesangial cell proliferative diseases, atherosclerosis and injuries to the nerve tissue are used.

The invention also relates to medicaments which contain at least one polypeptide sequence which is derived from the nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59 can be expressed.

The nucleic acid sequences according to the invention found can also be genomic or mRNA sequences. The invention also relates to genomic genes, their promoters, enhancers, silencers, exon structure, intron structure and their splice variants, obtainable from the cDNAs of the sequences Seq. ID No. 1 to Seq. ID No. 59, and their use together with suitable regulatory elements, such as suitable promoters and / or enhancers.

With the nucleic acids (cDNA sequences) according to the invention, genomic BAC, PAC and cosmid libraries are screened and specifically human clones are isolated via complementary base pairing (hybridization). The BAC, PAC and cosmid clones isolated in this way are hybridized with the aid of fluorescence in situ hybridization to metaphase chromosomes and corresponding chromosome sections are identified on which the corresponding genomic genes lie. BAC, PAC and cosmid clones are sequenced in order to elucidate the corresponding genomic genes in their complete structure (promoters, enhancers, silencers, exons and introns). BAC, PAC and cosmid clones can be used as independent molecules for gene transfer.

The invention also relates to BAC, PAC and cosmid clones containing functional genes and their chromosomal localization, according to the sequences Seq. ID. No. 1 to Seq. ID No. 59, for use as a vehicle for gene transfer.

Meanings of technical terms and abbreviations

Nucleic acids = nucleic acids are to be understood in the full invention: mRNA, partial cDNA, full length cDNA and genomic genes (chromosomes). ORF = Open Reading Frame, a defined sequence of

Amino acids that can be derived from the cDNA sequence.

The following examples illustrate the preparation of the nucleic acid sequences according to the invention, without the

To limit the invention to these examples and nucleic acid sequences.

example 1

1. Search for candidate genes relevant to angiogenesis

1.1 Cells used

Primary, human, microvascular endothelial cells (MVEC) were prepared from human foreskins and selected using biotinylated anti CD31 (PECAM) antibodies (reference).

Culture conditions: 37 ° C, 5% C0 ₂

Medium: M199, 10% FCS, 10% human serum, 6μg / ml ECGF, 1mM

Sodium pyruvate, 3 U / ml heparin, 100 U / mIPenicilin, 100μg / ml streptomycin, 1x non-essential amino acids

1.2 Cultivation and RNA preparation

For the culture form a), the cells are cultivated on plastic coated with collagen I. For the culture form b), the cells are applied on a gel made of extracellular matrix proteins. The Matrigel (Becton Dickinson) used was diluted 1 to 1 with M199 medium, poured into the culture vessel (60μl / cm2) in the cold and at 37 ° C for 30 min. gelled. The lines were then applied. For culture forms a) and b), MVEC were applied at a density of ² × 10 ⁴ / cm ² and incubated for 7 hours at 37 ° C., 5% CO ₂ . The total RNA preparation was made after the guanidinium thiocyanate

Method followed by centrifugation through a cesium chloride cushion (Sambrook J., Fritsch E.F. and Maniatis T .; 1989, Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press). The polyA ⁺ RNA selection was carried out using oligo (dT) cellulose columns (mRNA

Purification Kit, Pharmacia Biotech).

1.3 Creation of subtractive cDNA banks

The subtraction was carried out according to the method of Diatchenko et al. (Proc. Natl. Acad. Sci. U.S.A., 1996 Jun 11, 93: 6025-30) using the PCR-Select cDNA Subtraction Kit.

The polyA + RNA that contains the target sequences is called the tester, and the polyA + RNA to be subtracted from this is called the driver.

Two subtractions were carried out, the polyA + RNA of culture form a) and the polyA + RNA of culture form b) serving as testers. The following test description is only an example of a subtraction.

1.4 Synthesis of double-stranded cDNA (ds cDNA)

A double-stranded cDNA synthesis is carried out for both the tester and the driver.

1st strand synthesis

The strand synthesis is carried out with the following approach: polyA + RNA 2μg cDNA synthesis primer (10μM) 1 μl water add 5μl The reactions are for 2 min. incubated at 70 ° C and then on ice for 2 min.

The following was added to each reaction:

5x first-strand buffer (250mM Tris-HCL, pH8, 330mM Mg-Chloride, 375mM KCI)

2μl 10mM dNTP

1 μl of water

1 μl MMLV reverse transcriptase (200 U / μl)

1 ul

The reactions were incubated at 42 ° C for 90 minutes and then on ice for 2 minutes.

2nd strand synthesis The 2nd strand synthesis was carried out using the following approach: 1st strand synthesis 10μl

Water 48.4μl

5x Second-strand buffer (500mM KCL, 50mM ammonium sulfate, 25mM Mg chloride, 0.75mM ß-NAD, 100mM Tris-HCL, pH7.5, 0.25mg / ml BSA) 16μl

10mM dNTP 1, 6μl

20x Second-strand enzyme cocktail (DNA Polymerase 1 6U / μl RNase H 0.2U / μl, E. coli DNA Ligase 1, 2U / μl) 4μl

The reactions were incubated at 16 ° C for 2 h.

T4 DNA polymerase was added to each reaction as follows: T4 DNA polymerase 3U / μl 2μl

The reactions were incubated at 16 ° C for 30 min. The reactions were stopped with EDTA, the solution having the following composition:

20x EDTA / glycogen mix (200mM EDTA, 1 mg / ml glycogen) 4μl

Phenol / chloroform extraction and ethanol precipitation were performed for each reaction. The pellets were resuspended in 50 μl water.

1.5 Rsa I digestion of the ds cDNA

Rsa I digestion was performed for both the tester and the driver. The following solutions were used for this:

ds cDNA 43.5μl 10x Rsa I restriction buffer (100mM bis tris propane-HCl, pH7.0, 100mM Mg-Clorid, 1mM DTT) 5μl

Rsa l (10U / μl) 1.5 μl

The reactions were incubated at 37 ° C for 90 min.

The reactions were then stopped with EDTA, the solution having the following composition:

20x EDTA / glycogen mix (200mM EDTA 1 mg / ml glycogen) 2.5μl

Phenol / chloroform extraction and ethanol precipitation were then performed for each reaction. The resulting pellets were resuspended in 5.5μl water for further processing. 1.6 Adapter ligation to Rsa I digested the tester cDNA

The tester cDNA was divided into 2 fractions. An adapter was ligated to each tester fraction. The concentrations of the substances used for the two testers are listed in detail in the table below.

The reactions were incubated at 16 ° C. overnight and then stopped with EDTA (20 × EDTA / glycogen mix, 1 μl (200 mM EDTA, 1 mg / ml glycogen)).

The reactions were incubated for 5 min at 72 ° C.

1.7 Subtractive hybridizations The drivers and testers were then hybridized with each other in two steps.

Hybridization

The first hybridization was carried out for the two reactions with the solutions and compounds listed in the table below.

The reactions were incubated for 90 seconds at 98 ° C. and then directly for 8 hours at 68 ° C.

1. Hybridization:

For the second hybridization, reactions 1 and 2 were mixed and freshly denatured drivers were added as follows:

Driver 1 μl

4x hybridization buffer 1 μl

Water 2μl 1 μl of this mixture was incubated at 98 ° C. for 90 seconds and then fused with reaction 1 and reaction 2 as quickly as possible.

The 2nd hybridization was incubated at 68 ° C overnight. Then 200 .mu.l dilution buffer (20 mM HEPES-HCl (pH 8.3), 50 mM NaCl, 0.2 mM EDTA (pH 8.0)) were added for the second hybridization. Then the 2nd

Hybridization incubated for 7 min at 68 ° C. The batch prepared in this way was then used for the PCR.

Differentially expressed fragments in the subtracted cDNA pools were selectively amplified using two successive PCRs. The 1st PCR was carried out with the following approach:

10x PCR buffer (400mM Tricine-KOH, pH9.2, 150mM KOAc, 35mM MG (OAc) 2, 37.5μg / ml BSA) 2.5μl 10mM dNTP 0.5μl PCR Primer 1 (10μM) 1 μl

50x Advantage cDNA polymerase 0.5μl diluted 2nd hybridization 1 μl water 19.5μl

The PCR program was carried out as follows: 75 ° C., 5 min

Loop 94 ° C, 30 sec

66 ° C, 30 sec

72 ° C, 90 sec

A total of 27 cycles were carried out.

The second PCR was carried out using the following approach:

10x PCR buffer 2.5μl 10mM dNTP 0.5μl nested PCR primer 1 (10μM) 1 μ | nested PCR primer 2R (1 OμM) 1 _μ | 50x Advantage cDNA polymerase 0.5μl

PCR product 0.1 ul

H2O 19.4μl I

The PCR program was carried out as follows: 94 ° C, 30 sec

68 ° C, 30 sec

72 ° C, 90 seconds in total, 12 cycles were carried out.

The subtraction efficiency was checked by a semi-quantitative PCR for a known unregulated gene (SH3P18). There was a reduction in the subtracted cDNA pool by a factor of 150-200.

2. Ligation of the subtracted cDNA pools in pUC 18

The forward and backward subtracted cDNA pools were ligated in pUC 18 Sma l / BAP (SureClone Ligation Kit, Pharmacia Biotech) and then cloned into chemically competent E. coli DH5α.

The fragments of the subtracted cDNA pools were filled in to blunt ends and phosphorylated. The following compositions were used for this:

Subtracted cDNA pool 1, 5μg

Klenow fragment 1 ul

10x blunting / kinasing buffer 2μl

Polynucleotide kinase 1 ul

Water add 20μl The reactions were incubated for 30 min at 37 ° C., then purified via PCR purification columns and eluted in 30 μl water. The DNA concentration was then determined by means of OD measurement.

^

2.1 Ligation in pUC 18

The ligation in pUC 18 was carried out using the following approach:

0 Blunt-ended cDNA pool 50ng pUC 18 Sma l / BAP (50ng / μl) 1 _μ |

2x ligation buffer 10μl

DTT 1 μ |

T4 DNA Ligase (6U / μl) 3μl 5 water add 20μl

The reactions were incubated overnight at room temperature.

0 2.2 Transformation of the ligations in E. coli DH5α

The ligations were transformed into chemically competent E. coli DH5α. The transformed cells were spread on 2YT agarose plates with 100μg / ml ampicilin, 625μM IPTG and 0.005% X-Gal and grown overnight at 5 37 ° C.

Colony PCR with vector primers (M13 standard primer) was carried out on 17 randomly selected white clones. 15-16 clones showed inserts with a size distribution that corresponded to that of the cDNA pool used. 0 For each subtraction, 1536 clones were placed in 384-well plates with 50μl 2YT,

IxHMFM, 100μg / ml ampicilin transferred per well. The filled 384-well plates were incubated overnight at 37 ° C and could then be stored at -80 ° C.

3. Production of colony filters:

The 1536 clones of a subtractive cDNA library were inoculated onto a Hybond Nylon N + membrane (Amersham). The membrane was placed on a 2YT agarose plate with 100μg / ml ampicilin and incubated overnight at 37 ° C. The membrane was in with the colony side up for 4 min

Denaturing solution (0.5M NaOH, 1.5M NaCI) soaked Whatman 3MM paper. The membrane was then incubated for 4 min on Whatman 3MM paper soaked in neutralizing solution (1 M Tris-HCl (pH7.5), 1.5M NaCl). The membrane was then treated with Proteinase K for 1 h at 37 ° C. For this purpose, the membrane was immersed in 300ml Proteinase K buffer (50mM NaCl, 5mM EDTA, 10mM Tris-HCl (pH8), 50mg / ml Proteinase K). Finally, the membrane was dried at 80 ° C. for 3 hours and was then used for the hybridizations.

4. Differential hybridization:

In order to detect the differential expression of the cloned fragments, a differential hybridization was carried out on colony filters of the subtractive cDNA banks with the aid of a PCR select differential screening kit.

For a specific hybridization of the forward and backward subtracted cDNA pools to the subtractive cDNA bank colony filters, it was necessary to remove the adapter sequences in the hybridization sample. As hybridization samples for the Rsa I restriction of the subtracted cDNA

Pools were used: cDNA pool 28μl

10x Rsa I restriction buffer (100mM bis Tris propane-HCl, pH7.0 100mM Mg chloride, 1mM DTT) 3μl

Rsa l (10U / μl) 2μl

The reactions were incubated at 37 ° C. for 5 hours and then purified using PCR cleaning columns and eluted in 30 μl of water. The DNA concentration was determined by means of OD measurement.

5. Radioactive labeling of the subtracted cDNA pools

The radioactive labeling of the subtracted cDNA pools was carried out using the following approach: cDNA Pooi 150ng in 9μl

Reaction buffer, - dCTP (333mM Tris-HCl, pH8, 33.3 Mg chloride, 10mM 2-mercaptoethanol, 170μM dATP, 170μM dGTP, 170μM dTTP) 3μl

Random Primer Mix (0.9mg / ml random nonamers, 50mM Tris-HCl, pH7.5, 10mM Mg chloride, 1mM DTT, 50μg / ml BSA) 2μl

AP32 dCTP 3μl

Klenow fragment (3U / μl) 1, 5μl

The reactions were incubated at 37 ° C. for 1 h, then purified using PCR cleaning columns and eluted in 30 μl of water. The specific activity of the reactions was determined to ensure that the same amount of labeled DNA was used in both hybridization reactions. 6. Prehybridization and hybridization of the filters and

Hybridization samples

The following solutions were used for the hybridizations:

20x SSC 50μl

Blocking solution (10mg / mi sheared salmon sperm DNA,

0.3mg / ml complementary oligos to adapters) 50μl

The solution was incubated for 5 minutes at 98 ° C., then placed on ice for 5 minutes and mixed with 5 ml of express hybridization solution. This solution was then prehybridized in the hybridization bottle with the filter at 72 ° C. for 1 h.

The hybridization samples were also mixed with the following solution: 20x SSC 50μl

Blocking solution (10mg / ml sheared salmon sperm DNA, 0.3mg / ml complementary oligos to adapters) 50μl

The mixture was then incubated for 5 minutes at 98 ° C. and for 2 minutes on ice. The hybridization samples were then added to the filter in the hybridization bottles and hybridized overnight at 72 ° C.

Then the procedure was as follows:

a) 4x 20min at 68 ° C with preheated 2xSSC, 0.5% SDS

b) 2x 20 min at 68 ° C with preheated 0.2xSSC, 0.5% SDS

c) then exposure in phosphor imager cassettes for 22 hours at room temperature 7. Evaluation of the differential hybridizations

The hybridizations were evaluated on a phosphor imager.

A clone was classified as differentially expressed if it only showed a detectable hybridization signal with the forward-subtracted cDNA pool or if the signal strength with the forward-subtracted cDNA pool was at least 5 times greater than with the backward-subtracted cDNA pool.

8. Confirmation of differential expression using semi-quantitative RT-PCR

To the differential expression of the clones with differential

To confirm the hybridization result, sequences were selected at random and appropriate primers were prepared.

The comparative multiplex RT-PCR according to Pilarsky et al. (The Prostate 36: 85-91 (1998)) applied. Primers for the 23kD highly basic protein were used as the internal standard. The sequence of interest and the standard fragment were amplified simultaneously in one reaction for a different number of cycles. The PCR products were then separated on a 6% sequencing gel and analyzed and quantified using software. First, the number of cycles was determined for which both the standard fragment and the sequence of interest were linearly amplified and which was then used for the quantifying PCR. Different RNA preparations were used for the quantifying RT-PCR and 3 reactions were set up. It was possible to use differential for 90% of the examined sequences

Hybridization result a difference in expression was found that was greater than a factor of 2.

9. Automatic extension of the nucleic acid sequences found

In order to obtain as much sequence information as possible for each differentially expressed clone, an automatic extension of the initial sequence was carried out using all available EST sequences.

The sequence S is automatically extended in three steps:

1. Determination of all sequences homologous to S from the total amount of all available ESTs from the LifeSeq database (as of October 1997) using the BLAST algorithm (Altschui S., Gish W., Miller W., Myers E., Lipman D. ( 1990) J. Mol. Biol., 215, 403-410).

2. Assembly of these sequences using the standard program GAP4 (Bonfield J., Smith K., Staden R. (1995), Nucleic Acids Research 23, 4992-4999).

3. Calculation of a consensus sequence from the assembled sequences.

Now an attempt is made to extend the consensus sequence in the same way. This iteration is continued with the consensus sequence obtained in each case until no further extension is possible.

10. Found nucleic acid sequences Analogously to the procedure described in 1 to 9, z. B. found the following sequences, some of which are overexpressed several times in culture form a) or culture form b) of the endothelial cells.

These nucleic acid sequences are also the subject of the present invention.

The possible function of these gene areas concerns angiogenesis.

The result is shown in Table I below:

TABLE

a), b) = forms of culture

11. Expression analysis

In order to investigate whether the regulated sequences are also involved in the formation of new blood vessels in vivo, their expression in human placental tissue of the δ week with high angiogenesis activity and in human placenta tissue of the 9th month with little angiogenesis was examined Activity determined. A stronger expression in the 8 week placenta was taken as an indication of an angiogenesis-relevant function of the sequence. A stronger expression in the 9 month old placenta was assessed as an indication of a vascular stabilizing function of the sequence. A semi-quantitative RT-PCR technique was used for this, the comparative multiplex RT-PCR. In this method, the expression of the sequence of interest is based on the expression of a non-differentially regulated so-called “household gene”, here the 23 kD highly basic protein. The expression of the VEGF receptor KDR was determined as a positive control. This endothelial cell-specific gene is known that it is on angiogenetically active endothelium is upregulated. Accordingly, a significantly increased KDR expression was detected in the 8 week placenta compared to the 9 month old placenta. The results are summarized in Table II

table

Sequence MVEC, placenta proliferating placenta 8th week 9th month

1 *** *** _

2 nd

3 **** * ★ * *

4 ** ** **

5 *** *** *

6 ** **

7 * **** * *

8 n.

9 ** * ***

10 *** **** *

11 ** - **

12 n.

13 - ** _

14 **** *** *

15 *** # * _

16 - - _

17 *** ** _

18 **** *** _

19 nd

20 *** **** **

21 nd

22 ** ** **

23 * **** **

24 *** **.

25 ** * *

26 n.

27 ** * *

28 ** * -

29 * * *

30 **** *** -

31 ** *** ***

32 **** *** *

33 *** ** **

34 ** ** - • ***

In the table mean:

**** = very strong expression

*** = strong expression

** = medium expression

= weak expression

= Expression below the detection limit n. D. = not carried out

The nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59 of the candidate genes determined are described in the following sequence listing. Due to the clear overexpression of sequence 34 in the tubular MVEC (> 8x) and a weak homology to thrombospondin-2, a gene that plays an important role in the maturation of the blood vessel system, sequence 34 became the further sequence for further analysis selected. Starting from the identified partial sequence, the complete mRNA sequence for sequence 34 was determined by means of 5 'and 3' RACE experiments. With a length of 6011 bp, the size for sequence 34 agrees very well with the size (~ 6 kb) determined in a Northern hybridization. The complete mRNA sequence contains an open reading frame that codes for 1036 amino acids. The derived protein has a molecular weight of ~ 114kD, is high in cysteine (12.5% cysteine content) and has a hitherto unique domain structure. The protein has an N-terminal signal peptide, part of a thiol protease domain, an RGD motif, 6 Von Willebrand factor type C domains, a potential transmembrane domain and 5 possible N-glycosylation sites. The genomic localization of sequence 34 to Chr. 2p21 and the complete intron / exon structure were also determined. Due to the domain structure of the protein, a type I transmembrane orientation can be assumed, with a long extracellular N-terminus and a short intracellular C-terminus. To test this, a rabbit antiserum was produced which is directed against a peptide from the extracellular part of the protein. With the help of this antiserum it could be shown that the protein actually has a type I transmembrane orientation.

This anti-sequence 34 serum was used for immunohistological examinations on sections of an ovarian carcinoma or a prepuce. It was shown that sequence 34 is expressed in the tumor by endothelial cells, but not by stromal cells. In contrast, no sequence 34 expression could be detected in the prepuce. Sequence 34 is therefore expressed in the angiogenetically active tumor endothelium of the ovarian carcinoma examined, but not in the resting endothelium of normal tissue. These results were confirmed by in situ hybridizations at the mRNA level. In order to determine the expression profile for sequence 34, Northem hybridization was carried out on various human tissues. This revealed an expression pattern for sequence 34, which speaks for a specific function of the protein in endothelial cells, with the strongest expression in placenta, followed by the kidney, heart and lung.

In order to test whether sequence 34 in the in vitro model on Matrigel has an important function in tubule formation, antisense oligonucleotides were produced. An oligonucleotide was found that inhibits sequence 34 expression. This oligonucleotide was not toxic to the cells and did not lead to an altered proliferation behavior of the treated cells. Endothelial cells that were transfected with this oligonucleotide, on the other hand, showed a dramatic inhibition of tubule formation on Matrigel (> 20% of the control value) in comparison to untransfected and transfected cells with a control oligonucleotide. Sequence 34 thus contributes significantly to the formation of capillary-like structures. These results are in the

Consistent with the data from the expression analysis in the two placenta samples for sequence 34. The stronger expression of sequence 34 in the 9-month-old placenta was taken as an indication of a vessel-stabilizing function of the sequence. The antisense oligonucleotide data clearly show that sequence 34 does not play a role during endothelial cell proliferation, but is significantly involved in the formation of stable capillary structures.

The invention thus relates in particular to the sequence Seq ID No. 34 and their use to form stable capillary structures. Furthermore, this sequence and the protein sequence derived therefrom also relates to the use, either alone or in formulation as a medicament for the treatment of psoriasis, arthritis, such as rheumatoid arthritis, hemangioma, angiofribroma, eye diseases, such as diabetic retinopathy, neovascular glaucoma, kidney diseases, such as glomerulonephritis, diabetic Nephropathy, malignant nephrosclerosis, thrombic microangiopatic syndromes,

Transplant rejection and glomerulopathy, fibrotic diseases such as Cirrhosis of the liver, mesangial cell proliferative diseases, atherosclerosis and nerve tissue injuries.

Claims

claims

1 . A nucleic acid sequence encoding a gene product or a part thereof

a) a nucleic acid sequence selected from the group Seq ID No. 1 to Seq. ID No. 59

b) an allelic variation of the nucleic acid mentioned under a)

Sequences

or

c) a nucleic acid sequence which is complementary to the nucleic acid sequences mentioned under a) or b).

2. A nucleic acid sequence according to one of the sequences Seq ID No. 1 to Seq. ID No. 59 or a complementary or allelic variant thereof.

Nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59, characterized in that they are expressed in increased endothelial cell tissue.

BAC, PAC and cosmid clones containing functional genes and their chromosomal localization, corresponding to the nucleic acid sequences Seq. ID. No. 1 to Seq. ID No. 59, for use as a vehicle for gene transfer.

5. A nucleic acid sequence according to claims 1 to 4, characterized in that it has a 90% homology to a human nucleic acid sequence.

6. A nucleic acid sequence according to claims 1 to 4, characterized in that it has a 95% homology to a human nucleic acid sequence.

7. A nucleic acid sequence comprising a part of the nucleic acid sequences mentioned in claims 1 to 6, in such a size that they hybridize with the sequences according to claims 1 to 6.

8. A nucleic acid sequence according to claims 1 to 7, characterized in that the size of the fragment has a length of at least 50 to 3000 bp.

9. A nucleic acid sequence according to claims 1 to 7, characterized in that the size of the fragment has a length of at least 150 to 2800 bp.

10. A nucleic acid sequence according to claims 1 to 7, characterized in that the size of the fragment has a length of at least 150 to 2600 bp.

11. A nucleic acid sequence according to any one of claims 1 to 10, which encodes at least a partial sequence of a biologically active polypeptide.

12. An expression cassette comprising a nucleic acid fragment or a sequence according to any one of claims 1 to 10, together with at least one control or regulatory sequence.

13. An expression cassette comprising a nucleic acid fragment or a sequence according to claim 12, wherein the control or regulatory sequence is a suitable promoter.

14. An expression cassette according to one of claims 12 and 13, characterized in that the DNA sequences on the cassette encode a fusion protein which comprises a known protein and a biologically active polypeptide fragment.

15. Use of the nucleic acid sequences according to claims 1 to 11 for the production of full-length genes.

16. A DNA fragment comprising a gene obtainable from the use according to claim 15.

17. Host cell containing, as a heterologous part of its expressible genetic information, a nucleic acid fragment according to one of claims 1 to 11.

18. Host cell according to claim 17, characterized in that it is a prokaryotic or eukaryotic cell system.

19. Host cell according to one of claims 17 or 18, characterized in that the prokaryotic cell system E. coli and the eukaryotic cell system is an animal, human or yeast cell system.

20. A method for producing a polypeptide or a fragment, characterized in that the host cells are cultivated according to claims 17 to 19.

21. An antibody directed against a polypeptide or a fragment which is derived from the nucleic acids of the sequences Seq. ID No. 1 to Seq. ID No. 59 is encoded, which is obtainable according to claim 20.

22. An antibody according to claim 21, characterized in that it is monoclonal.

23. Polypeptide sequence expressed by one of the nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59.

24. Polypeptide sequences according to claim 23, with at least 80% homology to these sequences.

25. Polypeptide sequences according to claim 23, with at least 90% homology to these sequences.

26. Polypeptide sequence, characterized in that it has the sequence Seq ID No. 34 includes.

27. Use of the polypeptide sequences according to claims 23 to 26 as tools for finding active substances against angiogenic

Diseases.

28. Use of the nucleic acid sequences according to the sequences Seq. ID No. 1 to Seq. ID No. 59 for the expression of polypeptides that can be used as tools for finding active substances against angiogenic diseases.

29. Use of the nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 59 in sense or antisense form.

30. Use of the polypeptide sequences according to claims 23 to 26 as a medicament in gene therapy for the treatment of angiogenic diseases.

31 Use of the polypeptide sequences according to claims 23 to 26 for the manufacture of a medicament for the treatment of angiogenic diseases.

32. Medicament containing at least one polypeptide sequence according to claims 23 to 26.

33. A nucleic acid sequence according to claims 1 to 11, characterized in that it is a genomic sequence.

34. A nucleic acid sequence according to claims 1 to 11, characterized in that it is an mRNA sequence.

35. Genomic genes, their promoters, enhancers, silencers, exon structure, intron structure and their splice variants, obtainable from the cDNAs of the

Sequences Seq. ID No. 1 to Seq. ID No. 59.

36. Use of the genomic genes according to claim 35, together with suitable regulatory elements.

37. Use according to claim 36, characterized in that the regulative element is a suitable promoter and / or enhancer.

38. Use of the nucleic acid sequences according to claims 1 to 1 1 and the peptides according to claims 23 to 26, either alone or in formulation as a medicament for the treatment of psoriasis, arthritis, such as rheumatoid arthritis, hemangioma, angiofribroma, eye diseases, such as diabetic Retinopathy, neovascular glaucoma, kidney diseases such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopatic

Syndromes, transplant rejection and glomerulopathy, fibrotic diseases such as cirrhosis of the liver, mesangial cell proliferative diseases, atherosclerosis and injuries to the nervous tissue.

39. Nucleic acid sequence Seq. ID No. 34, characterized in that it forms stable capillary structures.

0. Use of the nucleic acid sequences Seq. ID No. 34 and the peptides expressed via this sequence, either alone or in formulation as a medicament for the treatment of psoriasis, arthritis, such as rheumatoid arthritis, haemangioma, angiofribroma, eye diseases, such as diabetic retinopathy, neovascular glaucoma, kidney diseases, such as glomerulonephritis, diabetic nephropatiasis , thrombic microangiopatic syndromes, transplant rejections and glomerulopathy, fibrotic diseases such as cirrhosis of the liver, mesangial cell proliferative diseases, atherosclerosis and nerve tissue injuries.