CN1240359A - Therapeutic use of an agent that stimulates no or prostacyclin production and delivery device - Google Patents

Abstract

The present invention concerns vascular endothelial growth factor (VEGF) which has utility in the treatment of intimal hyperplasia, hypertension and atherosclerosis, and of conditions susceptible to treatment with agents that produce nitric oxide or prostacyclin. Instead of VEGF, an equivalent agent such as an agonist of VEGF receptors may be given, as may nucleic acid encoding such an agonist. The agent may successfully be administered via the adventitial surface of a blood vessel, e.g., using a device which defines a reservoir between the body wall and the vessel's adventitial surface, the reservoir being at least part-filled by a pharmaceutical formulation containing the agent to be delivered.

Description

The therapeutic use of somatomedin and the conveyer device that is used in particular for the neointimal hyperplasia treatment

Invention field

The treatment that the present invention relates to somatomedin is used, and relates in particular to vascellum endometrial hyperplasia and particularly hypertensive treatment of other disease and prevention.The present invention also relates to be used for a kind of device of delivery of active agents.

Background of invention

Neointimal hyperplasia is between blood vessel endothelium and the internal elastic membrane, the especially growth of the cell quantity in theca interna or in the tremulous pulse.Neointimal hyperplasia is normally caused by the smooth muscle cell in the blood vessel wall (SMC) propagation.

When neointimal hyperplasia takes place when, can cause from the beginning thickening of theca interna or tube wall, promptly narrow.So blood vessel wall can be changed into obturation.

Equally, after blocking in the blood vessel was eliminated, the neointimal hyperplasia that the operation back takes place can cause tremulous pulse to become obturation once more.This is called as restenosis.

When for example tremulous pulse distortion or when disturbed of intra-operative blood vessel, the propagation of arterial smooth muscle cell takes place usually.For example, after carrying out the bypass graft transplanting of vein and arterial anastomosis and after the surgical stapling art, neointimal hyperplasia can cause from stenocephaly usually.Two examples that can cause narrow operation method are crown bypass graft and above-knee strand of-popliteal artery bypass graftings.

Equally, be used for removing the balloon angioplasty method that blood vessel blocks and for example after the balloon angioplasty method restenosis can take place.

Whether neointimal hyperplasia causes narrow and restenosis after different operation methods, is still a subject matter so far.

The atherosclerosis cardiovascular disease is the most important reason of Europe and North America death, and causing the sickness rate of highly significant, it is by the lumen of artery obturation that stops or reduce blood flow, is superimposed on the thrombosis on the atheromatous plaque and causes aneurysm to expand and final disruptive tip thromboembolism, arterial wall slacken caused.According to the distribution of position and disease, there are several selections of treatment, artery bypass grafting is the most frequently used surgical intervention method.For coronary artery disease, this has become the most frequently used surgical method now, since nineteen ninety carries out＞200,000 example operation every year in the U.S., carries out＞20,000 example operation every year in Britain.In aorta, kidney, mesentery and peripheral blood vessel, the burden of surgery bypass therapy continues to increase, and is 35-70 example in per 100,000 populations in the US and European operability.Combine, the quantity of the surgery bypass therapy of carrying out every year is near 1,000,000.

In postoperative preceding 24 middle of the month, the artery bypass grafting failure (obturation) of highly significant quantity is arranged.The numerical range of quoting from 20% to 30%.This means the Britain of all hearts carry out in to(for) every year and peripheral arterial bypass therapy (about 25,000-30,000 example), expection has 6,000 to 7,000 example failures in two years.The mortality of " (re-do) reforms " method even higher.The economic cost of failure is like this equally, it is calculated that in the U.S., and mortality even from 33% to 25% appropriateness reduce and just can save 7.5 hundred million dollars from the health care budget after the crown therapy.

Performing the operation, graft failure has three main causes in back 5 years.First is considered to be in (＜5%) early takes place in 30 days of operation, represents error of performance (for example bad anastomosis).The result of the normally initial Atherosclerosis development of the failure in the later period after 24 months.Yet those inaccessible grafts have caused most failure (＜70%) between 1 to 24 months just.In these situations, cause the carrying out property contraction of lumen of artery by the SMC neointimal hyperplasia, promptly narrow, finally cause entirely shutting.Usually, the SMC neointimal hyperplasia be positioned at around the distal artery anastomosis with the natural blood vessel wall relative with the anastomosis around.Therefore this is the main pathology at this position, rather than as the restenosis at the generable former neointimal hyperplasia of postangioplasty position.The SMC neointimal hyperplasia can betide the more arterial anastomosis place of near-end, and takes place along graft itself.

The restenosis of postangioplasty can cause even higher mortality, is 20% to 50% in preceding 6 middle of the month of postangioplasty.Narrow and restenosis all is the subject matter that stays after the operation.

Up to now, having tested the method for many treatments or prevention neointimal hyperplasia, is satisfied clinically but do not have a kind of.

VEGF (VEGF) is the protein of natural generation.In the mankind, there are 121,165,189 and 206 amino acid whose four kinds of forms at least.People such as Houck, No. 5,12, molecular endocrinology (Molecular Endocrinology) (1991) volume, the 1806-1814 page or leaf has been delivered cDNA and the aminoacid sequence of the people VEGF of four kinds of forms.Also delivered portion gene group sequence.People such as Leung among science (1989) 246:1306-1309, have also delivered the cDNA sequence of people VEGF, and the cDNA sequence of cattle VEGF.

These four kinds of forms are referred to herein as VEGF-121, VEGF-165, VEGF-189 and VEGF-206.Be to be understood that this numbering refers to the aminoacid quantity of mature protein in each example.Protein after the translation also comprises 26 amino acid whose presequences, and in fact this presequence is cut during processing in cell.

Known VEGF works in angiogenesis, and it stimulates the division of vascular endothelial cell (EC), improves the permeability of endothelium, and serve as endothelium " survival factors " in retinal vessel.For example, maybe when expressing from plasmid, VEGF is in the growth that can induce neovascularity after the ischemic limb intra-arterial injection for the VEGF of recombiant protein form.This characteristic can make it be applied to repair the tremulous pulse that has damaged endothelium in operation.Therefore, people such as Asahara, circulation (Circulation) (1995) 91:2793 makes the exposed postangioplasty of arterial endothelium VEGF is delivered to the inside of rat carotid artery by intubate; As if find that VEGF stimulates the endothelialization again (reendothelialisation) of this tremulous pulse, this helps the inhibition of neointimal hyperplasia subsequently.

WO-A-9013649 discloses vegf protein matter and gene, and has proposed it and be used for the treatment of the purposes of blood vessel epithelium wound, diabetic ulcer and blood vessel wound.Described the VEGF fragment among the WO-A-9102058, and they are in for example application in the treatment in ulcer of endothelialization again of angiogenesis and interior blood vessel surface.

GB-A-2298577 discloses a kind of nonrestrictive, foraminous, the external stent that is used for the arteriovenous shut transplantation therapy.This stent has to the size in chamber and to the advantageous effect of middle and intimal thickening.

WO-A-9423668 discloses and a kind of reagent has been localized delivery to endovascular device, comprises a bin that forms between two element.Its application need is implanted, and promptly cuts blood vessel and then this device is fixed in blood vessel wall.This device is that part is foraminous.Its bin directly contacts with the intracavity blood flow.This has the danger of infection.

US-A-3797485 discloses a kind of with the device of drug conveying to the tunica adventitia surface.The transdermal pipe that it has permanent walls and is used to carry liquid form medicine.Purpose is that medicine should pass to another position.

Summary of the invention

The present invention manages to treat and/or prevent above-mentioned all diseases, because they are caused by neointimal hyperplasia.Surprisingly, identified the character of VEGF, shown that it can be used in different aspect antagonism neointimal hyperplasia.

A collar is positioned over the exterior circumferential of rabbit arterial.This method causes the neointimal hyperplasia in the rabbit arterial usually, causes thickening of arterial wall, and this is similar to take place among the human artery behind the shunt operation narrow.When the collar being used to use plasmid/liposome vectors when arterial wall is carried the DNA of coding VEGF, the VEGF gene comprises overexpression in the endodermis in arterial wall.Neointimal hyperplasia is suppressed.Found that the adventitia collar is applicable to the tremulous pulse gene transfer of the gene delivery system of all tests.

VEGF is except stimulating under the situation of endothelial injury the endothelialization again for this proof, can also occur suppressing neointimal hyperplasia under the situation of neointimal hyperplasia when complete when endothelium or complete basically.Therefore, it not only can be used to suppress the restenosis of postangioplasty potentially, and can be used for preventing or treat in other operative status from stenocephaly.Therefore new discovery and before discovery between exist difference, find the regrowth or the healing of VEGF stimulating endothelial in the past.May be that new discovery results from a kind of different mechanism of VEGF effect.

In addition, new discovery proves, effective agents can be transported to the blood vessel outside with the treatment neointimal hyperplasia.This has several advantages.Particularly, therapeutic agent can be as intracavity not be carried by blood flow from hypertrophy position flush away.A kind of conveying bin can remain in around the blood vessel, does not need any transluminal operation (can cause neointimal hyperplasia these operations) of injured blood vessel endothelium itself.

More specifically, the present invention makes the reagent of antagonism SMC neointimal hyperplasia can be directly used in the outer membrane face (cell in the promptly the most close outer middle film) of arterial wall.Any reagent that uses can be applied to the position of most probable development neointimal hyperplasia damage specifically, because these positions are easy to expose at intra-operative.

VEGF mediates its known effect by special high affine tyrosine kinase receptor flk-1/KDR and the flt-1 that only expresses on EC and mononuclear cell.Be not limited to theory, think that the effect of VEGF in hypertrophy suppresses also may be to mediate by identical receptor.Therefore, the present invention also expands to and other agonist of the bonded receptor of VEGF or the application of other material in treatment or prevention neointimal hyperplasia with same function mechanism.Compare with cytokine with many other somatomedin that are proposed to be used in the intimal thickening treatment, the advantage of VEGF is also given in the special location of vegf receptor; The effect of VEGF is more special to EC, because when not having mononuclear cell, the high-affinity vegf receptor in the arterial wall is only expressed on EC.

For example, found at endothelium fully or under the unmarred basically situation, the VEGF of neointimal hyperplasia suppresses mechanism at least in part by nitric oxide (NO) approach, because using of NO synthetic inhibitor L-NAME offset VEGF to the outgrowth effect of inner membrance in above-mentioned cover ring model.Therefore, VEGF stimulates NO to produce.

Also may be that VEGF has other biological action that suppresses neointimal hyperplasia owing to it.Particularly, generation, the cPLA2 A of the overexpression stimulation prostacyclin of VEGF have been found ₂Activation and EC secretion von willebrand's factor.Situation may be that VEGF suppresses neointimal hyperplasia to the stimulation of NO generation and to the stimulation synergism that prostacyclin produces.

The discovery that the VEGF effect stimulates NO and prostacyclin to produce also advises, VEGF and agonist with the bonded receptor of VEGF will be used for the treatment of conditions that the relevant disease of other NO and/or prostacyclin are correlated with.Particularly, people such as Forte, lancet (1997) 349:837-42 shows that the NO level is low in the hypertensive individuality of trouble.Therefore VEGF can be used for multi-form hypertensive prevention or treatment.Equally, VEGF can be used for atherosclerosis therapy.

According to an aspect of the present invention, have the reagent of the special properties of any VEGF of being found in, can be used for treating or prevent the manufacturing of neointimal hyperplasia such as narrow medicine.This reagent can provide with the form of implant.More specifically, this reagent stimulates the generation of NO or prostacyclin; It can be a kind of agonist with the bonded receptor of VEGF, as VEGF itself or its fragment, maybe can be the nucleic acid of this agonist of coding.

According to a second aspect of the invention, be used for a kind of device to patient vessel's delivering therapeutic agents, comprise that is adapted at the main body that blood vessel provides hermetic unit on every side, reagent is stored in this device or with this device and combines, so that in use this reagent can contact with the outer membrane face of blood vessel.This device can be biodegradable, and does not need nonvolatil transdermal delivery pipe.Invention is described

That as above advises reaches shown in the embodiment, and plurality of reagents comprises that nitric oxide synthetase and its nucleic acid of coding all are fit to purposes in the present invention.This reagent is described to vegf protein matter or nucleic acid usually at this, and the reference of those references and VEGF itself provides with the method for embodiment.Any form of above-mentioned VEGF can be used for purpose of the present invention.

At this, the reference of these vegf protein matter sequences is considered to be meant sequence that comprises presequence and the sequence that lacks presequence.The vegf protein matter that contains or do not contain presequence all is suitable for enforcement of the present invention.Equally, the reference of VEGF nucleic acid (DNA and RNA) sequence relates to the sequence of the encode sequence of presequence and the presequence of not encoding.

Should be pointed out that people such as Houck, the source is the same, has delivered the sequence of VEGF-165, and it comprises the aminoacid agedoite (N or Asn) (115 in people's such as Houck the representation, it is corresponding to mature protein) in site 141.People such as Houck point out that this aminoacid is lysine (K or Lys) in VEGF-121, VEGF-189 and VEGF-206, (VEGF-206's) cDNA sequence that people such as Houck quote has been supported this point.Therefore, in the present invention, the aminoacid in site 141 can be agedoite (N or Asn) or lysine (Lys or K).Each aminoacid is by suitable triplet codon coded (for DNA, can be AAA or AAG to the lysine codon, be AAT or AAC for agedoite) in nucleotide sequence of the present invention.This is applied to VEGF-165 especially.

Four kinds of forms are by identical coded by said gene, but produce by the different montages on the rna level.Therefore, people VEGF and three kinds of known clipped forms of having a kind of total length form.VEGF-121 and VEGF-165 are soluble, and are secreted forms.Equally, 26 amino acid whose presequences are hydrophobic, and think that it reduces proteinic dissolubility, and therefore, the form that does not contain the VEGF of presequence is preferred, because expect that they have higher dissolubility.The VEGF of form of ownership is suitable for enforcement of the present invention, although secreted form is preferred.Be suitable for vegf protein matter of the invention process and also can derive from other kind, although people VEGF is preferred.For example, cloned the VEGF of mice, rabbit and cow, its sequence is available.

For reference, should be pointed out that VEGF-121,165,189 and 206 is also referred to as VEGF-120,164,188 and 205 in the art.

Vegf protein matter and nucleic acid (DNA and RNA) are to be used for suitable reagent of the invention process.

When using vegf protein matter, the vegf protein matter with aminoacid sequence of SEQ ID No.2 (VEGF-121), 4 (VEGF-165), 6 (VEGF-189) or 8 (VEGF-206) is preferred.The secreted form of VEGF is preferred, therefore particularly preferably is VEGF-121 and VEGF-165.

In enforcement of the present invention, preferably use the VEGF DNA of coding VEGF-121, VEGF-165, VEGF-189 or VEGF-206, for example have the VEGF DNA of SEQ ID No.1,3,5 or 7 sequence.The DNA sequence of the secreted form of coding people VEGF is preferred.Therefore, SEQ ID No.1 and 3 DNA sequence are particularly preferred.

Yet, be suitable for VEGF DNA of the invention process and protein and be not limited to those specific sequences.And then the present invention also provides the application of other DNA that is closely related and protein sequence.

DNA sequence of the present invention is can be in many aspects relevant with SEQ ID No.1,3,5 or 7 sequence.For example, be suitable for the degenerate sequence that DNA sequence of the invention process can be coding SEQ ID No.2,4,6 or 8 proteinic same protein.

In addition, DNA sequence of the present invention can with SEQ ID No.1,3,5 or 7 sequence homology basically, and a kind of like this protein of coding, it is different from SEQ ID No.2,4,6 or 8 protein on aminoacid sequence, but coding has the active protein of VEGF.Usually, being used for DNA sequence of the present invention has and SEQ ID No.1,3,5 or 7 sequence 70%, 80%, 90%, 95% or 99% sequence homology at least at least at least at least at least.

Equally, be used for the fragment that VEGF DNA sequence codified of the present invention keeps the active VEGF of VEGF.That is, target fragment length is at least 15 aminoacid, for example can reach 40 or more aminoacid.Suitable segmental example is that length is 20 aminoacid, for example sequence 1-20,11-30,21-40,31-50,41-60,51-70,61-80,71-90,81-100,91-110,101-120,111-130,121-140,131-150,141-160,151-170,161-180,171-190,181-200,191-210 and the 196-215 of the active vegf protein matter shown in the SEQ ID No.6.

Be used for DNA sequence of the present invention and can be the hybrid of genomic DNA for example or cDNA or genomic DNA and cDNA, perhaps they can be synthetic or semisynthetic.They can derive from the biology of any kind of, although the DNA of coding people VEGF is preferred.They can be strand or two strands.Coding SEQ ID No.2,4,6 and 8 proteinic genomic DNA are particularly preferred.

Have the active protein of VEGF if be used for dna sequence encoding of the present invention, they can or substitute owing to the disappearance of one or more nucleotide, insertion and be different from the sequence shown in the SEQ ID No.1,3,5 or 7.Equally, if their codings have the active protein of VEGF, they can or extend one or more nucleotide by truncate with respect to SEQ ID No.1,3,5 or 7.

The RNA sequence also is suitable for enforcement of the present invention.Particularly, the invention provides application corresponding to SEQ IDNo.1,3,5 or 7 RNA sequence; These are preferred RNA sequences.The present invention also is provided at the application that above-mentioned any aspect about DNA sequence relates to the RNA sequence of these sequences.RNA sequence of the present invention can be strand or two strands.RNA of the present invention can be any source.For example, they can derive from the biology of any kind of, and the RNA with people VEGF of sequence shown in the SEQ ID No.2,4,6 or 8 is preferred although coding people VEGF particularly encodes.Also can use synthetic DNA, and semisynthetic RNA.In addition, can use DNA to transcribe the interior or external bacterial plasmid of body of form.

It will be understood by those of skill in the art that the T residue will be replaced by U in being suitable for RNA sequence of the invention process.

Be used for vegf protein matter of the present invention by DNA of the present invention or RNA sequence are coded as mentioned above.Preferred protein of the present invention is SEQ ID No.2,4,6 and 8 protein, although the present invention also provides other the proteinic application with the sequence that is closely related, this sequence is different from SEQ ID No.2,4,6 or 8 sequence, has the VEGF activity.

According to the present invention, because it relates to the treatment or the prevention of neointimal hyperplasia in this scope, so the VEGF activity is the ability that completely or partially suppresses or prevent in the blood vessel neointimal hyperplasia in the tremulous pulse particularly.As mentioned above, the protein that is different from the VEGF of natural generation on sequence slightly keeps this character, although the fixation VEGF that differs on the degree is the same.Equally, these protein can show the VEGF activity stronger than the VEGF of natural generation.The agonist of VEGF, for example peptide, class peptide or other micromolecule also have similar situation.

Owing in this scope, the present invention relates to other character of VEGF,, the VEGF activity reproduces the ability of those character so being molecule except that VEGF.For example, produce to stimulate NO owing in this scope, the present invention relates to the activity of VEGF antagonism NO-associated conditions, so the VEGF activity comprises the ability that stimulates NO to produce.Owing to the present invention relates to the activity of VEGF antagonism prostacyclin-associated conditions in this scope, the VEGF activity comprises the ability that stimulates prostacyclin to produce.Be suitable for one or more biological properties that vegf protein matter of the invention process generally also shows VEGF known in the art, as in external and/or body, promoting the ability of tremulous pulse EC propagation, perhaps with the ability that activates them with the bonded receptors bind of VEGF and in the mode of VEGF.

Be suitable for vegf protein matter of the invention process can with SEQ ID No.2,4,6 or 8 VEGF homology, generally at least 70%, at least 80%, at least 90%, at least 95% or at least 99% homology basically.

Have the activity of VEGF if be suitable for vegf protein matter of the invention process, they can or substitute owing to one or more amino acid whose disappearances, insertion and be different from the sequence shown in the SEQ ID No.2,4,6 or 8.Equally, if they have the activity of VEGF, they can or extend one or more aminoacid by the one or more aminoacid of truncate with respect to SEQID No.2,4,6 or 8.About substituting, conservative substituting is preferred.Usually, conservative substituting is a kind of like this substituting, and the aminoacid that exists among the VEGF of wherein alternate aminoacid and natural generation has similar character, for example aspect electric charge and/or size and/or polarity and/or hydrophobicity.Equally, conservative substituting generally has slight influence or not influence to proteinic VEGF activity.

The vegf protein matter that is used for VEGF of the present invention, be different from natural generation on sequence can be transform as the VEGF that is different from natural generation on activity by protein engineering.For example, they can be transform as and have stronger VEGF activity.General use recombinant technique known in the art carries out these operations on nucleic acid level.

As a kind of selection method that is equipped with that uses vegf protein matter as mentioned above, it is possible using the VEGF agonist.It is applied to all medical applications, particularly atherosclerosis therapy described herein.

Usually, the VEGF agonist is a kind of molecule same and the bonded receptors bind of VEGF, and has the active same function of the VEGF of causing as described herein basically.Particularly, agonist can be in conjunction with flk-1/KDR or flt-1 receptor.Therefore agonist of the present invention be called as VEGF agonist and with the agonist of the bonded receptor of VEGF.

The VEGF agonist can have any chemical constitution.For example, the VEGF agonist can be for example can reach 10, can reach 20, can reach 50 and maybe can reach 100 amino acid whose peptides or polypeptide.Agonist can be peptide or the class peptide of modifying equally.Can carry out any suitable modification, comprise glycosylation, sulfuration, COOH-amidatioon and acetylation, for example the acetylation of N-end.In addition, or select ground fully, can have the aminoacid and/or the L-aminoacid of modification.

Some preferred agonist is the fragment with VEGF VEGF active, that randomly modify as mentioned above.The agonist fragment of a particularly preferred VEGF is made up of the amino acid/11 to 20 (M-H) of SEQ ID No.4; People such as Ahmed, laboratory research (Lab.Invest.) (1997) 76:779 report, this peptide is a kind of agonist of Flt-1 receptor among the human trophocyte cell.

Other relevant agonist also can be derived from the N-petiolarea of VEGF.For example, with regard to SEQ ID No.4, the peptide agonists of VEGF can comprise the N-end (amino acid/11 number) of VEGF, and has the aminoacid sequence of the VEGF that can reach 25 to 30,30 to 40,40 to 50 or 50 to 100 aminoacid scopes.Equally, preferred agonist can be derived from the N-petiolarea of VEGF, but comprises the N-end of clipped form.For example, replace starting from the amino acid/11 number of SEQ ID No.4, they can start from No. 2,3,4,5,6,7,8,9 or 10, the aminoacid of SEQ ID No.4, and have the aminoacid sequence of the VEGF that can reach 25 to 30,30 to 40,40 to 50 or 50 to 100 aminoacid scopes.

Peptide agonists of the present invention also can be derived from the other parts of VEGF sequence.For example, further preferred a kind of peptide agonists is the peptide that the amino acid/11 45 to 169 (R-P) by the VEGF-189 sequence of SEQ ID No.6 constitutes.

Other relevant agonist also can be derived from this district of VEGF.For example, with regard to SEQ ID No.6, the peptide agonists of VEGF can have the aminoacid sequence of zone from about 135 to 155 aminoacid to about 160 to 180 amino acid whose VEGF.For example, by this distinguish deutero-peptide agonists can have the zone from about 135 to 140,140 to 145 or 145 to 150 aminoacid to about 160 to 165,165 to 170 or 170 to 175 amino acid whose VEGF sequences.

The fragments of peptides of aforesaid VEGF preferably has 10 to 20,20 to 25,25 to 30,30 to 40 or 40 to 50 amino acid whose total lengths.

Other preferred agonist is the proteinic fragment of HIV Tat.The agonist effect of HIV Tat protein simulation VEGF, and can be by the angiogenesis in the Flk-1/KDR receptor acting stimulating endothelial cell; See people such as Albini, oncogene (Oncogene) (1996) 12:289-297 and Nature Journal medicine fascicle (Nature Medicine) (1996) 2 (12): 1321-1375.Therefore, the growth and the migration of stimulating endothelial cell have been shown by deutero-peptide of HIV-1Tat sequence such as the proteinic aminoacid 46-60 of HIV Tat; See people such as Albini, oncogene (1996) 12:289-297.The peptide that is made of the proteinic aminoacid 41 to 65 of HIV-1Tat is the further preferred peptide agonists of the present invention.

Agonist of the present invention also can have above-mentioned be different from the aminoacid sequence of the VEGF of natural generation aspect vegf protein matter any, as long as keep their agonist character.

When agonist of the present invention was peptide, in order to realize treatment according to the present invention, they can be produced in vivo by their nucleotide sequence of coding.Therefore, as described here, can carry the nucleic acid of coding agonist by gene therapy.

In addition, can use the VEGF agonist of non-peptide.For example, can use the micromolecule of the VEGF form partly of simulation and its acceptor interaction.

In enforcement of the present invention, the nucleic acid of VEGF, coding VEGF or the nucleic acid of VEGF agonist or coding VEGF agonist can be delivered in the blood vessel with any suitable form, preferably in the tremulous pulse.Can be not carry nucleic acid with carrier-bound " exposing " form or by gene therapy vector.Preferably carry them by any suitable gene therapy vector.Particularly, can use virus or non-virus carrier.

Suitable viral vector comprises adenovirus, retrovirus, pseudotyped retroviral virus, herpesvirus, vaccinia virus and baculovirus.Suitable non-virus carrier comprises oligonucleotide, plasmid, liposome, cationic-liposome, pH-sensitive lipid body, liposome-protein complex, immunoliposome, liposome-protein-polylysine derivant, water-oil emulsion, polymine and dendrimer.Preferred carrier comprises Moloney muroid leucovirus (MMLV)-deutero-retrovirus, comprises retrovirus, adenovirus, plasmid and the plasmid/liposome complex of the herpetic oral cavity of false type viroid protein-G (VSV-G).

In the time of suitably, can use the carrier of two or more types together.For example, a kind of plasmid vector can be used in combination with liposome.Suitable liposome comprises; for example; comprise dioleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE), 3 β-[N '-[N '; N '-dimethylamino ethane] carbamyl] (N-[1-(2 for cholesterol (DC-Chol) or positively charged fat; 3-two oily alkene oxygen bases) propyl group]-N; N, the liposome of N-triethyl ammonium (DOTMA).

Viral vector of the present invention is anergy preferably, for example, is to duplicate-deficient.That is, they lack duplicates required one or more functional genes, and this has stoped their uncontrolled propagation in vivo, and has avoided undesirable side effect of viral infection.Preferably, remove all viral genome except that the minimal genome element, the minimal genome element is that the viral genome of will mix VEGF nucleic acid is packaged into virus coat and housing is required.For example, wish deletion all viral genome except that long terminal repeat (LTR) and packaging signal.As for adenovirus, generally in the E1 district, randomly in one or more E2, E3 and/or E4 district, delete.

Also can make viral anergy of the present invention by any suitable technique.For example, genomic deletion can comprise the removal fully of duplicating required gene, or only part is removed.It is preferred removing fully.Usually, preferred disappearance is the disappearance of the required gene of viral gene early transcription.

Also can use replication form from the body restriction or from the destructive viral vector of body.

Usually, be used for VEGF nucleic acid of the present invention and be contained in a kind of expression construct, this construct guarantees that it preferably can express in vivo after being transported to tremulous pulse by aforesaid carrier.These constructs generally comprise a promoter that can guide the VEGF expression of nucleic acid (with the regulator gene of this promoter randomly), translation initiation codon and the VEGF nucleic acid that effectively is connected with promoter.Preferably, these components are arranged with 5 '-3 ' direction.

This construct also can comprise any other suitable component.For example, this construct can comprise the nucleic acid of coded signal sequence, then be positioned this position with respect to VEGF nucleic acid, so that in its when translation, can guide to specific cellular type or cellular compartment with the vegf protein matter of expressing.Any sort signal sequence generally is positioned to be close to 3 of VEGF nucleic acid ' end or 5 ' end, holds the single fusion rotein that contains signal sequence so that signal sequence and vegf protein matter are translated at C-or N-.

This construct also can comprise the enhancer that strengthens the expression degree that promoter provides.Can use any enhancer that strengthens the expression that selected promoter provides.For example, for CMV early gene promoter, can use CMV early gene enhancer.

Randomly, this construct can comprise the transcription terminator of VEGF nucleic acid 3 ' end.Can use any suitable terminator.

Randomly, this construct can comprise the polyadenylation signal sequence that effectively is connected to VEGF nucleic acid 3 ' end.

Randomly, this construct can comprise one or more selected markers, and antibiotics resistance gene for example is to allow the screening of transformant in cultivation.For example, can screen cell in order according to antibiotic resistance.

Randomly, this construct can comprise one or more introns, or other non-coding sequence, for example is positioned at 3 of VEGF nucleic acid ' end or 5 ' end.

Can use any suitable promoter to control expression of nucleic acid of the present invention.Usually, the promoter of preferably using viral promotors or be suitable in the individual population of being treated, to work.Therefore,, preferably use viral promotors, particularly by the viral deutero-promoter that infects the mankind, or by the deutero-promoter of human gene for human patients.Randomly, a kind of promoter can be used in combination with any suitable enhancer.

Wish ground, use " by force " promoter, promptly guarantee the promoter of vegf protein matter high level expression of the present invention.Wish to realize the promoter of vegf protein matter overexpression.Preferred promoter comprises cytomegalovirus (CMV) promoter, randomly combines with cmv enhancer; People's beta-actin promoter; Simian virus 40 (SV40) early gene promoter; Rous sarcoma virus (RSV) promoter; With long terminal repetition (LTR) promoter of retrovirus.

Promoter is connected with VEGF nucleic acid effectively with other construct component.So be located, so that they can bring into play the effect to the VEGF expression of nucleic acid.For example, for promoter, promoter is located with respect to VEGF nucleic acid, so that it can guide the VEGF expression of nucleic acids.Wish ground, location construct component is to allow the maximum effect of they performances to expressing.

Nucleic acid or the construct that uses among the present invention can be mixed viral genome by any appropriate method known in the art.Viral genome can be packaged into virus coat or housing by any suitable method then.Particularly, can use any suitable package cell line to produce viral vector of the present invention.These packings are complementary replication-defective virus genome of the present invention, mix its gene genomic, that delete because they generally comprise from duplicate the deficient genome.Therefore, the use of packing system allows viral vector of the present invention to produce in culture.Suitable packing system comprises that PA317 cell, ψ-2 cell, CRE cell, CRIP cell, E-86-GP cell, Fly cell, 293 are the derivant of cell and 293GP cell.

For non-virus carrier, can nucleic acid be mixed non-virus carrier by any suitable method known in the art.

As desired, can select carrier particularly viral vector or nucleic acid or construct are stayed in the kytoplasm freely realizing nucleic acid or the integration of construct in patient's cellular genome to be treated.Integrating vector is preferred.

For realizing to outgrowth treatment, can be in any suitable manner to tremulous pulse use aforesaid preferably with virus or bonded vegf protein matter of the present invention or the VEGF nucleic acid of being used for of non-virus carrier.For example, can for example pass through the chamber, to the outer wall of blood vessel such as tremulous pulse or blood vessel endothelium such as arterial endothelium are used the nucleic acid of VEGF or coding VEGF.Local gene transfer the using of vegf protein matter that may be better than recombinating, because the complex that injects is rinsed out rapidly by blood flow, half life, is short in blood.

In case carry, VEGF nucleic acid of the present invention is promptly expressed, produce vegf protein matter, this protein is realized the treatment or the prevention of neointimal hyperplasia subsequently.Expression can betide in any cellular type or in the vascular type as in the arterial wall.

Preferably, express to betide such position, that is, make the VEGF of expression can arrive the endothelium of blood vessel such as tremulous pulse.For example, expression can betide in smooth muscle cell and/or the endothelium.Most preferably, expression betides in the endothelium of blood vessel such as tremulous pulse at least.

For example, can by around the outgrowth position of direct injection with treatment or prevention, or by in the chamber that is injected into blood vessel such as tremulous pulse, vegf protein matter or nucleic acid are delivered to the outside of blood vessel such as tremulous pulse.

Preferred, the blood vessel by being positioned over contiguous hypertrophy position of being treated, as the implant of tremulous pulse outside is carried vegf protein matter or nucleic acid, and this implant comprises vegf protein matter or nucleic acid or carrier, and the bin of reagent is provided.Can before or after implant is introduced the patient who is treated, vegf protein matter or nucleic acid (preferably combining with carrier) be introduced implant.For example, this implant can be fixed near the blood vessel, by for example injecting vegf protein matter or nucleic acid is introduced implant subsequently.

Preferably, placing implant directly contacts with blood vessel such as tremulous pulse.When using retroviral vector to carry VEGF nucleic acid, this is particularly preferred.Because the physical deformation of blood vessel can be induced smooth muscle cell proliferation, this has improved the efficient by the gene transfer of retroviral vector.This propagation as the inductive propagation of hypertrophy itself, can be overcome by the conveying of vegf protein matter or nucleic acid or can improve at least.Equally, when when its target cell division, using other carriers that show the gene transfering efficiency that improves, preferably contact with tremulous pulse for implant.For example, cell proliferation also can improve the gene transfering efficiency of plasmid/liposome complex.

These implants can be any suitable forms.Preferably, this implant is the form of the collar, its partially or even wholly, preferably fully to be treated or the prevention the hypertrophy position or near this position around tremulous pulse.

The blood vessel alia gene is carried the method for having avoided inserting as balloon catheter art or highly pressurised liquid, and these methods can cause the damage of endothelium or expose.Preferably use the gene of transfection, preferably the collar by placing, preferably the collar by placing in the blood vessel outer periphery near the blood vessel outside by silica gel or biodegradable implant.Endothelium suffers very little damage or not damage.This is the topmost advantage of this transporting pattern.

When directly carrier being applied to overlap intra-annular tunica adventitia surface according to the present invention, keep contacting with the tight of adventitia.In rabbit arterial, the formation of neointima in 7-14 days after an independent collar generally causes performing the operation.This collar also keeps the carrier of high concentration at outer membrane face.

The preferred collar of implant can be made by any suitable material.Silica gel implant, the implant that promptly comprises silica gel are a kind of preferred selections.Most preferably biodegradable implant.Can use any suitable biodegradable material.

In the implant such as the collar, can comprise vegf protein matter or nucleic acid by any way.Preferably, the structure of the implant such as the collar makes vegf protein matter or nucleic acid directly contact with blood vessel wall.Therefore, in one embodiment, the structure of implant leaves a space between blood vessel wall and graft wall.As for the collar, so implant forms the container of a sky around blood vessel.VEGF nucleic acid or protein can be introduced this space, so that they contact with blood vessel wall.Preferably, the end of implant contacts with blood vessel wall, thereby has prevented VEGF nucleic acid or proteinic overflowing.Preferably, the outer wall of the collar is impermeable or impermeable basically for VEGF nucleic acid or protein, thereby prevents from or limited it at least to enter surrounding tissue, and guarantees its vasotropic conveying.

Randomly, comprise VEGF nucleic acid or proteinic space can by one or more layers to VEGF or nucleic acid penetrating or semipermeable material separate with blood vessel wall.If plan progressively to carry and wish to limit the speed that vegf protein matter or nucleic acid are carried to blood vessel wall, this can wish.

Randomly, can design the implant such as the collar to be used as osmotic pumps.

Randomly, VEGF can be contained in the intra-annular medium of cover, as solid or gel media.This helps to prevent that vegf protein matter or nucleic acid from entering tissue.If so, the outer wall of the collar needn't need to contact with the blood vessel of implant end.

In addition, can be with VEGF nucleic acid or protein bag by on implant surface, this implant in use contacts with blood vessel.In addition, VEGF nucleic acid or protein can be scattered in the whole implant structure.

Implant in this mode particularly with the mode advantages of application of the collar is: (i) they provide a conveying bin that allows to continue conveying; (ii) do not need transluminal operation, and arterial endothelium is kept perfectly; And (iii) as mentioned above, the distortion (for example contraction under the situation of the collar) that implant produces can improve the efficient that gene is carried.

Device of the present invention generally includes a main body, this main body comprises first impermeable basically main part at least, the shape of this main part is suitable in use extending longitudinally, and at least in part around first this blood vessel, first main part comprises the hermetic unit of the longitudinal separation that is suitable in use sealing first tunica adventitia surface, and a mid portion between hermetic unit, sealing partly is suitable in use comprising at least a reagent and the outer membrane face of first blood vessel is carried this reagent.

Now with reference to accompanying drawing, only preferred embodiment according to device of the present invention is described in the mode of embodiment, wherein:

Fig. 1 is a sketch map, is the longitudinal sectional view at a circumvascular device of the present invention;

Fig. 2 is a sketch map, is the cutaway view along the identical embodiment of A-A line shown in Figure 1;

Fig. 3 shows the main body sketch map of second embodiment that is positioned at connected head-to-tail anastomotic position device on every side;

Fig. 4 is the longitudinal sectional view of the embodiment of Fig. 3;

Fig. 5 is the view similar to Fig. 4, but shows the modified forms of this embodiment; And a kind of structure of selecting fully of hermetic unit;

Fig. 6 shows the schematic perspective view of the 3rd embodiment that is positioned at terminal and the bonded anastomotic position in side device on every side;

Fig. 7 is the longitudinal sectional view of the embodiment of Fig. 6;

Fig. 8 shows the main body sketch map of the 4th embodiment that is positioned at the bonded anastomotic position in side and side device on every side;

Fig. 9 is the longitudinal sectional view of the embodiment of Fig. 8;

Figure 10 shows the main body sketch map of the 5th embodiment that is positioned at the bonded anastomotic position in side and side device on every side, shows the blood vessel of a part embedding; And

Figure 11 is the longitudinal sectional view of the embodiment of Figure 10.

As a setting, when natural blood vessel is stopped up by the tremulous pulse gruel type or remarkable when narrow usually, usually with the artery bypass grafting thing to recover or to improve the blood flow that flows to tissue.No matter use vein or tremulous pulse, or synthetic material such as terylen (Dacron) or polytetrafluoroethylene (PTFE) from body, usually in one of three kinds of modes with graft and natural vascular anastomosis: " headtotail " (Fig. 4); " terminal combine with the side " (Fig. 7); Or " side and side are bonded " (Fig. 9).In these technology, terminal combine with the side and the side bonded more commonly used with the side than connected head-to-tail.Blood flow direction is represented with arrow.

Fig. 1 and Fig. 2 show blood 1 and the adventitia collar that comprises space 3 within the blood vessel wall 2, and space 3 is surrounded by the wall 4 of biological example degradation material.The collar 5 is in the end contact blood vessel wall of the collar.Can the present embodiment be used for other embodiment with same way as described below.

The described embodiment of Fig. 3 and Fig. 4 is applied to connected head-to-tail anastomosis according to the show.This device comprises that is generally a piped main body 12, and this main body is in use along by graft 13 and the natural blood vessel 14 connected head-to-tail formed blood vessel longitudinal extensions that coincide, and around this blood vessel.

As the most clear finding of Fig. 4, this device main body 12 has the hermetic unit 15 of the longitudinal separation that is provided to the opposite end.When this device in use is positioned at connected head-to-tail anastomotic position 16, the outer membrane face of these hermetic units 15 sealing grafts 13 and natural blood vessel 14.The material of main body 12 at the radial thickness of hermetic unit 15 greater than thickness at mid portion 17.Therefore, when the outer membrane face of hermetic unit 15 sealing grafts 13 and blood vessel 14, space of formation between the outer membrane face of the end that inner face and the graft 13 and the blood vessel 14 of this apparatus main body 12 surrounds.This space constitutes bootstrap reservoir 18, and demonstration and anastomotic position 16 vertically align.

This bin can make the pharmaceutical preparation that comprises one or more reagent contact with the outer membrane face of graft with blood vessel 13,14 at anastomotic position 16.As preparation is the form of liquid or gel, and the wall that for example can use hypodermic needle and syringe to pass main body 12 is injected into bootstrap reservoir 18 with it.The contained reagent of said preparation advantageously has anti--proliferative effect, with the smooth muscle cell neointimal hyperplasia of antagonism at anastomotic position 16 and adjacent area place.

Pharmaceutical preparation needs not to be the form of liquid or gel, and for example it can be the ointment with the denseness that is similar to toothpaste.Thereby it keeps contacting vasoconstrictive with the outer membrane face of the pulsation that is contacted.

For device being positioned on the anastomotic position 16, cylinder 12 can endwisely slip on one of graft 13 and blood vessel 14 before coincideing.Thereby the doctor can link together graft 13 and blood vessel 14 at anastomotic position 16 places, then main body 12 slid back on anastomotic position 16, to occupy position shown in Figure 4, makes hermetic unit 15 can seal separately outer membrane face.

In addition, as show that this device main body 12 can have the vertical slit 19 along its whole length.Like this, the doctor does not need device 12 is introduced in patient's body and graft 13 and blood vessel 14 can be coincide.In case complete successfully anastomosis, the doctor can select a kind of main body 12 of suitable size, around the graft and blood vessel that is applied to coincide, method is to open pliability main body 12 along slit 19, on graft that coincide and blood vessel 13,14, slide, for example use then conventional " tissue glue ", be called thrombin glue that Tisseal sells or based on the glue of cyano group methacrylate, the opposing longitudinal edges of main body is sealed at slit 19 places as name.

For the effect of contained drug preparations in the concentrated bin 18, and the seepage of avoiding its reagent to organize towards periphery, 12 pairs of preparations of main body are impermeable basically.Advantageously, its material also is biodegradable through certain hour, and stage of 1 to 5 day for example, the activating agent in this moment preparation may exhaust.Also select its material, so that do not cause the too serious reaction of surrounding tissue.

The example that is used as the suitable material of main body comprises gelatin, alginate or collagen protein.These materials are also given the pliability of main body, and device can be made by method of molding or squeezing and pressing method.

The wall material of main body 12 advantageously also is self sealss (self-sealing), so that when the bin 18 of sealing need be bored a hole by hypodermic needle, keep its integrity.Select fully ground or in addition, remove any leak that shows in the syringe needle rear wall and can use sealings such as " tissue glues ".

The main body 12 that a series of different sizes can be provided is to adapt to the blood vessel of different sizes.Lower limb vascular has the external diameter of about 6-8mm usually.Coronary vasodilator has the external diameter of about 3-5mm usually.Therefore, a series of size of main body of the about 3-10mm diameter of scope can obtain from surgical sterile bag.In addition, can change the size of main body to influence the volume of bin 18.The suitable dimension of bin 18 can be up to 10ml, preferably 2-5ml.

For adapting to the expansion of the blood vessel 13,14 that pulsatile blood flow causes, the hermetic unit 15 of main body can be stretched easily at least, so that adapt to the expansion of blood vessel wall.Wish very much to avoid the contraction of blood vessel wall and keep sealing complete simultaneously by this device.

Fig. 5 has illustrated different sealing positions.The radial thickness of main body 12 materials is constant along the length of main body, and mid portion 17 is inner capsule with respect to the internal diameter of main body 12 at hermetic unit 15 places.Two hermetic units 15 all are that the tail by longitudinal extension constitutes, and for example, on the axial length " X " of about 8-15mm, each hermetic unit is the outer membrane face of swaging graft thing 13 and blood vessel 14 respectively.The long tail of these of hermetic unit 15 can work in the mode of " clack valve ", to help bootstrap reservoir 18, although wish to determine not have to pass through the blood flow of " clack valve ".In addition, these tails can be to be similar to the inwardly folding (not shown) of mode shown in Figure 4, to double main body in end portion thickness, forms radially body thickness greater than the hermetic unit of mid portion body thickness.

In order to form or help form the sealing of liquid impermeable, the doctor can for example use the glue of the above-mentioned type as " tissue glue " hermetic unit 15 to be adhered to outer membrane face.Yet this is not crucial.For example, if the size of main body 12 of selecting hermetic unit 15 places is enclosed length, any glue of so unnecessary use with coupling blood vessel 13,14.Alternatively, the doctor can rely on main body 12 in the internal diameter at hermetic unit 15 places and the diametral interference between the outer membrane face diameter.The long-tail hermetic unit embodiment of Fig. 5 is especially true.Yet hermetic unit should be too not tight on blood vessel, so that vasoconstrictive.

Fig. 6 and Fig. 7 have illustrated an embodiment with the terminal device that is used in combination with the bonded anastomosis in side.These accompanying drawings have shown a main body 20 with first main part 21 and second main part 22, and they form a normally main body of gamma-form with the angle branch less than 90 °.Should be appreciated that first and second main part 21,22 can be can reaching 90 ° and comprise that other branched angulation of 90 ° is intersected with each other, main body T-shape normally in the latter event.The doctor can obtain a series of different sizes and difform main body easily, and therefrom he can select the distribution of suitable anastomosis of blood vessel and the device of size.For example, first main part 21 can be that about 1-10cm is long; Second main part 22 can be that 1-5cm is long.

First main part 21 is normally piped, and shows around natural blood vessel 23.Second main part 22 also is piped usually, and shows the graft 24 that matches at anastomotic position 29 and natural blood vessel 23 around with end and the bonded mode in side.As Fig. 7 finding, the end that the opposite end of first main part 21 has 25, the second main parts 22 of hermetic unit has hermetic unit 26.In embodiment early, can use easily " tissue glue " with these hermetic units 25,26 respectively with the outer membrane face sealing of blood vessel 23 and graft 24.

Fig. 7 is presented at the bootstrap reservoir 27 that forms between the inner face of first main part 21 and blood vessel 23 outer membrane faces.Bin 27 extends to the inside of second main part 22.So sealing bin 27 aligns with anastomotic position 29.Can use hypodermic needle and syringe easily bin 27 to be injected the liquid pharmaceutical formulation that comprises activating agent.

For ease of this device to the installation of blood vessel 23 and graft 24, but be provided at this device main body 20 in the aseptic packaging Xiang the doctor, this aseptic packaging comprises two symmetric half parts, it divide on the plane of section shown in Figure 7 and is symmetrical.In this case, after graft 24 and blood vessel 23 were coincide, the doctor need be assemblied in two identical body halves branches together, and the opposite edges of using for example aforesaid glue that identical halves is divided are enclosed in together.

In addition, as shown in Figure 6, have only first main part 21 can have vertical slit 28.Doctor's second main part 22 of on the end of graft 24, sliding like this.So the doctor can coincide the free-end of graft 24 and blood vessel 23, then with second main part, 22 slip travelling backwards plants 24, covers anastomotic position 29, use slit 28 that blood vessel 23 is put into the center of first main parts 21, thus with its around.The doctor can seal the relative longitudinal edge of first main part 21 mutually at slit 28 places then, forms bootstrap reservoir 27.

Should be appreciated that other structure can be used for main part 21 and 22.For example, can separately provide first and second main part 21 and 22, and they can be only fixed to one another to form the bin 27 of sealing in patient's original position.

Fig. 8 and Fig. 9 explanation are applicable to an embodiment of the device of the bonded anastomosis situation in side and side.This device main body 30 shows and comprises 31, the second of first main parts and the 3rd main part 32,33 from this part branch.This branch has formed the main body of common X-shaped as shown in Figure 8.

All three main parts 31,32,33 are normally piped in shape.Except that other the 3rd main part 33, this device is similar to Fig. 6 and device shown in Figure 7 usually.

First main part 31 is around the natural blood vessel 34 of obturation, and by hermetic unit 35 and its outer membrane face sealing.Second and the 3rd main part 32,33 be around graft 36, and in separately hermetic unit 37,38 places and the outer membrane face sealing of graft.Show the most clearly that as Fig. 9 institute the effect of hermetic unit 35,37,38 is formed in the bootstrap reservoir 39 between the outer membrane face of blood vessel of the inner face of main body 30 and encirclement, the pharmaceutical preparation of can aforesaid mode having packed at least in part of this bin 39.

For ease of Fig. 8 and Unit Installation shown in Figure 9, the form with at least two parts provides this device Xiang the doctor easily.For example, can separate with second assembly that comprises second and the 3rd main part 32,33 first main part 31 is provided.By the main part that contains vertical slit (not shown) is provided, main part can be installed with around blood vessel 34 and graft 36, slit sealing longitudinally then, and seal mutually along contact wire is to be provided at the bin 39 of the sealing around the point 40 that coincide.

A kind of variant of Figure 10 and Figure 11 displayed map 8 and device shown in Figure 9 (using identical parameter) for common segment.A specially suitable application of device of the present invention is in the coronary artery bypass transplantation.In this case, as show that first blood vessel 34 can be the coronary artery that partly is embedded in the heart wall 50.The device of Fig. 8 and form shown in Figure 9 can not be installed on the coronary artery 34 of part embedding, because first main part can not be in full extension around the tremulous pulse 34.Therefore, in the embodiment of Figure 10 and Figure 11, when the cross-sectional view of scope longitudinally, first main part 51 of main body 31 can not be described a complete circle; Alternatively, it is normally arciform.In the embodiment illustrated, it describes an about arc of 180 °.This makes first main part 51 can only be installed on the expose portion of the coronary artery 34 of part-embedding, with only partly around it.In this arranges, the doctor for example by tissue glue with the longitudinal extension edge 41 of first main part 51 as shown with the outer membranous wall sealing of coronary artery 34, or with the face seal of heart wall 50.

The device of Figure 10 and Figure 11 also is applied to other operation method, and wherein first blood vessel is a tremulous pulse, and this tremulous pulse partly is embedded in the wall of the organ that tremulous pulse supplies with.These organs comprise brain, bladder and uterus.

Although illustrated embodiment concentrate on this device at anastomotic position to blood vessel and to the application of contiguous position delivery of therapeutic agents, the invention is not restricted to these application.For example, this device can more generally be used for the outer membrane face delivery of therapeutic agents to non-anastomosis of blood vessel.For example, behind the balloon angioplasty, a kind of device form shown in Fig. 3 to 5 can be placed around the tremulous pulse outside, near the balloon angioplasty position, carry one or more reagent to it with outer membrane face by this tremulous pulse.

In the above-described embodiment, the bin that shows sealing is taked tunica adventitia surface and the radial space between the inner face of first main part or the form in gap at least, the pharmaceutical preparation of in use packing at least in part of this space.Yet this space is optional.For example, in an embodiment of selecting fully, main body can have a common impermeable pliability skin and a pliability internal layer that in use contacts with outer membrane face with said preparation infiltration and preparation.

For example, its skin can be made by the solid collagen protein, and internal layer is made by crosslinked with it spongioid collagen protein, and sponge sample layer can infiltrate in the enough pharmaceutical preparation that comprises institute's delivery of therapeutic agents.In this case, but provide this device of the preparation that had infiltrated Xiang the doctor, perhaps can be for example by moistening this device of foregoing injection preparation after installation.

In addition, can be with this pack by on body inner surface, this surface in use contacts with blood vessel just.In addition, reagent can be dispersed in the structure of entire body.

What wish is the sufficient intensity that this device main body has the opposing twisting resistance.For this purpose, for example this main body can be with internal layer such as collagen film or vertically, laterally or the rib of spiral constitute.Can provide the rib that bin is further divided into compartment, so that additional stability to be provided.

These protein or nucleic acid can be used for the treatment or the prevention of the neointimal hyperplasia that caused by any clinical setting.For example, the hypertrophy that produces behind the treatment any kind operation method is possible, and these operation methods comprise angioplasty, as balloon angioplasty; Shunt operation is as the crown shunt operation with vein and arterial anastomosis; Other identical method is as the anastomosis of shank; And endarterectomy, as carotid endarterectomy.The neointimal hyperplasia that treatment and arterial injury or hypertension such as pulmonary hypertension are relevant also is possible.The invention provides in any kind blood vessel treatment, preferably tremulous pulse as neointimal hyperplasia in tremulous pulse or the vein.

According to the present invention, treatment or the neointimal hyperplasia of improve determining or when taking place the prevention neointimal hyperplasia be possible.Equally, the neointimal hyperplasia determined of the probability of the neointimal hyperplasia that reduce to take place or reduce or the order of severity of contingent hypertrophy are possible.According to treatment of the present invention can betide before the operation method, during or afterwards, in order to reduce the operation back outgrowth chance takes place for example.

Preferably, be that purpose is used VEGF nucleic acid or protein with prevention or treatment from stenocephaly.Yet it also can be used for the treatment of or prevention of restenosis.

Preferably carry protein of the present invention or nucleic acid with the pharmaceutical dosage forms that comprises pharmaceutically acceptable carrier.Can use any suitable pharmaceutical preparation.

For example, appropriate formulation can comprise aseptic injection aqueous solution and non-aqueous solution, and this solution can comprise antioxidant, buffer, antibacterial, sterilization antibiotic and make preparation and the isoosmotic solute of receiver's blood; And can comprise the SAS and the non-aqueous suspensions that can comprise suspending agent and thickening agent.Said preparation can be present in the container of unit dose or multiple dose, for example in Mi Feng ampoule and the phial, and can be stored under refrigerated or cryodesiccated (freeze dried) condition, only needs to add sterile liquid carrier, for example water for injection before using.

Should be appreciated that except the above composition of mentioning especially, consider the type of described preparation, preparation of the present invention can comprise other the conventional reagent in this area.In possible preparation, aseptic aqueous solution that does not contain pyrogen and non-aqueous solution are preferred.

Can and use any proper dosage scheme to carry protein, nucleic acid and carrier with any proper dosage.It will be apparent to one skilled in the art that according to many factors dosage and scheme can make it to be suitable for to guarantee the optimal treatment to the special disease of being treated.These factors can be age, sex and the patient's that treated clinical symptoms.

For the exposed nucleic acid of coding VEGF or comprise the conveying of the construct of these nucleic acid, general dosage is to use 0.1-5000 μ g at every turn, and 50-2000 μ g for example is as 50-100 μ g, 100-500 μ g or 500-2000 μ g.For the conveying of vegf protein matter, proper dosage comprises 1-1000 μ g, as 1-10 μ g, 10-100 μ g, 100-500 μ g or 500-1000 μ g.

The dosage that is used for the conveying of VEGF nucleic acid by virus or non-virus carrier will depend on many factors, comprise that carrier is delivered to the efficient of cell and the efficient that VEGF nucleic acid is expressed with VEGF nucleic acid in cell.

For example, can be with 10 ⁴To 10 ¹⁴The dosage of cfu or pfu/ml is carried viral vector, and for example 10 ⁴To 10 ⁶, 10 ⁶To 10 ⁸, 10 ⁸To 10 ¹⁰, 10 ¹⁰To 10 ¹²Or 10 ¹²To 10 ¹⁴Cfu or pfu/ml.10 ⁵To 10 ⁹Dosage about cfu or pfu/ml is preferred.Term pfu (plaque forming unit) is used for some virus, comprises adenovirus, and corresponding to the infectivity of viral solution, its assay method is to infect suitable cell culture, and the mensuration of the infection cell plaque quantity after common 48 hours.Term cfu (colony forming unit) is applied to other virus, comprises retrovirus, and it is being measured after 14 days with selected marker thing incubation usually by methods known in the art.Measuring the cfu of viral solution or the technology of pfu titre is well known in the art.

For retrovirus, 10 ⁵To 10 ⁶Dosage about cfu/ml is particularly preferred.For pseudotyped retroviral virus, 10 ⁷Dosage about cfu/ml is particularly preferred.For adenovirus, 10 ⁹Dosage about pfu/ml is particularly preferred.

Equally, these dosage can be contained in and be used for progressively carrying (gradualdelivery) in the implant of the present invention.

Also can be as mentioned above with any proper dosage, carry and the bonded VEGF nucleic acid of non-virus carrier by any application process, or from implant, progressively carry.Proper dosage generally is the nucleic acid of 0.1-1000 μ g, for example 1-100 μ g, 100-500 μ g or 500-1000 μ g, 1000-2000 μ g, 2000-3000 μ g or 3000-5000 μ g.Preferred dosage is about 5-50 μ g, as 10-20 μ g.

The medication schedule also will be according to for example route of administration, receiver's kind and receiver's disease and is different.Yet, should imagine the continuity a couple of days, several weeks or period several months single administration and repeatedly use.Equally, as mentioned above, can realize the conveying of vegf protein matter and nucleic acid by being suitable for being mounted to blood vessel, the implant around the tremulous pulse preferably; Preferably, this implant is the form of the collar.This implant will realize progressively carrying.For example, conveying can betide the time of a few hours, a couple of days, several weeks or several months.

Protein of the present invention and nucleic acid also can be used by any administration form, for example part, skin, parenteral, intramuscular, subcutaneous or transdermal administration, or by being injected directly into blood flow, being injected directly into around arterial wall or its or by mucosal tissue is directly used.Preferably, use by implant as mentioned above.

Can in any mammal, use protein of the present invention, nucleic acid and vehicle treatment neointimal hyperplasia.Treatment to human patients is preferred.

The present invention also is provided for treating or preventing the test kit of neointimal hyperplasia.These test kits comprise (i) a kind of reagent, as previously discussed, preferably with carrier-bound vegf protein matter or nucleic acid; A kind of implant (ii) of the present invention, it can be introduced vegf protein matter or nucleic acid wherein with the form of the collar.Preferably, provide VEGF nucleic acid or protein with the pharmaceutical dosage forms that comprises pharmaceutically acceptable carrier as mentioned above.Component (i) and (ii) can packing in any suitable manner.Also can comprise other component known in the art, for example standard reagent and/or solution and/or device.

The present invention also provides the method for treatment or prevention neointimal hyperplasia, comprises vegf protein matter, nucleic acid or the agonist of the patient of this treatment of needs being used the present invention of avirulent effective dose.Realize this treatment with method described herein.

As mentioned above, implant of the present invention especially with the implant of collar form, also can be used for reagent outside the VEGF to the conveying of blood vessel such as tremulous pulse.Can carry any suitable reagent with the method, with the therapeutic purposes that realize wishing.

Found that plasmid/liposome complex, MMLV retrovirus, VSV-G retrovirus and adenovirus cause the expression in adding the tremulous pulse of the collar.The gene transfer rate of adenovirus is the highest, and false type VSV-G retrovirus also produces high relatively transfection efficiency.Proof was not duplicated-application of deficient VSV-G retrovirus in the tremulous pulse gene transfer in the past.In some endotheliocyte of Adenovirus Transfection tremulous pulse, see expression.Because the intrusion from the adventitia to the inner membrance takes place, these results draw the gene that uses outside the VEGF and shift the general probability that changes endothelial function in human diseases by the chamber alia gene.This also can be used for the expression of diffusible or excretory gene outcome in adventitia and outer middle film, so expression product works at other place of arterial wall.Preferably, as mentioned above, this conveying realizes the treatment of neointimal hyperplasia, although its therapeutic purposes also that can realize other or that select fully.

The preferred therapeutic agent of Shu Songing is included in the protein outside the VEGF that arterial wall moderate stimulation nitric oxide (NO) produces in this way.The treatment of realization neointimal hyperplasia or the NO synzyme of the prevention especially conveying of inductible NO-synthase (iNOS) are particularly preferred.

Other preferred therapeutic agent comprises the agonist (on seeing) that activates the endothelium vegf receptor.These agonist generally are synthesized micromolecules.Also can use the peptide that comprises vegf protein matter fragments of peptides.In this case, for as described in the VEGF, conveying that can be by peptide itself or their the transporting of nucleic acid of encoding are realized treatment as above.Preferably carry, promptly carry, although they can be carried by systematicness by implant described herein by identical approach.

Preferably, therapeutic agent will be the form of coding pharmaceutical active polypeptide or proteinic nucleic acid.More preferably, as mentioned above, this nucleic acid will be contained in the construct.Again more preferably, will nucleic acid or construct be delivered to tremulous pulse by aforesaid carrier (virus as previously discussed or non-virus carrier).

Therefore, the outer gene transfer of tremulous pulse can be used for hereditary material to the preferably conveying of arterial wall of blood vessel wall.From embodiment, can see, in the middle of realizing from adventitia one side the variation of SMC and even the variation of endothelium, this makes and is used for for example development of the new method of arterial disease treatment of blood vessel.

Therefore, the invention provides the application of the nucleic acid of NOS (randomly iNOS) or coding NOS (randomly iNOS), be used for the treatment of or prevent the manufacturing of the medicine of vascellum endometrial hyperplasia; Wherein as above for as described in the VEGF, preferably provide NOS protein or nucleic acid with implant in the mode of the collar.

The present invention also provides test kit, and it comprises (i) NOS (randomly iNOS) protein or nucleic acid; A kind of implant (ii) of the present invention.These test kits as above for as described in the VEGF.They are suitable for the treatment or the prevention of vascellum endometrial hyperplasia.

The present invention also provides a kind of method of treatment vascellum endometrial hyperplasia, comprises near the hypertrophy of the implant implantation of the present invention that comprises NOS protein or nucleic acid is to be treated or prevention, realizes the conveying of NOS protein or nucleic acid thus.

For as described in the VEGF, NOS nucleic acid preferably combines with carrier as above.Treat as described in VEGF as above, dosage and pharmaceutical preparation also as above for as described in the VEGF.

The present invention also provides the implant of the present invention that comprises NOS (randomly iNOS) protein or nucleic acid.

VEGF also points out in the discovery that arterial wall moderate stimulation NO and prostacyclin produce, the agonist of VEGF and vegf receptor will be used for other NO-relevant and/or prostacyclin-relevant treatment of conditions.

Particularly, VEGF and can be used for treating hypertension with the agonist of the bonded receptor of VEGF.People such as Forte, lancet (1997) 349:837-42 finds that low NO level is to suffer from the feature of the hypertensive individuality of constitutional (i.e. systematicness).Known NO loosens blood vessel wall; People such as Forte suggestion, the NO of weakening produces and has reduced this loosening, and causes the contraction of blood vessel, and has therefore improved blood pressure.In addition, in the individuality of suffering from essential hypertension, the level of prostacyclin may be suppressed.

Because VEGF stimulates NO and prostacyclin to produce, so it can be used for the treatment of hypertension.More clearly, a kind of reagent of the present invention can be used for interested especially three kinds of treatment of diseases.

First kind is essential hypertension, the systemic hypertension of promptly any position or whole body.So to the treatment of the essential hypertension of systemic viral disease, preferably carry vegf protein matter of the present invention or agonist by gene therapy in the mode of system, for example the system of the VEGF nucleic acid by coding vegf protein matter or agonist carries.

Second kind is pulmonary heart disease, i.e. the right heart failure that hypertension causes in the pulmonary artery.This is medicable, and method is to use VEGF nucleic acid, protein and agonist as described here, particularly through a tremulous pulse collar (on seeing) VEGF DNA is delivered to tremulous pulse, and DNA is expressed in and produces protein in the arterial wall subsequently.

The third is a primary pulmonary hypertension, i.e. hypertension in the lung.This finally causes heart failure and death usually, and only can inculcate or the method for lung transplantation is treated by continuous prostacyclin at present.At this, preferred treatment technology will be nucleic acid conversion or the transfection lung tissue with coding VEGF or its agonist, produce VEGF thus in vivo and stimulate NO and/or the generation of prostacyclin.

Therefore, the invention provides the application of a kind of reagent in medicine is made, this reagent is selected from the nucleic acid molecules of the agonist of the agonist of nucleic acid, VEGF of VEGF (VEGF), coding VEGF and coding and the bonded receptor of VEGF, and the medicine of manufacturing is used to stimulate nitric oxide (NO) or the intravital generation of prostacyclin.

Test kit of the present invention, comprise vegf protein matter or nucleic acid or with the agonist of the bonded receptor of VEGF or the nucleic acid of these agonist of encoding, also be applicable to hypertensive treatment.

The present invention also provides the method for a kind of treatment or prophylaxis of hypertension, comprises vegf protein matter of the present invention, nucleic acid or the agonist of the patient being used avirulent effective dose.As this treatment of said realization.For primary pulmonary hypertension, as said, preferable methods is the nucleic acid conversion lung tissue with the agonist of the nucleic acid of coding VEGF or coding and the bonded receptor of VEGF.

The another kind of disease that can treat according to the present invention is an atherosclerosis, and it can be that NO-is correlated with and/or prostacyclin-relevant disease.Relate to atherosclerosis therapy, preferably use VEGF agonist as described here.

Therefore, the invention provides the application of a kind of reagent in medicine is made, this reagent is selected from nucleic acid, the agonist of VEGF and the nucleic acid molecules of coding and the bonded receptor stimulating agent of VEGF of VEGF (VEGF), coding VEGF, and the medicine of manufacturing is used for producing atherosclerosis therapy by stimulating in NO and/or the prostacyclin body.

The present invention also provides a kind of treatment or the atherosis method of prevention of arterial, comprises vegf protein matter, nucleic acid or the agonist of the patient being used the present invention of avirulent effective dose.As this treatment of said realization.

For atherosclerosis therapy, a preferred transport model is, the oral administration of vegf protein matter of the present invention or VEGF agonist preferably of the present invention is carried, for example with the form of tablet.

Following examples have illustrated the present invention.Use following abbreviation:

BSA-bovine serum albumin

The Eagle culture medium of DMEM-Dulbecco improvement

FCS-hyclone

The HUVEC-Human umbilical vein endothelial cells

IgG-immunoglobulin G

Map kinase-mitogen-activated protein kinase

MAb-monoclonal antibody PBS-phosphate buffered saline(PBS) PGI ₂-prostacyclin cPLA ₂-cPLA2 A ₂PIGF-placental growth factor SDS PAGE-sodium dodecyl sulfate-polyacrylamide gel electrophoresis VEGF-VEGF vWF-von willebrand's factor VSMC-vascular smooth muscle cell embodiment 1

Use to insert the silicon collar around the carotid artery, studied endotheliocyte (EC)-special VEGF gene transfer is to the effect of intimal thickening, this collar is not only as the reagent that causes the intimal smooth muscle cells growth but also as the bin of gene and carrier.This model keeps the integrity of EC, and do not need to allow the direct blood vessel alia gene transfer of operation in any blood vessel.Embodiment 1.1 gene transfer:

By under general anesthesia, inserting an inertia silicon collar around the tremulous pulse, in 32 New Zealand white rabbit, induce intimal thickening people such as (, atherosclerosis (Atherosclerosis) (1989) 76:257-268) Booth.The cover loop mapping carries out gene transfer after 5 days, method is to open this collar gently under anesthesia, and injects 500 μ l plasmid/liposome complexes in this collar (promptly on the tunica adventitia of artery surface).Do not relate to operating procedure in the blood vessel in any step of this research.Plasmid/liposome complex:

((people such as Breier grows (Development) (1992) 114:521-32 to comprise mice VEGF cDNA with 25 μ g pCMV5-VEGF-164 plasmids; Nucleotide 1-583)) compound with 25 μ lLipofectin (BRL), be diluted to 500 μ l with Ringer's solution.Before gene transfer, complex was placed room temperature at least 15 minutes.Measure according to this, study on the employed concentration at this, plasmid/Lipofectin complex external to rabbit aorta EC avirulence.With the similar plasmid/liposome complex transfection contrast tremulous pulse that comprises escherichia coli lacZ cDNA people such as (, the source is the same) Kalnins (nucleotide 1-3100) expression plasmid.Use Qiagen Mega post from culture of Escherichia coli (DH5 α), to separate the plasmid that is used for this research, and with three phenol/chloroform extractings and an ethanol precipitation purification (people such as Ausubel, compile, " molecular biology general scheme " (Current Protocolsin Molecular Biology) New York, NY:Greene Publishing Associates andJohn Wiley﹠amp; Sons (1991) 4.2.3-4.2.4).It is adjusted to 1 μ g/ μ l, analyzes it and do not have any microorganism or contaminated with endotoxins (limulus test detects lower limit 0.2ng).Animal is put to death in 3 days (n=8) of gene transfer operation, 7 days (n=12) and 14 (n=12) days backs; The careful tremulous pulse of removing, it is divided into three moieties: with nearside 1/3rd submergence in 4% paraformaldehyde/PBS-fix 15 minutes, and be embedded in (Miles Scientific) in the OCT chemical compound (people such as Yla-Herttuala, Journal of Clinical Investigation (J.Clin.Invest.) (1995) 95:2692-2698).Middle-end 1/3rd is as above fixed 4 hours, and rinsing is 48 hours in 15% sucrose, and is embedded in the paraffin.Far-end 1/3rd directly is embedded in the OCT chemical compound, and freezing in liquid nitrogen.In 4 tremulous pulsies, far-end 1/3rd is used for mRNA and separates and RT-PCR (as follows).Do not know that with two of causes the independent observation person of sample source carries out the mensuration of inner membrance/media thickness ratio (people such as Yla-Herttuala, atherosclerosis (1989) 6:230-236) from 10 sections that the stage casing part is selected at random.Use two independent mean value calculation results that measure (meansigma methods ± SD).Analyze the respectively difference of inner membrance/media thickness ratio between the group by ANOVA, the t-that improves subsequently check ( ^*P＜0.05).RT-PCR：

The VEGF (n=2) that gene transfer was collected after 7 days and the tip of lacZ (n=2) transfection tremulous pulse partly are used for carrying out mRNA and separate (Micro-FastTrack, Invitrogen), and as state (people such as Hiltunen: circulation (Circulation) (1995) 92:3297-3303) use hexabasic at random basic primer (cDNA circulation test kit, Invitrogen) with AMLV reverse transcription (every reaction 5U, Boehringer) the reverse transcription first chain cDNA.With Taq polymerase (Boehringer) and the primer special, carry out 35 circulation PCR (5 '-primer: SEQ ID No.9 to the pCMV5-VEGF-164 construct of transfection; 3 '-primer: SEQ ID No.10; The PCR loop parameter: except that last the circulation 5 minutes, 1 minute 90 ℃, 1 minute 60 ℃, 1 minute 72 ℃).In the tremulous pulse of VEGF-transfection, can see amplified fragments with expection length (547nt).(the 1kb gradient BRL) is shown in the gel both sides to the big tick marks of DNA.

Take microphotograph, demonstration VEGF or the lacZ gene transfer carotid feature of rabbit after 7 days.The immunostaining of transfection tremulous pulse proves: the contrast tremulous pulse with lacZ-plasmid/liposome (MAbHHF-35 that SMC-is special, dilution in 1: 500, Enzo Diagnostics) transfection shows typical intimal thickening; Tremulous pulse with VEGF plasmid/liposome (the MAb HHF-35 that SMC-is special) transfection only shows that limited tremulous pulse thickens; (continuous part of A, the MAb CD31 that EC-is special diluted 1: 50, DAKO) endothelium in the blood vessel sections of all researchs; (the MAb RAM-11 that macrophage is special diluted 1: 500, DKO) not detect the evidence of inflammation in the tremulous pulse of VEGF-and lacZ-transfection; Neovascularization (hematoxylin-eosin dyeing) is seen in gene transfer in the tunica adventitia of artery of VEGF-transfection after 14 days.

Avidin-biotin horseradish peroxidase system (Vector Elite, Vector Labs) is used for immunostaining (people such as Yla-Herttuala, newspaper (1990) 87:6959-6963 of institute of NAS).The contrast of immunostaining comprises the incubation with the immunoglobulin of uncorrelated class and irrelevant kind-coupling, omits the incubation of first antibody.As state people (1990) such as (, the source is the same) Yla-Herttuala and use and carry out in situ hybridization from the synthetic antisense VEGF nuclear probe of pBluescript SK plasmid (Stratagene) (583nt).Briefly, with E.C. 3.4.21.64 pretreatment paraffin-embedded part, with its acetylation, and with 35-S-UTP (DuPont, NEN)-nuclear probe (6 * 10 of labelling ⁶Cpm/ml) 52 ℃ of hybridization 16 hours.Washed 30 minutes at 60 ℃ with 0.1 * SSC the hybridization back.Use autoradiography to carry out signal detection (Eastman-Kodak NAB-2).The contrast hybridization of carrying out with the adopted nuclear probe of having of non-hybridization people (1990) such as (, the source is the same) Yla-Herttuala shows negative findings.The section haematoxylin redyeing.Embodiment 1.2

As described in embodiment 1.1, carry out gene transfer.From VEGF (n=5) or lacZ (n=5) gene transfer the previous day, in drinking water, rabbit is used L-NAME (70mg/kg/ days).Gene transfer was put to death animal after 7 days, analyze as mentioned above inner membrance/media thickness than and histology (people such as Yla-Herttuala, arteriosclerosis (Arteriosclerosis) (1986) 6:230-236; People such as Yla-Herttuala, (1990), the source is the same).Eliminate the difference of intimal thickening.Embodiment 1.3

Wash the confluent culture twice of HUVEC with serum-free medium, with culture shown in the presence of the recombinant human VEGF of concentration with this culture medium incubation 15 minutes, or with the time shown in the 10ng/ml VEGF incubation.In some experiments, cell is with 100 μ M L-NAME pretreatment 1 hour, subsequently with or handled 10 minutes without 10ng/ml VEGF.Remove culture medium, by adding 10mMTris/HCl (pH7.6), 50mM EDTA, 50mM NaCl, 30mM tetrasodium pyrophosphate, 50mMNaF, 0.1mM Na ₃VO ₄, 1mM PMSF and 1%Triton X-100 (lysis buffer), make cell 4 ℃ of quick cracking.Centrifugal 10 minutes of 15000 * g clarification lysate, by clarifying lysate is resisted with PY20-phosphotyrosine mAb is 4 ℃ of incubations 2 hours, carries out immunoprecipitation.By with lysate and protein A-agarose again incubation collected immunoprecipitate in 1 hour.Wash immunoprecipitate three times with lysis buffer, use 2 * SDS-PAGE sample loading buffer to extract protein then.Behind the SDS-PAGE, the protein transduction of immunoprecipitation is moved on the film, carry out immunoblotting with PY20 mAb then.The result shows that VEGF induces the proteinic phosphorylation of main 205kD corresponding to vegf receptor (in the protein of the main tyrosine phosphorylation at 100,125,145,190 and 205 places).L-NAME has eliminated replying VEGF.

Concentration with 25ng/ml adds reorganization VEGF, and the time can reach 2 hours (nitrite produces from the c.16 inner membrance level increase of μ M, remains in 1.8-2 μ M).Perhaps with shown in 0,2.5, the concentration of 25ng/ml carried out 10 minutes altogether.Add 25ng/ml after VEGF10 minute, measure the influence that L-NAME (100 μ M) pretreatment (1 hour) is replied VEGF.Use the capillary detection method measure the generation of nitrite (people such as Leone, " nitric oxide research method " (Methodsin Nitric Oxide Research), Feelisch and Stanler compile, John Wiley﹠amp; Sons, NewYork (1996) 499-508).C.19 level rises to and μ M c.2.1 in the presence of VEGF; Add L-NAME and reduce to control level (c.17 μ M).The result:

Find, compare, one week of operation back VEGF gene transfer significantly reduction intimal thickening (be respectively, inner membrance/middle film is than 0.3 pair 1.1,＜0.05) with the tremulous pulse of lacZ-transduction.The back effect reduction of two weeks, this may be because such fact, the temporary transient expression of rotaring redyeing gene is generally only induced in the gene transfer of plasmid/liposome-mediation, maximum protein expression people such as (, physiology's year summary (Ann.Rev.Physiol.) (1994) 56:741-761) Nabel between 2-3 after the gene transfer days.

The immunohistochemical analysis of tremulous pulse shows that intimal thickening almost all is made up of SMC.Endodermis comes across the section of all researchs.In the tremulous pulse of transfection, do not detect opposite effect or inflammation.

Use is passed through RT-PCR and by in situ hybridization, has been proved conclusively the expression of the VEGF of transfection the special primer of transgenic.Most of VEGF and lacZ express adventitia and the outer middle film that betides fibroblast and SMC.The adventitia neovascularization is seen in gene transfer in the tremulous pulse of three VEGF-transfections after 14 days.In the tremulous pulse of lacZ-transfection, do not detect neovascularization.

Show in the past, use lacZ-plasmid/liposome gene transfer of cover ring model in 0.05% arterial cell, to cause local gene transfer.Although the gene transfer rate is low, the VEGF of the inner secreted form that produces of the collar causes biological action in the local intra-arterial microenvironment, and this is shown in the appearance of neovascularization in the tremulous pulse of gene transfer three VEGF-transfections after 14 days.In acute anoxia, think that excretory VEGF arrives EC by diffusion, and combine with vegf receptor on the EC.

Suppose that VEGF is owing to the direct or indirect inductive EC-derivative factor of VEGF-to the inhibitory action of intimal thickening, or owing to can suppress the activity of SMC propagation subsequently.Particularly, suppose that VEGF is to mediate by the NO approach to the effect of intimal thickening.In a series of New Zealand white rabbit (n=8),, tested this hypothesis by animal being used NO synthetase inhibitors L-NAME at the gene transfer experimental session.Find that L-NAME has eliminated the difference of intimal thickening between the tremulous pulse of the tremulous pulse of VEGF transfection and lacZ-transfection.The main target cell of VEGF is EC in the arterial wall.Other cellular type with vegf receptor only is mononuclear cell, but as judge with the immunocytochemistry of specific antibody, add in these cases in the tremulous pulse of the collar and do not have mononuclear cell.

These results (ability of intimal thickening difference) are with consistent by the inhibition that stimulates the inductive intimal thickening of NO generation VEGF-.Therefore to verify whether VEGF can directly stimulate NO to produce in the EC culture.VEGF induces the proteinic tyrosine phosphorylation of main 205kDa corresponding to vegf receptor in the 1-25ng/ml concentration range.Monitor as the mensuration of nitrite level, VEGF is to interpolation time of causing of the Human umbilical vein endothelial cells (HUVEC) of cultivating-and the increase of the NO generation of concentration-dependence.VEGF early can see after 30 seconds to the VEGF adding the effect that NO produces, and reaches maximum after 5 minutes, can keep reaching 2 hours.Obtain the half maximum effect (half maximal) of VEGF during 5ng/ml.Inductive phosphorylation of VEGF-and NO are created in when 100 μ M L-NAME exist and eliminate fully.Therefore, may be the generation that the VEGF gene transfer has stimulated NO among the tremulous pulse EC of transfection, and limit the propagation of SMC through the mechanism of NO-mediation at least in part.This find with former usefulness endothelial NO synzyme cDNA to the observation that the transfection of tremulous pulse has reduced intimal thickening consistent people such as (, institute of NAS reports (1995) 92:1137-1141) Von der Leyon.

People's (source is the same) such as people such as Callow (source is the same) and Asahara infer that using of vegf protein matter stimulates the propagation of exposing EC in the tremulous pulse, but research participates in the actual mechanism that intimal thickening suppresses.In adding the carotid artery of the collar, in the presence of the complete endothelium of anatomy, stimulate intimal thickening.Therefore, the tremulous pulse VEGF of this report gene transfer to intimal thickening inhibitory action can not be because the endothelialization again that VEGF-stimulates.According to these results, VEGF can directly induce the generation of NO in HUVEC, and this is a kind of mechanism that VEGF can suppress intimal thickening.VEGF also can stimulate the generation that can bear other factor of regulating SMC propagation, comprises TGF-β or prostacyclin.Embodiment 2

Using plasmid/liposome complex to be used for gene carries, use is applied to the silica gel collar of adventitia, and Moloney muroid leukemia virus-deutero-(MMLV) retrovirus, the retrovirus that comprises false type herpes stomatitis virus albumen-G (VSV-G) and adenovirus are delivered to rabbit carotid artery.Using the collar is because 1) it provides a kind of gene to carry bin; 2) need not carry out transluminal operation, it is complete that endothelium remains anatomy; And 3) propagation of arterial smooth muscle cell (SMC) has been induced in the installation of the collar, and has improved the retroviral gene transfer rate that needs target cell propagation place.Gene transfer:

Use New Zealand white rabbit (1.8-2.5kg).Anesthetis is fentanyl-haloanisone (0.3ml/kg)/midazolam (1mg/kg) (people such as Yla-Herttuala, Journal of Clinical Investigation (1995) 95:2692-2698).Center line neck otch exposes left neck artery.The 2cm silica gel collar (MediGene Oy, Kuopio, Finland) of an any biological inert is positioned over around the carotid artery, so that it at one end contacts adventitia (people such as Booth, the source is the same) gently.The cover annuloplasty surgery carries out gene transfer after 4-5 days.For gene transfer, animal is anaesthetized again.Again the collar that exposes by operation is opened the 500 μ l gene transfer liquid (as follows) of packing into lightly.Close otch, the gene transfer rate of subsequent analysis tremulous pulse.Histologic analysis:

The careful tremulous pulse that adds the collar of removing is divided into three moieties with it: with near-end 1/3rd submergence in 4% paraformaldehyde/PBS (pH7.4)-fix 15 minutes, be embedded in subsequently in the OCT chemical compound (Miles Scientific, USA).Middle-end 1/3rd submergence in 4% paraformaldehyde/PBS (pH7.4)-fix 4 hours, rinsing is 48 hours in 15% sucrose (pH7.4), and is embedded in the paraffin.Far-end 1/3rd is embedded in the OCT chemical compound, and is processed as frozen section.To the betagalactosidase activity dyeing of 10 sections selecting at random 12 hours, be used for mensuration (people such as Nabel, science (1990) 249:1285-1288 of gene transfer rate with X-gal; People such as Yla-Herttuala, (1995), the source is the same).The gene transfer rate is calculated as the percent of the cell that contains beta galactosidase, the ratio of 20 100 * visual field center sums of selecting at random.From the section of selecting at random the collar tremulous pulse that adds of per three/part, be used to carry out analysis people such as (, the source is the same) Booth of immunocytochemistry and cellular type and/or inner membrance/media thickness ratio.

Use following antibody identification of cell type: SMC:HHF-35mAb (dilution in 1: 500, EnzoDiagnostics, the U.S.), α-Ji Dongdanbai mAb (dilution in 1: 1000; Sigma ChemicalCo.); Macrophage: RAM-11mAb (dilution in 1: 1000; Dako, U.S.), anti--CD68mAb (dilution in 1: 250; Dako); Endotheliocyte: anti--CD31mAb (dilution in 1: 50; Dako); Polymorphonuclear leukocyte: anti--CD45mAb (dilution in 1: 100; Dako); And anti--rabbit T-cell: MCA805mAb (dilution in 1: 1000; Dako).Avidin-biotin-horseradish peroxidase system is used for signal detection (Vector laboratories) (source is the same for people such as Yla-Herttuala, (1995)).Behind the immunostaining, with tissue slice hematoxylin negative staining.The mensuration of proliferation index:

Use 5-bromo-2 '-BrdU (BrdU) marker determination to add proliferation index in the tremulous pulse of collar people such as (, arteriosclerosis and thrombosis (Arterioscler.Thromb.) (1993) 13:571-578) Soma.Briefly, put to death preceding 3 hours to New Zealand white rabbit (n=12) injection BrdU (40mg/kg body weight).Carotid artery is fixedly spent the night in 70% ethanol, and be embedded in the paraffin.Behind the iodate second ingot dyeing nuclear, anti--mice IgG (Dako) dyeing series section (20 sections of every animal) of using the FITC-labelling is to detect BrdU.Label index is calculated as the percent of BrdU-hylon.Opposing carotid carries out sham-operation with comparing.Plasmid/liposome:

PCMV-beta galactosidase (lacZ) expression plasmid (Promega) is following compound with Lipofectin (BRL): 25 μ g plasmids slowly mix with 25 μ l Lipofectin reagent, are diluted to 500 μ l with Ringer's solution.In plasmid/Lipofectin solution, do not find precipitation.Mixture is placed room temperature at least 15 minutes, and within two hours, be used for gene transfer.Check the pollution (limulus test, Sigma Chemical Co.) that does not have lipopolysaccharide in the plasmid preparation.Retrovirus:

(source is the same for people such as Yla-Herttuala, (1995) to contain the pLZRNL MMLV retrovirus of LacZ; People such as Miyanohara, institute of NAS newspaper (1988) 85:6538-6542) or LacZ VSV-G pseudotyped retroviral virus people such as (, newspaper (1994) 91:9564-9568 of institute of NAS) Yee be used for this research.Among both, the expression of LacZ is driven by 5 ' LTR.Duplicate-deficient LZRNL amphotropic retrovirus is packaged in the PA317 cell, and as state with 5 * 10 ⁵The titre of cfu/ml is used (source is the same for people such as Yla-Herttuala, (1995)).Use transient transfection people such as (, the source is the same) Yee in the 293GP cell, to produce and duplicate deficient VSV-G pseudotyped retroviral virus.Use super centrifugal concentrated pseudotyped retroviral virus, and with 1 * 10 ⁷The titre of cfu/ml is used.Before the use, check that the reverse transcription disease toxin preparation does not contain any bacterial pollutant or helper virus people such as (, the source is the same) Yee.Adenovirus vector:

The deficient adenovirus that duplicates of E1-disappearance is used for this research (people such as Gosh-Choudhury, gene (1986) 50:161-171; People such as Simari, Journal of Clinical Investigation (1996) 98:225-235).The E1-disappearance district that uses homologous recombination will be positioned at nuclear guiding beta galactosidase cDNA under a β actin promoter and the cmv enhancer to be cloned into the adenoviral gene group (people such as Gosh-Choudhury, the source is the same; People such as Simari, the source is the same).In 293 cells, produce and duplicate the deficient adenovirus, and by super centrifugal concentrating.1 * 10 ⁹The titre of pfu/ml is used for the gene transfer experiment.Analyze adenovirus preparation and do not contain helper virus or bacterial pollutant people such as (, the source is the same) Gosh-Choudhury.The result:

Neointimal hyperplasia after the adventitia collar caused performing the operation 7-14 days.Endothelium keeps anatomy complete in the whole research.23% peak hypertrophy index after the demonstration of BrdU labelling was performed the operation 3 days.Neointima is made up of SMC fully.Plasmid/liposome complex causes the detectable transfer of gene to adventitia and outer middle film, and efficient is less than 0.01%.The collar tremulous pulse that adds untransfected or liposome-processing shows that no betagalactosidase activity is painted.

The retroviral gene transfering efficiency of adventitia is lower during the no collar, and perhaps this is only to betide in the proliferative cell because reverse transcription virus gene shifts.Duplicate the retroviral gene transfer rate of deficient MMLV low (less than 0.01%).The gene transfering efficiency of VSV-G pseudotyped retroviral virus is 0.1%.Use MMLV and VSV-G retrovirus, it is painted to observe beta galactosidase in adventitia and outer middle film.

Duplicate-the deficient adenovirus shows effective gene transfer, it is painted to detect beta galactosidase in adventitia and outer middle film.Interesting is also to observe painted in some endotheliocyte and some endo cell.Because the lacZ adenovirus construct comprises the nuclear localization signal of a beta galactosidase, so the painted nuclear that is arranged in transfectional cell of intensive X-gal.The Estimate of Total Number of painted nuclear from the section of analyzing, the gene transfer rate approximately is 10%.In the tremulous pulse of VSV-G retrovirus and adenovirus-transfection, see some inflammatory cells.In the tremulous pulse of plasmid/liposome transfection, can't see inflammatory cell.

In the present embodiment, with all gene transfer systems the transfer of beta galactosidase (lacZ) marker gene to adventitia and outer middle film takes place all.Adenovirus also is transferred to beta-galactosidase gene some endotheliocyte.After 5 days, adenovirus vector produces the highest gene transfer rate, can reach 10% cell demonstration betagalactosidase activity.False type VSV-G retrovirus finish adventitia and outside also be effective aspect the gene transfer of 0.1% cell in the middle film.The cell of plasmid/liposome complex and MMLV retroviral infection＜0.01%.All do not observe opposite tissue reaction in any gene transfer system.

Therefore, duplicate-the deficient adenovirus, VSV-G pseudotyped retroviral virus and plasmid/liposome complex can be used to use collar method to the arterial wall metastatic gene.Interaction energy to middle SMC even endothelium is realized from the adventitia sidepiece.Embodiment 3

In the present embodiment, detected VEGF to PGI ₂Stimulation.Cell culture:

Obtain HUVEC from Clonetics, and in the culture medium of adding 2%FBS that manufacturer carries, cultivate, perhaps HUVEC grows from fresh umbilical cord by collagenase digesting, and be incubated on the flat board of the culture medium 199 of adding 20%FCS and endothelial cell growth additive containing of 1% gelatin bag quilt (people such as Wheeler-Jones, journal of biological chemistry (Biochem J.) (1996) 315:407-416).Be used for experiment purpose, disperseed the HUVEC primary culture in 5 minutes by handling in 37 ℃, and then coated plate be in 90mm, 60mm or 35mm plastics plate or on the flat board of 24-hole with 0.05% trypsin/0.02%EDTA.Culture remained in contain 5%CO ₂In 37 ℃ of wet environments of 90% air, use this culture after 6-8 days or when cell forms the monolayer that is paved with.PGI ₂Measure:

The confluent culture of HUVEC in the 24-orifice plate is washed twice in serum-free M199 (pH7.4), and be exposed to the culture medium that contains VEGF.By 6-ketone group-PGF _1aThe quantitative cell conditioned medium liquid of radioimmunoassay in PGI ₂Content, 6-ketone group-PGF _1aBe the PGI of (people such as Wheeler-Jones, the source is the same) as previously mentioned ₂Stable catabolite.Arachidonic acid discharges:

Basically measure arachidonic release as stating (Domin and Rozengurt, journal of biological chemistry (1993) 268:8927-8934).The HUVEC culture that is paved with [5,6,8,9,11,12,14,15- ³H] (1mCi/ml, 211Ci/mmol) incubation is 24 hours for arachidonic acid.Wash cell twice with culture medium 199 then, and in adding this culture medium of 1ml of 0.3%BSA (being substantially free of fatty acid) and VEGF or thrombin incubation.After the processing, remove culture medium, in microcentrifuge centrifugal 5 minutes, measure radioactivity in the supernatant by the scintillation counter counting with 16,000 * g.The Western blotting:

With described in result and the legend, carry out the processing and the lysis of factor pair cell dormancy culture as mentioned above.Behind the SDS-PAGE, protein transduction is moved to Immobilon film (MilliporeInc.).For map kinase is measured, use 5% defatted milk powder closing membrane in PBS, pH7.2, and in the PBS/0.05% tween 20 of first antibody shown in containing (1mg/ml) incubation 3-5 hour.For with antibody to cPLA ₂Carry out immunoblotting, film was sealed 3 hours in the TBST 50mM Tris/HCl, 150mM NaCl, 0.02% (v/v) the polysorbas20 pH7.4 (TBST) that contain 0.2% (w/v) I-sealing (Tropix), then with first antiserum incubation in TBST.In TBST, wash film then 6 times (washing 10 minutes) at every turn, and in the TBST that contains the bonded second antibody of HRP-incubation 1 hour.According to manufacturer explanation use HRP-bonded anti--mice or anti--rabbit igg and ECL ^TMReagent develops the color immune response belt by chemoluminescence method.Map kinase is measured:

As state with the factor and handle cell, ice-cold PBS washes twice fast, immediately by adding 2 * SDS-PAGE sample loading buffer extraction cell that 100ml boils.Scrape and get the collection cell extract, be heated to 95 ℃ 10 minutes, electrophoresis on 12.5% acrylamide SDS-PAGE gel.After being transferred to the Immobilon film, with a kind of antibody protein is carried out immunoblotting, this antibody is discerned specifically by the phosphorylation of the Tyr204 activated p42 of institute and p44 map kinase (Erk-1 and Erk-2) people such as (, European molecular biology association's magazine (EMBO J.) (1991) 10:885-892) Payne.CPLA ₂Mobility shift assay:

The dormancy HUVEC that is paved with in the 60mm plate is washed twice in serum-free medium 199 (pH7.4), as legend in detail is described in detail the specified time of culture medium that contact contains the factor subsequently.Prepare cell lysate (people such as Wheeler-Jones, the source is the same) as previously mentioned.Through SDS-PAGE (10% acrylamide) isolated protein, use cPLA after being transferred to film ₂Polyclonal antiserum carry out immunoblotting (people such as BorschHaubold, journal of biological chemistry (1995) 270:25885-25892; With people such as Kramer, journal of biological chemistry (1996) 271:27723-27729).The excretory mensuration of vWF:

As state with ELISA and measure vWF secretion in the media samples that from the HUVEC confluent culture of handling with the factor, obtains people such as (, the source is the same) Wheeler-Jones.Flat board wraps quilt with anti--vWFmAb CLBRAg35, and the low detection limit of this algoscopy is about 1.0mU/ml.Material:

VEGF is from UBI or R﹠amp in reorganization; D system obtains.Reorganization PIGF is Werner Risau professor's a present, and also from R﹠amp; D system obtains.Polyclone cPLA ₂(Eli Lilly Indianapolis) is so kind as to give antiserum by doctor RuthKramer.Anti--vWF mAb CLBRAg35 is the present of J.A.Van doctor Mourik (center blood laboratory (Central Blood Laboratory), Amsterdam (Amsterdam), Holland).The antibody of the activated phosphorylation form of p42/p44 map kinase is available from New England Biolabs Inc..[5,6,8,9,11,12,14,15- ³H] arachidonic acid, ECL ^TMReagent and HRP-bonded anti--mice IgG is from Amersham, Britain.Goat is anti--and the bonded IgG of rabbit HRP-obtains from Pierce Inc.All other reagent that use are the purest obtainable grades.The result:

With the confluent culture of VEGF processing HUVEC, handle the different time that was 2 hours, when these times, remove culture medium, measure 6-ketone group-PGF _1aExistence, 6-ketone group-PGF _1aBe PGI ₂Stable metabolism catabolite.VEGF causes PGI ₂Time-generation that dependency increases, this early can detect after the factor adds 15 minutes, and sustainable growth reaches 60 minutes, can keep thereafter reaching 2 hours.Control cells only shows PGI in the time-histories of being checked ₂The less growth that produces.VEGF stimulates PGI ₂Produce also is concentration-dependent.Behind 60 minutes the incubation, can when 5ng/ml concentration, detect PGI ₂Synthetic growth is half-maximum when 10ng/ml, reaches maximum when 15ng/ml concentration.Point out the inductive PGI of VEGF-always ₂Synthesize the less reduction of experience when 25ng/ml.

Checked also whether the relevant factor PIGF (a kind of ligands specific of Flt-1 vegf receptor) of VEGF-can cause PGI in HUVEC ₂Synthetic.In 5 are independently tested, the PGI that PIGF stimulates ₂The synthetic VEGF that obviously is weaker than.Also replying with thrombin of VEGF and PIGF compared, thrombin is the synthetic a kind of effective derivant of prostaglandins in endotheliocyte and the platelet.In several independent experiments, directly acting in the parallel culture of VEGF, PIGF and thrombin contrasted, with 25ng/ml VEGF, 60ng/ml PIGF and 1U/ml thrombin 6-ketone group-PGF that incubation produced in 60 minutes _1aThe balanced growth multiple, be respectively 2.0 times, 1.3 times and 4.4 times of the average control level that do not stimulate.

If PGI ₂It is by isotype PLA that synthetic VEGF stimulates ₂Activation mediated, expection VEGF will cause arachidonic acid employing fast from cell.Be the test this point,, attack different time with VEGF subsequently HUVEC and radiolabeled arachidonic acid precincubation 24 hours.VEGF causes that the label in the culture medium that is released into the preliminary making cell increases, and this is obvious after the factor adds 10 minutes, and reaches maximum after 30 minutes.As measuring in parallel culture, time-histories and thrombin that the arachidonic acid that VEGF-stimulates discharges are closely similar.Concentration-dependency that the arachidonic acid that VEGF-stimulates discharges with from PGI ₂Produce obtained closely similar, when the half maximum effect is 2.5-5ng/ml in concentration, when maximum is 10-20ng/ml in concentration.Be similar to thrombin and VEGF and stimulate PGI ₂The relative ability that produces, the inductive maximum arachidonic acid of VEGF-discharge to be lower than always and obtain from thrombin.At 4 independently in the experiment, VEGF and thrombin cause that foundation level that the arachidonic release ratio of labelling does not stimulate has the balanced growth of 1.6 times and 3.4 times respectively.What PIGF did not cause that arachidonic acid discharges from the HUVEC of preliminary making can detected phenomenal growth.

A kind of possible mechanism of the arachidonic acid rapid release that VEGF-stimulates is PLA ₂The catalytic arachidonic direct enzyme of kytoplasm form institute discharge.CPLA ₂Can be activated by the phosphorylation of map kinase-dependence, be similar to the phosphorylation and the activation of other enzyme that comprises map kinase, cPLA ₂Can in to the SDS-PAGE gel, detect to its conversion that activates form from the mobility shifting of fast migration to slow migration form.Therefore, use cPLA ₂The Western engram analysis of specific antibody by the HUVEC extract measure the cPLA that replys VEGF ₂Activation (people such as BorschHaubold, the source is the same; With people such as Kramer, the source is the same).Do not stimulating in the extract of contrast HUVEC cPLA ₂The separating belt of two about iso brightness of antibody recognition, and with about Mr 97,000 migrations.Although cPLA ₂Predicted molecular weight with 85kDa, but reported in the past this protein in SDS-PAGE with 97kDa band migration (people such as BorschHaubold, the source is the same; With people such as Kramer, the source is the same).

The VEGF of 25ng/ml causes cPLA ₂The immunoreactivity phenomenal growth of slow migration form, and the relative reduction of the fast migration form of following, this can detect after VEGF adds 2 minutes, reaches maximum after 15 minutes, and can keep reaching 60 minutes.In 5 were independently tested, VEGF always caused cPLA ₂The immunoreactivity phenomenal growth of slow-migration form.The inductive cPLA of VEGF- ₂Electrophoretic mobility to reduce also be concentration-dependences, the phenomenal growth of slow migration form is arranged, the maximum maximum concentration 5-10ng/ml that is testing that increases when 2.5ng/ml.Although VEGF causes cPLA ₂Very fast migration form level obviously reduce, but the type all is tangible in all VEGF concentration and the treatment time detected.Thrombin also causes cPLA ₂Near the remarkable change of the mobility of migration form slowly, two kinds of forms all just in time are total to migration with detected those in the HUVECs extract of VEGF-processing from.The directly relatively demonstration of the effect of VEGF and thrombin is similar to from PGI in identical experiment ₂Synthetic and arachidonic acid discharges the result who is obtained, and thrombin causes cPLA ₂The gel blocking increase more remarkable, and actual this enzyme very fast-migration form complete obiteration.PIGF does not cause immunoreactivity cPLA ₂From near the detectable mobility shifting of slow migration form.

Checked further whether VEGF also induces HUVEC secretion vWF.The VEGF stimulation time-and the vWF of concentration-dependence produce.Early in the HUVEC culture medium, can detect vWF after 30 minutes, and in the time-histories that is detected, continue to increase, reach 2.5 times of levels after 3 hours to control cells in cell, adding 25ng/ml VEGF.Concentration-the dependency of VEGF effect is similar to by PGI ₂Generation is obtained, and the half maximum is replied at the about 10ng/ml of concentration, the maximum concentration 25ng/ml that maximum effect is being detected.Be similar to by PGI ₂The synthetic result who is obtained is although have comparability (Figure 12 A) with it always the effect of VEGF is weaker than thrombin.Opposite with VEGF, PIGF does not cause can detectedly be stimulated vWF among the HUVEC is excretory.VEGF and PIGF are to the relatively demonstration of HUVEC migration in the chemotactic chamber, although VEGF stimulates chemotactic concentration-dependency to increase, PIGF does not cause and can detectedly increase at concentration range 5-60ng/ml.

VEGF activates the p42/p44 map kinase in HUVEC.Suppose that map kinase participates in other factor pair cPLA ₂Activation, this causes that the map kinase cascade reaction can mediate the inductive PGI of VEGF-so ₂Synthesize and cPLA ₂Activated probability.Checked the concentration-dependency that is paved with the active activation of p42/p44 map kinase that VEGF-stimulates among the HUVEC.Cell contacts 15 minutes with the VEGF that is low to moderate 0.5ng/ml concentration causes detectable active the growth, the maximum concentration that increases of half 1 and 5ng/ml between, maximum effect continues to reach 50ng/ml when 10ng/ml concentration.Opposite with the remarkable effect of VEGF, the PIGF in the 1-60ng/ml concentration range causes the active raising of detectable map kinase hardly in HUVEC, even if the words that have are also very little.With the kinase whose a kind of selective depressant PD98059 of map kinase pretreatment 30 minutes, the VEGF that suppresses the p42/p44 map kinase fully stimulates (people such as Dudley, institute of NAS newspaper (1995) 92:7686-7689), the map kinase kinases is a phosphorylation and activate the bispecific threonine and the tyrosine kinase of p42/p44 map kinase specifically.The effect of inhibitor is concentration-dependence, has at 5mM to be higher than 50% inhibition, and the map kinase activity is reduced to not stimulated control level when 10-20mM concentration.

Initial by detecting PD98059 to PGI ₂Synthetic effect research map kinase cascade reaction is employed effect in the approach at the inductive arachidonic acid of VEGF-., suppressed fully HUVEC pretreatment 30 minutes with 30mM PD98059 by using 25ng/ml VEGF or 60 minutes inductive PGI of 1U/ml thrombin incubation subsequently ₂Produce.PD98059 is to the inductive PGI of VEGF- ₂Synthetic effect is concentration-dependence, and half maximum effect concentration is approximately 5mM, is that 10mM has the PGI to stimulating in concentration ₂The maximum inhibitory action that produces.Should point out that also PD98059 makes the PGI in the cell that VEGF-handles ₂Generation is reduced in control cells institute below the measured value, although this effect only is only significantly during greater than 10mM in inhibitor concentration.

Whether next has tested PD98059 to the inductive cPLA of VEGF- ₂The reduction of electrophoretic mobility has effect.Blocked the cPLA that VEGF-stimulates fully with 25mM PD98059 pretreatment ₂Slowly-the increasing of migration form.Be similar to PD98059 to PGI ₂Synthetic effect, this inhibitor have not only reversed the cPLA that VEGF-relies on ₂The reduction of gel shift rate, and the immunoreactivity of slow-migration form is reduced to below the control level, and the immunoreactivity of very fast-migration form is increased on the control level.

Whether the generation of the vWF that stimulates by test VEGF-is also to the inhibition sensitivity of PD98059, and the effect of having studied PD98059 is for the inductive cPLA of VEGF- ₂Activate and PGI ₂Synthetic selectivity.With PD98059 to cPLA ₂Activate and PGI ₂Synthetic effect is opposite, neither prevents from also significantly not reduce by the secretion that adds the caused vWF of VEGF subsequently with the pretreatment of 25mM map kinase inhibitors of kinases to HUVEC.PD98059 does not have to act on to basic vWF secretion or to the vWF secretion of thrombin-stimulations.Sum up:

The PGI of VEGF stimulation time dependency and concentration dependent ₂Synthetic growth, this can detect in 15 minutes, and the half maximum was arranged when concentration 10ng/ml after 60 minutes, and concentration has maximum when 15ng/ml.At 10 independently in the experiment, during 25ng/ml concentration after 60 minutes, the average maximum PGI that VEGF-stimulates ₂The synthetic foundation level that doubles.VEGF-correlation factor placental growth factor (PIGF) is induced 1.3 times more weak growth.Thrombin (1U/ml, 60 minutes) is induced 4.4 times of average maximum growths to control level.VEGF stimulates the release of arachidonic acid from HUVEC, has and PGI ₂The synthetic similar concentration-dependency that is obtained, but have kinetics faster: after the maximum arachidonic acid of half is employed and betided 10 minutes, maximum after 30 minutes.Use the cPLA2 A of the mobility shifting of SDS-PAGE as activation tagging ₂(cPLA ₂) activate and measure demonstration, VEGF stimulates cPLA in the mode that time-dependent and concentration rely on ₂Activity: cPLA early promptly took place to 2 minutes ₂Slow-migration and the increasing of the form of activation, concentration is low to moderate 2.5ng/ml and can detects, after 15 minutes and concentration reach maximum during for 5ng/ml.Be similar to and induce PGI ₂Synthetic other reagent, VEGF also causes the phenomenal growth that excretory time-dependent of von willebrand's factor among the HUVEC (vWF) and concentration rely on, this can detect in 30 minutes, concentration has the half maximum when 10ng/ml, handle 2.5 times that reached control level in back 3 hours with 25ng/ml VEGF.VEGF induces the quick and of short duration activation of p42/p44 map kinase, and this can detect when concentration is low to moderate 1ng/ml, and reaches maximum at 5-10ng/ml.On the contrary, PIGF is to the almost not effect of map kinase activity.PD98059 (the kinase whose a kind of selective depressant of map kinase) causes map kinase activity, the PGI that VEGF-stimulates ₂Synthesize and cPLA ₂The inhibition fully of gel blocking, but to not effect of the inductive vWF secretion of VEGF-.These discoveries provide VEGF can pass through cPLA ₂The arachidonic release of-mediation stimulates PGI ₂Synthetic first-hand evidence.These find also to show that the VEGF of this biosynthesis pathway stimulates and can pass through, and are that the activation of partly passing through the p42/p44 map kinase takes place at least.

Result shown here shows that VEGF is at HUVEC moderate stimulation PGI ₂Tangible time-dependent that produces and concentration dependent increase.The effect of VEGF is weaker than thrombin, although these two kinds of factors are replied variation factor only 2.5.As measure through gel blocking judge that VEGF stimulates arachidonic acid from the employing fast of human endothelial cell, and cPLA ₂Quick active.The method that is used to measure the effect that VEGF employs arachidonic acid based on ³The H-label is from using ³Release in the cell of H-arachidonic acid preliminary making.Owing to be to carry out the research that arachidonic acid discharges with the BSA that exists in the culture medium, the primary product that discharges from HUVEC very may be ³The H-arachidonic acid.The inductive PGI of VEGF- ₂Generation, arachidonic acid discharge and cPLA ₂Activated concentration-dependency similar each other (all in the 5-10ng/ml scope).These presentation of results arachidonic acids discharge and PGI ₂Synthetic VEGF stimulate be by this factor in HUVEC high-affinity receptor mediated.

Arachidonic acid discharges and cPLA ₂Mobility shifting is all than PGI ₂Synthetic generation quickly, this meets this viewpoint, i.e. PGI of Zeng Changing ₂Generation is likely the cPLA that (to small part being) improves ₂Activity, and the direct result of the raising that arachidonic acid can be measured in the cell subsequently, arachidonic acid is the substrate of constitutive enzyme COX-1 in the cell.Because known VEGF stimulates phospholipase C-g tyrosine phosphorylation and phosphoinositide metabolism, so comprise phospholipase C-g (and/or Choline phosphatase) and two acyls-may also can help the inductive PGI of VEGF-fully with other mechanism of monoacylglycerol lipase continuous action ₂Synthetic arachidonic acid is employed.

Also study VEGF-correlation factor PIGF (a kind of sepcific ligands of Flt-1) and whether can stimulate PGI ₂Generation.Results suggest PIGF can induce PGI ₂Synthetic, but be weaker than VEGF.Not detecting PIGF discharges or cPLA arachidonic acid ₂The remarkable effect of mobility shifting.Since PIGF only with Flt-1 vegf receptor high-affinity combine, so these results meet such conclusion most, promptly among the HUVEC VEGF to PGI ₂The stimulation that produces is mainly mediated by the KDR/Flk-1 receptor, but Flt-1 also can help the stimulation of this approach.Yet, the PGI of any Flt-1-mediation ₂As if be different from cPLA synthetic comprising ₂A kind of mechanism of phosphorylation.Have at least two kinds to explain this relatively low the replying that can illustrate to PIGF.Flt-1 induces more weak replying, and/or Flt-1 exists with the level lower than KDR/Flk-1.

These results described herein show, the secretion that VEGF induces vWF in the mode that time-dependent and concentration rely on.VEGF is to the secretory concentration-dependency of vWF and to PGI ₂, it is very consistent to synthesize the concentration dependent of employing with arachidonic acid.With PIGF to PGI ₂Synthetic weak relatively effect is opposite, and PIGF does not stimulate HUVEC to discharge the detectable growth of vWF.

VEGF causes cPLA ₂Electrophoretic mobility is the discovery of change fast, and is consistent with enhanced phosphorylation of p42/p44 map kinase and activation thus.This point has also been supported in discovery described herein, and is promptly known to platelet moderate stimulation cPLA ₂Phosphorylation and activated thrombin cause cPLA among the HUVEC ₂The similar change of mobility.Result described herein shows that also VEGF activates the p42/p44MAP kinases, and the kinase whose selective depressant PD98059 of map kinase has blocked PGI to the inhibition of this approach ₂Generation and the inductive cPLA of VEGF ₂Gel blocking strengthen.The effect of PD98059 is optionally at least in part, because it does not all have effect to the vWF secretion that VEGF or thrombin stimulated.

Opposite with the remarkable effect of VEGF, PIGF can not significantly improve the activity of map kinase.PIGF stimulates the active obvious incapability of map kinase roughly to meet this situation: this factor pair PGI ₂A little less than the effect of effect that produces, and meet this factor promotion cPLA than VEGF ₂The obvious incapability of gel blocking.

PGI among the HUVEC ₂There is the synthetic of obvious foundation level in the mensuration demonstration that produces.Enjoyably, the map kinase inhibitors of kinases is also eliminated not PGI in the stimulated control cell ₂Generation.With activation by the map kinase cascade reaction to PGI ₂The mediation that generates is consistent, and PD98059 also makes cPLA ₂The level of slow-migration phosphorylation form be reduced to below the control level, and the level of fast-migration low phosphorylation form is increased on the control level.The basic activity of the active mensuration prompting of map kinase significant level also can be suppressed by PD98059.These activation of finding the prompting map kinase not only can be undertaken the cPLA of VEGF-and thrombin-dependence ₂Activation, and basic map kinase activity is for composing type cPLA ₂Activity and PGI ₂Generation also need.

Seeming possible is that other signal pass through mechanism can help PGI ₂VEGF synthetic and that arachidonic acid discharges stimulates, and comprises [Ca in the cell ²⁺] rising.

VEGF mainly is considered to a kind of angiogenesis factor that promotes endothelial cell growth and migration up to now.Discovery shown here shows other activity, and therefore can having, and the purposes that is used for angiopathy such as narrow, restenosis, atherosclerosis and hypertensive treatment and prevention for the adjusting of endothelial function and the important application of understanding for the VEGF function.

Sequence table (1) general information (i) applicant:

(A) name: EUROGENE company limited

(B) street: Marquis house, 67/68 Jermyn Street

(C) city: London

(D) state: N/A

(E) country: Britain

(F) postcode (ZIP): SW1Y 6NY is denomination of invention (ii): the therapeutic use of somatomedin and the conveyer device that is used in particular for neointimal hyperplasia treatment be the sequence number (iii): 10 (iv) computer-readable information:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, Version#1.30 (EPO) (v) current request for data:

(A) application number: the information of WO the unknown (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 441 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological classification: linearity is molecule type (ii): cDNA (iii) supposes: non-(iii) antisense: non-(ix) feature:

(A) title/key word: CDS

(B) position: 1..441, (xi) sequence description of SEQ ID NO:1: ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTC GCC TTG CTG CTC 48Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 15 10 15TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA CCC ATG GCA GAA GGA 96Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30GGA?GGG?CAG?AAT?CAT?CAC?GAA?GTG?GTG?AAG?TTC?ATG?GAT?GTC?TAT?CAG 144Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln

35 40 45CGC?AGC?TAC?TGC?CAT?CCA?ATC?GAG?ACC?CTG?GTG?GAC?ATC?TTC?CAG?GAG 192Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu

50 55 60TAC?CCT?GAT?GAG?ATC?GAG?TAC?ATC?TTC?AAG?CCA?TCC?TGT?GTG?CCG?CTG 240Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu?65 70 75 80ATG?CGA?TGC?GGG?GGC?TGC?TGC?AAT?GAC?GAG?GGC?CTG?GAG?TGT?GTG?CCC 288Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

85 90 95ACT?GAG?GAG?TCC?AAC?ATC?ACC?ATG?CAG?ATT?ATG?CGG?ATC?AAA?CCT?CAC 336Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

100 105 110CAA?GGC?CAG?CAC?ATA?GGA?GAG?ATG?AGC?TTC?CTA?CAG?CAC?AAC?AAA?TGT 384Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys

115 120 125GAA?TGC?AGA?CCA?AAG?AAA?GAT?AGA?GCA?AGA?CAA?GAA?AAA?TGT?GAC?AAG 432Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Cys?Asp?Lys

The information of 130 135 140CCG AGG CGG 441Pro Arg Arg145 (2) SEQ ID NO:2: (i) sequence signature:

(A) length: 147 aminoacid

(B) type: aminoacid

(D) topological classification: linearity is molecule type (ii): the sequence description of protein (xi) SEQ ID NO:2: Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 15 10 15Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln

35 40 45Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu

50 55 60Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu?65 70 75 80Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

85 90 95Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

100 105 110Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys

115 120 125Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Cys?Asp?Lys

The information of 130 135 140Pro Arg Arg145 (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 573 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological classification: linearity is molecule type (ii): cDNA (iii) supposes: non-(iii) antisense: non-(ix) feature:

(A) title/key word: CDS

(B) sequence description of position: 1..573 (xi) SEQ ID NO:3: ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTC GCC TTG CTG CTC 48Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

150 155 160TAC?CTC?CAC?CAT?GCC?AAG?TGG?TCC?CAG?GCT?GCA?CCC?ATG?GCA?GAA?GGA 96Tyr?Leu?His?His?Ala?Lys?Trp?Ser?Gln?Ala?Ala?Pro?Met?Ala?Glu?Gly

165 170 175GGA?GGG?CAG?AAT?CAT?CAC?GAA?GTG?GTG?AAG?TTC?ATG?GAT?GTC?TAT?CAG 144Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln180 185 190 195CGC?AGC?TAC?TGC?CAT?CCA?ATC?GAG?ACC?CTG?GTG?GAC?ATC?TTC?CAG?GAG 192Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu

200 205 210TAC?CCT?GAT?GAG?ATC?GAG?TAC?ATC?TTC?AAG?CCA?TCC?TGT?GTG?CCG?CTG 240Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu

215 220 225ATG?CGA?TGC?GGG?GGC?TGC?TGC?AAT?GAC?GAG?GGC?CTG?GAG?TGT?GTG?CCC 288Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

230 235 240ACT?GAG?GAG?TCC?AAC?ATC?ACC?ATG?CAG?ATT?ATG?CGG?ATC?AAA?CCT?CAC 336Thr?Glu?Glu?Ser?Ash?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

245 250 255CAA?GGC?CAG?CAC?ATA?GGA?GAG?ATG?AGC?TTC?CTA?CAG?CAC?AAC?AAA?TGT 384Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys260 265 270 275GAA?TGC?AGA?CCA?AAG?AAA?GAT?AGA?GCA?AGA?CAA?GAA?AAA?CCC?TGT?GGG 432Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Pro?Cys?Gly

280 285 290CCT?TGC?TCA?GAG?CGG?AGA?AAG?CAT?TTG?TTT?GTA?CAA?GAT?CCG?CAG?ACG 480Pro?Cys?Ser?Glu?Arg?Arg?Lys?His?Leu?Phe?Val?Gln?Asp?Pro?Gln?Thr

295 300 305TGT?AAA?TGT?TCC?TGC?AAA?AAC?ACA?GAC?TCG?CGT?TGC?AAG?GCG?AGG?CAG 528Cys?Lys?Cys?Ser?Cys?Lys?Asn?Thr?Asp?Ser?Arg?Cys?Lys?Ala?Arg?Gln

310 315 320CTT?GAG?TTA?AAC?GAA?CGT?ACT?TGC?AGA?TGT?GAC?AAG?CCG?AGG?CGG 573Leu?Glu?Leu?Asn?Glu?Arg?Thr?Cys?Arg?Cys?Asp?Lys?Pro?Arg?Arg

The information of 325 330 335 (2) SEQ ID NO:4: (i) sequence signature:

(A) length: 191 aminoacid

(B) type: aminoacid

(D) topological classification: linearity is molecule type (ii): the sequence description of protein (xi) SEQ ID NO:4: Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 15 10 15Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln

35 40 45Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu

50 55 60Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu?65 70 75 80Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

85 90 95Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

100 105 110Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys

115 120 125Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Pro?Cys?Gly

130 135 140Pro?Cys?Ser?Glu?Arg?Arg?Lys?His?Leu?Phe?Val?Gln?Asp?Pro?Gln?Thr145 150 155 160Cys?Lys?Cys?Ser?Cys?Lys?Asn?Thr?Asp?Ser?Arg?Cys?Lys?Ala?Arg?Gln

165 170 175Leu?Glu?Leu?Asn?Glu?Arg?Thr?Cys?Arg?Cys?Asp?Lys?Pro?Arg?Arg

The information of 180 185 190 (2) SEQ ID NO:5: (i) sequence signature:

(A) length: 645 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological classification: linearity is molecule type (ii): cDNA (iii) supposes: non-(iii) antisense: non-(ix) feature:

(A) title/key word: CDS

(B) sequence description of position: 1..645 (xi) SEQ ID NO:5: ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTC GCC TTG CTG CTC 48Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

195 200 205TAC?CTC?CAC?CAT?GCC?AAG?TGG?TCC?CAG?GCT?GCA?CCC?ATG?GCA?GAA?GGA 96Tyr?Leu?His?His?Ala?Lys?Trp?Ser?Gln?Ala?Ala?Pro?Met?Ala?Glu?Gly

210 215 220GGA?GGG?CAG?AAT?CAT?CAC?GAA?GTG?GTG?AAG?TTC?ATG?GAT?GTC?TAT?CAG 144Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln

225 230 235CGC?AGC?TAC?TGC?CAT?CCA?ATC?GAG?ACC?CTG?GTG?GAC?ATC?TTC?CAG?GAG 192Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu240 245 250 255TAC?CCT?GAT?GAG?ATC?GAG?TAC?ATC?TTC?AAG?CCA?TCC?TGT?GTG?CCG?CTG 240Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu

260 265 270ATG?CGA?TGC?GGG?GGC?TGC?TGC?AAT?GAC?GAG?GGC?CTG?GAG?TGT?GTG?CCC 288Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

275 280 285ACT?GAG?GAG?TCC?AAC?ATC?ACC?ATG?CAG?ATT?ATG?CGG?ATC?AAA?CCT?CAC 336Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

290 295 300CAA?GGC?CAG?CAC?ATA?GGA?GAG?ATG?AGC?TTC?CTA?CAG?CAC?AAC?AAA?TGT 384Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys

305 310 315GAA?TGC?AGA?CCA?AAG?AAA?GAT?AGA?GCA?AGA?CAA?GAA?AAA?AAA?TCA?GTT 432Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Lys?Ser?Val320 325 330 335CGA?GGA?AAG?GGA?AAG?GGG?CAA?AAA?CGA?AAG?CGC?AAG?AAA?TCC?CGG?TAT 480Arg?Gly?Lys?Gly?Lys?Gly?Gln?Lys?Arg?Lys?Arg?Lys?Lys?Ser?Arg?Tyr

340 345 350AAG?TCC?TGG?AGC?GTG?CCC?TGT?GGG?CCT?TGC?TCA?GAG?CGG?AGA?AAG?CAT 528Lys?Ser?Trp?Ser?Val?Pro?Cys?Gly?Pro?Cys?Ser?Glu?Arg?Arg?Lys?His

355 360 365TTG?TTT?GTA?CAA?GAT?CCG?CAG?ACG?TGT?AAA?TGT?TCC?TGC?AAA?AAC?ACA 576Leu?Phe?Val?Gln?Asp?Pro?Gln?Thr?Cys?Lys?Cys?Ser?Cys?Lys?Asn?Thr

370 375 380GAC?TCG?CGT?TGC?AAG?GCG?AGG?CAG?CTT?GAG?TTA?AAC?GAA?CGT?ACT?TGC 624Asp?Ser?Arg?Cys?Lys?Ala?Arg?Gln?Leu?Glu?Leu?Asn?Glu?Arg?Thr?Cys

The information of 385 390 395AGA TGT GAC AAG CCG AGG CGG 645Arg Cys Asp Lys Pro Arg Arg400,405 (2) SEQ ID NO:6: (i) sequence signature:

(A) length: 215 aminoacid

(B) type: aminoacid

(D) topological classification: linearity is molecule type (ii): the sequence description of protein (xi) SEQ ID NO:6: Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 15 10 15Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln

35 40 45Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu

50 55 60Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu?65 70 75 80Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

85 90 95Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

100 105 110Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys

115 120 125Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Lys?Ser?Val

130 135 140 Arg?Gly?Lys?Gly?Lys?Gly?Gln?Lys?Arg?Lys?Arg?Lys?Lys?Ser?Arg?Tyr145 150 155 160Lys?Ser?Trp?Ser?Val?Pro?Cys?Gly?Pro?Cys?Ser?Glu?Arg?Arg?Lys?His

165 170 175Leu?Phe?Val?Gln?Asp?Pro?Gln?Thr?Cys?Lys?Cys?Ser?Cys?Lys?Asn?Thr

180 185 190Asp?Ser?Arg?Cys?Lys?Ala?Arg?Gln?Leu?Glu?Leu?Asn?Glu?Arg?Thr?Cys

195 200 205Arg?Cys?Asp?Lys?Pro?Arg?Arg

The information of 210 215 (2) SEQ ID NO:7: (i) sequence signature:

(A) length: 696 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological classification: linearity is molecule type (ii): cDNA (iii) supposes: non-(iii) antisense: non-(ix) feature:

(A) title/key word: CDS

(B) sequence description of position: 1..696 (xi) SEQ ID NO:7: ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTC GCC TTG CTG CTC 48Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

220 225 230TAC?CTC?CAC?CAT?GCC?AAG?TGG?TCC?CAG?GCT?GCA?CCC?ATG?GCA?GAA?GGA 96Tyr?Leu?His?His?Ala?Lys?Trp?Ser?Gln?Ala?Ala?Pro?Met?Ala?Glu?Gly

235 240 245GGA?GGG?CAG?AAT?CAT?CAC?GAA?GTG?GTG?AAG?TTC?ATG?GAT?GTC?TAT?CAG 144Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln

250 255 260CGC?AGC?TAC?TGC?CAT?CCA?ATC?GAG?ACC?CTG?GTG?GAC?ATC?TTC?cAG?GAG 192Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu

265 270 275TAC?CCT?GAT?GAG?ATC?GAG?TAC?ATC?TTC?AAG?CCA?TCC?TGT?GTG?CCG?CTG 240Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu280 285 290 295ATG?CGA?TGC?GGG?GGC?TGC?TGC?AAT?GAC?GAG?GGC?CTG?GAG?TGT?GTG?CCC 288Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

300 305 310ACT?GAG?GAG?TCC?AAC?ATC?ACC?ATG?CAG?ATT?ATG?CGG?ATC?AAA?CCT?CAC 336Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

315 320 325CAA?GGC?CAG?CAC?ATA?GGA?GAG?ATG?AGC?TTC?CTA?CAG?CAC?AAC?AAA?TGT 384Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys

330 335 340GAA?TGC?AGA?CCA?AAG?AAA?GAT?AGA?GCA?AGA?CAA?GAA?AAA?AAA?TCA?GTT 432Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Lys?Ser?Val

345 350 355CGA?GGA?AAG?GGA?AAG?GGG?CAA?AAA?CGA?AAG?CGC?AAG?AAA?TCC?CGG?TAT 480Arg?Gly?Lys?Gly?Lys?Gly?Gln?Lys?Arg?Lys?Arg?Lys?Lys?Ser?Arg?Tyr360 365 370 375AAG?TCC?TGG?AGC?GTG?TAC?GTT?GGT?GCC?CGC?TGC?TGT?CTA?ATG?CCC?TGG 528Lys?Ser?Trp?Ser?Val?Tyr?Val?Gly?Ala?Arg?Cys?Cys?Leu?Met?Pro?Trp

380 385 390AGC?CTC?CCT?GGC?CCC?CAT?CCC?TGT?GGG?CCT?TGC?TCA?GAG?CGG?AGA?AAG 576Ser?Leu?Pro?Gly?Pro?His?Pro?Cys?Gly?Pro?Cys?Ser?Glu?Arg?Arg?Lys

395 400 405CAT?TTG?TTT?GTA?CAA?GAT?CCG?CAG?ACG?TGT?AAA?TGT?TCC?TGC?AAA?AAC 624His?Leu?Phe?Val?Gln?Asp?Pro?Gln?Thr?Cys?Lys?Cys?Ser?Cys?Lys?Asn

410 415 420ACA?GAC?TCG?CGT?TGC?AAG?GCG?AGG?CAG?CTT?GAG?TTA?AAC?GAA?CGT?ACT 672Thr?Asp?Ser?Arg?Cys?Lys?Ala?Arg?Gln?Leu?Glu?Leu?Asn?Glu?Arg?Thr

The information of 425 430 435TGC AGA TGT GAC AAG CCG AGG CGG 696Cys Arg Cys Asp Lys Pro Arg Arg440,445 (2) SEQ ID NO:8: (i) sequence signature:

(A) length: 232 aminoacid

(B) type: aminoacid

(D) topological classification: linearity is molecule type (ii): the sequence description of protein (xi) SEQ ID NO:8: Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 15 10 15Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys?Phe?Met?Asp?Val?Tyr?Gln

35 40 45Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu?Val?Asp?Ile?Phe?Gln?Glu

50 55 60Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys?Pro?Ser?Cys?Val?Pro?Leu?65 70 75 80Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu?Gly?Leu?Glu?Cys?Val?Pro

85 90 95Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile?Met?Arg?Ile?Lys?Pro?His

100 105 110Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe?Leu?Gln?His?Asn?Lys?Cys

115 120 125Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg?Gln?Glu?Lys?Lys?Ser?Val

130 135 140 Arg?Gly?Lys?Gly?Lys?Gly?Gln?Lys?Arg?Lys?Arg?Lys?Lys?Ser?Arg?Tyr145 150 155 160Lys?Ser?Trp?Ser?Val?Tyr?Val?Gly?Ala?Arg?Cys?Cys?Leu?Met?Pro?Trp

165 170 175Ser?Leu?Pro?Gly?Pro?His?Pro?Cys?Gly?Pro?Cys?Ser?Glu?Arg?Arg?Lys

180 185 190His?Leu?Phe?Val?Gln?Asp?Pro?Gln?Thr?Cys?Lys?Cys?Ser?Cys?Lys?Asn

195 200 205Thr?Asp?Ser?Arg?Cys?Lys?Ala?Arg?Gln?Leu?Glu?Leu?Asn?Glu?Arg?Thr

The information of 210 215 220Cys Arg Cys Asp Lys Pro Arg Arg225,230 (2) SEQ ID NO:9: (i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological classification: linearity is molecule type (ii): cDNA (iii) supposes: non-(iii) antisense: the sequence description of non-(xi) SEQ ID NO:9: the information of TCGATCCATG AACTTTCTGC 20 (2) SEQ ID NO:10: (i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological classification: linearity is molecule type (ii): cDNA (iii) supposes: non-(iii) antisense: the sequence description of non-(xi) SEQ ID NO:10: TCCGTTTAAC TCAAGCTGCC 20

Claims (36)

1. purposes that stimulates the reagent that NO or prostacyclin produce, it is used for the treatment of or prevents the manufacturing of the medicine of vascellum endometrial hyperplasia.

2. according to the purposes of claim 1, wherein this blood vessel is a tremulous pulse.

3. according to the purposes of claim 1 or claim 2, its be used for operation method inductive or relevant narrow treatment or prevention with pulmonary hypertension.

4. according to the purposes of claim 3, wherein this operation method is angioplasty, coronary by-pass surgery, surgical stapling art or endarterectomy.

5. according to the purposes of aforementioned any one claim, it is used for the treatment or the prevention of angiostenosis or restenosis.

6. according to the purposes of aforementioned any one claim, wherein this reagent be nitric oxide synthetase, with a kind of agonist of the bonded receptor of VEGF or the nucleic acid of encode this synzyme or agonist.

7. according to the purposes of aforementioned any one claim, wherein this reagent is protein or this proteinic nucleic acid of coding with people VEGF function.

8. according to the purposes of claim 7, wherein this protein has SEQ ID No.2,4,6 or 8 sequence, or its active fragment.

9. according to any one purposes in the claim 6 to 8, wherein this reagent is the nucleic acid that combines with virus or non-virus carrier.

10. implant that is used for the treatment of purposes, it is suitable for being placed on the hypertrophy position of to be treated or prevention or near this position, and comprises the described a kind of reagent of aforementioned any one claim.

11. the implant according to claim 10, it is silica gel implant or biodegradable implant.

12. the implant according to claim 10 or 11, it is used for being installed on around the hypertrophy position or the close blood vessel at this position of to be treated or prevention for the form of the collar.

13. one kind according to any one implant in the claim 10 to 12, it has for the impermeable basically outer wall of the reagent that is comprised.

14. the purposes of any one described reagent in the claim 6 to 9, it is used for the treatment of the manufacturing of the medicine of disease, the stimulation that described disease can produce by body intracellular nitric oxide (NO) and/or prostacyclin and treated or prevent.

15. according to the purposes of claim 14, wherein disease is a hypertension, for example essential hypertension, primary pulmonary hypertension or pulmonary heart disease.

16. one kind is used for the device of therapeutic agent to patient vessel's conveying, it comprises a main body that is suitable for providing hermetic unit around blood vessel, and its reagent is stored in this device or with this device and combines, so that in use this reagent contacts with the tunica adventitia surface.

17. the device according to claim 16, a bin between its definition main wall and the tunica adventitia surface, this bin is packed at least in part and is comprised a kind of pharmaceutical preparation for the treatment of delivery of therapeutic agents.

18. the device according to claim 17, wherein said preparation is the form that injectable is gone into the liquid or the gel of bin, or the form of paste.

19. the device according to claim 18, wherein the material of main part is self-packing.

20. one kind according to any one device in the claim 17 to 19, wherein this bin can comprise the liquid that can reach 10ml, preferably 2-5ml at least.

21. one kind according to any one device in the claim 17 to 20, wherein the thickness of material of main part is normally constant along its length, and its bin is in use formed by the capsule of first main part, its and the compartment of blood vessel sealing between.

22. one kind according to any one device in the claim 17 to 20, wherein material of main part the thickness of mid portion less than with the thickness of the separation seal part of blood vessel sealing, the thickness of reduction has formed bin.

23. one kind according to any one device in the claim 16 to 22, wherein the inner surface of main body comprises a kind of sponge-like material, and this material can infiltrate with the pharmaceutical preparation that comprises this reagent.

24. one kind according to any one device in the claim 16 to 22, wherein the inner surface of main body infiltrates with a kind of pharmaceutical preparation that comprises this reagent.

25. one kind according to any one device in the claim 16 to 24, wherein the material of main body is biodegradable.

26. one kind according to the device in the claim 25, wherein this material is gelatin, alginate or collagen protein.

27. one kind according to any one device of claim 16 to 26, wherein this main body be molding or extrude.

28. one kind according to any one device in the claim 16 to 27, wherein main body comprises flexible hermetic unit, and these parts can adapt to the vasodilation that pulsatile blood flow causes.

29. one kind according to any one device in the claim 16 to 28, wherein this main body comprises the elongation hermetic unit that 8-15mm is long.

30. one kind according to any one device in the claim 16 to 29, wherein this main body is normally piped.

31. one kind according to any one device in the claim 16 to 29, wherein this main body has two and is generally piped part, and their branches form the main body of common gamma-form or T-shape.

32. one kind according to any one device in the claim 16 to 29, wherein this main body has three and is generally piped part, and they are branch at least in use, forms the main body of common X-shape.

33. device according to claim 31 or claim 32, one of them main part is normally arcual at the cross section of its longitudinal extent, with convenient vasculature part be embedded in when organizing, can make its expose portion around first blood vessel, and in use arrange first main part vertically-extend the edge to make it to seal with the outer membranous wall of first blood vessel or with adjacent tissue.

34. one kind according to any one device in the claim 16 to 33, wherein this main body has a vertical slit, is beneficial to its installation on blood vessel.

35. one kind according to any one device in the claim 16 to 34, wherein this main body comprises an internal layer or helical form girth member, to strengthen torsional strength.

36. method that any one described reagent in the claim 1 to 10 is delivered to blood vessel, it comprises and will be positioned over according to any device in the claim 16 to 35 around the blood vessel, if and the device in do not have medicament in advance, reagent is introduced this device.

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