A new protein of endothelial cells of the brain and composition

 

(57) Abstract:

The invention relates to a new isolated and purified protein that is produced by endothelial cells of the brain, has a molecular weight of approximately 67 KD on SDS-RADA, and mentioned protein is able to stimulate the proliferation of smooth muscle cells of cerebral arterioles. Also described composition, including a new protein. The invention provides for obtaining new drugs for the prevention or treatment of a wide variety of vascular diseases of the brain. 2 C. p. F.-ly, 7 Il.

The invention generally relates to the field of neurology and chemistry of proteins. More specifically the present invention relates to a new membrane protein 67 KD, Sekretareva endothelial cells of the brain.

Growth factor nerve tissue (NGF) plays an important role in the development and survival of sensory, sympathetic and some of cholinergic neurons. Protooncogen trk encodes a protein gp 14trkincluded in the membrane protein to tyrosinekinase whose expression is limited to the nervous tissue. Neurotrophic receptors trk A, B and C are tyrosine kinases and is expressed in the peripheral and Central nervous systems, policiesto NGF in the area of damaged or inflamed tissues increases several times, and this phenomenon is observed within the time of initiation of damage. NGF is involved in the immunomodulation through the activation of fat cells, and also in the recovery process of the fabric. The endothelial cells of brain capillaries together with astrocytes play an important role in maintaining the blood-brain barrier (BBB). The role of NGF in the restoration of capillary endothelium after the defeat of the tissue requires the presence of functional NGF receptor (trk-A) on endothelial cells to initiate angiogenesis necessary to replace the affected tissues.

In regulatory mechanisms of growth involved two proto-oncogene that encode nuclear proteins (c-fos, c-myc), as they are rapidly induced after treatment of cells polypeptide growth factors and other agents. Induction of c-fos is associated with a variety of biological events, including mitosis, differentiation and depolarization of nerve cells. These observations led to the proposal that fos plays a major role in signal transduction as a nuclear mediator.

Atherosclerotic changes or atherosclerotic plaques were at the centre of research for many years, and histological features are well described in the show four glassratner cholesterol deposits, especially esters of cholesterol; (3) projections of macrophages, particularly those Laden with lipid, the so-called foam cells, because they look filled with fat, plus associated cytokines produced by macrophages among other cellular elements, including cellular elements in the blood; and (4) enhanced synthesis of the elements of connective tissue, such as elastin and glucosaminoglycans. Each of these four areas histological convexity is the focus of intensive research, and although the final sequence of atherogenesis is still being discussed and is not defined, it is clear that he is multifactorial and includes the combination of weak cholesterol metabolism with increased proliferation of smooth muscle cells and increased activity of cytokines. It is known that some cytokines are important in the regulation of adhesion of leukocytes, cell growth, vasomotor functions, the reconstruction of the vascular matrix and regulation of the compatibility of blood to minimize or eliminate the impact of thrombosis in arterial endothelium.

A unique element separately causes atherosclerosis, but, probably, the regulation of one of these interacting Luge led to a decrease in the incidence of coronary heart disease in people with an increased risk of such disease, with the level of serum cholesterol was reduced below 170 mg/DL. Damage to the blood vessels that occur in hypertension, Smoking, use of oral contraceptives, and other damage not associated with the level of cholesterol in the plasma, causing adhesion of platelets at the injury site. This adhesion causes "response selection" in platelets with the secretion of various compounds, but in particular of thromboxane A2 and adenosine diphosphate, and mobilize platelets to increase aggregation. In addition, platelets secrete platelet-derived growth factor (PDGF), which causes proliferation of smooth muscle cells and their migration to the endothelium, where the smooth muscle cells form the primary elements of atherosclerotic plaques.

The process of belascoaran limited, primarily large vessels such as the aorta and elastic arteries such as the carotid, renal, coronary artery or middle cerebral artery. However, smaller arterioles can be damaged proliferative process, as with lacunar strokes, while loaded with fat cells and proliferation called "lipohyalinosis" ("lipohyalinosis"). Moreover, in the case of Binswanger or in subcortical arteriopathy of unknown nature. Proliferative process these smaller vessels can be an unregulated proliferation under normal presence of growth factor. Normal production of nerve growth factor tissue (NGF) cells of smooth muscles of arteries and reaction NGF protein - trk-he - Cohen suggests paracrine function. Distortion or complete loss of regulation of this paracrine control can lead to enhanced cell proliferation of arteriolar smooth muscle and to a narrowing of the lumen and subsequently to decreased blood flow to ischemia. Thus, a normal cell proliferation, smooth muscle, probably, participates in atherosclerosis, and the same uncontrolled development in the cerebral arterioles may lead to luminal occlusion, cerebral ischemia conductive fibers and symptoms similar to the symptoms of stroke, and dimensie.

When prior art lacks an effective means of prevention or treatment of a wide variety of vascular diseases of the brain. The present invention fulfills this long-standing need and want for the equipment.

In one of the embodiments of the present invention provides a composition of matter containing isolated and purified belino 67 KD on SDS-PAGE, moreover, the mentioned protein is able to stimulate the proliferation of smooth muscle cells of cerebral arterioles.

In another embodiment, the present invention provides a pharmaceutical composition comprising isolated and purified protein that is secreted by endothelial cells of the brain, having a molecular weight of approximately 67 KD on SDS-PAGE, and mentioned protein is able to stimulate the proliferation of smooth muscle cells of cerebral arterioles, and a pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides a method of obtaining a protein of the present invention, including the stage of growth of endothelial cells in a nutrient medium at a temperature of about 37oC; collection of cells and isolation and purification of the protein of the present invention of the above-mentioned cells.

In another embodiment, the present invention provides a method of treating vascular diseases brain in humans, including the stage of the introduction of man pharmacologically effective dose of the oligonucleotide created for inhibiting production of a protein of the present invention.

In one embodiment, Oswestry stage introduction to human a pharmacologically effective dose of the pharmaceutical composition of the present invention.

In another embodiment, the present invention provides a method of determining the severity sosedstvovalo disease in humans, including the state of measurement in the serum concentration of the protein of the present invention.

Other and further aspects, features and advantages of the present invention will become apparent from the following further description presents preferred embodiments of the present invention, is provided only with an explanatory purpose.

For more in-depth perception and understanding of the above features, advantages and purposes of this invention, and others which will become clear, you can get more specific description of the invention, the essence of which is briefly described above, referring to some variants of its implementation, which are illustrated by figures. It should be noted, however, that the appended figures illustrate preferred embodiments of the present invention and, therefore, should not be construed as limiting its scope.

Fig. 1 shows the levels of DNA synthesis in the endothelial cells of the rat brain (RBE) and in pulmonary endothelial cells (PEC). VK is broblasts (bFGF), either platelet-derived growth factor (PDGF), or medium with 4% serum, or control environment.

In Fig. 2 shows the expression of TrkA in the endothelial cells of the rat brain. Cell lysates derived from endothelial cells of the brain and their cells PC-12, polypeptides cleaved during electrophoresis in polyacrylamide gel in the presence of 7.5% sodium dodecyl sulfate (SDS-PAGE) and transferred onto nitrocellulose membrane. Membranes incubated overnight with affinity purified polyclonal anti-trkA, being probed for one hour with the secondary antibody conjugated with horseradish peroxidase, and find using the ECL system and immunological analysis.

Fig. 3 refers to the product of polymerase (reverse transcriptase inhibitor) chain reaction (RT-PCR) endothelial cells of rats brain and control cells PC-12. Back transcribers five μg total RNA and cells PC-12, and then PCR amplified using trkA-specific primers. Tracks: 1) RNA PC-12; 2) RNA PC-12 + DMSO; 3) and 4) enzymes and matrix control; 5) RNA endothelial cells of the rat brain with DMSO; 6) RNA endothelial cells of the rat brain; and 7) a marker DNA. The product of two hundred (200) base pairs (indicated by arrow), the. 4 illustrates the activation of the complex AP-1 in the endothelial cells of the rat brain using NGF. Nuclear extracts derived from endothelial cells brain rats, untreated (control) or treated for 15 min with 4% serum, 300 mm TPA, 10 ng/ml bFGF or 100 ng/ml NGF. Nuclear extracts (2 μg protein) are incubated with 2 µg of poly(d1-DC) and32P-labeled API probe (0.5 ng) for 30 min at room temperature. The reaction mixture was loaded onto 5% polyacrylamide gel and carry out electrophoresis. The gel is dried and analyzed by autoradiography.

Fig. 5 shows the induction of c-fos with NGF on endothelial cells of the rat brain. Nuclear extracts (2 μg) from endothelial cells of rats brain pre-incubated with addition and no addition (control) antibodies to c - fos for 16 hours Then add labeled32P AP1 (0.5 ng), and analyze the complexes 5% UN-denatured gel.

Fig. 6 shows the secretion of protein in the 67 KD in the endothelial cells of the rat brain using NGF.

Fig. 7 shows the secretion of protein in the 67 KD in the endothelial cells of the rat brain using bFGF.

The present invention describes immunology rats. Endothelial cells of rats brain respond to the growth factor, nerve tissue, and these reactions are seen from the change increased the incorporation of thymidine, induction of genes, including transcription factors (AP1), and the expression of mRNA for trk And, probably through the activation of the fos gene. Conditioned medium treated with nerve growth factor tissue endothelial cells of the rat brain contain protein of about 67 KD, which causes increased inclusion (3H) thymidine into cells of smooth muscles of vessels. It is known that among cells that are not nerve cells only b cells are activated by NGF, and as a result increases the production of immunoglobulin and proliferation of b-cells. Thus, the present invention discloses a mechanism by which NGF is involved in the angiogenesis of endothelial cells of the brain during the development of collateral circulation and proliferation of smooth muscle cells.

The present invention shows that in response to the effects of NGF on endothelial brain cells increased DNA synthesis and trkA receptor protein and associated allocation of a unique protein having a molecular weight of 67 KD. In addition, the induction of ranni the tion of complex AP-1 in the endothelial cells of the rat brain after stimulation of NGF leads to the assumption, that fos can function in a system of signal transduction that associates a short time events induced extracellular signal-long changes in gene expression. The present invention shows that NGF is the main mediator secretion of a new protein in the 67 KD, as a six-hour exposure of endothelial cells of the rat brain with nerve growth factor tissue grown on the mattress in 25 cm2produces a sufficient amount of protein, which can be painted Coomassie blue, while the control cells are not stained. Such significantly high rates secreted protein did not change the earlier passages. Early culture passages 3-14 shows a similar amount of secreted protein, although with the passage 15, the picture changes.

The unique response of endothelial cells of rats brain growth factor nerve tissue, which is not detected with peripheral endothelial cells, such as lung endothelial cells, is involved in normal physiological processes, such as collateral circulation, and in pathological States, such as subcortical encephalopathy or b NGF allocation growth factor from endothelial cells of rats brain can lead to proliferation of the terminal arterioles and constriction of the lumen.

The present invention relates to a method of regulating proliferation of smooth muscle cells of cerebral arterioles with endothelial cells of the rat brain. With the provisions of the present invention the expert in this field of technology can manipulate genes controlling growth factors, and use of antisense technology. Thus, the present invention can be useful for creating a protective collateral circulation and minimize arteriole narrowing and ultimately to prevent shock.

A new protein of the present invention, by changing the growth of smooth muscle cells to affect the stability, elasticity, elongation of cerebral vessels and the integrity of the pulsating blood flow under normal conditions, modify, develop and maintain collaterale circulation.

These expected normal physiological functions, when there is distortion or reaction to external stimuli with increased or reduced expression of the protein of the present invention, can be set with cerebrovascular complications detected in various diseases and stress factors. And the more fragile the brain vessels, what is observed in some sosudistogo diseases, such as intracerebral hemorrhage, subarachnoid hemorrhage due to an aneurysm, and migraine.

In addition, the increased reactivity when excessive proliferation of smooth muscle cells may contribute to cerebral atherosclerosis, lipohyalinosis, disease Binswanger long subcortical arteriopathies encephalopathy, disease Moya-moya and reduced blood-brain barrier with the formation of brain edema.

When the protein in the 67 KD of the present invention is secreted by endothelial cells of the brain, circulation levels of this protein can be installed using radioimmunoassay. When painful conditions the content of this protein in the serum will comply with the conditions, such as disease Binswanger as abnormal reactivity to a protein. On the other hand, under normal physiological situations, such as the formation of collateral circulation associated with blood flow changes in vascular correction can initiate this role of protein in growth, but not in proliferation.

In normal physiology and in pathological States of the control and regulation of protein truly is as a means of optimizing the normal operations of vessels and minimize disease. The methods currently available that can be applied for modification of the protein of the present invention include methods of antisense oligonucleotides and methods of education oligonucleotide triplex structures (triplex).It is expected that in the future similar role can be played medicines, modifying specific promoter or enhancer region of genes.

Specifically, it is assumed that it is possible to obtain a pharmaceutical composition, using a new protein of the present invention. In this case, the pharmaceutical composition comprises the novel protein of the present invention and a pharmaceutically acceptable carrier. The specialist in this field of technology will easily be able to determine, without undue experimentation, the appropriate dosages and routes of administration of a new protein of the present invention.

The average level of a specialist in the field of molecular biology in recent times has increased significantly. The specialist in this field of technology will easily be able without undue experimentation to a new protein sequence of endothelial cells according to the present invention. Having information about the protein, the specialist can easily clone the gene encoding the protein. Knowledge about gene members is for inhibiting transcription of the gene encoding a new protein of the endothelium of the brain according to the present invention. Knowledge about protein protein sequence of endothelial brain according to the present invention will allow the specialist to easily, without undue experimentation to obtain antisense oligonucleotide for inhibition of protein translation.

Thus, the present invention relates to compositions of matter containing isolated and purified protein that is secreted by endothelial cells of the brain, having a molecular weight of approximately 67 KD on SDS-PAGE, and mentioned protein is able to stimulate the proliferation of smooth muscle cells of cerebral arterioles.

Also features a pharmaceutical composition comprising a protein according to the present invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention is suitable for use in a variety of drug-delivery systems. A brief review of existing methods of drug delivery, see Langer, Science, 249:1527-1533 (1990). Methods for obtaining compounds that can enter the known or obvious to a person skilled in this technical field and described in detail, for example, in Remington's, Pharmaceutical Science, 17 is p. 1, which includes the stage of growth of endothelial cells in nutrient media at a temperature of about 37oC; harvesting the cells; and the isolation and purification of the protein under item 1 of the above-mentioned cells. In General, as described in the present invention, producing the protein of the present invention can be stimulated by growth factors such as nerve growth factor tissue.

The present invention also relates to the treatment of vascular diseases brain in humans, including the stage of the introduction of man pharmacologically effective dose of the oligonucleotide created for inhibiting production of a protein of the present invention.

Examples of representatives of diseases of the brain vessels, which can be treated by the methods of the present invention, are intracerebral hemorrhage, subarachnoid hemorrhage due to an aneurysm, migraine, cerebral atherosclerosis, lipohyalinosis disease Binswanger or subcortical arteriopathies encefalopatia, Moyamoya disease and lower blood-brain barrier with the formation of brain edema.

The specialist in this field of technology can easily get oligonucleotides, such as forming tranceportation protein of the present invention.

The present invention also relates to a method of improving cerebral circulation, including the stage of the introduction of man pharmacologically effective dose of a composition according to p. 2. The present invention also relates to a method for determining the severity of cerebral vessels in humans, including the state of measurement in serum protein under item 1.

The following are examples to illustrate various embodiments of the present invention, and these examples are not intended to be any limitation of the present invention.

Example 1. The selection of endothelial cells of rats brain

Endothelial brain cells of the rat isolated from the brain of newborn rats, and cells passedout on gelatinizing Petri dishes in culture medium containing 2% depleted platelets human plasma, 2% fetal bovine serum and 100 μg/ml of growth factor endothelial cells. Colonies that detect endothelial morphology, subcloning, and then clone, and stored frozen cell strains. Determine endothelial properties of cells, including morphology, nitropropene cell surface and expropriating reaction of endothelial cells of brain and lung cells passedout 24-hole Petri dishes and grown to 80% confluence. Delay the growth of crops 48-hour incubation in DMEM (modified by way of Dulbecco Wednesday Needle) containing 0.1% bovine serum albumin (BSA) and 0.1% glucose. The experiments begin sequential incubation of the cells in DMEM without serum in the presence or in the absence of growth factors. Add [3H]-thymidine, and after 24-hour exposure of the cell layers are washed and examined for the inclusion of tritium in DNA. Fig. 1 shows that the degree of incorporation of thymidine increases growth factor nerve tissue in the endothelial cells of the rat brain, but not in pulmonary endothelial cells.

Example 2. The expression of trkA in the endothelial cells of the rat brain.

To determine the presence of NGF-receptor (gpl40trk) in the endothelial cells of the rat brain, carry out Western blotting using polyclonal trk-A, trk b and trk C. the Endothelial cells of rats brain cells and PC-12 primesouth NGF for 7 days. Cells are lysed on the rocking chair in the buffer for lysis NP-40 (20 mm Tris, pH 8.0, 137 mm NaCI, 10% glycerol, 1% NP-40) containing 1 mm PMSF (phenylmethylsulfonyl), 0.15 ug/ml Aprotinin and 1 mm orthovanadate sodium, 4oC for 20 minutes Get cell lysates, polypeptid irout overnight with affinity purified polyclonal anti-trkA. The membrane is washed and probe for 1 hour with the secondary antibody conjugated with horseradish peroxidase, and are using the system immunological analysis ECL (AMERSHAM) according to the manufacturer's recommendations. The specific immunoblot positive for trk And identified as a protein 140 KD (Fig. 2).

Example 3. Expression of trk-receptor gene

Expression of trk-receptor gene in endothelial cells of rat brain confirmed the presence of mRNA. Total RNA back transcribers using random GEK-samarrie primers. The mixture produced cDNA is then used as the source matrix to stretch part of trk-A-gene. Used primer of the cDNA encoding the extracellular region of the protein, is a 5'- GGTCCAGGTGCCCAATGCC-TCGGH 5'-AGCTGCTCTAGATCATCCTTCTTCTCCACCGG. Fig. 3 shows that the product 200 base pairs, generated by PCR, identical to the product generated trk-A positive cells PC-12.

Example 4. Activation complex AP-1

The effect of NGF and other promoting growth substances on the expression of AP-1 in the endothelial cells of the rat brain measured by analysis of the mobility shift in the gel (Fig. 4).

Extracts of nuclei obtained from indot serum 300 nm TPA, 10 ng/ml bFGF or 100 ng/ml NGF. In accordance with the description in Dignam et al, Nucl. Acids Res., 11:1475-1489 (1983), subconfluent endothelial cells of rats brain washed with ice SFR and scraped in 5 ml SPR. Cells precipitated by centrifugation (500g for 5 min), then resuspended in 5 ml of hypotonic solution (10 mm Tris-HCl [pH 7,9], 12.5 mm MgCl210 mm KCl, 0.5 mm DTT (dithiothreitol)) and give the ability to swell on ice for 10 min the cells are Then homogenized 20 by shaking in a glass homogenizer of the downs, and precipitate nuclei by centrifugation at 1000g for 5 minutes Then engine resuspending in resuspension buffer (20 mm Tris-HCI [pH to 7.9], 1.5 mm MgCl2, 20% glycerol, 0.5 mm DTT), then add 4 M KCl to a final concentration of 0.3 M KCl. The suspension is gently shaking on a rocking chair at 4oC for 30 min, then centrifuged at 13000g at 4oC for 15 minutes the Supernatant containing the nuclear extract, and stored until analysis at -70oC.

The binding site of AP-1 is obtained from two oligonucleotides, generalizing typical sequences are 5'- GATCTGTGACTCAGGGGA-3' and 5'-GATCTCGCGCTGACTCACA-3'. Synthetic oligomers mark on the ends by MAniatis et al. Add competitive DNA, and the ethir>P-labeled AP1 probe (0.5 ng) for 30 min at room temperature. The reaction mixture was loaded into a 5% polyacrylamide gel (30: 0,8/acrylamide: bisacrylamide/30:0.8 gel 0.25 TBE Trisma, 25 mm boric acid and 1 mm etc) and subjected to electrophoresis. The gel was dried and analyzed by autoradiography. Band density AP-1 determine quantitatively the densitometer for birationality (model GS-650). To determine the identity of proteins that contribute to the complex AP-1, check the action on these complexes of antibodies directed against c-fos and c-jun.

It is known that specific antibodies can either break or slow down the protein-DNA complexes in non gels. Antibodies added to nuclear extracts of endothelial cells of the rat brain, and before adding the labeled probe was incubated for 16 h at 4oC. Strip (Fig. 5), shown by arrows, represent specific binding sequences. Add cold probe shows the binding specificity, and c-fos-antibody prevents formation of normal complex DNA-protein and generates in the form of migrating slower.

Example 5. Stimulation of NGF protein in the 67 KD

Glycoproteins on the cell surface at the cellular processes. To show that the response of endothelial cells of rats brain growth factor nerve tissue associated with the phenomenon, conditioned medium from endothelial cells of rats brain harvested after exposure to nerve growth factor tissue. Basically, cells are grown to confluence, rinsed and incubated with NGF for 6 h (Fig. 6).

The environment is collected and concentrated using centricon (method) 30. Concentrated samples subjected to 7.5% SDS-PAGE for Laemmii, and identify the protein bands by staining Coomassie blue, and discolor in 30% methanol with 10% acetic acid (by volume). In environments endothelial brain cells in rats treated with NGF, the streak of a particular protein (67 KD), and represents about 80% of total protein. Spot painted Coomassie protein then cut out, and subjected to digestion with V8 protease, subjected to electrophoresis in 15% acrylamide gel and electroblotting. Using automatic sequencing to determine the intermediate amino acid sequence of the proteins separated by electrophoresis. The sequence of one of the peptides has the appearance of Pro-Glu-Pro-Asp-Asp-Glu-Ala-Leu-Glu-Ala-Asn-Val-Ala-GIn.

It turns out that h is ski proteins. Information about the sequence of truncated peptides is not combined with the sequence of any known protein in the database (GEN Bank).

Example 6. Stimulation of bFGF protein in 67 KD

The present invention also describes the allocation of secretory proteins from the conditioned media of endothelial cells of the rat brain by processing cells a small amount of 10 ng/ml - key growth factors fibroblast (Fig. 7). Electrophoretic mobility of this protein during SDS-PAGE is almost the same as in the protein obtained by treatment of endothelial cells of rats brain growth factor nerve tissue. It is known that basic fibroblast growth factor has an angiogenic effect on endothelial cells of rat brain. Thus, protein-stimulated bovine fibroblast growth factor, similarly included in the angiogenic process of vascular endothelium.

Protein stimulated the basic fibroblast growth factor, secreted immediately after processing b-FGF, and secretion continues for at least 48 hours This protein has a molecular mass of about 65 -70 KD, and has the properties of a glycoprotein.

All patents and publications, womenpetite and publications incorporated herein as references, to the extent that as if it was stated that each publication included in the present as a reference.

The specialist in this field of technology can easily understand that the present invention is well adapted to carry out the objectives and obtain the final results and benefits that are inherent in it. The examples together with described in methods, procedures, treatments, molecules, and specific compounds are typical representatives of the preferred embodiments of the invention are examples and are not intended to limit the scope of the invention. Specialists in this field of technology can make changes to them and to use otherwise, without leaving the essence of the present invention defined by the scope of the claims.

1. Isolated and purified protein that is secreted by endothelial cells of the rat brain, and this protein differs in that it has a molecular weight of approximately 67 kDa, determined by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS - PAGE), is induced by nerve growth factor, has activity, stimulating cell proliferation la - Leu - Glu - Ala - Asn - Val - Ala - Gln (SEQID No:5).

2. The composition comprising the protein under item 1 and a pharmaceutically acceptable carrier.

 

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