WO2015076475A1 - Composition containing hgf protein or gene thereof for preventing or treating erectile dysfunction and use thereof - Google Patents

Abstract

The present invention provides a composition for endothelial cell generation, containing HGF protein, a biologically active fragment thereof, or a fusion protein thereof, as an active ingredient; a composition for preventing or treating erectile dysfunction; a method for regenerating endothelial cells of an individual; and a method for preventing or treating erectile dysfunction of an individual.

Description

A composition for preventing or treating erectile dysfunction comprising a HWF protein or a gene thereof and use thereof

The present invention relates to a composition for endothelial cell regeneration, a composition for preventing or treating erectile dysfunction, a method for regenerating endothelial cells of an individual, or a method for preventing or treating erectile dysfunction of an individual.

HGF is a growth factor found, isolated and cloned as a growth promoter for primary cultured hepatocytes. HGF is produced by a variety of mesenchymal cells, most of which target epithelial cells, neurons, vascular endothelial cells, and some mesenchymal cells. HGF is involved in cell proliferation promoting activity and has cell motility promoting activity and epithelial morphogenesis inducing activity. HGF is a mediator of epithelial-mesenchymal interactions during development and is involved in the formation of internal organs such as liver, kidney, lung, placenta and skeletal system. It is expected that HGF functions as an organ regeneration factor that promotes regeneration of the liver, digestive tract, kidney, and lung in the adult. However, to date, there has been no teaching or description about the prevention or treatment of erectile dysfunction of HGF, and there is no research on this.

On the other hand, erectile dysfunction is a type of male sexual dysfunction, a phenomenon in which the male genitals are not erection or erection is not sustained, the sexual activity is not possible. The causes of erectile dysfunction are largely divided into psychogenic causes and organic causes. Psychogenic erectile dysfunction is due to excessive secretion of noradrenaline due to excessive effects of sympathetic nerve due to psychological and psychological effects, increased penile cavernous smooth muscle tension, and suppression of neurotransmitter secretion. In addition, organic erectile dysfunction is classified into neurogenic, vascular, and endocrine dysfunction depending on the cause.

The vascular erectile dysfunction is impaired due to hyperlipidemia, diabetes, hypertension, smoking, systemic cardiovascular diseases, etc., resulting in impaired release of loose substances such as nitric oxide (NO) from vascular endothelial cells. It is a disorder that occurs.

Recent studies have placed more emphasis on organic causes, and oral PDE-5 (phosphodiesterase-5) inhibitors, including viagra (component name sildenafil), have been used worldwide for its treatment. Such oral drugs are known to have an effect of treating erectile dysfunction by increasing the concentration of cGMP by inhibiting PDE-5, which is specifically distributed in the corpus cavernosum, thereby increasing the blood flow in the corpus cavernosum. However, not only side effects such as headache, hot flashes, indigestion, and heart attack have been reported, but drugs of the PDE-5 inhibitor family such as Viagra are fundamentally controlled by regulating transient protein expression and related factors at the molecular level. I can not say. Moreover, in case of erectile dysfunction due to diabetes, not only the effect of such a therapeutic agent does not appear well, but even if there is a disadvantage that its efficacy does not last long.

Therefore, there is a need for a treatment for an erectile dysfunction which fundamentally treats an abnormal vascular structure in an erectile dysfunction penis, and whose efficacy also lasts for a long time.

One aspect of the present invention is to provide a composition for endothelial cell regeneration.

Another aspect of the present invention is to provide a composition for preventing or treating erectile dysfunction.

Another aspect of the invention is to provide a method for regenerating endothelial cells of a subject.

Another aspect of the invention is to provide a method for preventing or treating erectile dysfunction in a subject.

One aspect of the present invention provides a composition for endothelial cell regeneration comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.

The composition is preferably a pharmaceutical composition.

In the present specification, "HGF protein" means hepatocyte growth factor. HGF is a heterodimeric protein consisting of a chain of 69 kDa and a chain of 34 kDa, and may have four Kringle structures in the chain. C-met with tyrosine kinase activity may be a receptor for HGF.

The HGF protein includes a protein having an amino acid sequence represented by SEQ ID NO: 1 or a protein encoded by a nucleotide sequence represented by SEQ ID NO: 2, and a functional equivalent of the protein. The term "functional equivalent" means at least 70%, preferably 80%, more preferably 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid. Preferably, it refers to a protein having a sequence homology of 95% or more, and having a substantially homogeneous physiological activity with a protein having an amino acid sequence represented by SEQ ID NO: 1. In addition, the HGF proteins of the present invention include not only proteins having their natural amino acid sequence, but also variants thereof, are also included in the scope of the present invention. The variant means a protein in which the amino acid sequence of the HGF protein and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and fragments that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). HGF proteins or variants thereof can be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by genetic recombination methods based on DNA sequences (Sambrook et al. , Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd Edition, 1989).

The HGF protein may be encoded by the nucleotide sequence of SEQ ID NO: 2, and a variant capable of functioning identically with the nucleotide is included within the scope of the present invention.

The composition may comprise not only all of the HGF protein, but also its biologically active fragments or fusion proteins thereof. The biologically active fragment may be one comprising substantially the vascular endothelial and smooth muscle cell regeneration activity of the HGF protein or a gene encoding the same. The biologically active fragment may comprise an active domain involved in vascular endothelial and smooth muscle cell activity of native HGF.

The fusion protein may include substantially the vascular endothelial and smooth muscle cell regeneration activity of the HGF protein or a gene encoding the same. The fusion protein may be one in which the HGF protein or fragment thereof is linked to a fusion partner useful for isolation of the HGF protein or fragment thereof, a fusion partner useful for delivery to a target site, a fusion partner for increasing stability in the body, and the like. The linkage may be linked via the N terminus or C terminus or side chain of the HGF protein or fragment thereof. The linkage may be made by a covalent or non-covalent bond. The fusion partner may be one comprising a polypeptide. Useful fusion partners for separation may be His sequences, such as His6 sequences. Fusion partners useful for delivery to a target site, or fusion partners for increasing stability in the body, may be polymers that confer resistance to degradation in the body, such as antibody constant regions, eg, Fc regions or PEG.

The polynucleotide encoding the HGF may be in a fused form with other substances as well as itself. For example, the other substance may be such that the polynucleotide encoding the HGF can be delivered into a cell, expressed in a cell by a gene expression apparatus within the cell, and maintained stable within and outside the cell. It may be one or more selected from the group consisting of. The fusion can be by covalent or non-covalent bonds. The fusion includes the form that is collected in the vesicles.

The polynucleotide encoding the HGF may be operably linked with gene expression regulators such as promoters, operators, enhancers and / or transcription terminators. The polynucleotide encoding the HGF may be, for example, inserted into a plasmid or viral genome to be expressed in a cell. The polynucleotide encoding the HGF may be a structure linked to a regulator that is specifically expressed in endothelial cells and smooth muscle cells of the penis, for example, cavernous endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. The polynucleotide encoding the HGF may be, for example, in the form of a structure linked to a regulator that is inserted into the adenovirus genome so as to be specifically expressed in corpus cavernothelium and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. The structure may be collected in a virus particle.

As used herein, the term "active ingredient" includes an agent which, when administered to a subject, has an activity that further promotes endothelial cell regeneration as compared to otherwise. The subject may be one or more mammals, eg, humans, mice, hamsters, dogs, cats, horses, cattle, pigs and goats.

In the present specification, the "endothelial cells" may be penile cavernous endothelial cells, penile vascular endothelial cells or smooth muscle cells, preferably penile cavernous endothelial cells or penile vascular endothelial cells.

The composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. When formulating the composition, it can be prepared using a commonly used diluent or excipient. The composition may be a preparation for oral administration or a preparation for parenteral administration.

As used herein, the term "administration" means providing a subject with any composition of the present invention in any suitable manner.

Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. For the desired effect, the composition of the present invention may be administered in an amount of 0.0001 mg / kg to 10000 mg / kg per day. Administration of the composition may be administered once a day, may be divided several times.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intracavernous injection.

The composition may be for preventing or treating erectile dysfunction of an individual.

Another aspect of the present invention provides a composition for preventing or treating erectile dysfunction comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.

The composition is preferably a pharmaceutical composition or a food composition.

The HGF protein and polynucleotides encoding it are as described above.

The term "active ingredient" when administered to a subject, includes those having the activity of preventing or treating erectile dysfunction, as compared to otherwise. The subject may be one or more mammals, eg, humans, mice, hamsters, dogs, cats, horses, cattle, pigs and goats.

The term "prevention" includes preventing the impotence from being reduced or the duration of the impotence in comparison with the composition not administered. The term “treatment” includes the further recovery of weakened or reduced erection duration as compared to when the composition is not administered. For example, in the diabetic erectile dysfunction animal model, the HGF protein according to the present invention increases the intracavernosal pressure and improves erection by inducing regeneration of penile cavernous endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. It can have an excellent effect.

The composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The composition may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods. In particular, the composition may be an injectable formulation. The carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polycellulose Vinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

When formulating the composition can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents or surfactants commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules and the like. The solid preparation may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin in the composition. In addition, when preparing the composition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

The composition may be a preparation for oral administration or a preparation for parenteral administration. Liquid preparations for oral administration include suspending agents, solvents, emulsions or syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations or suppositories. In addition, as the non-aqueous solvent or suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil or injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter or glycerogelatin may be used.

The composition may further contain one or more known active ingredients having a prophylactic or therapeutic effect of erectile dysfunction together with HGF protein.

The composition may be to increase the intracavernosal pressure.

The composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum. The endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.

The erectile dysfunction may be caused by damage to penile endothelial cells or smooth muscle cells. The damage of the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension, and penile nerve damage.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of erectile dysfunction.

Another aspect of the present invention comprises the step of administering to the subject an endothelial cell regeneration composition comprising administering to the subject a composition for endothelial cell regeneration comprising an effective amount of HGF protein or a polynucleotide encoding the same as an effective ingredient for endothelial cell regeneration. Provide a method.

The endothelial cell regeneration composition is as described above.

In the method, the term "individual" means a subject in need of treatment of a disease. The subject may be a subject in which endothelial cells are damaged. The subject may be, for example, one or more selected from the group consisting of mammals, eg, human or non-human primates, mice, rats, dogs, cats, horses and cattle.

In the method, “amount effective for endothelial cell regeneration” includes an amount that, when administered to an individual, further promotes endothelial cell regeneration compared to otherwise. “Amounts effective for endothelial cell regeneration” include, for example, factors including the type and severity of the disease administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route and rate of administration, the duration of treatment, and the drug used concurrently. And other factors well known in the medical field, and can be easily determined by those skilled in the art in such an amount that the maximum effect can be obtained without side effects in consideration of all the above factors. The effective amount is 0.00001 mg / kg to 1000 mg / kg, for example, 0.00001 mg / kg to 100 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 1000 mg / kg, 0.001 mg / kg to 1000 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 1000 mg / kg, or 100 mg / kg to May be 1000 mg / kg.

In this method, administration comprises administering the composition via any route that can reach the erectile tissue of the penis. Such routes of administration include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or penile corpus cavernosum.

The composition may be to increase the intracavernosal pressure.

The composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum. The endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.

Another aspect of the invention comprises administering to a subject a pharmaceutical composition for preventing or treating erectile dysfunction comprising an effective amount of an HGF protein or a polynucleotide encoding the same as an active ingredient for treating erectile dysfunction. It provides a method for preventing or treating erectile dysfunction.

The pharmaceutical composition for preventing or treating erectile dysfunction is as described above.

In the method, the term "individual" means a subject in need of treatment of a disease. The subject may be an individual who has or is likely to exhibit an erectile dysfunction symptom. The subject may be, for example, one or more selected from the group consisting of mammals, eg, human or non-human primates, mice, rats, dogs, cats, horses and cattle.

In the method, “amount effective to treat erectile dysfunction” means that when the composition is administered to a subject, an improvement in erectile dysfunction, eg, an increase in erection and / or an increase in erection duration, is compared to otherwise. Contains amount to let. “Amounts effective for treating erectile dysfunction” include, for example, the type and severity of the disease being administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route and rate of release, the duration of treatment, and the drug used concurrently. It is determined according to the elements included and other well-known factors in the medical field, and can be easily determined by those skilled in the art in such an amount that the maximum effect can be obtained without side effects in consideration of all the above factors. The effective amount is 0.00001 mg / kg to 1000 mg / kg, for example, 0.00001 mg / kg to 100 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 1000 mg / kg, 0.001 mg / kg to 1000 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 1000 mg / kg, or 100 mg / kg to May be 1000 mg / kg.

In this method, administration comprises administering the composition via any route that can reach the erectile tissue of the penis. Such routes of administration include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or penile corpus cavernosum.

The composition may be to increase the intracavernosal pressure.

The composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum. The endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.

The erectile dysfunction may be caused by damage to penile endothelial cells or smooth muscle cells. The damage of the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension, and penile nerve damage.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of erectile dysfunction.

Another aspect of the present invention provides a food composition for preventing or improving erectile dysfunction comprising an HGF protein or a polynucleotide encoding the same.

The HGF protein and polynucleotides encoding it are as described above.

The HGF protein of the present invention may be added to a dietary supplement for the purpose of preventing or improving erectile dysfunction. In the present invention, the "health functional food" refers to a food having a bioregulatory function, such as prevention and improvement of disease, biodefence, immunity, recovery of disease, inhibition of aging, and may be harmless to the human body when taken in the long term.

When the HGF protein of the present invention is used as a food additive, the HGF protein may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the manufacture of food or beverages, the HGF protein of the present invention is added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of foods to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may include various flavors or natural carbohydrates, and the like as an additional ingredient, as in a general beverage. The natural carbohydrate may be a monosaccharide such as glucose or fructose, a disaccharide such as maltose or sucrose, and a natural sweetener such as dextrin or cyclodextrin, or a synthetic sweetener such as saccharin or aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

The dietary supplement includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated beverages. And a carbonation agent used in the. The health functional food may include a pulp for the production of natural fruit juice, fruit juice drinks and vegetable drinks. The components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

HGF protein according to the present invention induces the regeneration of vascular endothelial cells, smooth muscle cells, perivascular cells reduced by diabetes mellitus, PECAM-1, SMA, PDGF- specific proteins, vascular endothelial cells, smooth muscle cells, perivascular cells increase beta expression; In addition, HGF protein has an effect of promoting tube production and apoptosis in vascular endothelial cells. Through the above mechanism, HGF protein has an excellent effect on improving erectile dysfunction by increasing intracavernosal pressure, and thus may be useful for preventing or treating erectile dysfunction.

1 is a diagram showing the results of measurement of intracavernosal pressure (erectile power) according to penile nerve electrical stimulation by HGF protein administration in a diabetic mouse model.

FIG. 2 shows the expression levels of PECAM-1, a vascular endothelial cell-specific protein, and SMA, a vascular smooth muscle cell-specific protein, by HGF protein administration in the penile tissues of a diabetic mouse model. It is a figure which shows the result analyzed by confocal microscope together.

Figure 3 is a diagram showing the results of the analysis of the hydroethidin (hydroethidin) staining by confocal microscopy to compare the production of peroxide anion by HGF protein administration in the penile tissue of the diabetic mouse model.

4 is a diagram showing the results of immunohistochemical staining for nitrotyrosine by confocal microscopy to compare the production pattern of peroxynitrite by HGF protein administration in the penile tissue of the diabetic mouse model.

5 is a confocal microscope analysis of immunohistochemical staining for PDGFR-b in order to compare the expression level of perivascular cells that play an important role in the stability of blood vessels by HGF protein administration in the penile tissue of the diabetic mouse model. Is a diagram showing.

6 is a diagram showing the results of analyzing the expression of HGF receptor c-MET by confocal microscopy by HGF protein administration after vascular endothelial cells obtained by primary culture in mouse penis tissues under high glucose conditions. .

7 is a diagram showing the results of analyzing the expression level of PECAM-1, a vascular endothelial cell-specific protein by HGF protein administration after treatment of primary cultures in mouse penile tissues under hyperglycemic conditions. .

8 is a diagram showing the results of analysis of the degree of tube formation by HGF protein administration by phase contrast microscopy after treatment of vascular endothelial cells obtained by primary culture in mouse penis tissue under hyperglycemic conditions.

9 is a diagram illustrating the degree of apoptosis by HGF protein administration after treatment of vascular endothelial cells obtained by primary culture in mouse penis tissues under hyperglycemic conditions by TUNEL staining.

Hereinafter, preferred examples, experimental examples, and preparation examples are provided to help understanding of the present invention. However, the following Examples, Experimental Examples and Preparation Examples are provided only to more easily understand the present invention, and do not limit the content of the present invention.

Example 1 Effect of HGF Protein on Erectile Dysfunction in a Diabetic Erectile Dysfunction Model

Two-month-old male mice (C57BL / 6J) were studied and divided into four groups (N = 10 / group; group 1, normal mice; group 2, normal mice + streptozotocin) Induction of diabetes mellitus [mouse 8 weeks after intraperitoneal administration at 50 mg / kg concentration] + administration of Phosphate buffered solution (PBS) [20 μl]; group 3, induction of diabetes mellitus with streptozotocin [50 mg 8 weeks after intraperitoneal administration at / kg concentration] + mice injected with HGF protein once [day 0; 4.2 μg / 20 μl]; group 4, induction of diabetes with streptozotocin [50 mg / kg concentration] 8 weeks after intraperitoneal administration] + 2 injections of HGF protein [day −3, 0; 4.2 μg / 20 μl] mice). The mice were anesthetized by intramuscular injection of ketamine (100 mg / kg) and xylazine (5 mg / kg) and injected with HGF protein in the cavernous corpus cavernosum for 2 weeks after penile cavernous nerve stimulation. Measured. Platelet-endothelial adhesion molecule-1 (PECAM-1) and smooth muscle cell-specific proteins SMA and c-MET, an HGF receptor, were used in the corpus cavernosum tissues to change the expression of vascular endothelial cells and perivascular Changes in the expression of pericyte, which regulates contraction and maintains morphology, were examined by immunohistochemical staining for plateletderived growth factor receptor-beta (PDGFR-beta). In addition, the production of superoxide anion was confirmed by hydroetitidine staining, and the expression level of peroxinitrite was evaluated by immunohistochemical staining for nitrotyrosine.

As a result, the group of two injections of HGF protein showed the highest improvement in erection, and it was confirmed that the erection was restored to the normal level. The amount of cavernous endothelial cells (including vascular endothelial cells) and smooth muscle cells in the penis of streptozotocin-induced diabetic animals was significantly reduced compared to the normal control group. The amount of was remarkably restored to almost normal level. In addition, HGF significantly increased the expression of perivascular cells and decreased peroxide anion and peroxynitrite production. It will be described in more detail below.

(1) Analysis of erection power according to electrical stimulation

In the experimental method for measuring the erection power, the left epidermis was opened in the lower abdomen of the prepared mouse, and the penile cavernous nerve (the penis nerve) located on the posterior and posterior side of the prostate gland was prepared. For electrical stimulation of the penis nerve, a platinum electrode was placed in the penis nerve, and then electrostimulated for about 1 minute at a strength of 1-5 volts 12 hertz. When electrical stimulation is applied, the penis will have an erection. At this time, the catheter inserted into the corpus cavernosum was measured through a pressure transmitter connected to a computer (BioPack system, USA) to collect data on the pressure inside the penis (intracavernosal pressure) during the erection. 1 is shown. The measured pressure values will reflect penile erection.

1 is a diagram showing the corpus cavernosum (erectile power) according to penile nerve electrical stimulation in the HGF protein-administered group and the control group in the diabetic mouse model. In FIG. 1A, the intracavernous pressure (ICP) of the vertical axis refers to the pressure inside the penis (penis cavernous pressure) during an erection and is generally an indicator of erectile power. The horizontal axis represents time after electric stimulation. In FIG. 1A, the electrical stimulation period of 1 minute is indicated by a black bar on the horizontal axis. Figure 1A is the result of measuring the erection after neurostimulation at 1V or 5V in the corpus cavernosum 2 weeks after the injection of HGF protein into the corpus cavernosum once or twice. 1B and 1C, the mean systolic blood pressure (MSBP) on the vertical axis represents mean systolic blood pressure. In general, the erection power is expressed by dividing the maximum ICP or area of the cavernosal pressure curve by the mean systolic blood pressure (MSBP), which is the blood pressure itself. This may affect the internal pressure. In Figure 1, the control (Control), DM + PBS, one HGF administration, two HGF administration are individuals corresponding to the fourth group in the first group, respectively.

As shown in FIG. 1, the HGF protein twice-administered group (HGF double) showed the highest improvement in erection compared to the PBS-administered group (DM + PBS). Two variables for penile erection, namely, the area of maximal corpus cavernosum and the penile pressure curve, were markedly increased after HGF protein administration.

(2) Analysis of expression of PECAM-1, a vascular endothelial cell-specific protein, and SMA, a smooth muscle cell-specific protein

The penis is a type of special vascular tissue that normally requires cavernous sinusoids and normal blood vessel activity for normal penile erection. The corpus cavernosum and blood vessels are the innermost monolayer cells, which consist of endothelial cells, which are the vascular wall cells that come into direct contact with blood, and the smooth muscle cells that are responsible for the contraction and relaxation of blood vessels. These vascular endothelial cells and smooth muscle cells play an important role in penile blood vessel relaxation and thereby penile erection. The cavernous corpus cavernosum and the abnormal structure or activity of blood vessels cause erection.

In order to investigate the structural changes of blood vessels in the penile erectile tissues after administration of streptozotocin to normal mice, the present inventors expressed the expression of PECAM-1, a protein specific for spongy bodies and endothelial cells of blood vessels, and the expression of SMA, a smooth muscle cell specific protein. I checked it.

The penile tissue was fixed at 4 ° C. in 4% paraformaldehyde for 24 hours, and then frozen in the freezing section using a freeze embedding agent to easily fix the tissue and cut it into 7 μm thickness to prepare a penile tissue section.

Next, the prepared penile tissue sections were placed on the slides and the tissues on the slides prepared for PECAM-1 and SMA expression analysis were fixed in 4% paraformaldehyde for about 5 minutes. Fixed penile tissue sections were washed three times in wash buffer (2% FBS + 0.1% Sodium Azide in PBS) and then blocked for 1 hour with non-specific protein blocking buffer (5% BSA in PBS). Removing the remaining antibody after reacting the first antibody (anti-PECAM-1 rat antibody and FITC-labeled anti-SMA antibody, anti-c-Met rabbit antibody) at 1: 50 ratio for 16 hours at 4 ° C Second wash (TRITC-labeled anti-rat antibody, FITC- and TRITC-labeled anti-antibodies), which were then washed three times with wash buffer again, followed by fluorescence to identify antibodies that specifically reacted to PECAM-1. Rabbit antibody) was reacted at room temperature in a ratio of 1: 1000 for 2 hours. After the reaction was completed, and washed again three times with a wash buffer to remove the remaining antibody, the expression level was analyzed by confocal microscopy to confirm the fluorescence microscope or fluorescent material, the results are shown in FIG.

FIG. 2A is a confocal micrograph showing the expression level of HGF protein c-Met and the expression level of PECAM-1, a vascular endothelial cell specific protein, in the penile tissues of diabetic mice. As shown in Figure 2A, it can be seen that the expression of the receptor c-Met is increased in diabetic mice administered with HGF protein. In the group administered with HGF protein once, the expression of c-Met was increased compared with the group administered with only PBS, and the expression of c-Met was most expressed in the group administered with HGF protein twice.

It can be seen that the number of vascular endothelial cells in the penis of the group injected with PBS to diabetic mice (DM + PBS) was significantly reduced compared to the control mice. Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Compared with the group treated with HGF protein (DM + HGF single) and twice (DM + HGF double), the group of vascular endothelial cell-specific proteins was compared with the group not treated with DM + PBS (PBS instead of HGF protein). Expression was increased. Figure 2B is a graph showing the statistical treatment of c-Met expression in the area of the corpus cavernosum. Figure 2C is a graph showing the statistical processing of the expression of vascular endothelial cells in the area of the corpus cavernosum. This is the result of demonstrating that vascular endothelial cells reduced by diabetes are induced by HGF protein and that erection is restored accordingly.

2D is a confocal micrograph showing the degree of expression of SMA, a smooth muscle cell specific protein, and the HGF receptor c-Met in the penile tissues of diabetic mice. As shown in Figure 2D, it can be seen that the number of vascular smooth muscle cells in the penis of the group (DM + PBS) injected diabetic mice PDM significantly reduced compared to normal mice. Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. The expression of smooth muscle cell-specific protein in the group treated with HGF protein once (DM + HGF single) and twice (DM + HGF double) compared to the group not treated with DM + PBS (PBS instead of HGF protein). This has been increased. 2D and 2E are results demonstrating that smooth muscle cells reduced by diabetes are induced to regenerate by HGF protein, thereby restoring erection.

(3) Analysis of hydroetitidine reactions to know the formation of peroxide anions

Hydroetitidine is a redox-sensitive probe used to detect peroxide anions in cells. When superoxide reacts with hydroethidine, two-electron oxidized product called ethidium is formed, and ethidium adheres to DNA and absorbs wavelengths of 500-530 nm, thus 590-620 nm. Fluoresce. Peroxide anion, one of the active oxygen, is a highly reactive anion free radical that is made in large quantities by NADPH oxidase, which is harmful and can cause DNA damage.

Therefore, the present inventors confirmed the degree of response through hydroetitidine to investigate whether peroxide anion is generated in the penile erectile tissue after administration of streptozotocin to normal mice.

Fluorescence immunostaining with anti-PECAM-1 antibody by the method described above, and after final washing, treated with hydroetitidine [1 mM in PBS] for 30 minutes, followed by expression with a confocal microscope to identify fluorescence microscope or fluorescent substance. The degree was analyzed and the results are shown in FIG. 3.

Figure 3 is a confocal micrograph showing the degree of luminescence after treatment with hydroethidine reacting with the peroxide anion of the HGF protein-administered group and the control group in diabetic mouse penis tissue. As shown in FIG. 3A, it can be seen that the peroxide anion is significantly increased in the penis of the group in which diabetic mice are injected with PBS (DM + PBS) compared to normal mice (Control). Here, Control is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Peroxide anion compared to the group administered with HGF protein twice (DM + HGF double) compared with the group not treated with DM + PBS: PBS instead of HGF protein, DM + HGF single: HGF protein once The production of is reduced. Figure 3B is a graph showing the statistical treatment of the expression level of hydroethidine in the area of the corpus cavernosum. This is the result of demonstrating that the peroxide anion increased by diabetes is reduced in its production by HGF protein and therefore erection is restored.

(4) Analysis of the nitrotyrosine reaction which can know the production of peroxynitrite

Nitric oxide (NO) is an important cytotoxic mediator produced by nitric oxide synthase (NO synthase, NOS). Recently, peroxynitrite (ONOO-) produced by the reaction of NO and superoxide (ONOO-) is reported to cause more damage to cells than direct damage by NO. Peroxynitrite nitrates tyrosine groups in proteins and oxidizes sulfahydryl groups to inhibit peroxidation of membrane lipids, cleavage of DNA, electrolyte migration in the mitochondria, and calcium leakage, which disrupts cell energy production. It is known to play a decisive role in cell damage such as causing necrosis. Since peroxynitrite changes to 3-nitrotyrosine by nitrifying the tyrosine group of the protein, immunostaining for nitrotyrosine may indirectly determine the presence and activity of peroxynitrite.

Thus, the present inventors stained nitrotyrosine to check whether or not peroxynitrite is produced in penile erectile tissue after administration of streptozotocin to normal mice.

Fluorescence immunostaining with anti-PECAM-1 antibody and anti-nitrotyrosine antibody and the second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-rabbit antibody) were reacted in the manner specified above. The degree of expression was analyzed by a confocal microscope that can identify the fluorescence microscope or fluorescent material, the results are shown in FIG.

4A is a confocal micrograph showing the expression level of nitrotyrosine in the HGF protein-administered group and the control group in the penile tissue of diabetic mice. As shown in Figure 4A, it can be seen that the nitrotyrosine is significantly increased in the penis of the group (DM + PBS) injected diabetic mice compared to the normal mouse (Control). Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Nitrotyrosine compared to the group treated with HGF protein twice (DM + HGF double) compared to the group not treated with DM + PBS: PBS instead of HGF protein, and DM + HGF single: HGF protein once. The production of is reduced. Figure 4B is a graph showing the statistical treatment of nitrotyrosine expression in the area of the corpus cavernosum. This is the result demonstrating that peroxynitrite increased by diabetes is reduced in production by HGF protein and therefore erection is restored.

(5) Analysis of PDGFR-beta expression, a pericyte-specific protein

Angiogenesis refers to the generation of microvessels from existing blood vessels.

In the final stage of the angiogenesis, pericyte constituting the blood vessels (pericyte) enters to form the structure of endothelial vascular periphery cells, new blood vessels mature and stabilize. At this time, platelet-derived growth factor (PDGF-BB) secreted from vascular endothelial cells is known to contribute to the recruitment and differentiation of perivascular cells.

The present inventors investigated the expression of PDGFR-beta by injecting HGF into the penile tissue. Therefore, the present inventors examined the expression level by staining PDGFR-beta in order to investigate the generation of perivascular cells in the penile erectile tissue after administration of streptozotocin to normal mice.

Fluorescence immunostaining with anti-PECAM-1 antibody and anti-PDGFR-beta antibody was carried out by the method specified above and a second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-rabbit antibody) was reacted. The degree of expression was analyzed by a confocal microscope capable of identifying a fluorescence microscope or fluorescent substance, and the results are shown in FIG. 5.

5A is a confocal micrograph showing the expression level of perivascular cells of the HGF protein-administered group and the control group in diabetic penis tissue. As shown in Figure 5A, it can be seen that the amount of perivascular cells is significantly reduced in the penis of the group (DM + PBS) injected diabetic mice compared to the normal mouse (Control). Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Vascular periphery compared to the group treated with HGF protein twice (DM + HGF double) compared to the group not treated with DM + PBS: PBS instead of HGF protein and DM + HGF single: HGF protein once The amount of cells increased.

Figure 5B is a graph showing the amount of perivascular cells in the corpus cavernosum area statistically processed. This is a result of proving that the perivascular cells reduced by diabetes are increased in amount by HGF protein and thus the erection is restored.

Example 2 Evaluation of the Effect of HGF Protein after Induction of Hyperglycemia by Endothelial Cells Obtained by Primary Culture of Penile Tissue

Vascular endothelial cells obtained from primary cultures of mouse penis tissues were subjected to in vitro experiments (group 1, NG (Normal Glucose), and vascular endothelial cells obtained from primary cultures of mouse penile tissues +). Glucose 5mmol, 48 hours treatment; Group 2, NG + HGF, vascular endothelial cells obtained from primary culture of mouse penis tissue + Glucose 5mmol, HGF protein 200ng / ml after 48 hours treatment, 24 hours treatment; Group 3, HG (hyperglycemia) , Endothelial cells + Glucose 30mmol obtained by primary culture of mouse penis tissue, 48 hours treatment; Group 4, HG + HGF, 30mmol of vascular endothelial cells + Glucose obtained by primary culture of mouse penis tissue, 48 hours treatment HGF protein 200ng / ml, 24 hours treatment).

After treatment with Glucose and HGF, immunohistochemical staining was performed on c-Met and platelet-endothelial adhesion molecule-1 (PECAM-1), and tube formation assay was used to determine the degree of tube formation. TUNEL (TdT mediated dUTP biotin nick end labeling method) analysis was performed to investigate the degree of apoptosis.

As a result, it was found that c-Met was expressed in vascular endothelial cells obtained by primary culture of mouse penis tissue, and c-Met was expressed more in the normal glucose group than in the hyperglycemic group. The HGF protein treated group was found to have a higher expression level of c-Met than the other group. PECAM-1 staining and tube formation analysis showed that the expression and tube formation of PECAM-1 in the hyperglycemic group was low, but it was recovered in the HGF protein treatment group. As a result of the TUNEL analysis showing the apoptosis, apoptosis occurred a lot in the hyperglycemic group, but the apoptosis was confirmed in the HGF protein treated group. It will be described in detail below.

(1) Analysis of expression patterns of c-MET, a receptor for HGF

The primary culture test method for obtaining endothelial cells from mouse penis tissue is as follows. The penis tissues of the 8-week-old C57BL / 6J mice are cut to remove the urethra and the arteriovenous vein and then placed in Hanks Balanced Salt Solution (HBSS). After washing with HBSS and PBS on a clean bench, the tissue is cut, seeded into a 60-mm cell culture dish, and matrigel (5 ng) containing bFGF (recombinant mouse FGF basic). / ml bFGF: matrigel = 1: 50). Incubate at 37 ° C for about 5 minutes in this state. When the matrigel is hardened, 3 ml of Complement medium 199 (including 20% Fetal bovine serum, 1% Penicillin / Streptomycin solution, 50 mg / ml heparin, and 5 ng / ml bFGF) is added and incubated in a 37 ° C, 5% CO ₂ incubator. After three weeks, the cells from the tissues are subcultured in a 0.2% gelatin coated petri dish.

The present inventors obtained endothelial cells from mouse penis tissue and carried out in vitro experiments.

Fluorescent staining of endothelial cells obtained from penile tissue is as follows.

Prepare a 12-well cell culture dish, place a cover slide and apply 0.2% gelatin coating. Cells are passaged into the wells and incubated for 1-2 days in a 37 ° C., 5% CO ₂ incubator. The cell-attached slides were removed from the wells, washed three times in wash buffer (2% FBS + 0.1% Sodium Azide in PBS), and the cells were fixed at room temperature for about 10 minutes in 4% paraformaldehyde. After washing three times with washing buffer, treatment with cold methanol for 1-2 minutes to increase permeability, washing with washing buffer, and blocking with non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. The first antibody (anti-c-Met antibody) was reacted for 16 hours at 4 ° C. in a ratio of 1: 100, and then washed three times again with a washing buffer to remove the remaining antibody, followed by specific c-Met specificity. A second antibody (FITC-labeled anti-rabbit antibody), which was designed to identify the reacted antibody by fluorescence, was reacted for 2 hours at room temperature in a ratio of 1: 200. After completion of the reaction, wash again with washing buffer three times to remove the remaining antibody, and then put the mounting solution containing DAPI to stain the nucleus and cover with a cover slide. Then, the degree of expression was analyzed by a confocal microscope that can confirm the fluorescence microscope or fluorescent substance, the results are shown in FIG.

6 is a confocal micrograph showing the expression of HGF receptor c-Met in HGF protein treated group and control group after inducing hyperglycemia by treating glucose (30 mmol) in endothelial cells obtained from mouse penis tissue. As shown in FIG. 6, the expression of c-Met was decreased in the hyperglycemic group than the normal glucose, and the expression of the HGF receptor c-Met was increased when HGF protein was treated in the normal and hyperglycemic groups. This result shows that HGF protein is effective on penile endothelial cells when HGF protein is treated in endothelial cells obtained from mouse penis tissue.

(2) Analysis of PECAM-1 Expression, a Vascular Endothelial Cell Specific Protein

The present inventors examined the expression level of PECAM-1, a protein specific for vascular endothelial cells, to investigate the changes in vascular endothelial cells after HGF protein administration to endothelial cells obtained from penile tissues. Fluorescence immunostaining with anti-PECAM-1 antibody by the method specified above, and after the final washing, a mounting solution containing DAPI containing a staining nucleus is added and covered with a cover slide. After that, the expression level was analyzed by a confocal microscope that can identify a fluorescence microscope or a fluorescent substance, and the results are shown in FIG. 7.

As shown in FIG. 7, there was no difference in PECAM-1 expression between the NG group and the NG + HGF group. In the HG group, PECAM-1 expression was significantly reduced compared to the NG group, and the HG + HGF group was found to recover to the control (NG group) level. This proves that the amount of endothelial cells reduced by hyperglycemic conditions is recovered by the HGF protein.

(3) evaluating the degree of tube formation during angiogenesis

Angiogenesis is not only a simple proliferation of vascular endothelial cells, but also consists of several complex and sequential processes, each of which is essential for the construction of a vascular network. The angiogenesis process can be divided into three types: proliferation, migration, and tube formation. The tube formation step is a step in which endothelial cells migrated and proliferated during angiogenesis continue to divide and grow into hollow tube shapes, differentiate into final blood vessels, and blood enters to complete blood vessel formation. The inventors conducted the following experiment to observe whether the HGF protein is involved in tube formation during angiogenesis through tube formation analysis.

Endothelial cells obtained by primary culture of penile tissues were subjected to serum starvation for 24 hours, then divided into four groups as described above, treated with glucose and HGF proteins, respectively, and seeded on matrigel in a 48-well cell culture dish. Incubate in 5% CO ₂ incubator. After 4, 8, 24 hours, the degree of tube formation was observed through a microscope.

As shown in FIG. 8, the tube was clearly generated after 8 hours, and the tube formation was significantly reduced in the HG group, and then recovered to the control level in the HG + HGF group. This is the result demonstrating that the formation of tubes reduced by hyperglycemic conditions is restored by HGF protein.

(4) TUNEL analysis to know the degree of cell death

TUNEL analysis is an experimental method to determine whether DNA fragmentation has occurred by attaching digoxigenin-labeled uridine triphosphate (UTP) to the end of a DNA fragment using an enzyme called terminal deoxynucleotidyl transferase (TdT). When DNA fragmentation occurs in the process of decomposing DNA in the cell nucleus, splitting occurs in the double helix structure and the 3'-OH group is exposed at the N-terminus. TUNEL analysis results are shown in FIG. 9. At this time, DAPI (4 ', 6-diamidino-2-phenylindole) was used to stain the nuclei of cells.

As shown in FIG. 9, apoptosis was increased in the HG group compared to NG and NG + HGF groups, and apoptosis was significantly reduced in the HG + HGF group. This is a result demonstrating that apoptosis increased by hyperglycemic conditions is reduced by HGF protein.

Based on the above experimental results, HGF protein according to the present invention induces the regeneration of vascular endothelial cells, smooth muscle cells, perivascular cells reduced by diabetes mellitus, PECAM- specific proteins, vascular endothelial cells, smooth muscle cells, perivascular cells 1, SMA, PDGF-beta was confirmed to increase the expression. In addition, HGF protein has been shown to promote tube production and inhibit cell death in vascular endothelial cells. Through the above mechanism, HGF protein has been found to have an excellent effect on improving erectile dysfunction by increasing intracavernosal pressure, and thus it may be usefully used for the prevention or treatment of erectile dysfunction.

Hereinafter, the preparation examples of the pharmaceutical composition and the food composition of the present invention, but this is not intended to limit the present invention.

Formulation Example 1 Preparation of Pharmaceutical Composition

1-1. Manufacture of powder

HGF Protein 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

HGF protein 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

1-3. Preparation of Capsule

HGF protein 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

1-4. Preparation of Injectables

HGF protein 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 ·} 2H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

1-5. Preparation of liquid

HGF Protein 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 2 Preparation of Food Composition

2-1. Manufacture of health food

HGF Protein 100 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Claims (12)

1. An endothelial cell regeneration composition comprising an HGF (hepatocyte growth factor) protein or a polynucleotide encoding the same as an active ingredient.
2. The composition of claim 1, wherein the endothelial cells are penile cavernous endothelial cells or penile vascular endothelial cells.
3. Pharmaceutical composition for preventing or treating erectile dysfunction comprising HGF protein or a polynucleotide encoding the same as an active ingredient.
4. The composition of claim 3, wherein the HGF protein consists of the amino acid sequence of SEQ ID NO: 1.
5. The composition of claim 3, wherein the polynucleotide encoding the HGF consists of a nucleotide sequence of SEQ ID NO: 2.
6. The composition of claim 3, wherein the composition increases the internal pressure of the corpus cavernosum.
7. The composition of claim 3, wherein the erectile dysfunction is caused by damage to penile endothelial cells or smooth muscle cells.
8. The composition of claim 7, wherein the damage to the penile endothelial cells or smooth muscle cells is caused by one or more selected from the group consisting of hyperlipidemia, diabetes, hypertension and penile nerve damage.
9. A method for regenerating endothelial cells of a subject, comprising administering to the subject a composition for endothelial cell regeneration comprising an HGF protein or a polynucleotide encoding the same as an effective ingredient effective for endothelial cell regeneration.
10. Preventing an erectile dysfunction of an individual, comprising administering to the subject a pharmaceutical composition for preventing or treating erectile dysfunction comprising an effective amount of HGF protein or a polynucleotide encoding the same as an active ingredient for treating erectile dysfunction. How to treat.
11. The method of claim 10, wherein the administration is oral, rectal, intravenous, intramuscular, subcutaneous or corpus cavernosum.
12. Food composition for preventing or improving erectile dysfunction comprising a HGF protein or a polynucleotide encoding the same.

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