JP7748281B2 - APJ expression promoter - Google Patents

Description

The present invention relates to an APJ expression promoter.

Skin elasticity has a significant impact on apparent age and appearance, and maintaining and improving skin elasticity is a major cosmetic challenge. Much research has been done on the dermal layer matrix, including collagen, elastin, and hyaluronic acid, regarding skin elasticity. For example, it is well known that the amount of collagen in the dermis layer has a significant impact. In conventional cosmetic methods, efforts have focused on increasing elastic fibers by activating dermal fibroblasts in order to improve skin elasticity. Ingredients that have the effect of activating dermal fibroblasts and promoting collagen production have been discovered and are being applied to cosmetics.

To date, natural ingredients such as isoflavone compounds and phytosterols have been reported to promote collagen production and thereby prevent and improve skin aging. Plant extracts such as Peucedanum japonicum (WO 2013/099378), Oriental Arborvitae seed (WO 2012/057123), Ryukyu bamboo, Ficus batatas, and Tsuruna japonica (JP 2011-195505) have also been identified. It is also known that fibroblasts interact with the extracellular matrix via adhesion molecules such as integrins expressed in fibroblasts, and that such interactions contribute to skin firmness (Non-Patent Document 1). Previous findings have focused on the relationship between skin elasticity and collagen or fibroblast adhesion molecules.

Patent No. 5808769 Japanese Patent Application Laid-Open No. 2012-20942 Japanese Patent Application Laid-Open No. 2013-209339 International Publication No. 2012/133825 Japanese Patent Application Publication No. 2018-172410 Special Publication No. 2019-501899 International Publication No. 2007/072980 Patent No. 5648149

J. Inv. Dermatology (2013) vol. 133, 899-906 BLOOD, 2010, VOLUME 115, NUMBER 15, 3166-3174 DIABETES, VOL. 62, JUNE 2013, 1970-1980 J Cell Physiol. 2019;234:61-74 Kidoya et al., EMBO J. 2008 Feb 6;27(3):522-34 Kajiya et al., J Dermatol Sci. 2018 Oct;92(1):3-5 Nakatani et al., Skin Res Technol. 2013 Feb;19(1):e332-8

The objective of the present invention is to provide substances that improve skin elasticity using a mechanism different from that of conventional skin elasticity improving agents and methods, as well as a method for screening substances that have the effect of improving skin elasticity.

As a result of intensive research into improving skin elasticity, the inventors discovered that skin elasticity can be improved by stabilizing and improving the function of blood vessels by increasing the expression of APJ, and/or by inhibiting the decrease in skin blood vessels due to aging. They also discovered a novel substance that promotes the expression of APJ, leading to the present invention.

The present invention relates to the following inventions:
(1) APJ expression promoter to improve skin elasticity.
(2) An APJ expression promoter consisting of one or more herbal medicines selected from the group consisting of herb extract, cherry leaf extract, and neem leaf extract.
(3)
A skin elasticity improving agent comprising the APJ expression promoter according to (1) or (2).
(4)
A screening method for skin elasticity improving agents using APJ expression as an indicator.
(5)
culturing the biological sample in a medium containing the candidate drug;
measuring APJ expression in a biological sample; and determining the candidate drug as a substance having a skin elasticity improving effect when APJ expression is increased compared to a control;
The screening method according to (4), comprising:
(6)
A kit for carrying out the screening method described in either (4) or (5), which comprises a reagent for determining APJ expression.
(7)
A cosmetic method for improving skin elasticity in a subject, comprising promoting APJ expression in the subject.
(8)
The method according to (7), wherein the promotion of APJ expression in a subject is achieved by administering the skin elasticity improving agent according to (3) to the subject.

The APJ expression promoter of the present invention can promote APJ expression. Promotion of APJ expression stabilizes blood vessels and improves skin elasticity. An APJ expression promoter containing one or more herbal medicines selected from the group consisting of Herb Extract, Cherry Leaf Extract, and Neem Leaf Extract is effective in improving skin elasticity through stabilization of blood vessels. Furthermore, the screening method of the present invention can be used to select substances that have the effect of improving skin elasticity. Skin elasticity improvers selected by the screening method of the present invention improve skin elasticity through stabilization of blood vessels by promoting APJ expression. Improving skin elasticity can alleviate skin problems such as wrinkles and sagging.

Figure 1 shows the construction of a 3D vascularized dermis model. FIG. 2 is a micrograph of an angiogenesis model showing the localization of blood vessels (green) stained with anti-PECAM-1/CD31 antibody and type I collagen (red) stained with anti-type I collagen antibody. The left image in Figure 3 is a superimposition of an image of the sound speed (stiffness) (m/s) of a human skin section measured using an ultrasound sound velocity microscope and an immunostained image of the same section visualizing blood vessels (red). Representative examples are shown for a young person (20 years old) and a middle-aged person (50 years old). The right image is a graph showing the quantified sound speed (stiffness) values around blood vessels, averaged over young people (10-20 years old) and middle-aged people (50-70 years old). In the figure, *** indicates a significant difference based on Student's t-test (***p<0.001). FIG. 4 is a graph showing the relationship between collagen concentration (mg/ml) and storage modulus G' (Pa). FIG. 5 is a graph showing the relationship between collagen concentration (mg/ml) and contact force (mN). Figure 6 shows micrographs of a 3D angiogenic dermal model cultured in collagen gels of various concentrations (mg/ml), stained with anti-PECAM-1/CD31 antibody to visualize blood vessels (green) and with Cy3αSMA antibody to visualize pericytes (red). The left panel of Figure 7 is a graph showing the relationship between collagen gel concentration and APJ mRNA expression levels in vascular endothelial cells cultured in collagen gels of various concentrations. The values are expressed as relative values (%), with the expression level at a gel concentration of 0 mg/ml (-) set to 100. In the figure, * and ** indicate significant differences based on Student's t-test (*p<0.05, **p<0.01). The right panel is a micrograph showing the localization of blood vessels (green) stained with anti-PECAM-1/CD31 antibody, cell nuclei (blue) stained with Hoechst, and APJ (red) stained with anti-APJ antibody in a 3D angiogenic dermal model formed in collagen gel. The left panel of Figure 8 shows a schematic diagram of the construction of a 3D angiogenic dermal model in which APJ was highly expressed by transfection with an APJ high-expression vector. The right panel shows micrographs of vascular structures (red) stained with anti-PECAM-1/CD31 antibody in the control and APJ high-expression 3D angiogenic dermal models. FIG. 9 is a schematic diagram illustrating the concept of the relationship between skin elasticity and APJ expression, and the associated vascular stabilization, derived from the present application. Figure 10 shows the APJ expression when using Herbaceous Herb extract, Cherry Leaf extract, and Neem Leaf extract, expressed as a relative value (%) with the control set at 100. In the figure, * and ** indicate significant differences from the control by Student's t-test (*p<0.05, **p<0.01).

APJ (also known as AGTRL1: Angiotensin receptor like 1) is a seven-transmembrane G protein-coupled receptor that has been reported to be widely expressed in the vascular system, nervous system, and adipocytes. APJ expression has been shown to be involved in myocardial contractility in the heart, regulation of vasopressin expression in the nervous system, stabilization of blood and lymphatic vessels, and fluid regulation. In the vascular system, APJ is reported to be expressed in endothelial cells and mural cells, and is thought to play an important role in angiogenesis in the cardiovascular system and peripheral blood vessels. In blood vessels, apelin (APJ endogenous ligand) has been reported to be involved in blood pressure reduction, angiogenesis, arteriosclerosis, and recovery from ischemia by binding to the APJ receptor. Furthermore, various APJ agonists are being explored, and their use in the treatment of various diseases, such as cardiovascular failure, vascular disorders, and mood disorders, is being studied (Patent Documents 1-7, Non-Patent Documents 1-5).

However, previous findings did not reveal a relationship between skin elasticity and APJ expression. Surprisingly, the inventors discovered that promoting APJ expression stabilizes blood vessels and promotes collagen synthesis around blood vessels, thereby improving skin elasticity. Furthermore, they discovered that APJ expression changes depending on the physical properties of the surrounding environment, such as matrix elasticity, which is closely related to skin elasticity. In other words, APJ expression in blood vessels closely interacts with physical properties such as skin elasticity, which is determined by collagen synthesis, and they discovered a mechanism by which APJ expression is enhanced in vivo in response to the physical properties of the surrounding matrix environment, resulting in stabilization of vascular structure, promotion of collagen synthesis in the surrounding area, and maintenance of appropriate skin elasticity.

The present invention is based on the inventors' findings and relates to an APJ expression promoter for improving skin elasticity. In one embodiment, the APJ expression promoter of the present invention consists of or contains one or more herbal medicines selected from the group consisting of Herbaceous Herb extract, Cherry Leaf extract, and Neem Leaf extract. However, the present invention is not limited to these, as long as the substance can promote APJ expression. For example, APJ activators such as those described in Patent Documents 3, 4, and 7, Non-Patent Document 4, etc. may also be used.

Houttuynia cordata, also known as Houttuynia cordata, is a perennial plant of the genus Houttuynia in the family Houttuyniaceae, which is widely distributed in Asia, including Japan, China, and Vietnam. The plant's leaves, stems, flowers, and roots are used medicinally for diuretic effects, high blood pressure, and arteriosclerosis. Houttuynia cordata extract is an extract of the plant, particularly the above-ground parts.

Sakura leaf refers to the leaves of plants belonging to the cherry genus (Prunus), such as Somei-Yoshino (Prunus yedoensis Matsum.), Oshima cherry (Prunus lannesiana var. speciosa.), and Sato cherry (Prunus serrulata). The cherry genus is a deciduous broadleaf tree of the Rosaceae family found throughout Japan, East Asia, North America, and other areas. Sakura leaf is known to have moisturizing, whitening, and anti-inflammatory properties. Sakura leaf extract refers to an extract of the leaves of the genus Prunus.

Neem (Azadirachta indica), also known as the Indian neem, is a tree of the Meliaceae family native to India. In India, Sri Lanka, and other countries, neem is used in Ayurveda for its antiseptic and moisturizing properties. Neem leaf extract is an extract made from neem leaves.

The above-mentioned plant extracts may be commercially available as cosmetic or health food ingredients, or may be obtained by conventional methods. While the extraction method is not particularly limited, solvent extraction is preferred. The plant material can be soaked or heated under reflux with the extraction solvent at room temperature or elevated temperatures, followed by filtration and concentration. While the plant material can be used as is, crushing it into granules or powder prior to extraction allows for more efficient extraction of active ingredients under milder conditions in a shorter time. The extraction temperature is not particularly limited and can be set appropriately depending on factors such as the particle size of the crushed material and the type of solvent. It is typically set between room temperature and the boiling point of the solvent. The extraction time is also not particularly limited and can be set appropriately depending on factors such as the particle size of the crushed material, the type of solvent, and the extraction temperature. Furthermore, the extraction may be performed with or without stirring, or with the addition of ultrasound.

As the extraction solvent, any solvent normally used for extraction can be used, for example, aqueous solvents such as water, saline, phosphate buffer, borate buffer, or organic solvents such as alcohols such as ethanol, propylene glycol, 1,3-butylene glycol, glycerin, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc., can be used alone or in combination.

Through this extraction process, the active ingredients are extracted and dissolved in the solvent. The solvent containing the extract may be used as is, or may be subjected to conventional purification processes such as sterilization, washing, filtration, bleaching, and deodorization before use. If necessary, it may be concentrated by freeze-drying or diluted with any solvent before use. Furthermore, the solvent may be completely evaporated to form a solid (dried product), or the dried product may be redissolved in any solvent before use.

In addition, the squeezed liquid obtained by squeezing the raw plant material contains the same active ingredients as the extract, so the squeezed liquid can also be used instead of the extract.

The APJ expression promoter of the present invention may consist of one or more herbal medicines selected from the group consisting of herb extract, cherry leaf extract, and neem leaf extract, or may contain these as active ingredients. Furthermore, the skin elasticity improving agent of the present invention contains the APJ expression promoter of the present invention. For example, the skin elasticity improving agent of the present invention promotes APJ expression, and the promoted APJ acts on and stabilizes blood vessels, thereby promoting collagen synthesis around blood vessels and improving skin elasticity.

Improving skin elasticity refers to restoring skin to an optimal state, neither too soft nor too hard. For example, excessive softness may indicate conditions such as swelling or edema, while excessive hardness may indicate conditions such as lumps, warts, or tumors. Therefore, while not limited to, a contact force centered around approximately 17.5 mN, which is the average contact force on the human cheek as described in the Examples, or a contact force range of approximately 10.0 mN to 22.0 mN or approximately 12.0 mN to 20.0 mN, or a storage modulus centered around a collagen concentration corresponding to such a contact force range and corresponding to a collagen gel concentration of approximately 0.5 to 1.00 mg/ml or approximately 0.6 to 0.9 mg/ml, where APJ expression levels are high, such as approximately 5.0 to 30.0 Pa or approximately 5.0 to 20.0 Pa. It is also known that skin loses elasticity and becomes softer with aging. Therefore, improving skin elasticity may mean increasing elasticity that has been reduced due to, for example, aging or swelling, such as a contact force of less than 5.0 mN, less than 6.0 mN, less than 7.0 mN, less than 8.0 mN, less than 9.0 mN, less than 10.0 mN, less than 11.0 mN, less than 12.0 mN, less than 13.0 mN, less than 14.0 mN, less than 15.0 mN, less than 16.0 mN, or less than 17.0 mN, or may mean further increasing current elasticity that is within an appropriate value range, such as approximately 10.0 mN to 22.0 mN or approximately 12.0 mN to 20.0 mN, within the above appropriate value range. The increase may be, for example, an increase in contact force or storage modulus that has a statistically significant difference (e.g., Student's t-test) with a significance level of 5% and/or an increase of, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more. Alternatively, the increase may be maintaining the contact force or storage modulus of the skin within the above-mentioned optimum value range.

Promotion of APJ expression may mean, for example, that the amount of APJ mRNA or protein in a biological sample increases when the APJ expression-promoting agent of the present invention is added compared to when it is not added. The increase may be, for example, an increase that is statistically significant (e.g., Student's t-test) at a significance level of 5% and/or an increase of, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more.

The APJ expression promoter and skin elasticity improving agent of the present invention (hereinafter collectively referred to as "the agent of the present invention") may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.

The agent of the present invention can also be a composition in which the active ingredient described above is combined with one or more other ingredients, such as excipients, carriers, and/or diluents. The composition may have any composition and form, and may be appropriately selected depending on the active ingredient, intended use, and other conditions. The composition can be manufactured using conventional methods in a formulation in which excipients, carriers, and/or diluents, etc., and other ingredients are appropriately combined depending on the dosage form.

The agent of the present invention may be administered orally or parenterally (transdermally, intravenously, intraperitoneally, etc.) as appropriate, and may be administered by any route, but transdermal administration is preferred so that it acts on the blood vessels in the skin.

The agent of the present invention may be used by humans and animals by being incorporated into cosmetics, pharmaceuticals, quasi-drugs, etc., or may be incorporated into various foods and beverages, such as nutritional supplements, and ingested by humans and animals, or may be administered to humans and animals as a pharmaceutical preparation.

When applying this invention to external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, the amount (dry mass) of active ingredients such as the extracts described above can be determined appropriately depending on their type, purpose, form, and method of use. For example, Jewel Herb extract, Cherry Leaf extract, and Neem Leaf extract can each be blended in amounts of approximately 0.000001% to 50.0% (dry mass equivalent) of the total amount of the cosmetic, and may be added in amounts of approximately 0.0001% to 10.0% (dry mass equivalent).

In addition to the above ingredients, if necessary, ingredients typically used in topical skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, such as antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, etc., can be added as needed within the scope that does not impair the effects of the present invention.

In addition, sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, preservatives such as methylparaben, ethylparaben, and butylparaben, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, glabridin, hot water extract of quince fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts, whitening agents such as vitamin C, magnesium ascorbyl phosphate, ascorbic acid glucoside, arbutin, and kojic acid, and sugars such as glucose, fructose, mannose, sucrose, and trehalose may also be added as appropriate.

The topical skin preparation of the present invention can be applied to the outer skin as a cosmetic or quasi-drug, and is particularly suitable as a cosmetic. There are no limitations on its formulation, so long as it can be applied to the skin. Any formulation can be used, including solution, solubilized, emulsified, powder dispersion, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc.

When the agent of the present invention is used as a cosmetic, it may be used in the form of, for example, face cream, massage cream, body cream, emulsion, lotion, serum, gel, pack, cleansing cream, hand cream, lotion, foundation, lipstick, lip balm, hand powder, body shampoo, bath cosmetics, etc.

When the agent of the present invention is incorporated into foods, beverages, etc., the amount of plant matter or its extract (dry mass) to be incorporated can be determined appropriately depending on the type, purpose, form, method of use, etc. For example, it can be incorporated so that the daily intake of the plant matter or its extract by an adult is approximately 0.00001 mg to 10.0 g (dry residue) or approximately 0.001 mg to 5.0 g (dry residue).

Food, beverage, or feed can be in any form, such as granules, particles, paste, gel, solid, or liquid. These forms can contain various known substances approved for inclusion in foods, beverages, etc., such as excipients such as binders, disintegrants, thickeners, dispersants, resorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers, and pH adjusters, as appropriate.

Any dosage form can be used, including oral solid preparations such as tablets, granules, powders, and capsules; oral liquid preparations such as oral liquids and syrups; and parenteral liquid preparations such as injections. Any of these forms can be prepared appropriately using known methods. Topical preparations can be used in a variety of forms, including lotions, suspensions/emulsions, solutions, ointments, and patches. These preparations may contain appropriate excipients, such as commonly used binders, disintegrants, thickeners, dispersants, resorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers, and pH adjusters.

However, the forms that the agent of the present invention can take are not limited to those described above.

The present invention also relates to a method for screening for skin elasticity-improving agents using APJ expression in a biological sample as an indicator. The screening method of the present invention includes, for example, the following steps: culturing the biological sample in a medium containing a candidate drug; measuring APJ expression in the biological sample; and, if APJ expression increases compared to the control, determining the candidate drug as a substance with the effect of improving skin elasticity. Here, the control is APJ expression in a case where the sample is cultured in a medium that does not contain the candidate drug. Experiments on the control may be conducted in parallel with the screening method of the present invention, or may be conducted in advance.

The biological sample used in the screening method of the present invention may be any suitable sample, such as vascular endothelial cells or adipocytes, as long as APJ expression can be measured. For example, as described in the Examples, the sample may be vascular endothelial cells or pericytes, or other vascular component cells, in a vascular model created by inducing angiogenesis from the subcutaneous adipose tissue of an animal such as a human. Alternatively, the sample may be vascular endothelial cells or pericytes obtained from a living organism, or passaged cells thereof, or established cell lines, such as HUVEC, HAEC, or HMVEC. Alternatively, APJ-expressing cells may be used, as described in Patent Documents 3 and 4.

The method may also include a pre-culture step of culturing the biological sample as described above before the step of culturing the biological sample in a medium containing a candidate drug. In the pre-culture step, angiogenesis may be induced from subcutaneous adipose tissue to create a vascular model. Furthermore, after the step of culturing the biological sample in a medium containing a candidate drug, a post-culture step of further culturing in a medium not containing the candidate drug may be included. The step of culturing the biological sample in a medium containing a candidate drug may involve directly adding the candidate drug or a dilution thereof to the culture obtained in the pre-culture step and culturing, or the culture may be carried out by replacing the medium with a medium containing the candidate drug.

APJ expression can be measured by measuring the amount of APJ mRNA or protein in a biological sample. Measurement of mRNA levels can be performed using techniques known in the art, such as quantitative PCR or Northern blotting. For example, as described in the Examples, a probe for APJ mRNA may be used. Protein levels can be measured using any technique known in the art, such as Western blotting, immunostaining, or FACS. For example, an antibody that specifically binds to APJ may be used. Alternatively, a screening system using APJ-expressing cells, such as those described in Patent Documents 3 and 4, may be established and used. The present invention also provides a kit for carrying out the screening method of the present invention, which contains the above-mentioned reagents for determining APJ expression.

Substances screened for by the screening method of the present invention that have the activity of promoting APJ expression may be any substance as long as they can promote APJ expression. One embodiment of the screening method of the present invention involves screening using any library of cosmetic, food, or pharmaceutical materials, using APJ expression in biological samples such as vascular endothelial cells as an indicator. Substances thus determined to have the activity of promoting APJ expression include one or more herbal medicines selected from the group consisting of Jewel Herb extract, cherry leaf extract, and neem leaf extract.

As explained above, it has been found that promoting APJ expression improves skin elasticity. Therefore, the present invention also relates to a method for improving skin elasticity, which comprises applying an APJ expression promoter. Improved skin elasticity is expected to lead to improvements in wrinkles and sagging. The method for improving skin elasticity described herein relates to a cosmetic method for cosmetic purposes, and can be distinguished from treatments performed by doctors or medical professionals. Such cosmetic methods may be performed privately or at beauty salons, cosmetic stores, esthetic salons, etc.

All documents referred to in this specification are incorporated herein by reference in their entirety.

The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications to the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, may be made without departing from the spirit of the present invention.

Experiment 1: Creation of a 3D vascularized dermal model and visualization of vascular structure and produced collagen. Type I collagen gel solution was prepared using a collagen gel culture kit (Nitta Gelatin). Specifically, 2 ml of 3 mg/ml cell matrix collagen solution was suspended in 2 ml of 1 mM dilute hydrochloric acid in a 15 ml tube on ice, and 500 μl of 10x concentrated MEM and 500 μl of C buffer (included in the collagen gel culture kit) for neutralization were added while stirring. 100 μl of this solution was placed on a 35 mm glass-based dish (IWAKI) and incubated at 37°C for 30 minutes to allow gelation.

Normal human subcutaneous adipose tissue was minced into 1 mm squares in 10% FBS-containing PBS, and four pieces were placed on the gelled collagen gel. Then, 100 μl of the remaining type I collagen gel solution, prepared on ice, was added to cover the tissue pieces and incubated at 37°C for 30 minutes to allow gelation. The tissue pieces were cultured for 7 to 21 days in EBM-2 (Cambrex; Verviers, Belgium) supplemented with growth factors and other additives, plus VEGFA at a final concentration of 50 ng/ml. The medium was changed every 2 to 3 days. For visualization of type I collagen and analysis of APJ expression localization, this model was created using a collagen gel with a concentration of 0.9 mg/ml.

After incubation, the cells were fixed with 1 ml of 4% paraformaldehyde solution at 4°C for 30 minutes and then immunostained. Primary antibodies used were sheep anti-PECAM-1/CD31 antibody (AF806, R&D Systems, MN) for endothelial cells, Cy3α SMA antibody (c-6198, Sigma, MO) for pericytes, rabbit anti-APJ antibody for APJ, and rabbit anti-type I collagen antibody (ab34710, Abcam, UK) for type I collagen. Secondary antibodies used were Alexa Fluor 488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR) and Alexa Fluor 594 donkey anti-rabbit antibody (R37119, Molecular Probes, Eugene, OR). Hoechst 33342 (H3570, Invitrogen, CA) was used for nuclear staining. After staining, 3D vascular structures were detected and imaged using a confocal microscope LSM880 (CarlZeiss, Germany).

Figure 1 shows the creation of a 3D angiogenic dermal model stained with anti-PECAM-1/CD31 antibodies. As can be seen in Figure 1, vascular endothelial cells formed lumens, confirming the formation of a 3D vascular structure. Figure 2 shows the angiogenic model stained with anti-PECAM-1/CD31 antibodies and anti-type I collagen antibodies. As can be seen in Figure 2, collagen was found to be synthesized around the blood vessels. In particular, as indicated by the arrows in the right image, collagen was observed around the blood vessels, with a particularly large amount of collagen being formed in areas thought to be the scaffolding from which the blood vessels will extend. This suggests that the presence of vascular structures contributes to collagen synthesis and, ultimately, skin elasticity.

Experiment 2: Reduction of blood vessels and reduction of surrounding stiffness in aging The above experiments demonstrated that the presence of vascular structures contributes to skin elasticity, but it is known that blood vessels decrease in aging skin (Non-Patent Document 6). Therefore, with the aim of suppressing the reduction of blood vessels due to aging, we analyzed how vascular structures are maintained, focusing on the interaction with the stiffness of the surrounding tissue.
Eyelid skin samples from young (10-20 years old) and middle-aged (50-70 years old) human subjects were used to observe the stiffness of the area around capillaries located 200 μm below the epidermis using an ultrasound sound velocity microscope (medical ultrasound microscope, AMS-50SI, Honda Electronics Co., Ltd.). Specifically, frozen sections of the skin were prepared, and the sound velocity across the skin cross section was measured using a conventional ultrasound sound velocity microscope to obtain a sound velocity distribution image (Patent Document 8). Furthermore, the same sections were immunostained to visualize vascular structure. Vascular endothelial cells were stained with sheep anti-Pecam-1 antibody (AF806, R&D Systems, MN) and pericytes with rabbit anti-NG2 antibody (Milipore) as primary antibodies. The secondary antibodies used were Alexa Fluor 594 donkey's anti-sheep antibody (A-11016, Molecular Probes, Eugene, OR) and Alexa Fluor 488 donkey's anti-rabbit antibody (A-21206, Molecular Probes, Eugene, OR). The sound speed distribution image and the immunostained image were then overlaid, and sound speed values within a 50 μm perivascular range were quantified. Ten specimens were used for quantitative analysis, and 15 perivascular locations were quantified per tissue.
The results are shown in Figure 3. Figure 3 shows that in aged skin, the sound speed in the area around the blood vessels decreases significantly as the number of blood vessels decreases. This suggests that the decrease in skin elasticity and the decrease in blood vessels due to aging are related.

Experiment 3: Measurement of collagen gel stiffness. Type I collagen gel solutions with various concentrations (0.24, 0.6, 0.9, 1.2, and 1.8 mg/ml) were prepared on ice using the same method as in Experiment 1. The solutions were then allowed to gel in a 24-well plate at 37°C for 30 minutes to produce collagen gels. The storage modulus (Pa) of the prepared collagen gels was measured using a Physica MCR 300 Modular Compact Rheometer (Anton Paar, Germany), and the stiffness of the gel was determined.
The results are shown in Figure 4. Figure 4 shows that there is a corresponding relationship between collagen gel concentration and storage modulus.

Experiment 4: Relationship between collagen gel hardness and skin physical properties To investigate the relationship between the collagen concentration of the collagen gel and the actual physical properties of skin, the contact force of type I collagen gels with various concentrations (0.24, 0.6, 1.2, 1.8 mg/ml) was measured using a skin softness sensor (Non-Patent Document 7) in the same manner as in Non-Patent Document 7.
The results are shown in Figure 5. As Figure 5 shows, there is a roughly proportional relationship between collagen concentration and contact force up to approximately 2.0 mg/ml. Furthermore, since it has been confirmed using the same device that the average contact force of cheek skin in healthy humans (ages 20-50, N=168) is approximately 17.5 mN, the following experiment was conducted by varying the collagen concentration around the concentration corresponding to 17.5 mN.

Experiment 5: Analysis of vascular structure changes in response to collagen gel stiffness. Type I collagen gels (0.24, 0.9, and 1.8 mg/ml) were prepared by varying the collagen gel concentration using the same method as in Experiment 1. These gels had storage moduli of 2.6 ± 0.8, 14.0 ± 5.4, and 48.1 ± 17.0 (Pa), respectively. Using these gels, 3D angiogenesis was induced from normal human subcutaneous adipose tissue using the same method as in Experiment 1. After 13 days of culture, vascular endothelial cells and pericytes were visualized by immunostaining as in Experiment 1.

The results are shown in Figure 6. Figure 6 shows that, in the case of a low-concentration gel (0.24 mg/ml) with an elastic modulus of 2.6 ± 0.8 Pa, thin, unstable blood vessels were formed. However, in the case of a gel with a higher concentration (0.9 mg/ml) and a high elastic modulus (14.0 ± 5.4 Pa), thick, stable blood vessels were formed. However, in the case of a gel with an even higher concentration (1.8 mg/ml) and a high elastic modulus (48.1 ± 17.0 Pa), many thin, unstable blood vessels were observed. The results in Figure 6 indicate that the structure of blood vessels formed changes in response to the physical properties of the surrounding environment, such as matrix elasticity. Furthermore, it is suggested that there are optimal physical properties of the surrounding environment for stabilizing blood vessels. Considering these results together with the results in Figure 3, blood vessels sense the stiffness of their surroundings to maintain their stable structure. However, in aging skin, the decrease in surrounding stiffness may cause vascular instability and lead to a decrease in blood vessel size.

Experiment 6: Analysis of vascular cell expression factors (APJ) in response to collagen gel stiffness. Type I collagen gel solutions were prepared on ice using the same method as in Experiment 1. Human umbilical vein endothelial cells (HUVECs) were seeded at ¹⁰⁵ cells/well in a 12-well plate and cultured to confluence. 300 μl of each type I collagen gel solution was added to each well and incubated at 37°C for gelation. After 6 hours of incubation, Trizol was added to each well to disrupt the cells. Total RNA was extracted with chloroform from the resulting solution. The concentration of the resulting total RNA was measured using a nanodrop and adjusted to 35 ng/ml with RNase-free water. Then, the mRNA expression levels of APJ were compared using TaqMan ^™ RNA-to-C _T ^™ 1-Step Kit (Applied Biosystems, CA) and a Taqman probe for human APJ mRNA (Hs00270873, Applied Biosystems, CA).

The results are shown in Figure 7. The left graph shows the relationship between collagen gel concentration and APJ mRNA expression. APJ mRNA expression peaked under 0.9 mg/ml collagen gel conditions, with expression decreasing in collagen gels with lower and higher concentrations. This indicates that APJ expression changes in response to the physical properties of the surrounding environment, such as matrix elasticity. This relationship between physical properties and expression was not observed for other vascular stabilizing factors, such as Tie2 (data not shown), and was specific to APJ. Furthermore, the collagen gel elasticity determined in this experiment was nearly consistent with the gel concentration corresponding to normal human skin elasticity. Therefore, APJ expression appears to change depending on matrix elasticity, contributing to vascular stability. It is suggested that elasticity that is too low or too high is inappropriate, and that there is an appropriate range. The right graph in Figure 7 shows micrographs showing APJ localization in a vascular model formed in a 0.9 mg/ml collagen gel. It was found that APJs are localized at the sites of cell protrusions, such as filopodia, that extend from vascular endothelial cells toward the periphery, suggesting that APJs may directly sense the surrounding matrix environment and stabilize blood vessels.

Experiment 7: Creation and Structural Analysis of a 3D Angiogenic Dermal Model with High APJ Expression . The human APJ gene sequence was inserted into the pEGFP-N1 vector (Clontech, CA) to obtain an APJ-high expression vector for lentiviral transduction. Subsequently, an APJ-high expression lentivirus was generated using this vector (Oriental Yeast). A 3D angiogenic dermal model was created using the same method as in Experiment 1. The model was then transfected with the APJ expression vector via lentiviral infection on day 21 of culture and cultured for 5 days. A control vascular model was cultured under the same conditions but without transfection. After culture, the vascular models were fixed with 4% paraformaldehyde solution at 4°C for 30 minutes and then immunostained. The primary antibody used for vascular cells was sheep anti-Pecam-1 antibody (AF806, R&D Systems, MN), and the secondary antibody used was Alexa Fluor 594 Donkey anti-sheep antibody (A-11016, Molecular Probes, Eugene, OR). After staining, 3D vascular structures were detected and imaged using a confocal microscope LSM880 (CarlZeiss, Germany).

The results are shown in Figure 8. Figure 8 shows that increasing APJ expression leads to thicker and more stable blood vessels, which is consistent with previous reports (Non-Patent Document 5).

From the above results, as shown in the schematic diagram in Figure 9, it is possible that skin elasticity and APJ expression interact, with APJ acting as a sensor of skin elasticity, stabilizing blood vessels and promoting collagen synthesis around blood vessels, which then feeds back into skin elasticity in a cycle. Therefore, even in skin whose elasticity has decreased due to aging, it is possible to strengthen the cycle and improve skin elasticity by enhancing APJ expression. Therefore, in the following experiment, we screened for substances that have the effect of promoting APJ expression.

Experiment 8: Screening method for substances with skin elasticity-improving effects that promote APJ expression . A total of 49 raw materials, including plant extracts known to have anti-aging properties, were screened as candidate drugs. We used Jewel Herb Extract (Jewel Herb Extract ET-50) manufactured by Koei Kogyo Co., Ltd., cherry leaf extract (Falcorex Sakura Leaf B) manufactured by Ichimaru Falcos Co., Ltd., and neem leaf extract (Neem Leaf Liquid B) manufactured by Ichimaru Falcos Co., Ltd. These extracts were dissolved in dimethyl sulfoxide (DMSO) to prepare samples at a final concentration of 0.1% (w/w). As a control, a DMSO solution without these extracts was used. HUVECs seeded at ¹⁰⁵ cells/well in a 12-well plate were cultured until confluent, and the above samples were added to a final concentration of 0.1%. The cells were incubated at 37°C for 4 hours, and APJ mRNA expression levels were compared using the same method as in Experiment 6.

The results are shown in Figure 10. Figure 10 shows that when Jewel Herb extract, Cherry Leaf extract, and Neem Leaf extract were used as candidate drugs, APJ expression significantly increased compared to the control. These extracts were selected as substances that promote APJ expression.

Claims (1)

An APJ expression promoter consisting of one or more herbal medicines selected from the group consisting of herb extract, cherry leaf extract, and neem leaf extract.