KR102633897B1 - Biomarker composition for diagnosing blood vessel calcification comprising pannexin3 and diagnostic method using the same - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing vascular calcification containing Pannexin3 and a method for diagnosing vascular calcification using the same. More specifically, it was confirmed that Pannexin3 expression was suppressed in vascular smooth muscle cells that induced calcification, using high-dose vitamin D. As a result of suppressing Pannexin3 expression in a vascular calcification model, it was confirmed that vascular calcification increased in a calcification mouse model. As a result, Pannexin3 can be provided as a biomarker composition for diagnosing vascular calcification, and a regulator that can enhance the expression or activity of Pannexin3 Can be provided as a treatment for vascular calcification disease.

Description

Biomarker composition for diagnosing blood vessel calcification comprising pannexin3 and diagnostic method using the same}

The present invention relates to a biomarker composition for diagnosing vascular calcification containing Pannexin3 and a method for diagnosing vascular calcification using the same.

Blood vessels are circulatory organs that continuously regulate the movement of body fluids, supplying oxygen and nutrients to tissues, and receiving and processing carbon dioxide and waste products from tissues. Decreased vascular function causes various diseases such as cardiovascular disease, blood pressure, diabetes, and Alzheimer's disease. Therefore, it is very important to protect blood vessel health. In particular, vascular calcification is the deposition of minerals such as calcium and phosphoric acid in blood vessels, which increases the stiffness of blood vessels and causes blood vessels to rupture, causing various diseases such as cardiovascular disease. Loss of calcium accumulation inhibitors, induction of bone formation, nucleation complexes in the blood, and apoptosis are considered to be the main causes of vascular calcification, but the exact mechanism is not yet known, and prevention and treatment methods have not yet been developed.

Accordingly, there is a need to develop technology for compositions for preventing, improving, or treating vascular calcification.

Republic of Korea Patent Publication No. 10-2016-0087037 (published on July 21, 2016)

The present invention provides a composition containing Pannexin3 (Panx3) as a biomarker to effectively diagnose vascular calcification, and provides a regulator of the expression or activity of Pannexin3 as a preventive or therapeutic agent for vascular calcification disease.

The present invention provides a biomarker composition for diagnosing vascular calcification containing Pannexin3.

The present invention provides a kit for diagnosing vascular calcification including an agent capable of measuring the expression level of Pannexin3.

The present invention includes the steps of confirming the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level from a sample isolated from an individual; and comparing the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level with a control sample.

The present invention provides a biomarker composition for predicting the prognosis of vascular calcification containing Pannexin3.

The present invention provides a pharmaceutical composition for preventing or treating vascular calcification disease containing a Pannexin3 expression promoter or activator as an active ingredient.

In addition, the present invention includes the steps of contacting a test substance with vascular cells in which calcification is induced; Confirming the level of Pannexin3 expression or activation in cells contacted with the test substance; and selecting a test substance with increased expression or activation level of Pannexin3 compared to a normal control sample.

According to the present invention, it was confirmed that Pannexin3 expression was increased in vascular smooth muscle cells that induced calcification, and as a result of suppressing Pannexin3 expression in a vascular calcification model using high-dose vitamin D, it was confirmed that vascular calcification increased in the calcification mouse model. Accordingly, Pannexin3 can be provided as a biomarker composition for diagnosing vascular calcification, and a regulator that can enhance the expression or activity of Pannexin3 can be provided as a treatment for vascular calcification disease.

Figure 1 shows the results of confirming the expression change of Panx3 in vascular smooth muscle cell calcification.
Figure 2 shows the results of confirming the expression changes of osteogenic differentiation markers in soft tissue cells due to overexpression of Panx3.
Figure 3 shows the results confirming vascular calcification caused by Panx3 deletion ex vivo.
Figure 4 shows the results of confirming changes in osteogenic differentiation markers ex vivo using real-time PCR.
Figure 5 shows the results confirming the change in body weight of Panx3-deleted mice in a vascular calcification model caused by high-dose Vit D administration.
Figure 6 shows the results confirming vascular calcification caused by Panx3 deletion.

Hereinafter, the present invention will be described in more detail.

The present invention conducted a study using a calcification model using high-dose vitamin D to observe the effect of Pannexin3, which is a matrix vesicle protein and plays an important role in maintaining bone homeostasis, on ectopic calcification. As a result, vascular calcification was found in mice with a deletion of Pannexin3. As the increase was confirmed, the present invention was completed.

The present invention can provide a biomarker composition for diagnosing vascular calcification containing Pannexin3.

The present invention can provide a kit for diagnosing vascular calcification including an agent capable of measuring the expression level of Pannexin3.

An agent that can measure the expression level of Pannexin3 may be a primer or probe that specifically binds to the Pannexin3 gene, an antibody, peptide, aptamer, or compound that specifically binds to the Pannexin3 protein.

The present invention includes the steps of confirming the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level from a sample isolated from an individual; and comparing the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level with a normal control sample.

The sample may be any one selected from the group consisting of cells, tissues, blood, saliva, and urine isolated from an individual in which vascular calcification is predicted.

If the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level increases compared to the control group, it may be determined that the possibility of vascular calcification is high.

Pannexin3 of the present invention may be NP_443191.1, but is not limited thereto.

In the present invention, “diagnosis” refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or condition, and determining whether an object currently has a specific disease or condition. Includes determining the prognosis of a subject, or therametrics (e.g., monitoring the condition of a subject to provide information about treatment efficacy).

The present invention can provide a biomarker composition for predicting the prognosis of vascular calcification containing Pannexin3.

When the expression of Pannexin3 is reduced, the prognosis may be predicted as there is a high possibility of worsening vascular calcification.

In the present invention, “prognosis prediction” refers to the act of predicting the course and outcome of a disease in advance. More specifically, the course of the disease after treatment may vary depending on the patient's physiological or environmental condition, and it can be interpreted to mean all actions that predict the course of the disease after treatment by comprehensively considering the patient's condition. .

The present invention can provide a pharmaceutical composition for preventing or treating vascular calcification disease containing a Pannexin3 expression promoter or activator as an active ingredient.

The expression promoter or activator may be any one selected from the group consisting of miRNA, low-molecular-weight compounds, peptides, aptamers, antibodies, and natural products that specifically bind to the Pannexin3 gene or protein.

The vascular calcification disease may be any one selected from the group consisting of atherosclerosis, hypertension, myocardial infarction, and thrombosis.

In one embodiment of the invention, the pharmaceutical composition for preventing or treating vascular calcification disease containing the Pannexin3 expression promoter or activator as an active ingredient is administered as injections, granules, powders, tablets, pills, capsules, suppositories, etc. according to conventional methods. Any one formulation selected from the group consisting of gel, suspension, emulsion, drops, or liquid can be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating vascular calcification disease containing a Pannexin3 expression promoter or activator as an active ingredient may contain appropriate carriers, excipients, disintegrants, sweeteners, and blood commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of agents, leavening agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants.

Specifically, carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agents, etc., and such solid preparations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered to the subject.

The preferred dosage of the pharmaceutical composition according to the present invention may vary depending on the subject's condition and weight, type and degree of disease, drug form, administration route and period, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, but is not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal, including humans, but is not limited to these examples.

In addition, the present invention includes the steps of contacting a test substance with vascular cells in which calcification is induced; Confirming the level of Pannexin3 expression or activation in cells contacted with the test substance; And it is possible to provide a screening method for a treatment for vascular calcification, including the step of selecting a test substance with an increased level of Pannexin3 expression or activation compared to a normal control sample.

The level of Pannexin3 expression or activation is determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, Western blotting, and flow cytometry (FACS). It can be measured with any one selected from the group.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Reference example>

The following reference examples are intended to provide reference examples commonly applied to each embodiment according to the present invention.

1. Reagents

Ascorbic acid, benzoyl peroxide, DMSO (Dimethyl sulfoxide), ethanol, fast red violet LB, glycerol, naptol As-Mx phosphate, N,N-dimethylformamide, Nitric acid, nonylphenyl-polyethyleneglycol acetate, Paraformaldehyde (PFA), picric acid solution, silver nitrate, sodium acetate, sodium carbonate, sodium thiosulfate, β -Glycerophosphate (β-glycerophosphate) was purchased from Sigma (St. Louis, MO, U.S.A.). Acetone and formaldehyde solutions were purchased from JUNSEI (Nihonbashi-honcho, Chuo-ku, Tokyo). DMEM (Hyclone) and permount were purchased from Thermo scientific (Rockford, IL, U.S.A.). Fetal bovine serum (FBS) was from GIBCO (Grand Island, NY, U.S.A.), oligo(dT) from Promega (Madison, WI, USA), penicillin/streptomycin from Lonza (Rockland, ME, USA), SuperScript II Reverse Transcriptase was purchased from Intron (iNtRON Biotechnology, Gyeinggi-do, Korea), and SYBR green master mixture was purchased from ABI (Carlsbad, CA, U.S.A.).

2. Laboratory animal management

The progression of vascular calcification was confirmed using Pannexin3 12-week-old male mice. They were used in this study after being acclimatized for one week in an SPF (Specific Pathogen Free) laboratory animal breeding room (temperature 25°C, relative humidity 60%). The feed was regular food (Super bead Co, Korea), and all animal experiments were performed in accordance with the guidelines of the Animal Experiment Ethics Committee of Kyungpook National University. (Animal testing approval number: KNU-2018-0156).

3. Experimental equipment

Real-time PCR used ViiA7 (Applied Biosystems, CA, U.S.A.), Nano drop 2000 was used for DNA and RNA quantification, and Thermo scientific (Rockford, IL, U.S.A.) was used as an incubator. Protein quantification was performed using an ELISA reader (TECAN, Mannedorf, Switzerland).

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell culture

Vascular smooth muscle cells were added to DMEM with 10% (v/v) FBS, penicillin (100 units/ml), and streptomycin (100 units/ml), and incubated in a constant temperature and humidity incubator at 37°C and 5% (v/v) CO _{2 .} Cultured. The vascular smooth muscle cell differentiation experiment was induced by adding ascorbic acid (50 μg/ml) and β-glycerophosphat (10 mM) when the cells were 100% filled in the culture dish. During the differentiation period, the differentiation medium was changed every 3 days.

2. Von kossa staining

Vascular smooth muscle cells were distributed in a 48-well multi-plate at 5×10 ⁴ cells/well and cultured for 14 days while being replaced with differentiation induction medium 2 days later. Washed three times with PBS and fixed at room temperature for 10 minutes using 4% formaldehyde fixative. It was washed three times using tertiary distilled water, 1% AgNO ₃ was added and reacted under UV for 5 minutes, then washed several times with PBS and observed under a microscope.

To confirm the progress of mineralization of blood vessels, Vonkossa staining was performed. On the 9th day after high-dose vitamin injection, mouse arteries were isolated, fixed in 4% PFA and 4°C for one day, and paraffin samples were prepared. After rehydration of the paraffin section, 1% AgNO ₃ was added and reacted for 5 minutes under UV light. After the reaction was completed, the reaction was washed twice with tertiary distilled water, 5% sodium thiosulfate was added and reacted for 5 minutes to remove non-specific signals, counter-stained with Eosin, observed, and analyzed under a microscope.

3. Western blotting

Western blotting was performed to confirm changes in protein expression of osteoblast differentiation markers. Differentiation of osteoblasts was induced, and proteins were extracted on days 8, 15, and 28 of differentiation, proteins were quantified using the Bradford method, and 20 μg of proteins were loaded on 8% and 15% acrylamide gels. After blocking with 5% non-fat milk for 1 hour and washing with TBS-T (0.1% tween 20/Tris-buffered saline) three times for 5 minutes each, the primary antibody was reacted at room temperature for 1 hour and 30 minutes. Afterwards, after washing three times with TBST, the secondary antibody was reacted at room temperature for 1 hour, and then color was developed using ECL solution to analyze the protein expression level of osteoblast differentiation marker.

4. Real-time PCR analysis

Real-time PCR was performed to confirm changes in gene expression of osteoblast differentiation markers. Bone marrow stem cells were induced to differentiate into osteoblasts, and total RNA was extracted using an RNA extraction kit (Easy-blue) on days 1, 8, and 13 of differentiation, and then 2 ㎍ of total RNA was converted to cDNA using reverse transcriptase. It was synthesized. Real-time PCR was performed by mixing 0.5 ㎕ of synthesized cDNA with 2× SYBR green PCR master mixture (5 ㎕) and specific primer (0.2 ㎕). The primers used for real-time PCR are listed in Table 1, and the primers used were designed using Primer Express software (ABI).

mPanx3-F AAC TGC CCC TGG ATA AGA TGG T mPanx3-R TGA TCG GAG ACC CTG ATG AGA mRunx2l-F ACA TGG CCA GAT TCA CAG TGG mRunx2-R TGG TGC CCG TTA GCA ATT G mOsteopontin-F CAT GAG ATT GGC AGT GAT TTG C mOsteopontin-R TGC AGG CTG TAA AGC TTC TCC T mOsteocalcin-F TTC TGC TCA CTC TGC TGA CCC T mOsteocalcin-R CCT GCT TGG ACA TGA AGG CTT mmGP-F ACA CCT TTA TGT CCC CTC AGC A mmGP-R ATC GCA GGC CTC TCT GTT GAT

<Example 1> Confirmation of changes in expression of Panx3 in soft tissue (skeletal muscle, vascular smooth muscle cell) calcification

To confirm changes in the expression of Panx3 in vascular smooth muscle cell calcification, calcification was induced using vascular smooth muscle cells isolated from the arteries of 8-week-old male mice.

Vascular smooth muscle cells were inoculated into a 6-well plate at 2× ¹⁰⁵ cells/well, and when the cells were sufficiently confluent, the osteoblast induced differentiation medium [10% FBS/DMEM + ascorbic acid (50 μg/ml) + β-glycerophosphate ( 10 mM) + 1,25(OH)2D3 (10 ^-7 mol/l)] was replaced once every 3 days to confirm calcification on the 21st day of differentiation.

Afterwards, as a result of Ponkossa staining, it was confirmed that there was a lot of staining in the vascular calcification induction group. Referring to Figure 1A, in the group where calcification was induced, the expression of osteogenic differentiation markers Runx2, Osteocalcin, and Osteopontin was confirmed to be increased, and MGP, an inhibitor of calcification, was also confirmed to be increased. Additionally, as shown in Figure 1B, Panx3 gene expression was also confirmed to be significantly increased in the calcification-induced group.

From the above results, it can be suggested that Panx3 may be significantly involved in vascular calcification.

<Example 2> Confirmation of changes in expression of osteogenic differentiation markers due to overexpression of Panx3 in soft tissue cells

To confirm changes in the expression of osteogenic differentiation markers due to overexpression of Panx3 in vascular smooth muscle cells, Panx3 was overexpressed in vascular smooth muscle cells.

As a result, as shown in Figure 2, it was confirmed that the expression of Runx2 and Osteocalcin, which are osteogenic differentiation markers, were reduced by overexpression of Panx3.

From the above results, it was confirmed that Panx3 can suppress ectopic calcification of blood vessels.

<Example 3> Confirmation of vascular ectopic calcification due to Panx3 deletion ex vivo

Ectopic calcification was induced by treating arteries isolated from Panx3-depleted mice with phosphate. As a result, as shown in Figure 3, it was confirmed that vascular calcification in the blood vessels of Panx3 ^-/- mice was increased compared to Panx3 ^+/- .

From the above results, it was confirmed that the Panx3 gene exhibits an effect of suppressing vascular calcification.

Additionally, gene expression changes in ex vivo vascular calcification were confirmed through real-time PCR. As a result, as shown in Figure 4, it was confirmed that the osteogenic differentiation markers Runx2 and Osteocalcin were decreased due to Panx3 expression in the blood vessels of Panx3 ^-/- mice, while the expression of Osteopontin was confirmed to be increased.

<Example 4> Confirmation of induction of vascular calcification in vivo in mice in which the Panx3 gene has been deleted

To confirm the effect of Panx3 on vascular calcification in vivo, a high-dose vitamin D (vit D) administration vascular calcification model established in the laboratory was used (Han MS et al., vol: page, PLos One, 2013). As a result of checking the body weight of mice in which vascular calcification was induced, it was confirmed that when Panx3 was deleted, as shown in Figure 5, body weight loss was further accelerated by high-dose Vit D administration, but viability was increased, and high-dose Vit D returned to normal. Mice died after 9 days, but mice with Panx3 deletion were found to survive up to 10 days.

In addition, as a result of confirming the degree of calcification of blood vessels by performing Vonkossa staining on paraffin sections prepared after inducing vascular calcification, it was confirmed that vascular calcification was promoted in blood vessels of Panx3 ^-/- mice rather than Panx3 ^+/- , as shown in Figure 6. there was.

From the above results, it was confirmed that the Panx3 gene exhibits an effect of suppressing vascular calcification.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A biomarker composition for diagnosing the possibility of worsening vascular calcification induced by phosphate containing Pannexin3,
A biomarker composition for diagnosing the possibility of worsening vascular calcification, characterized in that if the Pannexin3 is not detected, the possibility of worsening vascular calcification due to phosphate is determined to be high. A kit for diagnosing the possibility of worsening vascular calcification induced by phosphate containing an agent capable of measuring the expression level of Pannexin3,
A kit for diagnosing the possibility of worsening vascular calcification, characterized in that if the expression level of Pannexin3 is not detected, the possibility of worsening vascular calcification due to phosphate is determined to be high. The method according to claim 2, wherein the agent capable of measuring the expression level of Pannexin3 is a primer or probe that specifically binds to the Pannexin3 gene, an antibody, peptide, aptamer, or compound that specifically binds to the Pannexin3 protein. A kit for diagnosing the possibility of worsening vascular calcification induced by phosphate. Confirming the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level from a sample isolated from the individual; and
A method of providing information for diagnosing the possibility of worsening vascular calcification induced by phosphate, comprising comparing the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level with a normal control sample,
A method for providing information for diagnosing the possibility of worsening vascular calcification, characterized in that if the mRNA expression level of the Pannexin3 gene or the Pannexin3 protein expression level is not detected, it is determined that there is a high possibility of worsening vascular calcification due to phosphate. The method of claim 4, wherein the sample is any one selected from the group consisting of cells, tissues, blood, saliva, and urine isolated from an individual in which vascular calcification is predicted. Possibility of worsening vascular calcification induced by phosphate. How to inform diagnosis. delete delete delete delete delete delete delete