KR102148892B1 - Role of NF-κB-responsive miR-31-5p and miR-155-5p on inflammatory cytokine-induced impairment of endothelial progenitor cell function, and use thereof - Google Patents

Abstract

The present invention relates to a role of NF-κB-dependent miR-31-5p and miR-155-5p in inflammatory cytokine-induced endothelial progenitor cell dysfunction, and use thereof. Particularly, the present invention relates to a composition for preventing, treating or alleviating inflammation-induced blood vessel dysfunction, or a composition for inducing angiogenesis or improving blood flow, which includes an inhibitor against expression or activity of miR-31-5p and miR-155-5p, or an inhibitor against expression or activity of NF-κB, as an active ingredient. The composition according to the present invention recovers inflammation-induced endothelial progenitor cell-related disorder, i.e. disorder of differentiation from monocytes into endothelial progenitor cells and endothelial progenitor cell dysfunction, and thus can effectively prevent, treat or alleviate inflammation-induced blood vessel dysfunction, and can effectively induce angiogenesis of inflammation tissues or patients suffering from inflammatory diseases, or can improve blood flow.

Description

Role of NF-κB-dependent miR-31-5p and miR-155-5p and their use in dysfunction of vascular endothelial progenitor cells by inflammatory cytokines {Role of NF-κB-responsive miR-31-5p and miR- 155-5p on inflammatory cytokine-induced impairment of endothelial progenitor cell function, and use thereof}

The present invention relates to the role and use of NF-κB-dependent miR-31-5p and miR-155-5p in dysfunction of vascular endothelial progenitor cells by inflammatory cytokines, specifically miR-31-5p or miR -155-5p expression or activity inhibitor, or NF-κB expression or activity inhibitor as an active ingredient for preventing, treating or improving vascular dysfunction caused by inflammation, and inducing angiogenesis in inflammatory tissues or inflammatory diseases or It relates to a composition for improving blood flow.

Vascular endothelial cells and endothelial progenitor cells (EPCs) not only affect fetal development and blood pressure control through angiogenesis, but also wound healing, myocardial infarction, stroke, diabetes and microvascular complications, arteriosclerosis, It affects a variety of pathological symptoms including rheumatoid arthritis.

In 1997, Asahara et al. isolated CD34 ⁺ cells from human peripheral blood and cultured them for a week. As a result, they differentiated into cells having the characteristics of vascular endothelial cells to form colonies, and these cells were called vascular endothelial progenitor cells. It was confirmed that these cells are involved in angiogenesis in an animal model of lower limb ischemia. These cells are mainly derived from the bone marrow, and are known to be involved in restoring damaged blood vessels by circulating with blood or in creating blood vessels in ischemic tissues.

Vascular endothelial progenitor cells are classified into early vascular endothelial progenitor cells (early EPCs) and late vascular endothelial progenitor cells (late EPCs). Early vascular endothelial progenitor cells are cells that have a spindle shape differentiated within 3 weeks from mononuclear cells in peripheral blood, and lose their proliferative capacity after 4 to 6 weeks and are gradually killed, but the ability to secrete angiogenic factors. It turns out to be excellent. On the other hand, late vascular endothelial progenitor cells are cells that form colonies when mononuclear cells are cultured for 4 to 6 weeks, and show a gravel-like cell shape similar to human umbilical vein endothelial cells. These cells have been reported to proliferate continuously for more than 10 weeks and have angiogenic ability similar to that of vascular endothelial cells.

It has been reported that the number of endothelial progenitor cells in the blood varies depending on the disease. For example, it is reported that the number of endothelial progenitor cells in blood in the blood of patients with inflammation-related diseases such as rheumatoid arthritis, arteriosclerosis, obesity, and type 2 diabetes is very low compared to that of normal subjects. In addition, it has been reported that the number of endothelial progenitor cells in blood in patients with pregnancy addiction known as inflammatory disease is lower than that of healthy pregnant women. Interestingly, in patients with pregnancy addiction, endothelial progenitor cells in blood are known to have very low angiogenesis and revascularization efficacy due to premature aging. These results suggest that the number of endothelial progenitor cells decreases due to inflammation, and differentiation inhibition and dysfunction occur, leading to various vascular diseases. Therefore, it is considered that vascular complications caused by inflammation can be prevented or treated by promoting the differentiation of endothelial progenitor cells or restoring their function in patients with these diseases.

When NF-κB is activated due to various causes, expression of various cytokines such as TNF-α (tumor necrosis factor-α) and IL-1β (interleukin-1β) increases, thereby promoting inflammatory diseases. In particular, these cytokines cause vascular disease, and are known to be deeply associated with inhibition of angiogenesis and blood vessel regeneration. For example, inflammatory cytokines reduce the function of vascular endothelial cells by inhibiting the expression of vascular endothelial nitric oxide synthase (eNOS), and various vascular diseases (high blood pressure, arteriosclerosis, pregnancy addiction, etc.) Cause of. However, no clear study results have been reported on the correlation between inflammatory NF-κB activation and vascular endothelial progenitor cell differentiation and dysfunction.

According to a number of recent studies, microRNA (miRNA) composed of about 22 bases is known to bind to the mRNA of a specific gene to reduce its stability or inhibit gene expression by interfering with protein synthesis. In particular, microRNAs expressed in inflammatory conditions are known to cause various inflammatory vascular diseases (arteriosclerosis, myocardial infarction, hypertension, pregnancy addiction, etc.) by regulating the expression of genes related to the etiology of inflammatory diseases. However, there are no studies showing that these microRNAs are directly involved in vascular endothelial progenitor cell differentiation and function regulation. Therefore, discovering microRNAs that regulate vascular endothelial cell differentiation and confirming the efficacy will be very important in establishing a new treatment strategy for inflammatory vascular disease.

The present inventors noted that miR-31-5p and miR-155-5p dependent on NF-κB activated by TNF-α, a representative inflammatory cytokine, can regulate the activity of endothelial progenitor cells. In particular, concentrations of TNF-α, miR-31-5p, and miR-155-5p were increased in blood and mononuclear cells of patients with inflammatory pregnancy addiction, whereas the number of endothelial progenitor cells was decreased.

Accordingly, the present inventors confirmed that TNF-α inhibits the differentiation of mononuclear cells into vascular endothelial progenitor cells, and this phenomenon is effectively recovered by inhibition of NF-κB activation and miR-31-5p and miR-155-5p inhibitors. Based on this, as a result of studying NF-κB and its dependent functions of miR-31-5p and miR-155-5p in various ways, the differentiation of endothelial progenitor cells due to inflammation and inhibition of angiogenesis in ischemic tissue Recovered by NF-κB inhibitors and miR-31-5p and miR-155-5p inhibitors, therefore, inhibitors of NF-κB and inhibitors of NF-κB-dependent miR-31-5p and miR-155-5p are inflammatory environments. It was confirmed that differentiation of vascular endothelial progenitor cells can be promoted, and the present invention was completed.

Nature. 2005;438(7070):932-936. Cell. 1996;86(3):353-364. Nature. 2005;438(7070):937-945. Nat Rev Mol Cell Biol. 2007;8(6):464-478. J Hematother Stem Cell Res. 2002;11(2):171-178. Science. 1997;275:964-967. J Clin Invest. 2000;105:71-77. Arterioscler Thromb Vasc Biol. 2004;24:288-293. Ann Rheum Dis. 2007;66(10):1284-1288. Atherosclerosis. 2008;201(2):236-247. Int J Obes (Lond). 2009;33(2):219-225. J Am Coll Cardiol. 2005;45(9):1449-1457. Clin Sci (Lond). 2016;130(6):409-419. Gynecol Obstet Invest. 2007;64(2):103-108. Hypertension. 2014;64(1):23-25. J Clin Endocrinol Metab. 2005;90(9):5329-5332. Nat Rev Immunol. 2009 Nov;9(11):778-88. Clin Sci (Lond). 2010 Feb 23;118(10):593-605. Biochem Biophys Res Commun. 2014;448(1):101-107. J Biol Chem. 2018;293(49):18989-19000. Free Radic Biol Med. 2017;104:185-198. Cell. 2009;136(2):215-233. Cell. 2009;136(1):26-36. Angiogenesis. 2018;21(4):699-710. J Cell Physiol. 2019.doi: 10.1002/jcp.28942.

The main object of the present invention is to provide a novel composition that can be used to prevent, treat, or improve inflammatory vascular disease by using microRNAs and related factors involved in the differentiation and function regulation of vascular endothelial progenitor cells in an inflammatory environment. .

Another object of the present invention is to provide a new composition that can be used to induce angiogenesis or improve blood flow in inflammatory tissues or inflammatory diseases by using the above microRNAs and related factors.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating vascular dysfunction caused by inflammation containing an inhibitor of expression or activity of miR-31-5p or miR-155-5p as an active ingredient.

In the pharmaceutical composition for preventing or treating vascular dysfunction caused by inflammation of the present invention, the inhibitor is preferably a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-31-5p or miR-155-5p. Do.

In the pharmaceutical composition for preventing or treating vascular dysfunction caused by inflammation of the present invention, the inhibitor is preferably a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In the pharmaceutical composition for preventing or treating vascular dysfunction due to inflammation of the present invention, the vascular dysfunction is selected from pregnancy addiction, wound inflammation, myocardial infarction, stroke, diabetes, microvascular complications, arteriosclerosis and rheumatoid arthritis. It is desirable to have vascular dysfunction caused by disease.

According to another aspect of the present invention, the present invention provides a food composition for preventing or improving vascular dysfunction caused by inflammation containing an inhibitor of expression or activity of miR-31-5p or miR-155-5p as an active ingredient.

In the food composition for preventing or improving vascular dysfunction caused by inflammation of the present invention, the inhibitor is preferably a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-31-5p or miR-155-5p. Do.

In the food composition for preventing or improving vascular dysfunction caused by inflammation of the present invention, the inhibitor is preferably a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In the food composition for preventing or improving vascular dysfunction caused by inflammation of the present invention, the vascular dysfunction is selected from pregnancy addiction, wound inflammation, myocardial infarction, stroke, diabetes, microvascular complications, arteriosclerosis and rheumatoid arthritis. It is preferable that the vascular function is impaired due to the disease.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases containing an inhibitor of expression or activity of miR-31-5p or miR-155-5p as an active ingredient. to provide.

In the pharmaceutical composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases of the present invention, the inhibitor is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-31-5p or miR-155-5p It is desirable.

In the pharmaceutical composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases of the present invention, the inhibitor is preferably a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

According to another aspect of the present invention, the present invention provides a food composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases containing an inhibitor of expression or activity of miR-31-5p or miR-155-5p as an active ingredient. to provide.

In the food composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases of the present invention, the inhibitor is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-31-5p or miR-155-5p. It is desirable.

In the food composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases of the present invention, the inhibitor is preferably a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating vascular dysfunction caused by inflammation containing an inhibitor of NF-κB expression or activity as an active ingredient.

In the pharmaceutical composition for preventing or treating vascular dysfunction caused by inflammation of the present invention, the inhibitor of NF-κB expression or activity is preferably a molecule that inhibits the expression or activity of NF-κB p65.

In the pharmaceutical composition for preventing or treating vascular dysfunction due to inflammation of the present invention, the vascular dysfunction is selected from pregnancy addiction, wound inflammation, myocardial infarction, stroke, diabetes, microvascular complications, arteriosclerosis and rheumatoid arthritis. It is preferable that the vascular function is impaired due to the disease.

According to another aspect of the present invention, the present invention provides a food composition for preventing or improving vascular dysfunction caused by inflammation containing an inhibitor of NF-κB expression or activity as an active ingredient.

In the food composition for preventing or improving vascular dysfunction caused by inflammation of the present invention, the inhibitor of NF-κB expression or activity is preferably a molecule that inhibits the expression or activity of NF-κB p65.

In the food composition for preventing or improving vascular dysfunction caused by inflammation of the present invention, the vascular dysfunction is selected from pregnancy addiction, wound inflammation, myocardial infarction, stroke, diabetes, microvascular complications, arteriosclerosis and rheumatoid arthritis. It is desirable to have vascular dysfunction caused by disease.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases containing an inhibitor of NF-κB expression or activity as an active ingredient.

In the pharmaceutical composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases of the present invention, the inhibitor of NF-κB expression or activity is preferably a molecule that inhibits the expression or activity of NF-κB p65.

According to another aspect of the present invention, the present invention provides a food composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases containing an inhibitor of NF-κB expression or activity as an active ingredient.

In the food composition for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases of the present invention, the inhibitor of NF-κB expression or activity is preferably a molecule that inhibits the expression or activity of NF-κB p65.

The composition of the present invention effectively prevents, treats or effectively prevents vascular dysfunction caused by inflammation by restoring vascular endothelial progenitor cell-related disorders due to inflammation, that is, differentiation disorders from monocytes to vascular endothelial progenitor cells and dysfunction of vascular endothelial progenitor cells. It can improve, and can effectively induce angiogenesis in inflammatory tissues or people with inflammatory diseases or improve blood flow.

1 is a result of analyzing the expression of TNF-α, miR-31-5p, and miR-155-5p in serum and mononuclear cells of healthy pregnant women and pregnant addiction patients. A: As a result of confirming the ratio of endothelial progenitor cells (CD34 ⁺ /KDR ⁺ ) in the blood of healthy pregnant women (HP) and pregnancy addiction patients (PE), B: blood from healthy pregnant women (HP) and pregnancy addiction patients (PE) As a result of measuring the concentration of TNF-α, C: a result of measuring the concentration of miR-31-5p and miR-155-5p in the blood of a healthy pregnant woman (HP) and a pregnancy addiction patient (PE), D: a healthy pregnant woman As a result of measuring miR-31-5p and miR-155-5p expressed in mononuclear cells isolated from blood of (HP) and pregnancy addiction patients (PE), *** p <0.001.
Figure 2 shows the degree of differentiation into vascular endothelial progenitor cells according to the treatment of an inhibitor of miR-31-5p, an inhibitor of miR-155-5p, and a TNF-α neutralizing antibody when mononuclear cells of a gestational addiction patient are cultured in autologous serum. Is the result of analysis. A: Mononuclear cells isolated from the blood of healthy pregnant women (HP) and pregnancy addiction patients (PE) were cultured in autologous serum for 4 days, and then negative control (NC), inhibitor of miR-31-5p, miR-155-5p Inhibitors of and miR-31-5p and miR-155-5p mixture (31i/155i+αTNF-α) and TNF-α neutralizing antibody were treated, and the degree of differentiation into vascular endothelial progenitor cells was determined by the absorption of Ac-LDL and Fluorescence micrographs confirmed by binding with UCA-1, B and C: As a result of quantifying the number and degree of differentiation into vascular endothelial progenitor cells from fluorescence micrographs, D and E: expression of KDR in differentiated vascular endothelial progenitor cells As a result of analyzing and quantifying the degree with a cytometer, * p <0.05; ** p <0.01; *** p <0.001.
Figure 3 is a condition of treating mononuclear cells isolated from umbilical cord blood of a healthy pregnant woman with NF-κB p65 siRNA, miR-31-5p mimic, and miR-155-5p mimic, and cultured in a medium containing TNF-α This is the result of analyzing the degree of differentiation into vascular endothelial progenitor cells in. A: A negative control (NC), siRNA of NF-κB p65 was introduced into mononuclear cells isolated from umbilical cord blood of a healthy pregnant woman (sip65) or synthesized with siRNA of NF-κB p65, miR-31-5p mimic and miR-155 After introducing the -5p mimetic (sip65+31m/155m), culture in a medium containing or without TNF-α, the degree of differentiation into vascular endothelial progenitor cells, absorption of Ac-LDL and binding with UCA-1 Fluorescence micrograph, B: The expression levels of hematopoietic stem cell marker CD34 and vascular endothelial cell markers CD31, KDR, VE-cadherin, and vWF in differentiated vascular endothelial progenitor cells were analyzed and quantified with a cytometer. * p <0.05; ** p <0.01; *** p <0.001.
FIG. 4 is a vascular endothelial progenitor under conditions of treatment with an inhibitor of miR-31-5p, an inhibitor of miR-155-5p, or a combination thereof on mononuclear cells isolated from umbilical cord blood of a healthy pregnant woman, and culture in a medium containing TNF-α. This is the result of analyzing the degree of differentiation into cells. A: Negative control (NC), inhibitors of miR-31-5p (miR-31i, 31i), inhibitors of miR-155-5p (miR-155i, 155i) or inhibitors of these mononuclear cells isolated from cord blood of healthy pregnant women After introducing the combination of (31i/155i), cultured in a medium containing or not containing TNF-α, and the degree of differentiation into vascular endothelial progenitor cells was confirmed by absorption of Ac-LDL and binding with UCA-1 under a fluorescence microscope. Photo, B: The expression levels of CD31, CD34, KDR, and VE-cadherin in differentiated vascular endothelial progenitor cells were analyzed and quantified with a cytometer, * p <0.05; ** p <0.01; *** p <0.001.
5 is a vascular endothelial progenitor by culturing in a medium containing TNF-α after treating a mononuclear cell isolated from umbilical cord blood of a healthy pregnant woman with an inhibitor of miR-31-5p, an inhibitor of miR-155-5p, or a combination of these inhibitors. This is the result of analyzing the degree of blood flow regeneration and angiogenesis by differentiating into cells and then injecting it into the ischemic tissue of the mouse. A: Negative control (NC), inhibitors of miR-31-5p (miR-31i, 31i), inhibitors of miR-155-5p (miR-155i, 155i) in mononuclear cells (MNCs) isolated from umbilical cord blood of healthy pregnant women , Or a combination of these inhibitors (miR-31i/155i, 31i/155i), cultured in a medium containing or without TNF-α to differentiate into vascular endothelial progenitor cells, and these cells or saline ) Was injected into the lower limb ischemic tissue of an immunodeficient (nude) mouse, and then the degree of recovery of blood flow was taken with a laser Doppler hemometry, B: As a result of quantifying the degree of recovery of blood flow measured with a laser Doppler hemometry, C: Photographs of formation of new capillaries (green) in ischemic tissue and endothelial progenitor cells (red) embedded in these capillaries, D: As a result of quantifying the degree of capillary formation from these photographs, * p <0.05; ** p <0.01; *** p <0.001.

In the present invention, inhibitors of miR-31-5p or miR-155-5p dependent on NF-κB recover from the inhibition of differentiation from mononuclear cells to vascular endothelial progenitor cells due to inflammation and inhibition of the function of vascular endothelial progenitor cells. It was conceived based on the new research results that it can be done.

miR-31 and miR-155 are small-sized RNA molecules, and their information is well known through the micro RNA database miRBase (http://www.mirbase.org). Representatively, in humans ( Homo sapiens ), the stem-loop sequence of miR-31 is the same as SEQ ID NO: 3, and the mature form of miR-31-5p is SEQ ID NO: 4, another mature form of miR- 31-3p consists of the sequence of SEQ ID NO: 5, the stem-loop sequence of miR-155 is the same as SEQ ID NO: 6, and the mature form of miR-155-5p is SEQ ID NO: 7, another The mature form, miR-155-3p, consists of the sequence of SEQ ID NO: 8.

The inhibitor of miR-31-5p or miR-155-5p contained as an active ingredient in the composition of the present invention refers to a substance that inhibits the expression or activity of miR-31-5p or miR-155-5p in a living body or cell. As such, typically miR-31-5p or antagomir for miR-155-5p may be applicable. In addition, antisense molecules against miR-31-5p or miR-155-5p, shorthairpin RNA molecules (shRNA), short interfering RNA (siRNA) molecules, seed-targeting locked nucleic acid (LNA), aptamers , Ribozymes, or DNA-RNA hybrids.

Preferably, it is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-31-5p or miR-155-5p, and this nucleic acid molecule is DNA, RNA, peptide nucleic acids (PNA), phosphothioester nucleic acid molecule, phosphorothioated It may be in the form of a nucleic acid molecule, a 2'-O-methyl-modified nucleic acid molecule, or a locked nucleic acid (LNA).

More preferably, it is a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may be a vector containing the nucleic acid molecule and expressing the nucleic acid molecule in an animal cell. In addition, even if some of the nucleotide sequences in the nucleotide sequence of SEQ ID NO: 1 or 2 are modified by deletion, substitution, or insertion, a variant capable of functionally equivalent function may also be included.

The inhibitor of NF-κB contained as an active ingredient in the composition of the present invention refers to a substance that inhibits the expression or activity of NF-κB in a living body or cells, for example IMD-1041, SAR113945, VGX-1027, Bay11 -7082, siRNA may be applicable. Preferably, it is a molecule that inhibits the activation of NF-κB, and more preferably, it may be a molecule that inhibits the expression of p65, a subunit of NF-κB.

More preferably, it may be an siRNA of NF-κB p65, for example, a double-stranded siRNA consisting of a sense strand of SEQ ID NO: 9 and an anti-sense strand of SEQ ID NO: 10, of SEQ ID NO: 11 Double-stranded siRNA consisting of a sense strand and an anti-sense strand of SEQ ID NO: 12 or a sense strand of SEQ ID NO: 13 and an anti-sense strand of SEQ ID NO: 14 It may be a double-stranded siRNA, or a combination of these siRNAs. In addition, for the stability of siRNA, siRNA protective molecules such as deoxythymidine dinucleotide (dTdT) may be included at the 3'end of the siRNA.

According to the present invention, such inhibitors of miR-31-5p or miR-155-5p, or inhibitors of NF-κB, are differentiated into vascular endothelial progenitor cells caused by inflammation (monocytes are differentiated into vascular endothelial progenitor cells. ) Is inhibited and by restoring the dysfunction of differentiated vascular endothelial progenitor cells, vascular dysfunction caused by inflammation can be prevented, treated or improved.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating vascular dysfunction caused by inflammation, and a food composition for preventing or improving vascular dysfunction caused by inflammation.

At this time, the type of vascular dysfunction caused by inflammation is not limited, but it may be a vascular dysfunction due to a disease selected from among pregnancy addiction, wound inflammation, myocardial infarction, stroke, diabetes, microvascular complications, arteriosclerosis and rheumatoid arthritis. have.

In addition, according to the present invention, such an inhibitor of miR-31-5p or miR-155-5p, or an inhibitor of NF-κB may induce angiogenesis or improve blood flow in inflammatory tissues or inflammatory diseases.

Accordingly, the present invention also provides pharmaceutical compositions and food compositions for inducing angiogenesis or improving blood flow in inflammatory tissues or inflammatory diseases.

According to the present invention, when an inhibitor of miR-31-5p and an inhibitor of miR-155-5p are used together, a synergistic effect can be exhibited. Therefore, if the composition of the present invention contains both an inhibitor of miR-31-5p and an inhibitor of miR-155-5p as active ingredients, a more excellent effect can be expected.

The composition of the present invention may be an active ingredient, the inhibitor itself, or a composition mixed with a pharmaceutically or food pharmaceutically acceptable carrier.

In addition, the inhibitors of the present invention are pharmaceutically acceptable to the extent that they can exhibit the inherent function of the inhibitor, that is, inhibit the expression or activity of miR-31-5p, miR-155-5p, or NF-κB, depending on the type. It can also be used in the form of an acceptable acid addition salt or metal complex.

The composition of the present invention may be administered by a known administration method depending on the form of the inhibitor during clinical administration, may be administered orally or parenterally, and may be administered systemically or locally. For example, when the inhibitor of miR-31-5p or miR-155-5p is antagomir and the inhibitor of NF-κB is siRNA, the composition of the present invention may be administered by injection into blood or topically to tissues. Can be administered.

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

The dosage of the composition of the present invention varies depending on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, and the appropriate dosage is the type or efficacy of the inhibitor. It can vary depending on the degree. For example, when the inhibitor of miR-31-5p or miR-155-5p is antagomir, and is administered by injection into blood, about 1 mg to 200 mg per 1 kg body weight based on the inhibitor contained in the composition It may be, and may be administered once to several times a day. In addition, the inhibitor of NF-κB is siRNA, and when administered by injection into the blood, it may be about 1 mg to 200 mg per 1 kg of body weight based on the inhibitor contained in the composition, and may be administered once to several times a day. have.

For clinical administration, it can be formulated into various forms of oral or parenteral formulation, and at this time, it can be prepared using generally used diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, excipients, or cell membrane penetration aids. There will be.

In addition, the composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredient inhibitor.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Experiment method

1-1. Experimental Materials and Research Ethics

All materials related to the micro RNA used in the experiment were purchased from QIAGEN (Germany), and the cell culture solution EGM-2 SingleQuots was purchased from Lonza (Switzerland). The blood used in this experiment was supplied from healthy pregnant women and patients with pregnancy addiction with the consent of the donor under the approval of the Bioethics Review Committee of Kangwon National University Hospital. Pregnancy addiction patients had systolic blood pressure of 140 mmHg or more, diastolic blood pressure of 90 mmHg or more, and proteinuria of 0.3 g/24 hours or more. In addition, all animal experiments were conducted in accordance with the guidelines of the ethics committee on the protection and use of experimental animals at Kangwon National University.

1-2. Isolation and culture of mononuclear cells and differentiation into endothelial progenitor cells

Mononuclear cells were isolated from blood by a density gradient centrifugation method using Ficoll-Hypaque (specific gravity: 1.077), suspended in EGM-2 SingleQuots medium (4x10 ⁶ cells/ml), and fibronectin (50 Mg/ml; Sigma-Aldrich) was transferred to a plastic cell culture container and incubated for 4 days in a 5% CO ₂ incubator (37° C.). Cells not attached to the culture vessel were removed, and NF-κB p65 siRNAs (80nM), micro RNA (miR-31-5p or miR-155-5p) inhibitors (80nM, mixed 40nM each, Quiagen), micro RNA (miR-31-5p or miR-155-5p) mimics (80 nM each, 40 nM in the case of mixing, Quiagen) were introduced into cells using Lipofectamine RNAiMAX or Lipofectamine 3000 (Invitrogen). These cells were cultured for an additional 4 days in a medium containing or without TNF-α (10 ng/ml, R&D Systems) to differentiate into vascular endothelial progenitor cells. Meanwhile, some cells were cultured for 4 days in a medium containing 30% autologous serum isolated from healthy pregnant women and patients with pregnancy addiction. In the case of cell culture fluid containing autologous serum, a neutralizing antibody of human TNF-α (10 ng/ml, R&D Systems) was also added.

At this time, the inhibitor of miR-31-5p is a synthetic RNA molecule consisting of a complementary sequence of the miR-31-5p sequence (SEQ ID NO: 1), and the inhibitor of miR-155-5p is a complementary sequence of the miR-155-5p sequence ( Synthetic RNA molecule consisting of SEQ ID NO: 2), miR-31-5p miR-31-5p synthetic RNA molecule consisting of sequence (SEQ ID NO: 4), miR-155-5p miR-155-5p A synthetic RNA molecule consisting of the sequence (SEQ ID NO: 7) was used.

NF-κB p65 siRNA is a double-stranded siRNA consisting of a sense strand containing dTdT (deoxythymidine dinucleotide) at the 3′ end of SEQ ID NO: 9 and an antisense strand containing dTdT at the 3′ end of SEQ ID NO: 10, SEQ ID NO: 11 Double-stranded siRNA consisting of a sense strand containing dTdT (deoxythymidine dinucleotide) at the 3′ end of the sequence and an antisense strand containing dTdT at the 3′ end of SEQ ID NO: 12, dTdT (deoxythymidine) at the 3′ end of SEQ ID NO: 13 dinucleotide) and a double-stranded siRNA consisting of an antisense strand containing dTdT at the 3'end of SEQ ID NO: 14 were mixed in a ratio of 1:1.

1-3. Analysis of the degree of differentiation of endothelial progenitor cells under cell culture conditions

Cells were treated with 10 mg/ml of Dil-ac-LDL (Molecular Probes) solution for 2 hours (CO ₂ constant temperature, 37° C.), washed twice and fixed with 2% formaldehyde. The fixed cells were treated in a 10 mg/ml FITC-UEA-1 (Sigma) solution for 2 hours and washed, and cells that absorbed or bound Dil-ac-LDL and FITC-UEA-1 were confirmed by fluorescence microscopy. .

In addition, after recovering the cells attached to the cell culture vessel with a non-enzymatic separation solution (C5789; Sigma-Aldrich), an antibody with phycoerythrin (PE) (CD31, cat no. 560983: CD34, cat no) 555822; KDR, cat no. 560872; VE-cadherin, cat no. 560410: BD PharMingen) and vWF antibody [(cat no. ab154193: Abcam) and fluorescein isothiocyanate (FITC)-attached rabbit IgG] 4 After staining for 30 minutes at °C, washing with physiological saline, it was fixed with 2% formaldehyde.

The level of expression of each target protein in the stained cells was analyzed and quantified using a flow cytometer (FACSCalibur, BD).

1-4. Blood vessel endothelial progenitor cell analysis

Human CD34 antibody with FITC (Clone 581, BD Biosciences) and human KDR antibody with PE (cat no. 560872, BD Bioscences) were added to 200 µl of blood, stained for 30 minutes in the dark, and then Pharm Lyse ( BD Pharmingen) solution 2 ml was added and kept at room temperature for 7 minutes to hemolyze red blood cells. The remaining cells were washed with physiological saline and fixed with 400 µl of a 2% formaldehyde solution. The fixed cells were analyzed and quantified using a flow cytometer (FACSCalibur, BD). CD34 ⁺ KDR ⁺ cells in blood were defined as endothelial progenitor cells.

1-5. Micro RNA and TNF-α quantification

Total microRNAs in cells and blood were isolated using the miRNeasy Mini kit or the miRNeasy serum/plasma kit (QIAGEN). CDNA was synthesized using 1 μg of total micro RNA, and the concentrations of miR-31-5p and miR-155-5p were analyzed by qRT-PCR using the miScript SYBR Green PCR Kit (QIAGEN). The primer used at this time was purchased from QIAGEN. Serum TNF-α concentration was measured using a Quantikine ELISA kit (R&D Systems).

1-6. Mouse lower limb ischemia experiment

Mixed anesthetics [ketamine (50 mg/kg) and xylazine (20 mg/kg) in the abdominal cavity of nude mice without thymus (7 weeks old, 17-20 g, Jackson Laboratory); Bayer Korea] was administered, and ischemia was induced by ligation of the femoral artery of the left hind limb. Endothelial progenitor cells ( ⁵ ×10 ⁵ cells) stained with a fluorescent substance CM-Dil (cat. no. C7000, Invitrogen Molecular Probes) were injected into the muscle tissue of the ischemic site with a 26-gauge needle. As a control group, physiological saline was injected. Blood flow of both hind limbs was measured using a laser Doppler hemometer.

1-7. Tissue staining

The skeletal muscle tissue of the lower extremity ischemic tissue of the mouse was isolated, wrapped in a frozen tissue embedding agent (OCT™ compound) and frozen, and a 6 µm sectioned tissue was made. After staining the section tissue with FITC-attached isolectin B4, the formation of total capillaries (FITC-isolectin B4, green) and incorporation of endothelial progenitor cells into the vascular structure (CM-Dil, red) were observed with a confocal fluorescence microscope. I did.

1-8. Statistical analysis

The results of quantitative experiments performed independently at least three times were expressed as mean-standard error (SEM). Statistical significance was determined using ANOVA or independent Student's T test (1-tailed) depending on the number of experimental groups analyzed. Statistical significance was set to P value of 0.05 or less.

2. Experiment result

2-1. Expression of TNF-α, miR-31-5p and miR-155-5p in serum and mononuclear cells of patients with pregnancy addiction

As a result of analysis of serum and mononuclear cells (PBMCs) obtained from peripheral blood of healthy pregnant women and patients with pregnancy addiction, the number of vascular endothelial progenitors, CD34 ⁺ /KDR ⁺ cells, was low in the patient's blood (Fig. 1). A), the concentration of the inflammatory cytokine TNF-α was high (see B in FIG. 1), and the concentrations of miR-31-5p and miR-155-5p were also high (see C in FIG. 1). On the other hand, it was found that the concentrations of miR-31-5p and miR-155-5p were also high in mononuclear cells isolated from the patient's peripheral blood (see D in FIG. 1). These results indicate that there is a reverse correlation between the concentration of TNF-α, miR-31-5p, and miR-155-5p in patient blood and the number of endothelial progenitor cells (EPCs). .

2-2. Effects of TNF-α neutralizing antibodies, miR-31-5p inhibitors, and miR-155-5p inhibitors on the differentiation of vascular endothelial progenitor cells from mononuclear cells in pregnancy addiction patients

After culturing mononuclear cells isolated from peripheral blood of healthy pregnant women and patients with pregnancy addiction in autologous serum, the degree of differentiation into vascular endothelial progenitor cells was determined by the absorption of Ac-LDL (acetylated low density lipoprotein) and UCA-1. (lex europaeus agglgutinin-I lectin) was analyzed through binding. As a result, mononuclear cells cultured with autologous serum of patients had significantly lower absorption of Ac-LDL and ability to bind to UCA-1 than mononuclear cells cultured with autologous serum of healthy pregnant women (see A to C in Fig. 2). , It was also found that the expression of vascular endothelail growth factor receptor-2 (VEGFR-2/KDR), which is a marker molecule of vascular endothelial progenitor cells, was also reduced (see D and E of FIG. 2). This inhibition was found to be restored to a healthy cell level by introducing an inhibitor of miR-31-5p and an inhibitor of miR-155-5p into the mononuclear cells of the patient and by treatment with a TNF-α neutralizing antibody in autologous serum (Fig. 1). See A to E of). These results indicate that increased TNF-α, miR-31-5p, and miR-155-5p in the blood of pregnant addiction patients inhibit the differentiation of mononuclear cells into vascular endothelial progenitor cells.

2-3. Expression control of NF-κB p65 and the effects of miR-31-5p and miR-155-5p mimics in an environment where TNF-α inhibits differentiation into vascular endothelial progenitor cells

When mononuclear cells isolated from umbilical cord blood of a healthy pregnant woman were treated with commercial TNF-α, the number of cells differentiated into vascular endothelial progenitor cells having the ability to absorb Ac-LDL and bind to UCA-1 was found to be reduced (Fig. 3). See A). However, inhibiting the activation of NF-κB by introducing siRNA of NF-κB p65 into mononuclear cells to control the expression of NF-κB p65 restored the ability to differentiate into vascular endothelial progenitor cells reduced by TNF-α. (See Fig. 3A). It was found that the recovery of differentiation into vascular endothelial progenitor cells due to the introduction of NF-κB p65 siRNA disappeared again by mixing the miR-31-5p mimic and miR-155-5p mimic (31m/155m) into the mononuclear cells. (See Fig. 3A). This vascular endothelial progenitor cell differentiation regulation was reconfirmed by an analysis method using a cytometer for the expression of vascular endothelial cell markers CD31, CD34, KDR, VE-cadherin, and vWF (von Willebrand factor) (see FIG. 3B). .

These results indicate that the inhibition of vascular endothelial progenitor cell differentiation by TNF-α is due to the biosynthesis of miR-31-5p and miR-155-5p through NF-κB activation, and inhibition of NF-κB activation and miR-31-5p. And inhibition of miR-155-5p activity means that it is possible to restore the inhibition of vascular endothelial progenitor cell differentiation that may be induced in inflammatory conditions.

2-4. Effects of miR-31-5p inhibitors and miR-155-5p inhibitors in an environment in which differentiation into vascular endothelial progenitor cells is inhibited by TNF-α

In order to confirm the role of miR-31-5p and miR-155-5p in the process of inhibiting vascular endothelial progenitor cell differentiation by TNF-α, an inhibitor of miR-31-5p or an inhibitor of miR-155-5p was used in cord blood mononuclear cells. After introduction, the degree of differentiation into vascular endothelial progenitor cells was analyzed. When an inhibitor of miR-31-5p or an inhibitor of miR-155-5p was introduced into mononuclear cells, it was found that the inhibition of endothelial progenitor cell differentiation by TNF-α was significantly restored, and these inhibitors were mixed into mononuclear cells. As a result of the introduction, it was found that synergistic effect was induced and the differentiation efficiency was restored to a normal level (see A in FIG. 4). Meanwhile, as a result of analyzing the expression of vascular endothelial cell marker molecules using flow cytometry, the expression levels of CD31, CD34, KDR, VE-cadherin, vWF, etc., significantly reduced in TNF-α, were inhibitors of miR-31-5p. Alternatively, a certain level was recovered by the inhibitor of miR-155-5p, and as a result of mixing treatment with these inhibitors, it was found to be recovered to a normal level due to a synergistic effect (see FIG. 4B).

These results imply that the inhibition of vascular endothelial progenitor cell differentiation due to inflammation can be more effectively restored when mixed treatment than treatment with an inhibitor of miR-31-5p or an inhibitor of miR-155-5p alone.

2-5. Effects of miR-31-5p Inhibitors and miR-155-5p Inhibitors on Vascular Endothelial Progenitor Cells Inhibited by TNF-α

In order to confirm the revascularization activity of vascular endothelial progenitor cells induced differentiation from mononuclear cells, vascular endothelial progenitor cells are injected into the ischemic tissue of the lower extremity of a mouse without thymus, and the degree of recovery of blood flow is measured with a laser Doppler flowmeter. It was measured using. When vascular endothelial progenitor cells, which normally induce differentiation from umbilical cord blood mononuclear cells, were injected into the ischemic tissue of the lower extremity of the mouse, it was found that regeneration of blood flow was more effectively induced than the control group administered with physiological saline (see A and B in FIG. 5). ). However, vascular endothelial progenitor cells treated with TNF-α induce differentiation significantly inhibited the blood flow regeneration effect, and this inhibitory effect was achieved by treatment with an inhibitor of miR-31-5p or an inhibitor of miR-155-5p alone. It was found to recover significantly (see A and B in Fig. 5). On the other hand, when these two inhibitors were mixed and treated, it was found that synergistic effects were induced and blood flow regeneration reached a normal level (see A and B of FIG. 5).

Since blood flow regeneration in ischemic tissue is closely correlated with new angiogenesis, the degree of new angiogenesis in ischemic tissue was confirmed with a confocal fluorescence microscope. Capillary formation (stained with isolectin B4, green) was more effectively promoted in the control group to which physiological saline was administered in the ischemic tissue of the lower extremity in which the vascular endothelial progenitor cells that were normally differentiated from the mononuclear cells were injected (Fig. Reference), it was found that a number of endothelial progenitor cells were incorporated into the new capillaries (labeled as Dil-Ac-LDL, red) (see C bottom of FIG. 5). However, in the vascular endothelial progenitor cells treated with TNF-α to induce differentiation, the degree of capillary formation and the incorporation of vascular progenitor cells into the new capillaries is significantly inhibited, and this inhibitory effect is an inhibitor of miR-31-5p It was significantly recovered by the inhibitor of miR-155-5p alone, and when the two inhibitors were mixed, there was a synergistic effect (see C and D of FIG. 5).

These results show that treatment with an inhibitor of miR-31-5p or an inhibitor of miR-155-5p can effectively promote angiogenesis and vascular remodeling by restoring the inhibition of vascular endothelial progenitor cell differentiation caused by inflammation. This means that if you mix it, you can get a synergistic effect. Therefore, these inhibitors can effectively improve vascular dysfunction and endovascular damage frequently caused in inflammatory diseases.

<110> KNU-Industry Cooperation Foundation <120> Role of NF-kB-responsive miR-31-5p and miR-155-5p on inflammatory cytokine-induced impairment of endothelial progenitor cell function, and use thereof <130> PA-D19192 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-31-5p inhibitor <400> 1 agcuaugcca gcaucuugcc u 21 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-155-5p inhibitor <400> 2 accccuauca cgauuagcau uaa 23 <210> 3 <211> 71 <212> RNA <213> Homo sapiens <400> 3 ggagaggagg caagaugcug gcauagcugu ugaacuggga accugcuaug ccaacauauu 60 gccaucuuuc c 71 <210> 4 <211> 21 <212> RNA <213> Homo sapiens <400> 4 aggcaagaug cuggcauagc u 21 <210> 5 <211> 22 <212> RNA <213> Homo sapiens <400> 5 ugcuaugcca acauauugcc au 22 <210> 6 <211> 62 <212> RNA <213> Homo sapiens <400> 6 cuguuaaugc uaaucgugau agggguuuuu gccucaagac uccuacauau uagcauuaac 60 ag 62 <210> 7 <211> 24 <212> RNA <213> Homo sapiens <400> 7 uuaaugcuaa ucgugauagg gguu 24 <210> 8 <211> 22 <212> RNA <213> Homo sapiens <400> 8 cuccuacaua uuagcauuaa ca 22 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of NF-kB p65 siRNA <400> 9 ccugagcacc aucaacuau 19 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> anti-sense sequence of NF-kB p65 siRNA <400> 10 auaguugaug gugcucagg 19 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of NF-kB p65 siRNA <400> 11 cugaugugca ccgacaagu 19 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> anti-sense sequence of NF-kB p65 siRNA <400> 12 acuugucggu gcacaucag 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of NF-kB p65 siRNA <400> 13 gauugaggag aaacguaaa 19 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> anti-sense sequence of NF-kB p65 siRNA <400> 14 uuuacguuuc uccucaauc 19

Claims (24)

It contains an inhibitor of expression or activity of miR-31-5p and an inhibitor of expression or activity of miR-155-5p as active ingredients, and the expression or activity inhibitor of miR-31-5p is all of the nucleotide sequence of miR-31-5p. Or a nucleic acid molecule capable of binding to a part, and the expression or activity inhibitor of miR-155-5p is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-155-5p. A pharmaceutical composition for preventing or treating vascular dysfunction. delete The method of claim 1,
The expression or activity inhibitor of miR-31-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1,
The composition, characterized in that the inhibitor of expression or activity of miR-155-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2. The method of claim 1,
The vascular dysfunction is a vascular dysfunction due to a disease selected from among pregnancy addiction, wound inflammation, myocardial infarction, stroke, diabetes, microvascular complications, arteriosclerosis and rheumatoid arthritis. It contains an inhibitor of expression or activity of miR-31-5p and an inhibitor of expression or activity of miR-155-5p as active ingredients, and the expression or activity inhibitor of miR-31-5p is all of the nucleotide sequence of miR-31-5p. Or a nucleic acid molecule capable of binding to a part, and the expression or activity inhibitor of miR-155-5p is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-155-5p. Food composition for preventing or improving vascular dysfunction. delete The method of claim 5,
The expression or activity inhibitor of miR-31-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1,
The composition, characterized in that the inhibitor of expression or activity of miR-155-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2. The method of claim 5,
The vascular dysfunction is a vascular dysfunction due to a disease selected from among pregnancy addiction, wound inflammation, myocardial infarction, stroke, diabetes, microvascular complications, arteriosclerosis and rheumatoid arthritis. It contains an inhibitor of expression or activity of miR-31-5p and an inhibitor of expression or activity of miR-155-5p as active ingredients, and the expression or activity inhibitor of miR-31-5p is all of the nucleotide sequence of miR-31-5p. Or a nucleic acid molecule capable of binding to a part, and the expression or activity inhibitor of miR-155-5p is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-155-5p, or A pharmaceutical composition for inducing angiogenesis or improving blood flow in a person with an inflammatory disease. delete The method of claim 9,
The expression or activity inhibitor of miR-31-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1,
The composition, characterized in that the inhibitor of expression or activity of miR-155-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2. It contains an inhibitor of expression or activity of miR-31-5p and an inhibitor of expression or activity of miR-155-5p as active ingredients, and the expression or activity inhibitor of miR-31-5p is all of the nucleotide sequence of miR-31-5p. Or a nucleic acid molecule capable of binding to a part, and the expression or activity inhibitor of miR-155-5p is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-155-5p, or Food composition for inducing angiogenesis or improving blood flow in people with inflammatory diseases. delete The method of claim 12,
The expression or activity inhibitor of miR-31-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1,
The composition, characterized in that the inhibitor of expression or activity of miR-155-5p is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2.
delete delete delete delete delete delete delete delete delete delete

Images