KR101813324B1

KR101813324B1 - Use of human fibroblast growth factor 11 relative to angiogenesis

Info

Publication number: KR101813324B1
Application number: KR1020150106936A
Authority: KR
Inventors: 이서연; 이정현; 임형순; 정재연; 이경원
Original assignee: 서울대학교병원; 한국해양과학기술원
Priority date: 2015-07-29
Filing date: 2015-07-29
Publication date: 2017-12-29
Also published as: KR20170014106A

Abstract

The present invention relates to a composition for regulating angiogenesis comprising an agent for modulating the expression or activity of human FGF 11, wherein mRNA and protein expression of human FGF11 is increased in human vascular endothelial cells under hypoxic conditions, and human vascular endothelial cells transfected with pFGF11 Overexpression of human FGF11 increases capillary-like tube formation and TJ protein but does not affect cell migration activity. HREs in the human FGF11 promoter region are sensitive to hypoxic conditions, and human FGF11 expression is regulated by HIF-1 Binding region, it is anticipated that human FGF11 may act as a modulator of hypoxia-guided pathological processes such as tumor progression or ischemic diseases. Therefore, the agent for modulating the expression or activity of human FGF 11 of the present invention can be useful for inhibiting or promoting angiogenesis.

Description

Use of human FGF11 related to angiogenesis {Use of human fibroblast growth factor 11 relative to angiogenesis}

The present invention relates to a composition for regulating angiogenesis, which comprises a human FGF (fibroblast growth factor) 11 expression or activity modulator.

Oxygen is an essential nutrient for cell respiration, and organisms should closely monitor changes in oxygen concentration to maintain homeostasis. Changes in oxygen concentration can be signal during embryonic development, determine the fate of stem cells, and contribute to pathological conditions.

Low oxygen partial pressure due to insufficient oxygen supply to tissue cells, hypoxia, induces adaptive responses through regulators such as HIFs (hypoxia-inducible factors). Hypoxia is observed in the center of solid tumors and is a common feature of malignant tumors. When the size of the tumor reaches 1-2 cc, the center of the tumor becomes hypoxic due to a lack of adequate blood supply, and tumor growth is disturbed (Folkman J: Dependence of tumors on angiogenesis, Journal of the National Cancer Institute 82: 4-6, 1990.).

Such hypoxia is a powerful stimulus signal of angiogenesis that produces new blood vessels in the existing blood vessels. The blood vessels created through angiogenesis can provide nutrients and oxygen to help the growth of the tumor. Cellular responses to hypoxia vary widely, including changes in metabolism, antioxidant gene expression, cell proliferation, apoptosis, and angiogenesis.

Angiogenesis is an essential factor for tumor growth and progression, and inhibition of angiogenesis may effectively inhibit tumor growth. Angiogenesis is a multistep process that begins when dormant endothelial cells are activated by signals from ischemic tissue or hypoxic solid tumors. Activated vascular endothelial cells degrade the extracellular matrix, migrate to the proliferating and stimulating circles, and form an immature vascular network. The newly formed vascular network undergoes the process of maturation and stabilization of the vascular network, including the recruitment of supporting vascular wall cells and the placement of new basement membranes. The angiogenic process is precisely regulated through angiogenic factors and anti-angiogenic factors.

On the other hand, various factors including the heparin-binding domain such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF / FGF-2), acidic FGF (aFGF / FGF- (heparin-binding EGF-like growth factor) has an angiogenic function. In addition, the hypoxia-inducible factor-1 (HIF-1) transcription factor is a major regulator of hypoxia-induced angiogenesis and regulates genes that affect angiogenesis such as VEGF, placental growth factor (PGF) do.

There are 22 FGFs family (FGF1-FGF23) reported in humans and rodents. FGFs (Fibroblast growth factors) can be classified into secretory FGFs (FGF1-FGF10 and FGF15-FGF23) and intracellular nonspecific FGFs (FGF11-FGF14). Most secretory FGFs and their surface FGF receptors are well known for their specific biological roles in cell growth, differentiation, and development, whereas intracellular nonspecific FGFs, also referred to as FGF analogues (FGF11-FGF14) And the research on this matter is also insignificant.

Therefore, there is a need to study the functions of intracellular nonspecific FGFs, particularly human FGF 11, as an angiogenesis-related factor that is hypoxic-sensitive.

While studying the functions of intracellular nonspecific FGFs, particularly human FGF 11 as an angiogenesis-sensitive factor hypoxic-sensitive, the expression of human FGF11 mRNA and protein in human vascular endothelial cells under hypoxic conditions is increased, and pFGF11 Overexpression of human FGF11 in human transfected human vascular endothelial cells increases capillary-like tube formation and TJ protein but does not affect cell migration activity, hypoxia response elements (HREs) in the human FGF11 promoter region are sensitive to hypoxia , Confirming that the expression of human FGF11 can be induced by HIF-1 transcription factors, and that human FGF11 can act as a regulator of hypoxia-induced pathological processes such as tumor progression or ischemic diseases, thus completing the present invention.

Accordingly, the present invention provides a composition for regulating angiogenesis, which comprises an agent for modulating the expression or activity of human FGF 11.

The present invention also provides a method for providing information for screening an angiogenesis regulator.

The present invention also provides a method for regulating angiogenesis, which comprises administering to a subject a human FGF 11 expression or activity modulating agent.

The present invention provides a composition for regulating angiogenesis, which comprises an agent for modulating the expression or activity of human FGF 11.

The present invention also provides a method of inhibiting angiogenesis comprising the step of administering to a subject a human FGF 11 expression or activity inhibitor.

In addition, the present invention provides a method for promoting angiogenesis comprising the step of administering to a subject an expression or activity promoter of human FGF 11.

The present invention is based on the finding that mRNA and protein expression of human FGF11 is increased in human vascular endothelial cells under hypoxic conditions and overexpression of human FGF11 in human vascular endothelial cells transfected with pFGF11 increases capillary-like tube formation and TJ protein, Human FGF11 is sensitive to a hypoxic state and the expression of human FGF11 can be induced through the HIF-1 binding site in its promoter region, so that human FGF11 is not involved in tumor progression or ischemic diseases Hypoxia-guided pathological processes. Therefore, the agent for modulating the expression or activity of human FGF 11 of the present invention can be useful for inhibiting or promoting angiogenesis.

FIG. 1 shows the degree of mRNA expression of human FGF11 [(A) end-point PCR and (B) real-time PCR] and the degree of protein expression [(C) Western blot] in human vascular endothelial cells under hypoxic conditions.
2A is a graph showing the expression level of human FGF11 when human FGF11 overexpression vector is produced and transfected into human vascular endothelial cells,
FIG. 2B shows the effect of overexpression of human FGF11 on capillary-like tube formation on matrigel in human vascular endothelial cells transfected with pFGF11,
FIG. 2C shows the effect of overexpression of human FGF11 on cell migration activity in human vascular endothelial cells transfected with pFGF11.
FIG. 3 is a graph showing the extent of expression of human FGF11 and TJ proteins occludin, ZO-1 and claudin-5 in human vascular endothelial cells transfected with pFGF11 by Western blotting.
FIG. 4A is a diagram showing a promoter region of human FGF11 in which two regions of HRE (hypoxia response element) capable of binding hypoxia-inducible factor-1 (HIF-1)
FIG. 4B is a graph showing the effect of hypoxic conditions on human FGF11 promoter luciferase activity,
4C is a graph showing the effect of HIF-1 on human FGF11 promoter luciferase activity under normal oxygen conditions.

In addition,

1) treating the biological sample with a candidate substance, measuring the expression or activity level of human FGF 11,

2) comparing the expression or activity level of human FGF 11 obtained in the above step 1 with the expression or activity level of human FGF 11 in a biological sample not treated with the candidate substance, and

3) If the expression or activity level of human FGF 11 obtained in the above step 1) is lower than the expression or activity level of human FGF 11 in a biological sample not treated with the candidate substance, the candidate substance is judged to be an angiogenesis inhibitor A method for providing information for screening of an angiogenesis regulator.

In addition,

1 ') measuring the expression or activity level of human FGF 11 after treating the biological sample with the candidate substance,

2 ') comparing the expression or activity level of human FGF 11 obtained in step 1') with the expression or activity level of human FGF 11 in a biological sample not treated with the candidate substance, and

3 ') If the expression or activity level of human FGF 11 obtained in step 1') is higher than the expression or activity level of human FGF 11 in a biological sample not treated with the candidate substance, the candidate substance is used as an angiogenesis promoter The method comprising the steps of: a)

Hereinafter, the present invention will be described in detail.

The modulator of human FGF 11 in the composition of the present invention comprises an expression or activity inhibitor of human FGF 11 or an expression or activity promoter of human FGF 11.

The human FGF 11 expression inhibitor includes at least one selected from the group consisting of antisense oligonucleotides complementary to mRNA of human FGF 11, siRNA, shRNA, microRNA, and ribozyme, but is not limited thereto.

The activity inhibitor or promoter of human FGF 11 includes at least one selected from the group consisting of a compound specifically binding to human FGF 11, a peptide, an aptamer, and an antibody, but is not limited thereto.

The human FGF 11 expression-promoting agent includes, but is not limited to, HIF-1 (hypoxia-inducible factor-1) protein.

The present invention is based on the finding that mRNA and protein expression of human FGF11 is increased in human vascular endothelial cells under hypoxic conditions and overexpression of human FGF11 in human vascular endothelial cells transfected with pFGF11 increases capillary-like tube formation and TJ protein, But not the other. Also, under hypoxic conditions, reporter gene activity was markedly increased for cells transfected with pGL3-FGF11-HREs (WT), whereas for cells transfected with the HRE-deletion-fragment pGL3-FGF11- DELTA HREs (DELTA HRE) It was found that the HREs in the human FGF11 promoter region are sensitive to the hypoxic state and the expression of human FGF11 can be induced through the HIF-1 binding site in its promoter region. Thus, human FGF11 is expected to act as a modulator of hypoxia-induced pathological processes such as tumor progression or ischemic diseases, so that the human FGF 11 expression or activity modulator of the present invention is useful for inhibiting or promoting angiogenesis .

The composition of the present invention may contain at least one known active ingredient having an effect of inhibiting or promoting angiogenesis together with an agent for modulating the expression or activity of human FGF 11.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the patient's weight, age, , Diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the human FGF 11 expression or activity regulator is about 0.0001 to 100 mg / kg, preferably about 0.001 to 10 mg / kg, and is preferably administered once a day or several times a day.

The composition of the present invention can be used alone or in combination with methods for the treatment or prophylaxis of angiogenesis or using surgery, hormone therapy, drug therapy and biological response modifiers.

In addition, the present invention provides an information providing method for screening an angiogenesis regulating agent, comprising the steps of measuring the expression or activity level of human FGF 11 after treating a candidate substance in a biological sample, and measuring the expression or activity level of human FGF 11 in a biological sample When the expression or activity level of human FGF 11 in the biological sample treated with the candidate substance is lower than the expression or activity level of human FGF 11 in the biological sample not treated with the candidate substance, as compared to the expression or activity level of FGF 11 The candidate substance is judged to be an angiogenesis inhibitor or the expression or activity level of human FGF 11 in the biological sample treated with the candidate substance is higher than the expression or activity level of human FGF 11 in the biological sample not treated with the candidate substance , The candidate substance is judged to be an angiogenesis promoter, so that an angiogenesis inhibitor or an accelerator can be easily It can be screened.

The expression or activity of human FGF 11 can be assayed intracellularly or in vitro.

Such biological samples include, but are not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine.

The expression or activity level of human FGF 11 may be determined by PCR, RT-PCR, competitive RT-PCR, real-time RT-PCR, , RNase protection assay (RPA), Northern blotting, DNA chip, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, But not limited to, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, Immunoprecipitation Assay, Complement Fixation Assay and Protein Chip, and the like. Do not.

In addition, the method for inhibiting angiogenesis of the present invention can effectively inhibit angiogenesis by administering to a subject a human FGF 11 expression or activity inhibitor.

In addition, the method of promoting angiogenesis of the present invention can effectively promote angiogenesis by administering to a subject the expression or activity promoter of human FGF 11.

The subjects were selected from the group consisting of canine, feline, wild boar and animal, cow animal, deer and animal, giraffe and animal, fecal and animal, camel and animal, hippopotamus, horse and animal, rhinoceros, weasel, rabbit, rodent and primate But are not limited thereto.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1: hypoxia Under the conditions In human endothelial cells, Of FGF11 Expression measurement

In order to confirm the expression level of human FGF11 mRNA and protein in human vascular endothelial cells under hypoxic condition, the following experiment was conducted.

Example 1-1. Cell culture and culture under hypoxic condition

Human vascular endothelial cells (HUVECs) (passage 5-8; Lonza) were incubated with 20% FBS (Lonza), bFGF (3 ng / ml, Invitrogen), heparin (5 U / ml, Gibco) and 1% penicillin / streptomycin Gibco) in M199 medium (Gibco). Human embryonic kidney 293 cells (HEK293a cells, ATCC CRL-1573) were cultured in DMEM medium (Gibco) containing 10% FBS.

For hypoxic condition composition, cells were incubated for 16 hours in a formala oxygen chamber (Forma Scientific) maintaining low oxygen partial pressure (1% O ₂ , 5% CO ₂ , balance with N ₂ ). The forma hypoxic chamber is an anaerobic system that regulates the hypoxic condition more tightly through a closed hypoxic workspace. The oxygen partial pressure in the normal oxygen state was 20% O ₂ .

Example 1-2. real - time PCR And end - point PCR

Total RNA was isolated using QIAshredder and RNeasyPlus Mini kits (Qiagen Inc.). The cDNA was synthesized from 1 μg of total RNA using the PrimeScript ™ 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer's instructions.

Real-time PCR was performed using SYBR Green PCR Master Mix (Roche) using primers of human FGF11 [forward, 5'-TGTCGCTTTAAGGAGTGCGT-3 'and reverse, 5'-AGAGAAGGCTCCCGGTACAT-3']. Real-time PCR data was obtained using an ABI PRISM-7500 sequencing system (Applied Biosystems). 18S rRNA gene was used as a positive control for standardization. End-point PCR was also performed to identify human FGF11. GAPDH was used as a control group for standardization. The oligonucleotide primers for PCR are as follows: human FGF11 [forward, 5'-GTCACCATCCAGAGTGCCAA-3 '; Reverse, 5'-CACTGTGGAGAGAAGGCTCC-3 ']; GAPDH [forward, 5'-CATGACAACTTTGGCATTGTG-3 '; Reverse, 5'-GTTGAAGTCGCAGGAGACAAC-3 ']. The PCR product was confirmed by electrophoresis on 1.2% agarose gel.

The degree of mRNA expression of human FGF11 in human vascular endothelial cells under hypoxic conditions is shown in FIGS. 1A and 1B. [(A) end-point PCR and (B) real-time PCR.

As shown in Figs. 1A and 1B, it was confirmed that mRNA expression of human FGF11 was increased in human vascular endothelial cells under hypoxic conditions.

Example 1-3. Plasmid Cloning , Transfection, and Western Blot analysis

Whole-length human FGF11 was synthesized by PCR and cloned into pcDNA3.1 / HA vector (Invitrogen). Transfection was performed using Metafectene Pro (Biontex). For Western blotting, the cells were harvested and lysed in lysis buffer containing the protease inhibitor (Roche). Total protein (20-30 μg) was immunoblotted with primary antibody specific for FGF11 (R & D). α-Tubulin (Calbiochem) was used as an internal control. The intensity of the band was quantified and standardized using Image-J (NIH).

The degree of protein expression of human FGF11 in human vascular endothelial cells under hypoxic conditions is shown in Fig.

As shown in Fig. 1C, it was confirmed that protein expression of human FGF11 was increased in human vascular endothelial cells under hypoxic condition.

As described above, human FGF11 is expected to function in hypoxia-mediated angiogenesis by increasing the expression of human FGF11 mRNA and protein in human vascular endothelial cells under hypoxic conditions.

Example 2: Human endothelial cells in human Of FGF11 Tube formation effect and cell migration confirmation

In order to confirm whether human FGF11 overexpression in human vascular endothelial cells is involved in angiogenesis essential for tumor formation, the following experiment was conducted.

Example 2-1. Tube formation Assay

Tube formation assay was performed by a known method.

Specifically, 200 μl of growth factor-reducing matrix (BD Biosciences) was applied to a 24-well plate and polymerized at 37 ° C for 30 minutes. Human vascular endothelial cells (1 x 10 ⁴ cells) transfected with pFGF11 or pMock were seeded onto the polymerized matrigel and cultured continuously for 16 hours in M199 medium containing 2% FBS and heparin (10 U / ml) Observations were made every hour. The cultures were observed and quantitated using an Olympus TH4-200 optical microscope (magnification: × 100).

The results are shown in Figures 2a and 2b.

As shown in FIG. 2A and FIG. 2B, when human FGF11 overexpression vector was transfected into human vascular endothelial cells, it was confirmed that human FGF11 protein was overexpressed. Overexpression of human FGF11 in human vascular endothelial cells transfected with pFGF11, And increased capillary-like tube formation significantly on the lipid compared to the control.

Example 2-2. Cell migration Assay

Human vascular endothelial cells were transfected with pFGF11 or pMock for 24 hours, and cells were plated on a 60 mm culture dish. After incubation until 90% saturation, cell migration assay was performed by a known method Respectively.

Specifically, human vascular endothelial cells were scratched with a razor blade and scraped off and then cultured in M199 medium containing 2% FBS and 1 mM thymidine. After 16 hours, human vascular endothelial cells were fixed with anhydrous methanol for 2 minutes, stained with Giemsa solution for 3 minutes, and then observed with an optical microscope. The mobile activity was quantified by counting the number of cells migrated beyond the baseline. BFGF (basic FGF) was used as a positive control.

The results are shown in Figure 2c.

As shown in FIG. 2C, human FGF11 overexpression in human vascular endothelial cells transfected with pFGF11 did not stimulate cell migration activity, and human vascular endothelial cells treated with bFGF as a positive control were found to migrate normally.

As described above, human FGF11 overexpression in human vascular endothelial cells transfected with pFGF11 increases capillary-like tube formation but does not affect cell migration activity, and thus human FGF11 is involved in stabilizing the vascular structure of blood vessels .

Example 3: Human endothelial cells Of FGF11 Expression TJ Effect on protein expression

In order to confirm whether the overexpression of human FGF11 in human vascular endothelial cells affects the expression of TJ (tight junction) protein, which is important for stabilizing the vascular structure, the following experiment was conducted.

Human vascular endothelial cells were transfected with pFGF11 or pMock for 48 hours and immunoblotted with primary antibodies specific for human FGF11 and TJ proteins occludin (Invitrogen), ZO-1 (Invitrogen), claudin-5 (Abcam) The expression of these proteins was observed and quantified.

As a result, as shown in FIG. 3, expression of occludin, ZO-1, and claudin-5, which are TJ proteins, in human vascular endothelial cells transfected with pFGF11 increased with overexpression of human FGF11. Thus, it is expected that overexpression of human FGF11 will be involved in stabilizing the vasculature structure.

Example 4: Human FGF11 Promoter Luciferase Assay

Since the expression of human FGF11 was increased under hypoxic conditions, the promoter region of human FGF11 was examined and HIF-1 (hypoxia-inducible factor-1), which is a typical transcription factor regulating gene expression in hypoxic conditions, HREs (hypoxia response elements, 5'-ACGTG-3 ') were present at two sites (-1254 to -1250, -695 to -691). HIF-1 regulates cell survival, metabolism, and transcription of genes that affect tumor angiogenesis, such as VEGF, erythropoietin, placental growth factor, and bFGF. Therefore, in order to confirm whether HIF-1 actually binds to the human FGF11 promoter, the following promoter luciferase assay was performed.

A partial sequence of a genomic DNA including a human FGF11 promoter region having a site predicted to be HRE was amplified by PCR and cloned into the luciferase pGL3 promoter vector (Promega). Here, the primer sequence for PCR is as follows: forward, 5'-CTGCTAGC CCAACCTCTCCTTCCTACC-3 '(pGL3-FGF11-HREs); Forward, 5'-GTGCTAGC GGGGCTGGTTAGATTGGAG-3 '(pGL3-FGF11-ΔHREs); And reverse, 5'-ATAGATCT ACTAGGGCATGCTCTTGACG-3 '.

Human embryonic kidney 293 cells (HEK293a) cells to 2 x 10 ⁵ cells / plated in 6-well plates at a density of wells, pCMV-β-gal, pGL3 -FGF11-HREs (WT) or deletion HRE--fragment of co-transfected with a full-length promoter sequence containing pGL3-FGF11-? HREs (? HRE) for 24 hours and then cultured for 16 hours under hypoxic conditions. Transfection with pGL3-FGF11-HREs (WT) or pGL3-FGF11-? HREs (? HRE), pCMV-? -Gal, pEGFP-HIF-1? And pEGFP-HIF-1? Plasmids under normal oxygen conditions . After transfection, reporter gene activity was performed according to the manufacturer's instructions, using Luciferase Assay System Kit (Promega) and a luminescence analyzer (GloMax luminometer, Promega). Relative luciferase activity was normalized to light unit and? -Galactosidase activity.

As a result, as shown in Fig. 4, the reporter gene activity under hypoxic condition was markedly increased for cells transfected with pGL3-FGF11-HREs (WT), but the HRE-deletion-fragment pGL3-FGF11-? HREs HRE) (Fig. 4B). In addition, when HIF-1 was overexpressed under normal oxygen conditions to test the expression of human FGF11 by HIF-1, the reporter gene activity was also significantly increased for cells transfected with pGL3-FGF11-HREs (WT) , But not for cells transfected with the HRE-deletion-fragment pGL3-FGF11-? HREs (? HRE) (Fig. 4C).

As described above, it can be seen that the HREs in the human FGF11 promoter region are sensitive to the hypoxic state, and the expression of human FGF11 can be induced through the HIF-1 binding site in its promoter region. Thus, it can be expected that human FGF11 may act as a modulator of hypoxia-guided pathological processes such as tumor progression or ischemic diseases.

Claims

A pharmaceutical composition for the treatment of ischemic diseases comprising an expression promoter for human fibroblast growth factor (FGF) 11,
Wherein the expression promoter is HIF-1 (hypoxia-inducible factor-1) protein, and the ischemic disease is at least one selected from the group consisting of stroke, peripheral vascular disease caused by arteriosclerosis, and myocardial infarction .

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