JPH05247097A - Blood vessel growth-depressing factor - Google Patents

Abstract

PURPOSE:To provide a factor depressing the growth of blood vessels, the wandering of the blood vessels, etc. CONSTITUTION:The blood vessel growth-depressing factor is contained in the supernatant of the cultured solution of dendritic cells originated from artheral synovial membrane and has the following properties: (1) a mol.wt. of 0.5-10kd, (2) unstable against thermal treatments at >=56 deg.C, and (3) the depression of the growth and wandering of vascular endothelial cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vascular growth inhibitory factor. More specifically, the present invention relates to a growth-suppressing factor having a growth-suppressing action on vascular endothelial cells and useful as a medicine, a clinical test reagent, or the like.

[0002]

2. Description of the Related Art It is known that various growth factors are contained in living tissues, organs, body fluids, etc., and many growth factors have been isolated. These growth factors show various actions such as cell proliferation, hypertrophy and elongation, and regulation of expression of differentiation function. The factor having a vascular endothelial cell growth action among these growth factors is called an endothelial cell growth factor. Significant neovascularization, often observed in certain immune responses and tumorigenesis, the formation of new capillaries, is a hallmark of synovitis and synovial superficial cell hyperplasia in rheumatoid arthritis. Proliferative synovium composed of heterogeneous cell populations such as fibroblast-like cells, macrophage-like cells, and dendritic cells is composed of interleukin 1, interleukin 6, prostaglandin E ₂ , granulocyte-macrophage colony activating factor, and collagenase. It spontaneously produces various joint destructive substances. It is possible that the proliferation of synovial cells that precedes pannus formation depends not only on cell division and the aforementioned chemical substances in a paracrine or autocrine mechanism, but also on new capillaries. There is also.

Few reports have been made on the mechanism of angiogenesis control in inflamed rheumatoid synovium and the identification of its vascular growth inhibitory factor. Low molecular weight (20
0-300) several vascular growth factors, such as tumor vascular growth factor-like substances, transforming growth factor beta, platelet-derived growth factor, acidic and basic fibroblast growth factor, in osteoarthritis and rheumatoid arthritis. It has been detected in the synovial fluid collected from patients, and some reports have been made on its activity and action.

Leibovich et al. Found vascular growth factor activity derived from rheumatoid synovial macrophage-like cells [Koch et al., "Arthritis and rheumatism"].
29 p. 471-479 (1986) (Koch,
A. E. , P .; J. Polverini, and
S. J. Leibovich, 1986, St
imitation of neovasculari
zation by human rheumatoi
d synovial tissue macroph
ages, Arthritis Rheum. , 29:
471-479) and Koch et al., “Rheumatology Magazine” 1
5 p. 1058-1063 (1988) (Koch,
A. E. , P .; J. Polverini, and
S. J. Leibovich, 1988, Func
regional heterogeneity of h
uman rheumatoid synovial
issue macrophages, J. et al. Rhe
um. , 15: 1058-1063. )].

Further, Melnik et al. Reported that basic fibroblast growth factor was present in the culture supernatant of synovial cells derived from not only rheumatoid arthritis but also patients with osteoarthritis and trauma. [Mernik et al., "Arthritis and rheumatism" 33
p. 493-500 (1990) (Melnyk,
V. O. , G. P. Shipley, M .; D. S
ternfield, L .; Sherman, and
J. T. Rosebaum, 1990, Synovi
ocytes synthesize, bind an
d respond to basic fibrob
last growt factor, Arthr
itis Rheum. , 33: 493-50
0. )]

On the other hand, as a blood vessel growth suppressing factor for suppressing blood vessel growth, a cartilage-derived molecular weight of 16,000
Metalloproteinase inhibitors (such as Langer,
"Science" 193 p. 70-73 (1976) (La
nger. R. , H .; Bren, K .; Fal
terman, M .; Klein, and J.K. F
olkman, 1976, Isolation of
a carrage factor thatt
inhibit tumor neovascular
ization, Science, 193: 70-
73). ], A bovine vitreous-derived protein having a molecular weight of 6200 [Raymond et al., "Experimental Ophthalmology Research" 3
4 p. 267-286 (1982) (Raymon)
d. L. , And B. Jacobson, 198
2, Isolation and identifier
Cation of stimulatory and
inhibitory cell grow f
actors in bovine vitreous,
Exp. Eye Res. , 34: 267-28
6)], protamine sulfate (molecular weight 2000 to 1200)
0) and the like have been reported as factors having an inhibitory action, but have not yet been put to practical use.

[0007] Further, no research has been conducted so far on the vascular growth inhibitory factor and its activity in rheumatoid arthritis, which is involved in the control mechanism of angiogenesis. By culturing cloned rheumatoid arthritis synovial cells, the inventor has already found that heparin-free, thermostable, molecular weight of about 67 k derived from fibroblast-like cells and macrophage-like cells.
The vascular growth factor of d has been isolated. This time, the present inventor has conducted extensive studies on synovial cells derived from rheumatoid arthritis, and as a result of culturing dendritic cells cloned from synovial cells, proliferates vascular endothelial cells in the culture supernatant. It was found that a suppressor having a suppressive effect exists, and it was successfully isolated.

[0008]

The problem to be solved by the present invention is to provide a factor for suppressing the proliferation of blood vessels and the migration of blood vessels.

[0009]

In order to solve the above-mentioned problems, the present invention has a molecular weight of 0.5 to 10 kd from a culture supernatant of articular synovium-derived dendritic cells, and has a molecular weight of 56 ° C or higher. It is intended to isolate and provide a suppressor which is unstable to heat treatment and has a property of suppressing vascular proliferation and migration. The vascular growth inhibitory factor of the present invention exerts an action of inhibiting cell proliferation and migration properties such as hypertrophy and elongation based on its physiological activity. Inhibitors of the present invention, arthritis synovial cells, preferably during the culture of dendritic cells isolated from cloned synovial cells obtained from rheumatoid arthritis, chronic osteoarthritis, meniscus damage, etc., preferably It is contained in the supernatant during the culture by the serum-free culture method and can be obtained by isolating it.

The above-mentioned synovial cells can be preferably obtained from the knee joint of a rheumatoid arthritis patient. For example, synovial cells obtained at the time of surgery are isolated by trypsin treatment, then cultured by the limiting dilution method and cloned. From the cloned synovial cells, in order to obtain the suppressor of the present invention, a supernatant of dendritic cell-like cells is collected, and the collected supernatant is used in a medium usually used in a cell culture method, such as HAMF-12,
Culture with RPMI 1640 or the like, or serum-free medium. In particular, serum components such as fetal bovine serum, etc.
%, Preferably 4 to 10%, and optionally an antibiotic, for example, penicillin about 100 U / ml and / or streptomycin about 100 μg / ml, or a serum-free medium is preferably used. The culturing conditions are those that are employed in ordinary cell culture and are not particularly limited.
C. to 38.degree. C., preferably 36.degree. To 37.degree. C., and carbon dioxide concentration of 1 to 10%, preferably 3 to 6%.

The dendritic cell supernatant is concentrated by means such as ultrafiltration and ammonium sulfate salting out, if necessary, and then separated and purified by gel filtration, affinity chromatography, ion exchange chromatography and the like. By attaching to
It is also possible to obtain the inhibitor of the present invention. The suppressor of the present invention has the following properties. 1) Molecular weight: 0.5 to 10 kd 2) Unstable at temperatures above 56 degrees Celsius. That is, it partially decomposes in 20 to 30 minutes under heating conditions of 56 to 60 degrees Celsius. 3) Inhibits proliferation and migration of vascular endothelial cells. That is, the vascular growth inhibitor of the present invention is contained in the supernatant during the culture of dendritic cells isolated from cloned synovial cells obtained from rheumatoid arthritis, osteoarthritis, meniscus injury, etc. Is characterized by being isolated.

The vascular growth inhibitor of the present invention acts particularly effectively in the presence of vascular cell growth factor. The suppressor of the present invention acts as a growth suppressor on vascular endothelial cells and can be used as a therapeutic or diagnostic agent for rheumatoid arthritis, solid tumors and the like. In general, diseases such as cancer, inflammation, and diabetic retinopathy are exacerbated by vascular proliferation. However, administration of the factor of the present invention to these patients is useful as a therapeutic drug, and the suppression of vascular proliferation It is also useful for diagnosis. The activity of the vascular growth inhibitory factor was confirmed by measuring vascular proliferation activity both in vivo and in vitro using human umbilical vein endothelial cells and chorioallantoic membrane vascular proliferation system of fertilized chicken eggs. These results suggest that in rheumatoid synovium, the angiogenesis that precedes pannus formation may be controlled in part by the synovial cells themselves in a paracrine fashion.

The growth inhibitory factor of the present invention has a vascular endothelial cell growth inhibitory activity (hereinafter referred to as ECGSF (Endothelial).
Cell Growth Suppressor F
Actor) activity. ) Can be measured by a known activity measuring method, for example, a sample is placed between the blood vessels of the chorioallantoic membrane of a fertilized egg of a chicken, and the capillaries induced in an axle shape around the sample are measured. Method (CAM method), a method in which a polymer pellet containing a sample is embedded in the cornea of a rabbit to measure the formation of capillaries, an air bag is made under the skin of a mouse, and the sample is administered there to Examples include methods that stimulate new birth [Taylor et al., "Natural" 2
97 p. 307 (1982); Folkman et al., "Science" 221 p. 719 (1983); Zimbron et al., "National Cancer Institute Journal" 52 p. 413 (197)
4); Farnier et al., "Ophthalmic Visual Studies" 21 p.
351 (1981); Langer et al., "Nature" 263.
p. 797 (1976); Murray et al., "In vitro" 19
p. 743 (1983) (S. Taylor et al.
l. , Nature, 297: 307, 198.
2. J. Folkman et al. , Scien
ce, 221: 719, 1983. M. A. Gi
mbrone et al. , J. Natl. Can
cer Inst. , 52: 413, 1974.
G. A. Fournier et al. , Inve
st. Ophthalmol. VisualSci. ，
21: 351, 1981. R. Langer et
al. , Nature, 263: 797, 19
76. J. B. Murray et al. , In
Vitro, 19: 743, 1983) and
Mitsui et al., "Japanese clinical practice" 44 p. 169, (1986)
Etc.].

More preferably, a method of measuring the proliferation of HUVEC using human umbilical vein vascular endothelial cells (HUVEC) with the amount of ³ H-thymidine incorporation as an index can be mentioned. [Suga et al., "Cell Physiology Magazine" 111 p. 155
(1982) (M. Kan et al., J. Cel.
l. Psysiol. , 111: 155, 198
2)]

[0015]

【Example】

A. Preparation and cloning of synovial cells Nine patients (7 male, 2 female; age 39-60 years) with clear rheumatoid arthritis symptoms were used in the study.
All patients received non-steroidal anti-inflammatory drugs and low doses (up to 5 mg / day) of prednisolone. The synovial cells used in this study were obtained from the knee joint during surgical synovectomy, and were treated as follows. That is, synovial cells were
The cells were treated with 25% trypsin at 37 ° C for 40 minutes, and the released cells were collected. The cells were washed 3 times with PBS, and HAM F-12 supplemented with 10% fetal bovine serum (FBS), 5 × 10 ⁻⁵ M2-mercaptoethanol, penicillin (100 U / ml), and streptomycin (100 μg / ml).
It was suspended in a medium (manufactured by GIBCO). The limiting dilution method was then used to clone the synovial cells thus obtained.

B. Preparation of Serum-Free Culture Supernatant Cloned synovial cells were analyzed for dendritic cell group (DC _S ), macrophage-like cell group (MC _S ) by morphological characteristics, cell surface antigen, cytohistochemical staining and functional characterization.
_S ) Fibroblast-like cell group (FC _S ). When the cloned DC became confluent in a 96-well flat bottom culture plate, the cells were washed twice with PBS, the medium was replaced with serum-free HAM F-12 medium, and after culturing for 48 hours, the culture supernatant was recovered. Then, centrifugation was performed at 3000 rpm for 10 minutes. The collected supernatant is Centricon-10
(Centricon-10, manufactured by Amicon) was concentrated 10 times and stored at -20 ° C until use.
When the cloned dendritic cells become confluent in the well, the approximate number of cells is about 200 to 50.
It was 0. No contamination of mycoplasma or endotoxin was observed in the culture supernatant.

C. Human umbilical vein endothelial cells (HUVEC)
Adjustment of HUVEC is a method in which the method of Juffe is slightly modified [Hashimoto et al., "Pediatric Study" 20 p. 9
43-946 (1986) (Hashimoto Y.,
S. Yoshinoya. T. Aikawa,
T. Mitamura, Y .; Miyashi,
M. Muranaka, T .; Miyamoto
and T.C. Kawasaki. 1986. En
Hangedendothelial cell pr
oliification in a cute Kawa
saki disease, Pediatr. Re
s. , 20: 943-946)] and cultured. That is, the healthy and fresh human umbilical vein vascular lumen was supplemented with 0.2% collagenase (Worshington) in PBS.
on Biochemical Co., Ltd.)
Digested for a minute. HUVECs were collected and washed 3 times with PBS.

HUVEC consisted of 20% FBS, L-glutamine (2 mM), streptomycin (100 μg / m).
1), penicillin (200 U / ml), porcine heparin (90 μg / ml, manufactured by Sigma), endothelial cell growth additive (ECGS.20 μg / ml.Collab)
orative Res. Manufactured by M199
Propagated in the medium and not passaged too many times (passage number IV
~ V). HUVEC became confluent in 3 to 5 days when the medium was replaced every 3 days.
This HUVEC was identified by its characteristic growth pattern and positive staining with rabbit anti-human factor VIII antiserum.

D. In vitro measurement of vascular proliferation inhibitory activity (1) Proliferation inhibitory assay Confluent HUVEC monolayer cultured cells were treated with 0.
The cells were suspended and recovered by treatment with a mixture of 05% trypsin and 0.53 mM EDTA at 37 ° C for 5 minutes. After washing three times with serum-free PBS, each well of a 96-well culture plate was suspended in M199 medium containing 10% FBS.
Dispense UVEC at a rate of 10 ⁴ cells / 0.1 ml,
Cultured for 5 hours. 100 μl of serum-free HAM F-12 medium or a test sample was added (as a result, the FBS concentration was 5%), and the mixture was cultured at 37 ° C. for 3 days. Two were randomly selected from the culture supernatant of each cloned synovial dendritic cell derived from 7 patients and used as a test sample (final concentration: 50%). Serum-free HAM F-12 medium incubated for 3 days under the same conditions as the culture of cloned cells was used as a negative control. In addition, ECGS (1
0 μg / ml) and recombinant human basic fibroblast growth factor (rh-bFGF) (Mallinckeo)
(manufactured by dt) was used as a positive control.

[ ³ H] -Thymidine (New Engla
nd Nuclear. 1 μCi / 10 μl / wel
1) was added and incubated for 4 hours. After removing the medium and washing twice with cold PBS, the cells were peeled off by treating with trypsin EDTA solution at 37 ° C. for 10 minutes, collected on a glass fiber filter, and incorporated [ ³ H] -thymidine was measured by a scintillation counter. .. HU
Since the proliferative potential of VEC varies greatly depending on the umbilical cord sample used, the present inventor throughout the present invention showed the results of the experiment only when two different HUVECs were used in each experiment and they showed similar tendencies. Adopted. The HUVEC growth inhibition assay results were normalized as stimulation index (SI) instead of actual cpm to facilitate comparison with results obtained from different experiments. Each experiment was performed three times. The average cpm value taken up by HUVEC cultured with an equal amount of serum-free HAM F-12 and M199 supplemented with 10% FBS (negative control: the final concentration of FBS was 5%) was expressed as 1.

That is, SI was calculated by dividing the average cpm value of each test sample by the average cpm value obtained from the negative control. For example, in a typical experimental example, the cpm value in the negative control was 307 ± 223 SD. The Student's t-test was used for the significance test. The results are shown in Table 1.

[Table 1] In Table 1, the results are shown by stimulation index, and the average value of three measurements was adopted. The stimulation index (SI) was calculated by the following formula. SI = (average amount of [ ³ H] uptake by HUVEC cultured by adding the culture supernatant of the indicated cloned cells)
/ (Average [ ³ H] uptake by HUVEC cultured with negative control medium)

As is clear from Table 1, when the culture supernatant was added so that the final concentration was 50%, there was a significant difference compared with the control (control example). The culture supernatant of fibroblast-like cells (FC) and macrophage-like cells (MC) significantly promoted HUVEC proliferation (FC:
P <0.001, MC: P <0.01). FC and MC
In contrast to the culture supernatant of C. dendritic cells, the culture supernatant of dendritic cells (DC) always significantly suppressed the proliferation of HUVEC (P <0.
0001). The activities of the culture supernatants of FC and DC were both concentration-dependent, and the activity of DC culture supernatant and TGFβ ₁ (1
(0 ng / ml) was also confirmed to significantly suppress the proliferative response elicited by the culture supernatant of autologous and non-autologous FCs.

(2) Chemotaxis inhibition assay For the chemotaxis inhibition assay, a gelatin-coated polycarbonate filter (pore size 5 μm, polyvinylpyrrolidone-free, manufactured by Nclepore) was used.
Was used in a 48-well modified Boyden chamber. A test sample (final concentration 12.5%) diluted with medium M199 containing 1% FBS was placed in the lower part of the chamber, and medium M19 containing 1% FBS was added.
9. Suspended 1 × 10 ⁵ HUVECs were sprinkled on the upper part. After culturing at 37 ° C. for 4 hours, the cells remaining on the surface of the filter were scraped off with a rubber policeman. Fix the filter, and use Diff-Qui
ck, manufactured by Harleco), and measured with an oil immersion microscope.

All measurements were performed 4 times each. The number of HUVECs that had migrated to the bottom side of the membrane was counted in 4 selected visual fields and expressed as the number of cells / mm ² . ECGS (5 mg / ml), transforming growth factor beta (0.5-50 ng / ml) (King J
manufactured by yozo). Serum-free HAM F-12 medium incubated for 3 days under the same conditions as the culture of cloned cells used in the experiment was used. The results are shown in Table 2.

[Table 2] As is clear from Table 2, the chemotaxis of HUVEC was determined by the culture supernatant of cloned FC and TGFβ ₁ (0.5 to 50 ng).
/ Ml), the culture supernatant of cloned DC and rh-bFGF (0.5 to 50 ng / m).
l) HAM F-12 medium alone had no effect.
H promoted by the culture supernatant of cloned FC
UVEC chemotaxis is a cloned D derived from self and non-self
It was significantly suppressed by the culture supernatant of C. It was also confirmed that the activity showed concentration dependency.

E. In vivo angiogenesis inhibition assay (1) Chicken fertilized egg chorioallantoic membrane In vivo angiogenesis is based on chicken fertilized chorioallantoic membrane (CA).
M) using the customary method of Knightton [Knighton et al., "British Cancer Magazine" 35 p. 347-3
56 (1977) (Knightton. D., D. A.
usprunk, D.I. Trapper and J.M. F
olkman, 1977, Avacularand
Vascular phases of tumor
grow in the check embr
yo, Br. J. Cancer. 35: 347-35
6. )] Was evaluated by a slightly improved method.

SPF (condition that does not infect a specific pathogen)
The fertilized chicken eggs produced under the conditions of 1. were purchased from Funabashi Farm (Chiba, Japan). Desalted sample solution 500
μl sterilized Thermanox
15-mm disk (Flow Laborato
(manufactured by Ries, Inc.) and dried under conditions where laminar flow was maintained. The disk containing the sample is 9 through the "window"
It was inserted into the CAM surface of a day-old embryo and used for the experiment. 2 after transplant
After 4, 48 and 72 hours, the reaction was positive or negative (for example, the presence or absence of "wheel-spoke" pattern having two or more new blood vessel loops), and the dilution ratio and survival of the sample. It was recorded as the number of positive angiogenic reactions per egg count.

Significant neovascularization was observed with FC supernatant under a light microscope. On the other hand, a fraction of the culture supernatant of synovial membrane FC,
When combined with the synovial DC culture supernatant,
No typical angiogenic response was observed when injected with AMF-12 medium alone. The following facts were confirmed in the angiogenesis assay using CAM. (A) When only the HAM F-12 medium was applied to the disc, a negative reaction was observed in CAM. (B) In the culture supernatant fraction of the cloned synovial membrane FC, it was confirmed that the newly formed capillaries extended from the medium-sized vessels toward the sample disc. (C) The culture supernatant fraction of cloned synovial membrane FC and DC
As a result of testing in combination, the formation reaction of capillaries was suppressed and was negative.

F. Measurement of Thermostability and Molecular Weight of Vascular Growth Inhibitory Factor The culture supernatant of DC was heated in a 63 ° C. water bath for 30 minutes. The culture supernatant of DC was passed through Centricon-10, and the passing fraction (molecular weight <10000) was further
It was passed through YC-05 (manufactured by Amicon). 50-fold concentrated culture supernatant (500 <molecular weight <10000)
It was dialyzed against 199 medium and adjusted to the same volume as the beginning.

G. Partial characterization of vascular growth factor The activity of the culture supernatant of DC was heat treated (56 ° C, 30 minutes).
Was unstable. The blood vessel growth inhibitory activity fractionated by the combination of Amicon membranes was detected in the fraction having a molecular weight of 500 to 10,000.

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Claims (1)

[Claims] 1. A vascular proliferation inhibitor, which is contained in the culture supernatant of synovial membrane-derived dendritic cells and has the following properties. (1) It has a molecular weight of 0.5 to 10 kd. (2) It is unstable with respect to heat treatment of 56 degrees Celsius or more. (3) Inhibits proliferation and migration of vascular endothelial cells.