JPWO2009044787A1 - Tendon-modulating agent containing tenomodulin as an active ingredient - Google Patents

Abstract

The present inventors have shown that inhibition of the expression or function of tenomodulin causes diseases related to rupture such as CTC. The tenomodulin protein or a substance that activates the expression or function of the protein is expected to have a therapeutic effect on tendon ruptured diseases.

Description

  The present invention relates to a therapeutic agent for tendon ruptured diseases using tenomodulin as an active ingredient, and a screening method for a therapeutic agent for tendon ruptured diseases using the expression of tenomodulin as an index.

  The heart valve and chordae tendineae cordis (CTC) are avascular tissues. Their avascularity is lost in some valvular heart diseases (VHDs) (see Non-Patent Documents 7 and 8). Chondromodulin-I (see Non-Patent Documents 9 and 10) is known to maintain normal valvular function by inhibiting angiogenesis (see Non-Patent Document 11).

  However, despite the clinical importance of heart disease, little is known about the mechanisms underlying heart valve disease.

  Atrioventricular leaflets and CTCs contain a variety of cell lines and highly organized substrates in the presence of cartilage (see Non-Patent Documents 15 and 16) and tendon (see Non-Patent Documents 17 and 18) cell types. The chondrocyte markers aggrecan and Sox9 are observed in the leaflet leaflets during embryogenesis, while the tendon-related genes Scleraxis and Tenascin are expressed in CTC It is known (see Non-Patent Document 19).

  Tenomodulin (Tem) is a 317 amino acid glycoprotein present in low blood vessel tissues including tendons, ligaments, outer myocardium and eyes (see Non-Patent Documents 1, 2, and 12), and chondromodulin-I And BRICHOS and a plurality of cysteine-rich domains (see Non-Patent Document 20). Tenomodulin is processed as efficiently as chondromodulin-I in several tissues in vivo, and the proteolytically cleaved 16 kD C-terminal domain is known to exhibit tendon cell proliferative activity (See Non-Patent Documents 3 and 4).

  However, details of the expression and function of tenomodulin were not yet known. The following patent documents or non-patent documents have been reported as related documents of the present invention.

WO 2007/034753 Chisa Shukunami et al., `` Molecular Cloning of tenomodulin, a Novel Chondromodulin-I Related Gene. '', Biochem.Biophys.Res.Commun., (2001) Vol.280, p.1323-1327 Yusuke Oshima et al., `` Expression and Localization of Tenomidulin, a Transmembrane Type Chondromodulin-I-Related Angiogenesis Inhibitor, in Mouse Eyes. '', Invest. Ophthalmol. Vis. Sci., (2003) Vol. 44, No. 5, p. .1814-1823 Yusuke Oshima et al., `` Anti-angiogenic action of the C-terminal domain of tenomodulin that shares homology with chondromodulin-I. '', J. Cell Science, (2004) Vol.117, p.2731-2744 Denitsa Docheva et al., `` Tenomodulin Is Necessary for Tenocyte Proliferation and Tendon Maturation. '', Mol. Cell. Biol., (2005) Vol. 25, No. 2, p. 699-705 Chisa Shukunami et al., `` Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes. '', Dev. Biol., (2006) Vol.298, p.234-247 Chisa Shukunami and Yuji Hiraki, “Chondromodulin-I and Tenomodulin: The Negative Control of Angiogenesis in Connective Tissue.”, Current Pharmaceutical Design, (2007) Vol.13, p.2101-2112 Soini, Y. et al., `` Angiogenesis is involved in the pathogenesis of nonrheumatic aortic valve stenosis. '', Hum.Pathol., (2003) Vol.34, p.756-763 Yamauchi, R. et al., `` Upregulation of SR-PSOX / CXCL16 and recruitment of CD8 + T cells in cardiac valves during inflammatory valvular heart disease. '', Arterioscler.Thromb.Vasc.Biol., (2004) Vol.24, p. 282-287 Hiraki, K. et al., `` Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes. '', Biochem.Biophys.Res.Commun., (1991) Vol.175, p. .971-977 Hiraki, Y. et al., `` Identification of chondromodulin I as a novel endothelial cell growth inhibitor.Purification and its localization in the avascular zone of epiphyseal cartilage. '', J. Biol. Chem., (1997) Vol.272, p. 32419-32426. Yoshioka, M. et al., “Chondromodulin-I maintaining cardiac valvular function by preventing angiogenesis.”, Nat. Med., (2006) Vol. 12, p.1151-1159 Shukunami, C. et al., `` Chondromodulin-I and tenomodulin: a new class of tissue-specific angiogenesis inhibitors found in hypovascular connective tissues. '', Biochem.Biophys.Res.Commun., (2005) Vol.333, p.299 -307 Millington-sanders, C. et al., `` Structure of chordae tenidineae in the left ventricle of the human heart. '', J. Anat., (1998) Vol.192, p.573-581 Hiraki, Y. et al., `` Inhibition of DNA synthesis and tube morphogenesis of cultured vascular endothelial cells by chondromodulin-I. '', FEBS Lett., (1997) Vol.415, p.321-324 Chimal-Monroy, J. et al., `` Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: Sox genes and BMP signaling. '', Dev. Biol., (2003) Vol.257, p.205-217 Lincoln, J. et al., `` Development of heart valve leaflets and supporting apparatus in chicken and mouse embryos. '', Dev.Dyn., (2004) Vol.230, p.239-250 Chiquet, M. and Fambrough, D.M., `` Chick myotendinous antigen: A monoclonal antibody as a marker for tendon and muscle morphogenesis. '', J. Cell. Biol., (1984) Vol. 98, p.1926-1936 Schweitzer, R. et al., `` Analysis of the tendon cell fate using scleraxis, a specific marker for tendons and ligaments. '', Development., (2001) Vol.128, p.3855-3866 Lincoln, J. et al., `` BMP and FGF regulatory pathways control cell lineage diversification of heart valve precursor cells. '', Dev. Biol., (2006) Vol.292, p.290-302 Hiraki, Y. and Shukunami, C., "Angiogenesis inhibitors localized in hypovascular mesenchymal tissues: chondromodulin-I and tenomodulin.", Connect.Tissue.Res., (2005) Vol.46, p.3-11 Lin, T.H. et al., `` Association of hypertenssion and primary mitral chordae tendineae rupture. '', Am.J.Hypertens., (2000) Vol.19, p.75-79 Jzsa, L.G. and Kannus, P., "Human tendons-anatomy, physiology, and pathology." Human kinetics, Champaign, III, USA. Movin, T. et al., "Tendon pathology in long standing achillodynia.Biopsy findings in 40 patients.", Acta.Orthop.Scand., (1997) Vol.68, p.170-175 Kannus, P. and Jozsa, L.G., `` Histopathological changes preceding spontaneous rupture of a tendon.A control study of 981 patients. '', J.Bone.Joint.Surg., (1991) Vol.73, p.1507-1525 Pufe, T. et al., `` The angiogenic peptide vascular endothelial growth factor is expressed in foetal and ruptured tendons. '', Virchows.Arch., (2001) Vol.439, p.579-585 Pufe, T. et al., `` The role of vasculature and angiogenesis for the pathogenesis of degenerative tendons disease. '', Scand.J.Med.Sci.Sports., (2005) Vol.15, p.211-222 Tallon, C. et al., `` Ruptured Achilles tendons are significantly more degenerated than tendinopathic tendons. '', Med.Sci.Sports.Exerc., (2001) Vol.33, p.1983-1990 Funaki, H. et al., `` Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye. '', Invest. Ophthalmol. Vis. Sci., (2001) Vol. 42, p. 1193-1200 Fujimoto, N. and Iwata, K., `` Use of EIA to measure MMPs and TIMPs. '', Methods Mol. Biol., (2001) Vol. 151, p.347-358 Zacks, S. et al., “Characterization of Cobblestone mitral valve interstitial cells.”, Arch.Pathol.Lab.Med., (1991) Vol.115, p.774-779 Pufe, T. et al., `` Mechanical factors influence the expression of endostatin-an inhibitor of angiogenesis- in tendons. '', J. Orthop. Res., (2003) Vol. 21, p.610-616

  An object of this invention is to clarify the onset mechanism of the tendon tearing disease in a chord or the like. More specifically, it is an object of the present invention to provide a therapeutic agent for tendon ruptured diseases and to provide a method for efficiently screening the therapeutic agent.

The present inventors have intensively studied to solve the above problems.
Based on the above observations (see Non-Patent Documents 15 to 19) and our previous knowledge on Chondromodulin-I (Chm-I) (see Non-Patent Document 11), the present inventors It was speculated to be involved in avascularity.

  The inventors examined whether tenomodulin is expressed in CTC, and in that case, whether the secreted form of tenomodulin has anti-angiogenic activity. We also investigated whether the absence of tenomodulin could induce abnormal angiogenesis and inflammatory cell infiltration, as well as expression of matrix metalloproteinases (MMPs), leading to CTC rupture.

  Specifically, we investigated the molecular mechanism underlying CTC's avascular basis and its relationship to valvular heart disease. In the autopsy specimen, CTC elastin-rich submucosal layer is rich in tenomodulin (see Non-Patent Documents 1, 2, and 12), which is one of chondromodulin-I-related anti-angiogenic factors (see Non-Patent Document 13) It was found to be expressed in. The conditioned medium of cultured CTC interstitial cells strongly inhibited tube formation and endothelial cell mobilization (see Non-Patent Documents 11 and 14). These effects were partially inhibited by the small interfering RNA (siRNA) of tenomodulin. Interestingly, tenomodulin was found to be locally lacking in the rupture area of patients with CTC rupture. In addition, a number of abnormal angiogenesis and strong expression of VEGF-A and matrix metalloproteinases were observed in the region where tenomodulin was down-regulated, but not in the non-ruptured region. When the tenomodulin layer of canine CTC was surgically removed, angiogenesis and VEGF-A and MMPs expression were gradually induced in the core layer. In addition, investigations into the causes of downregulation of tenomodulin in CTC interstitial cells have shown that various stimuli such as mechanical stretch, hypoxia, and oxidative stress cause downregulation of tenomodulin expression. It was.

  As noted above, we present for the first time evidence that tenomodulin plays an important role in maintaining normal function of CTC by inhibiting angiogenesis and that its local lack can lead to CTC rupture. Succeeded.

  That is, it is considered that the inhibition of the expression or function of tenomodulin results in many abnormal angiogenesis, and as a result, ruptured diseases such as tendons including CTC are caused. Therefore, the tenomodulin protein itself or a substance that activates the expression or function of the protein is expected to have an effective preventive and therapeutic effect on tendon tearing diseases.

  Also, as described above, the present inventors have found downregulation of tenomodulin in the rupture region of patients with CTC rupture. That is, it is possible to screen for therapeutic agents (candidate compounds) for tendon tearing diseases by using the expression level or activity of tenomodulin as an index.

  In addition, various findings obtained by the present invention provide new insights into the mechanism of onset of tendon tearing diseases. Understanding these mechanisms is extremely important in developing new therapeutic means for tendon tearing diseases, and the present invention greatly contributes from an academic point of view.

That is, the present invention relates to a therapeutic agent for tendon tearing diseases, and a method for efficiently screening the therapeutic agent, more specifically,
[1] A tendon tear-preventing agent containing any of the following (a) to (c) as an active ingredient,
(A) a tenomodulin protein (b) a protein functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the tenomodulin protein (c) DNA encoding the protein according to a) or (b)
[2] The tendon tear-preventing agent according to [1], which has a tendon tear-preventing action on a chord
[3] A therapeutic agent for tendon ruptured diseases comprising any of the following (a) to (c) as an active ingredient,
(A) a tenomodulin protein (b) a protein functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the tenomodulin protein (c) DNA encoding the protein according to a) or (b)
[4] A therapeutic agent for tendon ruptured diseases, containing a tennomodulin protein expression activator or function activator as an active ingredient,
[5] Tendon ruptured diseases include valvular heart disease, Achilles tendon rupture, patella tendon rupture, quadriceps tendon rupture, supraspinatus tendon rupture, subscapular tendon rupture, biceps long-head tendon rupture A therapeutic agent for tendon ruptured diseases according to [3] or [4], which is a disease selected from the group consisting of flexor tendon rupture and finger extensor tendon rupture,
[6] The drug according to any one of [1] to [5], wherein the tenomodulin protein is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2.
[7] A screening method for a therapeutic agent for tendon ruptured disease, which comprises selecting a compound that activates the expression or function of tenomodulin protein,
[8] A screening method for a therapeutic agent for tendon ruptured disease, comprising the following steps (a) to (c):
(A) contacting a test compound with a cell expressing the tenomodulin protein, (b) measuring the expression level of the tenomodulin protein in the cell, and (c) measuring in the absence of the test compound; A step of selecting a compound that increases the expression level in comparison [9] a method for screening a therapeutic agent for tendon ruptured disease, comprising the following steps (a) to (c):
(A) a step of contacting a test compound with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of a tenomodulin gene and a reporter gene are functionally linked; and (b) measuring the expression level of the reporter gene. (C) a step of selecting a compound that increases the expression level as compared with the case where it is measured in the absence of the test compound [10] a tendon including the following steps (a) to (c) Screening method for therapeutic agents for ruptured diseases,
(A) a step of contacting a test compound with a tenomodulin protein, or a cell or cell extract expressing the protein, (b) a step of measuring the activity of the protein (c) a measurement in the absence of the test compound A step of selecting a compound that increases the activity of the protein as compared with the above [11] Tendon tear disease is valvular heart disease, Achilles tendon tear, patella tendon tear, quadriceps tendon tear, supraspinatus tendon Any one of [7] to [10], wherein the disease is selected from the group consisting of rupture, subscapular tendon rupture, biceps long head tendon rupture, finger flexor tendon rupture, and finger extensor tendon rupture The screening method described in 1. is provided.

Furthermore, the present invention provides
[12] A method for preventing tendon rupture or a method for treating tendon ruptured disease, comprising a step of administering any of the following (a) to (c) to an individual (patient etc.):
(A) a tenomodulin protein (b) a protein functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the tenomodulin protein (c) DNA encoding the protein according to a) or (b)
[13] A method for treating tendon ruptured disease, comprising a step of administering an expression activator or function activator of tenomodulin protein to an individual (patient etc.),
[14] Use of any of the following (a) to (c) in the manufacture of a tendon tear-preventing agent or a tendon-ruptured disease therapeutic agent,
(A) a tenomodulin protein (b) a protein functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the tenomodulin protein (c) DNA encoding the protein according to a) or (b)
[15] Use of a tenomodulin protein expression activator or function activator in the manufacture of a therapeutic agent for tendon tearing diseases,
[16] A substance according to any one of the following (a) to (c) for use in a method for preventing tendon rupture or a method for treating a tendon ruptured disease:
(A) a tenomodulin protein (b) a protein functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the tenomodulin protein (c) DNA encoding the protein according to a) or (b)
[17] A tenomodulin protein expression activator or a function activator for use in a method for treating tendon ruptured diseases,
I will provide a.

FIG. 2 is a photograph showing the temporal and spatial expression of Tem in rodent (mouse), pig, and human hearts. (a) A photograph showing the temporal expression of Tem in the mouse heart. RT-PCR was performed on embryonic and adult hearts. A positive signal was seen after the E14.5 stage of the embryonic heart. Adult mouse eyes were used as positive controls. (b) Photograph of RT-PCR for Tem in pig heart. Tem mRNA was expressed specifically in the chordae (CTC), but not in the atrium, ventricle or valve. (c) It is a photograph showing the results of Western blot analysis using an antibody for detecting the C-terminal (upper figure) and N-terminal (middle figure) of Tem regarding porcine heart valve membrane and Tem in CTC. The eye was used as a positive control. The eyes expressed only 45 and 40 kD bands, whereas CTC expressed not only 45 and 40 kD bands, but also 16 kD bands. This suggested that in CTC, the C-terminus was processed but not in the eye. (d) Histological structure and immunohistochemistry of human CTC. HE and EVG represent hematoxylin-eosin staining and elastica-Wangeson staining, respectively. In the figure, TEM represents tenomodulin, CHM-I represents chondromodulin I, vWF represents von Willebrand factor, and VEGF-A represents vascular endothelial growth factor. FIG. 2 is a photograph and figure showing the expression of Tem in CTC interstitial cells (CTCs) and its effect on tube formation and migration in human coronary endothelial cells (HCAECs). (a) Photograph of rabbit CICs after culture after in vitro transplantation. On day 7, CICs showed a cobblestone-like (Cb) or spindle-like (Sp) appearance. On day 14, CICs had a fibroblast-like appearance. Ex: CTC explant. CICs were negative for acetyl-LDL-DiI staining; HCAECs are shown as positive controls (inset). Shown is Tem immunofluorescence staining for rabbit CICs and NIH3T3 cells; nuclei were stained with ToPro-3. (b) Photograph showing the effect of siRNA on Tem expression in cultured CICs. RT-PCR was performed on Tem. Lane 1, CIC; Lane 2, CIC + Tem specific siRNA; Lane 3, CIC + control siRNA; Lane 4, Tem as positive control for Tem. (c) Photograph showing tube formation analysis results. CIC-CM inhibited endothelial cell tube formation on Matrigel. Representative micrographs for tube formation of HCAECs are shown. CM is a conditioned medium. Tube formation was significantly suppressed by CIC-CM, but not by NIH3T3-CM (negative control). The suppression induced by CIC-CM was not reduced by control siRNA treatment, but was reduced by Tem-specific siRNA treatment of CICs. Recombinant C-terminal Tem represents the conditioned medium of NIH3T3 cells transfected with a C-terminal Tem expression plasmid. (d) A photograph showing the results of cell migration analysis. In vitro, CICs lost HCAEC chemotaxis. HCAECs migration was inhibited by co-culture with CICs, but not when cultured without cells (control) or with NIH3T3 cells (negative control). Although control siRNA treatment did not change, chemotaxis suppression was reduced by Tem-specific siRNA treatment of CICs. Recombinant C-terminal Tem represents co-culture with NIH3T3 cells transfected with a C-terminal Tem expression plasmid. (e) It is a graph which shows the quantitative analysis result regarding the length of the pipe | tube in a tube formation analysis. (f) It is a graph which shows the quantitative analysis result regarding the number of cells which migrated in cell migration analysis. * P <0.01 vs control; NS, not significant. (a) Representative histology and immunohistochemistry pictures for ruptured CTCs. Abbreviations in the figure are the same as in FIG. The ruptured region of CTC did not express TEM, but strongly expressed VEGF-A. Many abnormally large blood vessels were prominent in the ruptured area of CTC, which were covered with vWF ⁺ endothelial cells. EVG staining and immunostaining for elastin and type I collagen revealed that the layered structure was maintained in the CTC tear region despite being destroyed. (b) It is a graph which shows the quantitative analysis result about the number of cells in normal, non-ruptured area (NRA), and ruptured area (RA) of decay CTC. (c) It is a graph which shows the quantitative analysis result about vWF ⁺ cell number. (d) It is a graph which shows the quantitative analysis result about the ratio of TEM ⁺ in normal, NRA, or RA of CTC. (e) It is a graph which shows the quantitative analysis result about the ratio of VEGF-A ⁺ in normal, NRA or RA. * P <0.01 vs control; NS, not significant. (a) The above figure is a photograph of CTC in the tricuspid valve (septal leaflet) as seen by the surgeon in a dog model. The following figure is a picture of CTC with the surface layer and middle layer removed. (b) Photographs showing the results of HE staining for CTC after 1 month and 3 months. At 3 months, abnormally small blood vessels were observed. (c) It is a photograph which shows the result of immunohistochemistry about vWF on CTC. Abnormal blood vessels at 3 months were covered with vWF ⁺ endothelial cells. (d) It is a proof photograph of the immunohistochemistry about Vegf-A, Mmp1, Mmp2, and Mmp13. (e) It is a graph which shows the quantitative analysis result about the depth of the Vegf-A positive part compared with the depth (depth) of a core layer. (f) It is a graph which shows the quantitative analysis result about the depth of the Mmp1 positive part compared with the depth (depth) of the core layer. (g) It is a graph which shows the quantitative analysis result of the number of abnormal blood vessels. * P <0.01, ** P <0.01 vs control (sham surgery, 0 months). It is a photograph which shows the result of hematoxylin-eosin (HE) dyeing | staining about the Tem and Vegf-A in a mouse | mouth heart, and Elastica-Wangeson (EVG) dyeing | staining and an immuno-staining. Analysis of a series of sections indicated that Tem was confined to the extracellular matrix of CTC, not the myocardium (papillary muscle) or the heart valve. In contrast, Vegf-A expression was restricted to cardiomyocytes, not heart valve or CTC. MV: Mitral valve, PM: Papillary muscle. (a) Use anti-MMP-1, -MMP-2, -MMP-3, -MMP-9, -MMP-13, -CD11b, -CD14 and vimentin antibodies for human autopsy and surgical sample-derived CTC It is the photograph which shows the result of the immunohistochemistry which had. Normal represents a normal autopsy sample of CTC. Chordae rupture refers to the ruptured area of CTC from a surgical sample. (b) It is a graph which shows the quantitative analysis result about the ratio of the MMP-1 immuno-staining area | region in the non-rupture area | region and a rupture area | region of a normal autopsy sample and a surgical CTC specimen. (c) It is a graph which shows the quantitative analysis result about the ratio of a MMP-2 immuno-staining area | region. (d) It is a graph which shows the quantitative analysis result about the ratio of an MMP-13 immunostaining area | region. (e) It is a graph which shows the quantitative analysis result about the number of CD14 + cells in CTC. * P <0.01 vs control; NS, not significant. CTC interstitial cells were treated with several stimuli and RT-PCR for the tenomodulin gene was performed. (A) A photograph showing the effect of mechanical extension (extension degree 25%) on the expression of tenomodulin in CTC interstitial cells. After 6 hours, no tenomodulin expression was detected. (B) A photograph showing the effect of hypoxia on the expression of tenomodulin in CTC interstitial cells. (C) Photograph showing the effect of antimycin A mediated oxidative stress on tenomodulin expression in CTC interstitial cells. Tenomodulin expression was detected at antimycin A concentrations below 10 μg / ml, but not at antimycin A concentrations of 30 μg / ml.

Hereinafter, the present invention will be described in detail.
The present inventors have shown that inhibition of the expression or function of tenomodulin causes diseases related to rupture such as CTC. Therefore, the tenomodulin protein itself or a substance that activates the expression or function of the protein is expected to have an effective preventive and therapeutic effect on tendon ruptured diseases.

Connective tissue is one of the four major basic tissues that constitute a living body together with epithelial tissue, muscle tissue, and nerve tissue. It consists of cell components (connective tissue cells) derived from mesenchymal cells and intercellular substances surrounding these cells. Intercellular substances are composed of fibrous components (connective tissue fibers) and amorphous substrates. There are three types of fibrous components: collagen fibers, elastic fibers, and reticulofibers. A fiber component (mainly collagen fibers) having a particularly large amount is called a fibrous connective tissue.
Fibrous connective tissue is classified into tough connective tissue in which collagen runs densely and loose connective tissue in which irregularly runs. The tough connective tissue is a columnar, laced, or membranous structure, and is present in tendons, ligaments, fascia, sclera, cornea, and the like. In tough connective tissue, vegetative blood vessels are observed in parallel with fiber travel, but the number is extremely small. On the other hand, loose connective tissue is rich in vascular network and widely distributed throughout the body as connective tissue under the skin, submucosa and interlobule.
The “tendon” in the present invention refers to a tendon in the tough connective tissue, and is a tissue through skeletal muscle and bone. For example, chords (CTC), aponeurosis, periosteum, tendon sheath and the like are also included in the tendons in the present invention.

First, the present inventors provide a therapeutic agent for tendon ruptured diseases containing a tenomodulin protein as an active ingredient.
The tenomodulin protein of the present invention is preferably a human tenomodulin protein, but the species from which it is derived is not particularly limited, and is a protein equivalent to tenomodulin in non-human organisms (such as human tenomodulin homologs and orthologs). ) Is also included in the “tenomodulin protein” in the present invention.
As a homolog of tenomodulin, for example, chondromodulin-I which is an angiogenic factor is known.

For example, the present invention can be implemented as long as it has a tendon tissue and has a protein equivalent to human tenomodulin.
For example, the amino acid sequence of human tenomodulin protein is shown in SEQ ID NO: 2, and the base sequence of DNA encoding the amino acid sequence (tenomodulin gene) is shown in SEQ ID NO: 1.
Examples of organisms other than humans (accession number AF234259) having a protein corresponding to tenomodulin include, for example, mouse (accession number NM_022322, AF219993), rat (accession number NM_022290, AF191769), pig (accession number NM_001099934). EF503637), horses (accession numbers NM_001081822, AB059407), chickens (NM_206985, AY156693) and the like.

  Even proteins other than the above have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the sequences described in the sequence listing of the present application. And the protein which has the function (for example, tendon tear inhibiting activity etc.) which tenomodulin has is contained in tenomodulin of this invention.

  In the above protein, “amino acid sequence in which a plurality of amino acids are deleted, substituted, or added” refers to, for example, one or more amino acids added, deleted, substituted, or inserted in the amino acid sequence set forth in SEQ ID NO: 2. A protein consisting of an amino acid sequence, wherein the number of normally changing amino acids is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.

The “tenomodulin gene” in the present invention includes, for example, endogenous genes in other organisms (eg, homologs of human tenomodulin gene) corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1.
Moreover, the endogenous DNA of other organisms corresponding to the DNA consisting of the base sequence described in SEQ ID NO: 1 generally has high homology with the DNA described in SEQ ID NO: 1. High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, or 96%, 97%, 98% or 99% or more). ) Homology. This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7 ) Can be determined. Further, when the DNA is isolated from a living body, it is considered to hybridize with the DNA of SEQ ID NO: 1 under stringent conditions. Here, as “stringent conditions”, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C.” and “0.2 × SSC, 0.1% SDS, 65 ° C.” Can do. Those skilled in the art can appropriately obtain an endogenous gene corresponding to the tenomodulin gene in other organisms based on the base sequence of the tenomodulin gene. In the present specification, a protein (gene) corresponding to a tenomodulin protein (gene) in a non-human organism or a protein (gene) functionally equivalent to the above tenomodulin is simply referred to as “tenomodulin protein ( Gene) ”or“ Tem ”.

  The “tenomodulin protein” of the present invention can be prepared not only as a natural protein but also as a recombinant protein using a gene recombination technique. Natural proteins can be prepared by, for example, a method using affinity chromatography using an antibody against a tenomodulin protein to an extract of a cell (tissue) considered to express the tenomodulin protein. On the other hand, the recombinant protein can be prepared by culturing cells transformed with DNA encoding the tenomodulin protein. The “tenomodulin protein” of the present invention is suitably used, for example, as a control protein in the screening methods described below, for example.

In the present invention, “expression” includes “transcription” from a gene or “translation” into a polypeptide and “degradation inhibition” of a protein. “Expression of tenomodulin protein” means that transcription and translation of a gene encoding the tenomodulin protein occurs, or that the tenomodulin protein is generated by transcription and translation thereof. Examples of the “function of tenomodulin protein” include a function of suppressing angiogenesis, a function of suppressing (preventing) tendon rupture, and a function of promoting proliferation of tendon cells (Non-patent Document 4). .
The various functions described above can be appropriately evaluated (measured) by those skilled in the art using general techniques. Specifically, it can be carried out with reference to literatures and the like described regarding the various functions described above.
For example, the tendon modulin protein has a tendon rupture inhibitory activity, although it is not particularly limited, by appropriately evaluating (1) tissue structure analysis in tendon tissue, (2) immunohistochemical analysis in tendon tissue, etc. Can be measured.

  The present invention relates to a therapeutic agent for tendon ruptured diseases, which comprises a tenomodulin protein or a variant of the protein (modified, homolog of other organisms, etc.), which contains a protein functionally equivalent to tenomodulin. provide.

That is, as a preferable aspect of the present invention, it relates to a therapeutic agent for tendon ruptured diseases containing any one of the following substances (a) to (c) as an active ingredient.
(A) Tenomodulin protein (b) Functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (SEQ ID NO: 2) of the tenomodulin protein Protein (c) DNA encoding the protein described in (a) or (b) above

  The “tenomodulin protein” which is a component of the drug of the present invention can be prepared as a recombinant protein using a gene recombination technique in addition to a natural protein. Natural proteins can be prepared by, for example, a method using affinity chromatography using an antibody against a tenomodulin protein to an extract of a cell (tissue) considered to express the tenomodulin protein. On the other hand, the recombinant protein can be prepared by culturing cells transformed with DNA encoding the tenomodulin protein.

  Further, DNA encoding a tenomodulin protein which is a component of the drug of the present invention is also included in the present invention. The DNA encoding the tenomodulin protein of the present invention may be chromosomal DNA or cDNA. Chromosomal DNA encoding a tenomodulin protein can be obtained, for example, by preparing a library of chromosomal DNA from cells or the like and screening the library using a probe that hybridizes to DNA encoding the tenomodulin protein. it can. The cDNA encoding the tenomodulin protein is an RT-PCR using primers that hybridize to the DNA encoding the tenomodulin protein by extracting an RNA sample from cells (tissues) that are thought to express the tenomodulin protein. It can be obtained by gene amplification techniques such as the law.

  The tenomodulin protein of the present invention or the DNA encoding the protein may be a mutant having a modified base sequence or amino sequence as long as it is functionally equivalent to the tenomodulin protein. Such mutants may be natural or artificial. Methods for artificially preparing mutants are known to those skilled in the art. For example, Kunkel method (Kunkel, TA et al., Methods Enzymol. 154, 367-382 (1987)), double primer method (Zoller, MJ and Smith, M., Methods Enzymol. 154, 329-350 (1987)) , Cassette mutation method (Wells, et al., Gene 34, 315-23 (1985)), megaprimer method (Sarkar, G. and Sommer, SS, Biotechniques 8, 404-407 (1990)) Yes.

  The agent of the present invention may comprise a component expressing the DNA described in (c) above or the vector expressing the protein described in (a) or (b) above. That is, the present invention relates to a therapeutic agent for tendon ruptured diseases, comprising as an active ingredient the DNA described in (c) above or a vector capable of expressing the DNA.

  The DNA is preferably functionally linked to a promoter for efficient expression. As the promoter used in the present invention, the promoter of the original tenomodulin gene can be used, but besides this, various known promoters such as cytomegalovirus (CMV) promoter, SV40 promoter, actin promoter, metallothionein A promoter, heat shock promoter, SRα promoter, or the like can be used. Moreover, those skilled in the art can easily prepare a vector for expressing the protein of the present invention using various known expression vectors.

  The above vector of the present invention can also be used for gene therapy. Gene therapy refers to administration or treatment or prevention of a vector containing a DNA encoding a functional protein to a patient. Examples of vectors that can be used for gene therapy include, but are not limited to, adenovirus vectors (eg, pAdex1cw) and retrovirus vectors (eg, pZIPneo). General genetic manipulations such as insertion of the DNA encoding the protein of the present invention into a vector can be performed according to conventional methods. In vivo administration may be carried out by ex vivo method, but in vivo method is preferred.

In addition, by activating the expression or function of the tenomodulin protein of the present invention, tendon rupture is prevented or suppressed, and as a result, a prophylactic effect and a therapeutic effect on diseases related to tendon rupture are expected.
Therefore, the present invention provides a therapeutic agent for tendon ruptured diseases comprising a tenomodulin protein expression activator or function activator as an active ingredient.

  In the present invention, the “tendon tearing disease” refers to a disease related to tendon tearing. For example, the chords, Achilles tendon, patella tendon, quadriceps tendon, supraspinatus tendon, shoulder connected to the heart valve Mention may be made of tendon ruptured diseases in the inferior tendon, the long biceps tendon, the flexor tendon of the finger, the extensor tendon of the finger, and the like. Examples of tendon rupture diseases of the present invention include valvular heart disease such as mitral regurgitation, aortic regurgitation, Achilles tendon rupture, patella tendon rupture, quadriceps tendon rupture, supraspinatus tendon rupture, scapula Examples include lower tendon rupture, biceps long head tendon rupture, finger flexor tendon rupture, or finger extensor tendon rupture.

The “protein expression activator” in the present invention is a substance that significantly activates (increases) protein expression. The above “expression activation” of the present invention includes transcription activation of a gene encoding the protein and / or translation activation from a transcription product of the gene.
Examples of the substance that activates the expression of the tenomodulin protein of the present invention include substances that bind to the transcriptional regulatory region (eg, promoter region) of the tenomodulin gene and promote the transcription of the gene (such as a transcriptional activator). be able to.
In the present invention, measurement of the expression activity of the tenomodulin gene can be easily carried out by those skilled in the art by methods well known to the art, for example, Northern blotting, Western blotting and the like.

The “protein functional activator” in the present invention is a substance that significantly activates (increases) the function of the protein. It has been observed by the inventors that the tenomodulin protein is down-regulated, for example, in the area of tendon rupture. Therefore, examples of the function activating substance of the present invention include substances that enhance the tear-suppressing action of tenomodulin protein in tendon tissue.
In addition, a substance that activates the expression or function of tenomodulin protein has an action of preventing or suppressing tendon rupture (for example, tendon rupture in CTC). Therefore, the present invention provides a tendon rupture preventive agent (tendon rupture inhibitor or tendon rupture inhibitor) containing any of the above-mentioned (a) to (c) as an active ingredient.
The above-mentioned tendon tear-preventing agent of the present invention is preferably characterized by having a tendon tear-preventing action in a chord, a aponeurosis, a peritoneum, a tendon sheath and the like.

The “therapeutic agent” of the present invention can also be expressed as “medicine”, “pharmaceutical composition”, “therapeutic drug”, and the like.
In addition, since the “treatment” in the present invention includes a preventive effect that can suppress the occurrence of a disease in advance, the therapeutic agent of the present invention can also be expressed as a “prophylactic agent”. In addition, the present invention is not necessarily limited to the case of having a complete therapeutic effect, and the case of having a partial effect, the case where symptoms are improved, and the like are also included in the meaning of the “treatment” of the present invention.

  The drug of the present invention (tendon tear prevention agent, tendon tear disease treatment agent, etc.) is mixed with a physiologically acceptable carrier, excipient, diluent, or the like, and orally or parenterally as a pharmaceutical composition. Can be administered. Oral preparations include granules, powders, powders, powders, fine granules, tablets, coated tablets, pills, pellets, film coatings, soft / hard capsules, sublinguals, troches, pastes, syrups The dosage form may be an agent, a solvent, an emulsion, or a suspension.

  As the parenteral preparation, a dosage form such as an injection, an instillation, an external medicine (external preparation), or a suppository can be selected. Examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. Examples of external medicines include nasal administration agents, transdermal agents or ointments, plasters, eye drops, nasal drops, ear drops, poultices, lotions and the like. The preparation technology for making the above-mentioned dosage form so as to include the drug of the present invention as the main component is known.

  For example, a tablet for oral administration can be produced by adding an excipient, a disintegrant, a binder, a lubricant, and the like to the drug of the present invention, mixing, and compressing and shaping. As the excipient, lactose, starch, mannitol or the like is generally used. As the disintegrant, calcium carbonate, carboxymethyl cellulose calcium and the like are generally used. As the binder, gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used. As the lubricant, talc, magnesium stearate and the like are known.

  The tablet containing the drug of the present invention can be coated with a known coating for masking or enteric preparation. As the coating agent, ethyl cellulose, polyoxyethylene glycol, or the like can be used.

  An injection can be obtained by dissolving the drug of the present invention as a main component together with an appropriate dispersant, or dissolving or dispersing in a dispersion medium. Depending on the selection of the dispersion medium, either a water-based solvent or an oil-based solvent can be used. In order to use an aqueous solvent, distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium. In the oil solvent, various vegetable oils and propylene glycol are used as a dispersion medium. At this time, a preservative such as paraben may be added as necessary. In addition, known isotonic agents such as sodium chloride and glucose can be added to the injection. Further, a soothing agent such as benzalkonium chloride or procaine hydrochloride can be added.

  Moreover, it can be set as an external preparation by making the chemical | medical agent of this invention into a solid, liquid, or semi-solid composition. About a solid or liquid composition, it can be set as an external preparation by setting it as the composition similar to what was described previously. A semi-solid composition can be prepared by adding a thickener to an appropriate solvent as required. As the solvent, water, ethyl alcohol, polyethylene glycol, or the like can be used. As the thickener, bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone is generally used. A preservative such as benzalkonium chloride can be added to the composition. Also, a suppository can be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as a cellulose derivative as a carrier.

  When the agent of the present invention is used as a gene therapy agent, a method of administering a vector incorporating a nucleic acid in addition to a method of directly administering the agent of the present invention by injection can be mentioned. Examples of the vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, and the like, and can be efficiently administered by using these virus vectors.

  It is also possible to introduce the drug of the present invention into a phospholipid vesicle such as a liposome and administer the vesicle. That is, the endoplasmic reticulum holding the drug of the present invention is introduced into predetermined cells by the lipofection method. Then, the obtained cells are systemically administered, for example, intravenously or intraarterially. It can also be administered locally to tendon tissue or the like.

  The necessary amount (effective amount) of the drug of the present invention is administered to animals including humans within the safe dose range. The dosage of the drug of the present invention can be appropriately determined finally based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.

The present invention also provides a screening method for the agent of the present invention (tendon tear prevention agent or tendon tear disease treatment agent) using the expression level of the tenomodulin gene as an index.
A substance that increases (enhances) the expression level of the tenomodulin gene is expected to be a drug of the present invention. By the screening method of the present invention, a candidate compound for a tendon tear-preventing agent or a tendon-ruptured disease therapeutic agent can be efficiently obtained.

A preferred embodiment of the method of the present invention is a screening method for the drug of the present invention (tendon tear prevention agent or therapeutic agent for tendon tearing disease) comprising the following steps (a) to (c).
(A) contacting a test compound with a cell expressing the tenomodulin protein, (b) measuring the expression level of the tenomodulin protein in the cell, and (c) measuring in the absence of the test compound; Step of selecting a compound that increases the expression level in comparison

In the above method of the present invention, first, a test compound is brought into contact with a cell expressing a tenomodulin protein (gene).
The “cell” used in the present method is not particularly limited, but is preferably a human-derived cell. As the “cell expressing the tenomodulin protein”, a cell expressing an endogenous tenomodulin protein or a cell into which an exogenous tenomodulin gene is introduced and expressing the gene can be used. A cell in which an exogenous tenomodulin gene is expressed can usually be prepared by introducing an expression vector into which a tenomodulin gene has been inserted into a host cell. The expression vector can be prepared by general genetic engineering techniques.

There is no restriction | limiting in particular as a test compound used for the screening method of this invention. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts Products, plant extracts and the like, but are not limited thereto.
In addition, these test compounds can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.

  “Contact” of a test compound to cells expressing the tenomodulin protein (gene) is usually performed by adding the test compound to the culture medium of cells expressing the tenomodulin protein, but is not limited to this method. . When the test compound is a protein or the like, “contact” can be performed by introducing a DNA vector expressing the protein into the cell.

Next, in this method, the expression level of the tenomodulin protein is measured. Here, “protein expression” includes both transcription and translation. The expression level can be measured by methods known to those skilled in the art.
For example, mRNA is extracted from cells expressing tenomodulin protein according to a standard method, and the transcription amount of the gene is measured by performing Northern hybridization method, RT-PCR method, DNA array method, etc. using this mRNA as a template. It can be carried out. Alternatively, the amount of translation of a gene can be measured by collecting a protein fraction from cells expressing the tenomodulin protein and detecting the expression of the tenomodulin protein by electrophoresis such as SDS-PAGE. Furthermore, the amount of translation of a gene can be measured by performing Western blotting using an antibody against the tenomodulin protein and detecting the expression of the gene encoding the protein. The antibody used for detecting the tenomodulin protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.

  Antibodies that bind to tenomodulin protein can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. A serum is obtained by immunizing a small animal such as a rabbit with a recombinant (recombinant) tenomodulin protein expressed in a microorganism such as Escherichia coli or a partial peptide thereof as a fusion protein with natural tenomodulin protein or GST. This is prepared by, for example, purification by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with tenomodulin protein or synthetic peptide, or the like. In the case of a monoclonal antibody, for example, a small animal such as a mouse is immunized with a tenomodulin protein or a partial peptide thereof, the spleen is removed from the mouse, and this is ground to separate the cells. Are fused using a reagent such as polyethylene glycol, and a clone that produces an antibody that binds to the tenomodulin protein is selected from the fused cells (hybridomas) formed thereby. Next, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, tenomodulin protein. Or by purification using an affinity column coupled with a synthetic peptide.

  In this method, a compound that increases the expression level of the tenomodulin protein is then selected as compared with the case where the test compound is not contacted (measured in the absence of the test compound). It is also possible to select (c) above by comparing with a control.

  The compound isolated by this method has an action of preventing or suppressing tendon rupture (for example, tendon rupture in a chord) and is expected to be a therapeutic agent for tendon ruptured diseases.

Another aspect of the screening method of the present invention is a method using the activity (function) of the tenomodulin protein as an index. The said method is a screening method of the chemical | medical agent of this invention including the process of the following (a)-(c), for example.
(A) a step of contacting a test compound with a tenomodulin protein, or a cell or cell extract expressing the protein, (b) a step of measuring the activity of the protein (c) a measurement in the absence of the test compound Selecting a compound that increases the activity of the protein compared to

In this method, first, a test compound is brought into contact with a tenomodulin protein or a cell expressing the protein or a cell extract.
The activity of the tenomodulin protein is then measured. Examples of the activity of the tenomodulin protein include the various activities (functions) described above. The measurement of the activity can be appropriately evaluated by those skilled in the art using a known method or referring to various documents cited in the present specification.
Further, a compound that increases (enhances) the activity of the protein is selected as compared with the case where the test compound is not contacted (control).

Another embodiment of the screening method of the present invention is a method of selecting a compound that increases the expression level of the tenomodulin protein (gene) of the present invention using reporter gene expression as an indicator.
A preferred embodiment of the above method of the present invention is a method for screening a drug of the present invention, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of a tenomodulin gene and a reporter gene are functionally linked; and (b) measuring the expression level of the reporter gene. (C) a step of selecting a compound that increases the expression level as compared with the case of measuring in the absence of the test compound

  In this method, first, a test compound is brought into contact with a cell or a cell extract containing DNA having a structure in which a transcriptional regulatory region of a tenomodulin gene and a reporter gene are functionally linked. Here, “functionally linked” means that the transcriptional regulatory region of the tenomodulin gene and the reporter gene are bound so that expression of the reporter gene is induced by binding of a transcription factor to the transcriptional regulatory region of the tenomodulin gene. It means doing. Therefore, even when the reporter gene is bound to another gene and forms a fusion protein with another gene product, the transcription factor binds to the transcriptional regulatory region of the tenomodulin gene, so that the fusion protein Any expression that is induced is included in the meaning of “functionally linked”. Based on the cDNA base sequence of the tenomodulin gene, those skilled in the art can obtain the transcriptional regulatory region of the tenomodulin gene present in the genome by a well-known method.

  The reporter gene used in this method is not particularly limited as long as its expression can be detected, and examples thereof include CAT gene, lacZ gene, luciferase gene, and GFP gene. Examples of the “cell containing DNA having a structure in which the transcriptional regulatory region of the tenomodulin gene and the reporter gene are functionally linked” include cells into which a vector having such a structure inserted is introduced. Such vectors can be prepared by methods well known to those skilled in the art. Introduction of the vector into the cells can be performed by a general method such as calcium phosphate precipitation, electric pulse perforation, lipofection, microinjection and the like. “Cells containing DNA having a structure in which a transcriptional regulatory region of a tenomodulin gene and a reporter gene are functionally linked” also include cells in which the structure is inserted into a chromosome. The DNA structure can be inserted into the chromosome by a method generally used by those skilled in the art, for example, a gene introduction method using homologous recombination.

  “A cell extract containing DNA having a structure in which a transcriptional regulatory region of a tenomodulin gene and a reporter gene are functionally linked” refers to, for example, a cell extract contained in a commercially available in vitro transcription / translation kit containing the tenomodulin gene. Examples include those to which DNA having a structure in which a transcriptional regulatory region and a reporter gene are functionally linked is added.

  “Contact” in this method means that the test compound is added to the culture solution of “the cell containing DNA having a structure in which the transcriptional regulatory region of the tenomodulin gene and the reporter gene are functionally linked” or the DNA containing the DNA. The test compound can be added to a commercially available cell extract. When the test compound is a protein, for example, it can be performed by introducing a DNA vector expressing the protein into the cell.

In this method, the expression level of the reporter gene is then measured. The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.
In this method, the compound that increases (enhances) the measured reporter gene expression level is then selected as compared with the case where the expression level of the reporter gene is measured in the absence of the test compound (control).

The present invention also provides a kit containing various drugs / reagents, etc., used for carrying out the screening method of the present invention.
The kit of the present invention can be appropriately selected from, for example, the above-described various reagents of the present invention according to the screening method to be performed. For example, the kit of the present invention recognizes (binds) an oligonucleotide such as a probe for a tenomodulin gene or a primer for amplifying any region of the gene, or a tenomodulin protein that can be used for detection of the tenomodulin protein. ) (Anti-tenomodulin protein antibody) or the like as a constituent element.

  For example, the oligonucleotide specifically hybridizes to the DNA of the tenomodulin gene of the present invention. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the condition described in the second edition 1989), it means that cross-hybridization does not occur significantly with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide does not need to be completely complementary to the base sequence of the tenomodulin gene.

  As hybridization conditions in the present invention, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C.” and “0.2 × SSC, 0.1% SDS, 65 ° C.” Can do. More specifically, as a method using Rapid-hyb buffer (Amersham Life Science), prehybridization is performed at 68 ° C for 30 minutes or more, and then a probe is added and kept at 68 ° C for 1 hour or more for hybridization. Then wash 3 times for 20 minutes at room temperature in 2 x SSC, 0.1% SDS, then 3 times for 20 minutes at 37 ° C in 1 x SSC, 0.1% SDS, and finally 1 x SSC Washing can be performed twice in 0.1% SDS at 50 ° C for 20 minutes. In addition, for example, prehybridization in Expresshyb Hybridization Solution (CLONTECH) for 30 minutes or more at 55 ° C, add labeled probe, and incubate at 37-55 ° C for 1 hour or more, in 2 × SSC, 0.1% SDS In addition, washing for 20 minutes at room temperature can be performed three times, and washing for 20 minutes at 37 ° C. in 1 × SSC and 0.1% SDS can be performed once. Here, for example, by setting the temperature at the time of pre-hybridization, hybridization, or the second washing higher, the conditions can be made more stringent. For example, the prehybridization and hybridization temperatures can be 60 ° C., and the stringent conditions can be 68 ° C. Those skilled in the art can set conditions by considering other conditions such as probe concentration, probe length, probe base sequence configuration, reaction time, etc. in addition to such conditions as buffer salt concentration and temperature. can do.

  The oligonucleotide can be used as a probe or primer in the screening kit of the present invention. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the DNA of the tenomodulin gene of the present invention.

  The kit of the present invention can further contain various reagents, containers and the like used in the method of the present invention. For example, various reaction reagents, cells, culture solution, control sample, buffer solution, instructions describing how to use, and the like can be appropriately included.

The present invention also relates to a tendon tear-preventing agent or a tendon-ruptured disease therapeutic agent comprising a step of mixing (combining) any of the following (a) to (c) with a pharmaceutically acceptable carrier or medium. It relates to a manufacturing method.
(A) Tenomodulin protein (for example, human tenomodulin protein represented by the amino acid sequence set forth in SEQ ID NO: 2)
(B) a tenomodulin protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the tenomodulin protein (for example, the amino acid sequence of the human tenomodulin protein described in SEQ ID NO: 2). A protein functionally equivalent to (c) a DNA encoding the protein according to (a) or (b)

  The “pharmaceutically acceptable carrier or vehicle” is a material that can be administered together with any of the above (a) to (c) and does not significantly inhibit the action of preventing tendon rupture. Examples of such carriers or media include deionized water, sterilized water, sodium chloride solution, dextrose solution, dextrose and sodium chloride, lactic acid-containing Ringer solution, culture solution, serum, phosphate buffered saline (PBS), and the like. It is conceivable to formulate these in combination with any of (a) to (c) as appropriate. Further, it may be concentrated by centrifugation or the like, if necessary, and resuspended in a physiological solution such as physiological saline. Moreover, the membrane stabilizer of liposome (for example, sterols, such as cholesterol), may be included. Further, an antioxidant (for example, tocopherol or vitamin E) may be contained. In addition, vegetable oils, suspending agents, surfactants, stabilizers, biocides and the like may be contained. Preservatives and other additives can also be added. The composition of the present invention may be in the form of an aqueous solution, capsule, suspension, syrup and the like.

  Furthermore, the present invention relates to a method for preventing or treating a tendon rupture-related disease, comprising the step of administering any of the above (a) to (c) to an individual (for example, a patient). The individual in the prevention or treatment method of the present invention is preferably a human, but is not particularly limited and may be a non-human animal. The dose of any one of (a) to (c) of the present invention varies depending on the disease, the patient's weight, age, sex, symptom, administration purpose, form of administration composition, administration method, etc. It can be determined as appropriate. The administration route can be appropriately selected, and can be performed, for example, transcutaneously, intranasally, transbronchially, intramuscularly, intraperitoneally, intravenously, intraarticularly, or subcutaneously. Administration can be local or systemic. In the case of administration for animals other than humans, for example, an amount obtained by converting the human dose by the body weight ratio between the target animal and the human or the volume ratio (for example, average value) of the administration target site can be administered.

  Alternatively, the present invention provides use of any of the substances (a) to (c) in the manufacture of a tendon tear-preventing agent or a tendon-ruptured disease therapeutic agent.

  Furthermore, this invention relates to the substance in any one of said (a)-(c) for using for the prevention method of a tendon tear or the treatment method of a tendon tearing disease.

  It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

-Animals Wild-type ICR mice were purchased from Japan CLEA (Tokyo, Japan). Japanese white rabbits were purchased from Sankyo Laboratory Service Corporation (Tokyo, Japan). All experimental methods and protocols were approved by the Committee on Animal Handling and Use at Keio University and were in compliance with NIH's guide on laboratory animal care and use.

[Example 1] Expression of tenomodulin over time It was examined whether tenomodulin (Tem) was expressed in chordae (CTC).
Total RNA was isolated using Trizol reagent (Gibco-BRL) and treated with DNaseI (Roche). RT-PCR was performed as previously described using the following primers (see Non-Patent Document 11):
Mouse Tem, 5′-AGAATGAGCAATGGGTGGTC-3 ′ (forward, SEQ ID NO: 3), 3′-CTCGACCTCCTTGGTAGCAG-5 ′ (reverse, SEQ ID NO: 4);
Mouse Gapdh, 5′-TTCAACGGCACAGTCAAGG-3 ′ (forward, SEQ ID NO: 5), 3′-CATGGACTGTGGTCATGAG-5 ′ (reverse, SEQ ID NO: 6);
Porcine Tem, 5′-GGTGGTCCCTCAAGTGAAAG-3 ′ (forward, SEQ ID NO: 7), 3′-CTCGTCCTCCTTGGTAGCAG-5 ′ (reverse, SEQ ID NO: 8);
Porcine Gapdh, 5′-TGATGACATCAAGAAGGTGGTGAAG-3 ′ (forward, SEQ ID NO: 9), 3′-TCCTTGGAGGCCATGTGGACCAT-5 ′ (reverse, SEQ ID NO: 10).

  As a result, the Tem transcript was first detected in the mouse heart at 14.5 days after embryogenesis (E14.5) and was steadily expressed in adults (FIG. 1a). In particular, it was expressed in CTC, but not in the atrium, ventricle, and heart valve (FIG. 1b).

[Example 2] Spatial expression of tenomodulin
Whether the secreted form of Tem has anti-angiogenic activity was examined.
Pig tissue (heart valve, CTC, and eye) lysis buffer (50 mM Tris (pH 7.4), 1 mM DTT, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 10 ml buffer Homogenized in 100 μl protease inhibitor cocktail (registered trademark) (Nacalai Tesque). Western blot analysis was performed as previously described (see Non-Patent Document 11). The loading amount of each sample was varied to visualize the Tem protein. Rabbit polyclonal antibodies against C-terminal and N-terminal Tem proteins were established by Dr. Docheva (see Non-Patent Document 4) and Shukunami (see Non-Patent Document 2), respectively. Membranes were incubated overnight at 4 ° C. with antibodies against Tem. Membranes were incubated with horseradish peroxidase (HRP) -conjugated antibody to rabbit IgG (Chemicon international, Inc.) and signals were visualized using SuperSignal West Pico (PIERCE) according to the supplier's instructions.

  As a result, Western blot analysis using an antibody specific for the C-terminal portion of Tem identified 45 kD glycosylated and 40 kD non-glycosylated Tem in porcine CTC (FIG. 1 c). These were also present in the eyes. Interestingly, the eye was not immunopositive, but CTC is immunopositive for Tem's C-terminal truncated 16 kD domain and, like Chm-I, the C-terminal is truncated to produce a secreted form. Was suggested.

Western blot analysis for the N-terminal Tem protein revealed 29 kD and 24 kD strong bands in addition to the 45 kD and 40 kD weak bands. In contrast, the eyes showed mainly 45 kD and 40 kD fragments.
Taken together, these results suggested that cleavage of the C-terminal protein of Tem occurred in a tissue-specific manner, and the cleaved N-terminal protein still exists in CTC.

[Example 3] Histological structure and immunohistochemistry of CTCs Pregnant or non-pregnant adult mouse hearts were perfused from the apex with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS, as previously described. (See Non-Patent Document 28). CTCs were dissociated and fixed overnight in 4% paraformaldehyde at 4 ° C. and then embedded in paraffin. Prior to using the primary antibody, the paraffin was removed from the sections in xylene and heated in a microwave oven in 10 mM citrate buffer solution (pH 6.0) (Muto Pure Chemicals Co., Japan) for 3 minutes. Sections were washed with PBS, then incubated with ImmunoBlock® (Dainippon Sumitomo Pharma, Osaka, Japan) for 1 hour at room temperature, overnight at 4 ° C., 5% normal rabbit serum, and rabbit polyclonal antibody against Tem ( Non-patent documents 2 and 4) and rabbit polyclonal antibodies against the following components (VEGF-A (1: 200 dilution; Santa Cruz Biotechnology), vWF (1: 200 dilution; Lab Vision Corporation), elastin (1:50 dilution) Elastin Products Co.), type I collagen (1:50 dilution; Rockland), MMP-1 (ref. 44; Daiichi fine chemical), MMP-2 (Diichi fine chemical) (see Non-Patent Document 29), MMP- 3 (Daiichi fine chemical) (see non-patent document 29), MMP-9 (Daiichi fine chemical) (see non-patent document 29), MMP-13 (1: 100 dilution; Biogenesis) (see non-patent document 29), CD11b (1:20 dilution; BD PharMingen), CD14 (1:50 dilution; Santa Cruz Biotechnology), and vimentin (1 : 20 dilution; Sigma-Aldrich)).

  The immunohistochemical signal was obtained by using 0.05% 3,3'-diaminobenzine tetrahydrochloride (Sigma-Aldrich) containing 0.01% hydrogen peroxide in 0.05 M Tris-buffered saline (pH 7.6) as a chromogenic substrate. Detected. Sections were then counterstained with hematoxylin-eosin, dehydrated in a graded ethanol series, and mounted in Permount (Fisher Scientific).

  For immunofluorescence studies, sections were incubated with secondary antibodies conjugated to Alexa488 or Alexa546 (Molecular Probes). The slide was observed with a confocal laser scanning microscope (LSM510META; Carl Zeiss). Optical sections were obtained at a resolution of 1024 × 1024 pixels and analyzed using LSM software (Carl Zeiss). As a negative control, non-immunized rabbit serum was used in place of the primary antibody in each immunostaining experiment.

In mouse hearts, it was determined by immunohistochemistry that Tem is located in CTC (FIG. 5).
In normal human CTC, hematoxylin-eosin (HE) staining, elastica-Wangyson (EVG) staining, and immunohistochemistry have three layers: the superficial endothelial layer, the elastin-rich middle layer, and the type I collagen-rich core layer. Became clear (FIG. 1d). This structure was consistent with previous findings (see Non-Patent Document 13). Tenomodulin (TEM) is limited to the middle layer and was not detected in the other layers. Normal CTC showed no CHM-1 or VEGF-A (vascular endothelial growth factor A) expression or abnormal angiogenesis. TEM did not co-localize directly with elastin, but was deposited in the interstitial space of the elastin-rich layer.

[Example 4] Expression of Tem in CTC interstitial cells (CTCs) Next, it was examined whether CTC interstitial cells (CICs) produce Tem.
The heart was isolated from an anesthetized 12-week-old Japanese white rabbit. The primary culture of CTC was performed by modifying the protocol for heart valve gap cells (see Non-Patent Document 30) and Achilles tendon (see Non-Patent Document 31). That is, CTCs were quickly taken out, and surface layer endothelial cells were taken using a cotton swab, minced under a stereoscopic microscope, and used for explant culture. A 1 × 1 mm fragment was cut from the tissue, placed in a collagen-coated 12-well dish (Iwaki) and grown in DMEM (Sigma-Aldrich) containing 50% calf serum at 37 ° C. for 24 hours. An additional 1 ml of DMEM containing 50% FBS was added and left at 37 ° C. for 24 hours. After removing the medium and tissue fragments, DMEM containing 10% FBS was added, and the cells were cultured at 37 ° C. The medium was changed every 3 days. Conditioned media for confluent CICs was obtained 3 days after media change and used for subsequent analysis.

  CICs or HCAECs were treated with 10 μg / ml acetylated apoprotein (Ac-LDL) and labeled with DiI (Molecular Probes) at 37 ° C. for 1 hour. Fluorescent cells were observed under a Nikon Diaphot microscope (Ex554 nm, Em571 nm).

  Primary CICs were obtained from rabbit CTC explant cultures (Figure 2a). The explanted cells formed an orthogonal pattern due to abnormal growth. These cells were negative for the acetyl-LDL-DiI complex, consistent with CTC consisting of CICs. On immunostaining, Tem was found in the cytoplasm of CICs and was not expressed at all in NIH3T3 cells.

  The effect of siRNA on Tem expression in cultured CICs was also verified. Small interfering RNA (siRNA) duplex (Tem-siRNA, 5'-UUUAUUGGAAGUUCUUCCUCACUUG-3 ', SEQ ID NO: 11) or control siRNA (5'-UUUCAAGGUUUGAAUUCUCUCCUUGUG-3', SEQ ID NO: 12) against rabbit Tem (Invitrogen) was used to transfect CICs grown to 90% confluence. Medium from these cultures 3 days after transfection was used as CM for subsequent experiments.

  RT-PCR confirmed that cultured CICs expressed Tem and that this expression could be inhibited by specific siRNA treatment (FIG. 2b).

[Example 5] Tube formation in human coronary endothelial cells (HCAECs)
We investigated how Tem production of CICs affects the vascular development of human coronary artery endothelial cells (HCAECs).
Human coronary artery endothelial cells (HCAECs) were purchased from Takara Biotechnology, maintained according to the manufacturer's instructions, and used in the present invention at passage 3 or 5.

Tube formation analysis was performed as follows.
24-well culture plates (Costar) were coated with growth factor-reduced matrigel (0.4 ml; Becton Dickinson Labware) and incubated at 37 ° C. for 30 minutes. HCAECs that had been starved for 4 hours were treated with trypsin-EDTA and suspended in culture medium for 20 minutes. With or without CM of CICs or NIH3T3 cells, cells were seeded on polymerized Matrigel at a density of 30,000 cells in each well, and tube formation analysis was performed as previously described (see Non-Patent Document 11). ). After incubation at 37 ° C. for 6 hours, photographs were taken under a phase contrast optical microscope (Carl Zeiss).

  As a result, HCAECs formed capillary-like tubes on Matrigel (Fig. 2c). After treatment with conditioned medium from CICs (CIC-CM), these structures became less pronounced compared to pseudo medium or NIH3T3 cell conditioned medium (NIH3T3-CM). HCAECs cultured with conditioned media of siRNA-treated CICs restored the ability to form capillary-like structures. Conditioned medium of NIH3T3 cells transfected with C-terminal Tem (recombinant protein) similarly inhibited tube formation.

  Quantitative assessment of the capillary-like morphology of HCAECs also measures the total length of the tube-like structure per field of view by image processing and analysis software (Zion imaging computer program; available from the National Institutes of Health public domain) Was done. Each experiment was performed 5 times.

  As a result of statistical analysis, values were expressed as mean ± SEM. Statistical significance for the comparison of two mean values was assessed using the unpaired Student's t test. Multiple comparisons between three or more groups were performed using ANOVA. A value of P <0.05 was considered significant.

  As a result, quantitative analysis showed that CIC-CM inhibited tube formation by 58.3%, and that tube formation ability was restored by 38.7% by treatment with Tem-specific siRNA (FIG. 2e).

[Example 6] HCAECs' migration ability
Using a modified Boyden chamber (Becton Dickinson Labware) having a filter insert with a pore size of 8 μm for a 24-well plate, infiltration analysis was performed by the method described previously (see Non-Patent Document 31). That is, rhVEGF-A was dissolved in EBM2 at a concentration of 20 ng / ml and placed in the lower chamber of the Boyden apparatus. NIH3T3 cells or CICs (1 × 10 ⁵ cells / well) were added to the lower chamber 48 hours before HCAECs (5 × 10 ⁴ cells / well) were planted in the upper chamber. After 12 hours of incubation, cells that remained attached to the upper side of the filter were removed with a cotton tip, and cells on the lower side of the filter were stained with hematoxylin and counted using a light microscope. The analysis was performed 5 times and the average of the results was obtained.

  As a result, Tem inhibited the migration ability of HCAECs (FIG. 2d). HCAECs co-cultured with CICs in the modified Boyden chamber lost their ability to migrate compared to NIH3T3 cells. Treatment of CICs with Tem-specific siRNA partially restored the ability of HCAECs to migrate. Control siRNA had no effect. Co-culture with NIH3T3 cells transfected with C-terminal Tem similarly inhibited migration.

Quantitative analysis also showed that CICs reduced the number of migrating cells by 73.2%, and that migratory capacity was restored by specific siRNAs by 36.6% (FIG. 2f).
These results imply a central role for Tem as an angiogenesis inhibitor in CTC.

[Example 7] Histological structure and immunohistochemistry of CTC rupture region Next, biopsy was performed on 16 CTC rupture patients.
Human samples containing 16 CTCs were collected from 15 patients undergoing valve replacement or valvuloplasty for mitral valve rupture and 1 autopsy patient (8 males, 8 females, average age 62.4) ± 12.6 years old). Immediately after removal, the sample was fixed with formaldehyde and embedded in paraffin. As controls, 20 microscopically and macroscopically normal, non-calcified, smooth, and flexible mitral CTCs from 12 autopsy patients (10 males, 2 females, mean age 62.7 ± 16.5 years) collected. Necropsy of human tissues and use of surgical samples were approved by the Keio University Institutional Review Board and the National Cardiovascular Center.

As a result, the ruptured region contained innumerable large abnormal blood vessels in the middle layer and the core layer, and showed remarkable tissue degeneration (FIG. 3a). In the ruptured region, elastin was conserved, but TEM was significantly down-regulated and in the distant non-ruptured region it was not down-regulated. These TEM-deficient regions expressed VEGF-A and contained the large abnormal blood vessels described above.
Furthermore, their inner surface was covered with von Willebrand factor positive (vWF ⁺ ) endothelial cells rather than smooth muscle cells.

Quantitative analysis of stained areas was performed by converting the image to monochrome with optimal saturation and counting with black pixels and NIH image software. Computer image analysis shows a significant increase in the number of cells (Figure 3b), vWF ⁺ cell number (Figure 3c), and VEGF-A positive area (Figure 3e), whereas the TEM positive area in ruptured CTCs The proportion of (Fig. 3d) was shown to be significantly reduced.

  The ruptured region of CTC was also shown to express MMP-1 and MMP-2 strongly and moderately MMP-13 in response to VEGF-A expression (FIG. 6). In contrast, MMP-3 was only weakly expressed and MMP-9 was not detected. No MMP signal was detected in normal CTC or non-ruptured areas. Numerous inflammatory cells positive for CD11b, CD14 and vimentin infiltrated the ruptured area.

[Example 8] Influence of Tem layer deletion Next, it was examined whether an angiogenesis and Mmp activation were induced by the absence of a Tem positive layer in a collagen-rich core layer.
After light anesthesia by intravenous injection of pentobarbital (30 mg / kg), 14-14 kg (average 15.2 ± 0.6 kg) adult beagle dogs are intubated, and indoor air is mechanically ventilated using a Harvard spirator And anesthetized with 3% sevoflurane and nitrous oxide. Open the right fourth intercostal space, then create a pericardial cradle, and use an in-line oxygenator between the superior and inferior vena cava and the ascending aorta An extracorporeal bypass was formed. Both blood flow and gas exchange were monitored. The right atrium was opened and the septal cusp of the tricuspid valve was exposed directly. Some CTCs of the tricuspid septum were scraped off to remove the elastin layer (middle layer) (FIG. 4a). The heart was then closed, the extracorporeal bypass was removed, and the chest was closed. One or three months later, the dog was anesthetized with pentobarbital and killed with KCl. After scraping CTC (n = 5 for each group), histological and immunohistochemical experiments were performed.

  The CTC of the septal cusp of the tricuspid valve in an anesthetized dog model was examined and the tenomodulin-rich layer (middle layer) was scraped (FIG. 4). Immunohistochemistry was performed 1 and 3 months after acute inflammation.

  Vegf-A, Mmp1, Mmp2, and Mmp13 were observed from the first month, and the expression region reached approximately 15% of the core layer depth. In the third month, expression reached 37% of the depth, and expression became stronger. Furthermore, abnormal neovascularization was observed only at 3 months. It was suggested that angiogenesis and Mmp activation were not caused by surgery-induced inflammation, but by the loss of the Tem layer.

Example 9 Mechanical extension and hypoxia suppress the expression of tenomodulin in CTC interstitial cells To investigate the cause of the down-regulation of tenomodulin, we have determined that tenomodulin expression in CTC interstitial cells is mechanical. We investigated whether it was affected by various stimuli such as mechanical stretching, hypoxia or oxidative stress. CTC interstitial cells were considered to receive these types of stimuli when subjected to abnormal forces such as in hypertension and during inflammation such as in infective endocarditis.

  First, the present inventors stimulated CTC interstitial cells using a cell spreading apparatus. After 6 hours, no tenomodulin expression could be detected (FIG. 7A). CTC interstitial cells were then incubated under almost anoxic hypoxic conditions. The cells were alive, but tenomodulin expression was not detected as early as 1 hour (FIG. 7B). Finally, CTC interstitial cells were treated with several concentrations of antimycin A that mediate oxidative stress. Tenomodulin expression was observed at antimycin A concentrations below 10 μg / ml, but not detected at antimycin A concentrations of 30 μg / ml (FIG. 7C). These findings indicate that various stimuli such as mechanical stretch, hypoxia, and oxidative stress cause downregulation of tenomodulin expression in CTC.

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  Despite the clinical importance of heart disease, little is known about the mechanisms underlying heart valve disease. The present invention has shown that tenomodulin is not only a potent anti-angiogenic factor, but also an anti-tendon tear factor (tendon tear inhibitor). The findings regarding tenomodulin obtained by the present invention are considered to be effective in preventing atrioventricular regurgitation following CTC rupture.

  The inventors have also shown for the first time that both C-terminal and N-terminal tenomodulin proteins are persistently expressed in the middle layer of normal CTC but are down-regulated in the altered state. Furthermore, the truncated C-terminal domain of tenomodulin secreted from CTC interstitial cells (CIC) has been shown to be a critical angiogenic antagonist. Because the heart valve membrane is a flow-regulating tissue in a dynamic chamber pump, CTC is subject to mechanical stress and damage to the endothelial cells that cover the outer layer. To confirm the hypothesis that tenomodulin protects CTC from inflammation and angiogenesis resulting from mechanical damage, we analyzed for tenomodulin expression in normal and degenerate CTCs.

Similar to surface coating steel to prevent internal corrosion, expression patterns and biological activity suggested that tenomodulin serves to protect against angiogenesis and core layer degeneration. In fact, the observation of cleaved CTC revealed that VEGF-A and MMPs were strongly activated as abnormal blood vessel formation increased, leading to CTC degeneration. More importantly, the local lack of tenomodulin was completely consistent with these regions.
The hypothesis that angiogenesis and degeneration occur gradually in areas where the tenomodulin layer has been removed has been supported by experimental results using dogs.

These findings strongly suggested that CTC rupture does not occur accidentally, but occurs at the location of the absence of the tenomodulin layer when it is locally absent. The inventors have described the lack of tenomodulin as
(1) acute inflammation such as infective endocarditis and rheumatic fever, and
(2) It was estimated that it could be caused by excessive mechanical stress on the valve such as trauma, hypertension and mitral valve prolapse.

  The Achilles tendon is one of the most common sites where tears occur. Rupture occurs spontaneously in the majority of patients without substantial trauma (see Non-Patent Document 22). This pathogenesis is associated with local angiogenesis (see Non-Patent Documents 23 and 24), and VEGF-A is expressed at high levels in tendon cells of the torn Achilles tendon, but in normal adults It is known that this is not done (see Non-Patent Documents 25 and 26). Hypercellularity, prominent angiogenesis, and MMP expression lead to spontaneous rupture, leading to attenuation of normal tendon structure and subsequent reduction of mechanical tension (see Non-Patent Documents 26 and 27) .

  From these previous findings and the present invention, locally increased mechanical stress or inflammation causes local lack of tenomodulin, leading to ruptured MMP activation, angiogenesis and CTC degeneration leading to VEGF-A expression It was implied to induce. Also, in the present invention, the cause of the actual downregulation of tenomodulin in CTC interstitial cells was examined, and various stimuli such as mechanical extension, hypoxia, and oxidative stress caused the downregulation of tenomodulin expression. It was shown that.

  The discovery of the present invention aids preclinical research for using tenomodulin or a protein with a similar function as a therapeutic agent to prevent valvular disease ahead of rupture or CTC. Understanding these mechanisms is important in laying the foundation for new treatment programs for the treatment of valvular heart disease, and the present invention also contributes greatly from an academic perspective.

Claims (11)

A tendon tear-preventing agent containing any of the following (a) to (c) as an active ingredient.
(A) a tenomodulin protein (b) a protein functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the tenomodulin protein (c) DNA encoding the protein according to a) or (b)   The tendon tear-preventing agent according to claim 1, which has a tendon tear-preventing action on a chord. A therapeutic agent for tendon ruptured diseases comprising any of the following (a) to (c) as an active ingredient.
(A) a tenomodulin protein (b) a protein functionally equivalent to a tenomodulin protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the tenomodulin protein (c) DNA encoding the protein according to a) or (b)   A therapeutic agent for tendon ruptured diseases comprising a tennomodulin protein expression activator or function activator as an active ingredient.   Tendon ruptures include valvular heart disease, Achilles tendon tears, patella tendon tears, quadriceps tendon tears, supraspinatus tendon tears, subscapularis tendon tears, biceps longhead tendon tears, finger flexor tendon tears And a therapeutic agent for tendon ruptured diseases according to claim 3 or 4, which is a disease selected from the group consisting of finger extensor tendon ruptures.   The drug according to any one of claims 1 to 5, wherein the tenomodulin protein is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2.   A method for screening a therapeutic agent for a tendon ruptured disease, which comprises selecting a compound that activates the expression or function of tenomodulin protein. A screening method for a therapeutic agent for tendon tearing disease, comprising the following steps (a) to (c).
(A) contacting a test compound with a cell expressing the tenomodulin protein, (b) measuring the expression level of the tenomodulin protein in the cell, and (c) measuring in the absence of the test compound; Step of selecting a compound that increases the expression level in comparison A screening method for a therapeutic agent for tendon tearing disease, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of a tenomodulin gene and a reporter gene are functionally linked; and (b) measuring the expression level of the reporter gene. (C) a step of selecting a compound that increases the expression level as compared with the case of measuring in the absence of the test compound A screening method for a therapeutic agent for tendon ruptured disease, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a tenomodulin protein, or a cell or cell extract expressing the protein, (b) a step of measuring the activity of the protein (c) a measurement in the absence of the test compound Selecting a compound that increases the activity of the protein compared to   Tendon ruptures include valvular heart disease, Achilles tendon tears, patella tendon tears, quadriceps tendon tears, supraspinatus tendon tears, subscapularis tendon tears, biceps longhead tendon tears, finger flexor tendon tears The screening method according to any one of claims 7 to 10, which is a disease selected from the group consisting of ruptured finger extensor tendons.

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