JP2011231014A - Therapeutic agent for heart failure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new means for treating and preventing a heart failure by clarifying the abnormality of the sympathetic nervous system, particularly, the relation between the transdifferetiation of the sympathetic nervous system and the molecule mechanism or condition of cardiac insufficiency onset.SOLUTION: The therapeutic agent for a hear failure contains a substance inhibiting transdiffereniation of catecholaminergic neuron into cholinergic neuron as an effective component. Further, a method for screening the therapeutic agent is provided.

Description

  The present invention relates to a therapeutic agent for heart failure containing as an active ingredient a substance that inhibits the transformation of catecholaminergic neurons into cholinergic neurons, and a method for screening the therapeutic agent.

  The function of the heart is tightly controlled by the balance between the sympathetic and parasympathetic nervous systems. The sympathetic nervous system produces norepinephrine (NE), which increases heart rate, transmission rate, and myocardial contraction / relaxation, while the parasympathetic nervous system produces acetylcholine, which acts to suppress the heart's function.

  In congestive heart failure, the sympathetic nervous system is upregulated, and its excessive excitement damages the heart, resulting in severe arrhythmias and heart NE depletion. This NE depletion has been thought to be due to excessive NE secretion and inhibition of NE reuptake. On the other hand, the inventors recently reported that the decrease in NE in congestive heart failure is due to the down-regulation of NE synthesis accompanied by a decrease in NE reuptake (Non-patent Document 1).

  In vitro, leukemia inhibitory factor (LIF) is known to induce sympathetic nerves through gp130 and convert a neurotransmitter from catecholamine to acetylcholine (Non-patent Document 2). The inventors purified a cholinergic differentiation transforming factor that converts a sympathetic nerve transmitter from catecholamine to acetylcholinecholine from cultured rat heart cells, and reported that this is the same as LIF (Non-patent Document 3). ). LIF is a member of the IL-6 family and induces neurotransmitter conversion in vitro and in vivo. Gp130 is a component (β subunit) common to receptors such as IL-6, LIF, CNTF, OSM, and CT-1.

  Sweat glands are dominated by catecholaminergic nerves when born, but it is known that the neurotransmitter changes from catecholamines to acetylcholine as it grows. It has been confirmed that sympathetic nerves do not differentiate into cholinergic nerves in sweat glands by sympathetic nerve-specific gene targeting of gp130, which is a common component of IL-6 family receptors (Non-Patent Document 4). In transgenic mice that overexpress LIF in pancreatic islets, a decrease in catecholaminergic nerves and an increase in cholinergic nerves are observed (Non-patent Document 5). These results suggest that LIF / gp130 signaling is essential for differentiation transformation into cholinergic neurons.

  On the other hand, in congestive heart failure hearts, growth factors and cytokine levels are elevated, leading to upregulation of LIF and other IL-6 families that induce fetal gene expression (so-called rejuvenation) in adult heart cells In addition, it is known that strong expression of GAP43 and PSA-NCAM, which are immature neuronal markers specific to fetal sympathetic nerves, is observed. These facts suggest that abnormalities in the heart's sympathetic nervous system are associated with nerve rejuvenation.

Until now, there has been no sufficient report on the relationship between heart failure and sympathetic nervous system abnormalities. Currently, diuretics are used to improve venous stasis, and cardiotonic drugs are used to improve cardiac output, and vasodilators are used in combination as needed to treat heart failure. If the relationship between heart failure and cardiac sympathetic nervous system abnormalities is revealed, it can be a new target for the treatment of heart failure.
Kimura, K., et al., "Cardiac Sympathetic Rejuvenation: A Link between Nerve Function and Cardiac Hypertrophy.", Circ Res 100, 1755-64 (2007) Patterson, PH and Chun, LL, "The influence of non-neuronal cells on catecholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons.", Proc Natl Acad Sci US A. 71, 3607-3610 (1974) Yamamori, T. et al., "The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor.", Science 246, 1412-6 (1989) Stanke, M. et al. "Target-dependent specification of the neurotransmitter phenotype: cholinergic differentiation of sympathetic neurons is mediated in vivo by gp 130 signaling." Development 133, 141-50 (2006) Bamber, BA, Masters, BA, Hoyle, GW, Brinster, RL & Palmiter, RD "Leukemia inhibitory factor induces neurotransmitter switching in transgenic mice." Proc Natl Acad Sci USA 91, 7839-43 (1994)

  The object of the present invention is to provide a new means for the treatment and prevention of heart failure by elucidating the relationship between abnormalities of the sympathetic nervous system, in particular, the differential transformation of the sympathetic nervous system and the molecular mechanism or pathology of the onset of heart failure. There is to do.

  The inventors analyzed the distribution of the sympathetic nervous system and the expression of transmitters and various markers by human patients and animal models of congestive heart failure. As a result, congestive heart failure results in neurotransmitter transformation, ie, transformation of catecholaminergic neurons into cholinergic neurons, a process mediated by cholinergic differentiation transformation released from failing myocardium. It was confirmed that it was induced by gp130-mediated signaling.

  The present invention has been made based on the above findings, and relates to a therapeutic agent for heart failure containing, as an active ingredient, a substance that inhibits differentiation transformation of catecholaminergic neurons into cholinergic neurons.

  The differentiation transformation appears as the conversion of the neurotransmitter released from the sympathetic nerve from catecholamine to acetylcholine, and therefore the present invention is for heart failure containing a substance that inhibits the conversion of such a neurotransmitter as an active ingredient. Provide a therapeutic agent.

  In one embodiment, the substance that inhibits the differentiation transformation of catecholaminergic neurons into cholinergic neurons or the conversion of the neurotransmitter catecholamine to acetylcholine is a substance that inhibits leukemia inhibitory factor (LIF) signal. .

  Examples of the substance that inhibits the LIF signal include substances that bind to LIF and inhibit the binding between LIF and the LIF receptor.

  In another embodiment, the substance that inhibits the differentiation transformation of catecholaminergic neurons into cholinergic neurons or the conversion of the neurotransmitter catecholamine to acetylcholine is cardiotrophin-1 (CT). -1) It is a substance that inhibits signal.

  Examples of the substance that inhibits the CT-1 signal include substances that bind to CT-1 and inhibit the binding between CT-1 and the CT-1 receptor.

  The present invention also provides a therapeutic agent for heart failure containing, as an active ingredient, a substance that promotes differentiation transformation from cholinergic neurons to catecholaminergic neurons.

  The differentiation transformation also appears as the conversion of the neurotransmitter released from the sympathetic nerve from acetylcholine to catecholamine, and therefore the present invention contains heart failure containing a substance that promotes the conversion of such neurotransmitter as an active ingredient. These therapeutic agents are also included.

Examples of the substance that promotes the differentiation transformation from cholinergic neuron to catecholaminergic neuron or the conversion of neurotransmitter acetylcholine to catecholamine include nerve cell growth factor (NGF).
The heart failure that is the subject of the present invention is in particular congestive heart failure.
The present invention also relates to a screening method for a therapeutic agent for heart failure comprising the following steps:
(a) measuring the activity of inhibiting the transformation of a test substance from sympathetic nerve to cholinergic neuron,
(b) A step of selecting, as a therapeutic agent for heart failure, a substance having an activity of inhibiting differentiation transformation of sympathetic nerves to cholinergic neurons based on the measurement results.
Furthermore, the present invention relates to a screening method for a therapeutic agent for heart failure comprising the following steps:
(a) a step of measuring an activity of inhibiting the conversion of a neurotransmitter released from a sympathetic nerve of a test substance from catecholamine to acetylcholine,
(b) A step of selecting, as a therapeutic agent for heart failure, a substance having an activity of inhibiting the conversion of a neurotransmitter released from sympathetic nerves to acetylcholine based on the measurement result.
In one embodiment, the screening method of the present invention includes the following steps:
(a) measuring the LIF signal inhibitory activity of the test substance,
(b) A step of selecting a substance having LIF signal inhibitory activity.
In another embodiment, the screening method of the present invention comprises the following steps:
(a) measuring the CT-1 signal inhibitory activity of the test substance,
(b) A step of selecting a substance having CT-1 signal inhibitory activity.

  According to the present invention, there is provided a new method for the treatment and prevention of heart failure targeting the sympathetic nervous system transformation. The therapeutic agent for heart failure of the present invention has a mechanism of action different from that of conventional therapeutic agents for heart failure such as cardiotonic agents and diuretics, and may enable fundamental and preventive treatment of heart failure.

1. Definition 1.1 Sympathetic and parasympathetic nervous systems (SNS: Sympathetic nervous system)
The “sympathetic nervous system” is one of the nervous systems (autonomic nervous system) that regulate the autonomous functions of the living body such as digestion, breathing, sweating, and metabolism. The autonomic nervous system includes two sympathetic nervous systems and a parasympathetic nervous system, which will be described later. Generally, these two nervous systems act antagonistically for one organ. The sympathetic nervous system is generally activated during intense activity. Therefore, if the activation is the heart, the blood pressure increases, the heart rate increases, and the systolic force increases.

  The sympathetic axon changes neurons at the sympathetic ganglion and the post-node fiber reaches the target cell. The synaptic transmission of the sympathetic nerve is carried out through a nicotinic acetylcholine receptor, and noradrenaline (norepinephrine) is released as a transmitter from the end of the postnode fiber.

Parasympathetic nervous system (PNS)
The “parasympathetic nervous system” is another nerve that regulates the autonomous function of the living body. The parasympathetic transmitter is acetylcholine in both ganglia and nerve endings.

1.2 Cholinergic neurons and catecholaminergic neurons Autonomic nerves are classified into “cholinergic neurons” and “adrenergic neurons”.
A “cholinergic neuron” refers to a neuron that releases acetylcholine as a neurotransmitter. Cholinergic neurons are known to be blocked by large amounts of acetylcholine.

  “Catecholaminergic neurons” refer to neurons that release noradrenaline (norepinephrine) or adrenaline (epinephrine) as a neurotransmitter.

1.3 Neurotransmitters-Acetylcholine and Catecholamine "Neurotransmitters" are substances that mediate signal transmission at synapses and are synthesized in the cell body of presynaptic cells. When the action potential reaches the synaptic terminal, the precell opens, the neurotransmitter is released, binds to a receptor on the postsynaptic cell membrane, opens an ion channel, and causes depolarization (or hyperpolarization).

Catecholamines “Catecholamines” are neurotransmitters released from catecholaminergic neurons. “Catecholamine” is a general term for bioactive amines having a catechol nucleus, and includes adrenaline, noradrenaline, and dopamine.

Acetylcholine “Acetylcholine” is a neurotransmitter released from cholinergic neurons. “Acetylcholine” is an acetate compound of choline and is synthesized from choline and acetyl-CoA by the action of choline acetyltransferase.

1.4 transdifferentiation
“Differentiated transformation” means that a differentiated functional cell type changes to another normal cell type, and a differentiation-determining transformation in which a once determined differentiation trait changes to a foreign one, cell tumoriZation It is distinguished from metamorphosis, which is transformation that encompasses, transformation at the tissue level.

  In the present invention, “differentiation transformation of catecholaminergic neurons into cholinergic neurons” means that catecholaminergic neurons are transformed into cholinergic neurons upon differentiation transformation.

  The differentiation transformation can be understood as the conversion of the neurotransmitter catecholamine to choline in the neuron.

1.5 Substance that inhibits the differentiation transformation of catecholaminergic neurons into cholinergic neurons The substance that inhibits the differentiation transformation of catecholaminergic neurons into cholinergic neurons used in the present invention is not particularly limited, Any substance is acceptable. Whether or not a substance inhibits differentiation transformation from catecholaminergic neurons to cholinergic neurons can be determined by methods known to those skilled in the art.

  A method for measuring the inhibition of differentiation transformation from catecholaminergic neurons to cholinergic neurons is not particularly limited, and for example, catecholaminergic neuron markers or cholinergic neuron markers can be used as an index. A substance that increases a catecholaminergic neuron marker or a substance that decreases a cholinergic neuron marker is considered to be a substance that inhibits differentiation transformation from a catecholaminergic neuron to a cholinergic neuron. The cholinergic neuron marker is not particularly limited, and examples thereof include choline transporter (CHT), vesicular acetylcholine transporter (VAChT), and choline acetyltransferase. The catecholaminergic neuron marker is not particularly limited, and examples thereof include tyrosine hydroxylase (TH).

  For example, culturing stellate ganglion neurons in a medium containing cardiomyocyte cultures stimulated with angiotensin II promotes the transformation of catecholaminergic neurons to cholinergic neurons, such as CHT and VAChT. Cholinergic neuron markers increase and catecholaminergic neuron markers such as TH decrease. This increase in cholinergic neuron marker and decrease in catecholaminergic neuron marker were observed in the presence and absence of the test substance, and the increase in cholinergic neuron marker was suppressed and / or catecholamine in the presence of the test substance. When the decrease in the agonist neuron marker is suppressed, it is determined that the test substance has an activity to inhibit the transformation of catecholaminergic neurons into cholinergic neurons.

  Examples of materials that promote differentiation transformation from catecholaminergic neurons to cholinergic neurons include LIF, CT-1, cardiotrophin-like cytokine (CLC) and ciliary neurotrophic factor (CNTF) Examples thereof include neurotrophic factors such as neuropoietin, neurotrophin-3 (NT-3) and glial cell-derived neurotrophic factor (GDNF). Therefore, examples of substances that inhibit differentiation transformation from catecholaminergic neurons to cholinergic neurons include substances that inhibit these factors.

1.6 Substance that inhibits LIF signal As a preferred example of a substance that inhibits differentiation transformation of catecholaminergic neurons into cholinergic neurons used in the present invention, leukemia inhibitory factor (LIF) signal is inhibited. Can be mentioned.

  LIF is a glycoprotein having a molecular weight of about 40,000 to 60,000 having a sugar chain of about 20 kDa to 40 kDa bound to a peptide consisting of about 180 amino acids, and its amino acid sequence is also known (for example, GenBank Accession No: NP_002300, Proc. Nat. Acad. Sci. 85: 2623-2627, 1988, etc.).

  LIF transmits its signal via a heteroreceptor consisting of a LIF receptor (for example, GenBank Accession No: CAA43805) and gp130.

  The substance that inhibits the LIF signal of the present invention is not particularly limited, but a substance that inhibits the expression of any of LIF, LIF receptor, and gp130 (eg, a substance that inhibits LIF expression, expression of LIF receptor) Inhibitory substance), a substance that binds to LIF, LIF receptor, or gp130 and inhibits binding to LIF and its receptor (for example, a substance that binds to LIF, a substance that binds to LIF receptor), etc. Can be mentioned.

Examples of substances that inhibit the expression of any of LIF, LIF receptor, and gp130 include, for example, antisense oligonucleotides, dsRNA, and siRNA.
“Antisense oligonucleotide” can be prepared as an oligonucleotide complementary to a nucleotide constituting a predetermined region of DNA or mRNA encoding LIF, LIF receptor, gp130 according to a known method. “Antisense oligonucleotides” may contain some mismatches as long as they can stably hybridize to the predetermined region and inhibit the expression of LIF, LIF receptor, and gp130, and include oligonucleotide derivatives and modifications. (See JP 09-118623 A, etc.). Examples of modified products include lower alkyl phosphonate modified products such as methylphosphonate and ethylphosphonate types, phosphorothioate modified products, and phosphoramidate modified products.

  “DsRNA or siRNA” is a double-stranded RNA having a sequence identical or similar to a predetermined region of LIF, LIF receptor, or gp130 mRNA, and its expression system can be prepared according to a known method (Special Table 2006). / 016657 etc.). As long as “dsRNA or siRNA” can inhibit the expression of LIF, LIF receptor, and gp130, there may be some mismatch in the double-stranded RNA portion, and its length is also limited unless it shows toxicity in the cell. Not.

  In particular, as a preferred embodiment of the substance that inhibits the LIF signal of the present invention, a substance that binds to any of LIF, LIF receptor, and gp130 and inhibits the binding between LIF and its receptor can be mentioned, and particularly preferred. Can include substances that bind to LIF or LIF receptors and inhibit the binding between LIF and its receptors.

  LIF signal inhibitory activity can be measured by methods known to those skilled in the art, for example, the methods described below.

1.7 Substance that Inhibits CT-1 Signal As a preferred example of a substance that inhibits the differentiation transformation of catecholaminergic neurons into cholinergic neurons used in the present invention, cardiotrophin-1 (cardiotrophin-1: CT-1) can inhibit the signal.

  CT-1 is one of IL-6 family cytokines, and its amino acid sequence is known (for example, Cytokine 8: 183-189, 1996, GenBank Accession No: NP_001321).

  CT-1 also transmits its signal through a receptor containing gp130 as a β subunit. The substance that inhibits the CT-1 signal of the present invention is not particularly limited, but a substance that inhibits the expression of CT-1, CT-1 receptor, or gp130 (eg, inhibits the expression of CT-1). Substance, substance that inhibits the expression of CT-1 receptor), substance that binds to CT-1, CT-1 receptor, gp130 and inhibits binding to CT-1 and its receptor (for example, A substance that binds to CT-1 and a substance that binds to a CT-1 receptor).

  Examples of substances that inhibit the expression of CT-1, CT-1 receptor, or gp130 include, for example, antisense oligonucleotides, dsRNA, siRNA, and the like. Preparation of the antisense oligonucleotide, dsRNA or siRNA is as described above.

  In particular, as a preferred embodiment of the substance that inhibits CT-1 signal of the present invention, a substance that binds to CT-1, CT-1 receptor, or gp130 and inhibits the binding between CT-1 and its receptor Particularly preferred is a substance that binds to CT-1 or CT-1 receptor and inhibits the binding between LIF and its receptor.

  CT-1 signal inhibitory activity can be measured by methods known to those skilled in the art, for example, the methods described below.

  Substance that inhibits differentiation transformation of catecholaminergic neurons of the present invention into cholinergic neurons, substance that inhibits the conversion of catecholamines to acetylcholine, a neurotransmitter released from sympathetic nerves, substance that inhibits LIF signal, or The substance that inhibits CT-1 signal is not particularly limited, as long as it has inhibitory activity, protein, antibody, synthetic low molecular weight compound, natural compound, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, Examples include plant extracts.

  Substance that inhibits differentiation transformation of catecholaminergic neurons of the present invention into cholinergic neurons, substance that inhibits the conversion of catecholamines to acetylcholine, a neurotransmitter released from sympathetic nerves, substance that inhibits LIF signal, or Preferred examples of substances that inhibit CT-1 signal include antibodies.

For example, preferred examples of substances that inhibit the LIF signal of the present invention include anti-LIF antibodies, anti-LIF receptor antibodies, and anti-gp130 antibodies, and preferred examples of substances that inhibit CT-1 signal include anti-CT. -1 antibody, anti-CT-1 receptor antibody, and anti-gp130 antibody.
These antibodies are substances that block signal transduction via LIF receptor, CT-1 receptor or gp130 and inhibit biological activities such as LIF and CT-1.

  The origin of the antibody used in the present invention is not particularly limited, but is preferably an antibody derived from a mammal.

  The antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.

  Antibody-producing hybridomas can be basically produced using known techniques as follows. That is, using LIF, LIF receptor, CT-1, CT-1 receptor, gp130 or an immunologically active fragment thereof as a sensitizing antigen, immunization according to a normal immunization method, The resulting immune cells can be produced by fusing with known parental cells by a normal cell fusion method and screening monoclonal antibody-producing cells by a normal screening method.

Specifically, the antibody can be prepared as follows.
After inserting the gene sequence of the antigen into a known expression vector system and transforming an appropriate host cell, the target antigen protein is purified from the host cell or culture supernatant by a known method. A protein may be used as a sensitizing antigen. Further, a cell expressing an antigen or a fusion protein of an antigen protein and another protein may be used as a sensitizing antigen.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters and the like are used.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, it is performed by injecting a sensitizing antigen intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline and suspended, and then mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, after emulsification. Preferably, it is administered to mammals several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.

  After immunizing in this way and confirming that the desired antibody level rises in the serum, immune cells are removed from the mammal and subjected to cell fusion. Spleen cells are particularly preferred as preferable immune cells to be subjected to cell fusion.

  Mammalian myeloma cells as the other parental cells to be fused with the immune cells are already known in various cell lines such as P3X63Ag8.653 (Kearney, JF et al. J. Immnol. (1979) 123, 1548-1550), P3X63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511 -519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (De St. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G et al., Nature (1979) 277, 131-133) are used as appropriate.

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method such as the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It can be done according to this.

  More specifically, the cell fusion is performed, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used as the fusion accelerator, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.

  For example, the usage ratio of immune cells and myeloma cells is preferably 1 to 10 times that of myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture can be used. Serum supplements such as fetal calf serum (FCS) can be used in combination.

  For cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution pre-warmed to about 37 ° C., for example, a PEG solution having an average molecular weight of about 1000 to 6000 is usually used. The target fused cell (hybridoma) is formed by adding and mixing at a concentration of 30 to 60% (w / v). Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma can be removed by adding an appropriate culture solution successively and centrifuging to remove the supernatant.

  The hybridoma is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is continued for a time sufficient for the cells other than the target hybridoma (non-fused cells) to die, usually several days to several weeks. Subsequently, a normal limiting dilution method is performed, and screening and cloning of the hybridoma producing the target antibody are performed.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes are sensitized with a desired antigen protein or antigen-expressing cells in vitro, and sensitized B lymphocytes are human myeloma cells such as U266. And a desired human antibody having a binding activity to a desired antigen or antigen-expressing cell can be obtained (see Japanese Patent Publication No. 1-59878). Furthermore, antigens or antigen-expressing cells may be administered to a transgenic animal having a repertoire of human antibody genes, and desired human antibodies may be obtained according to the method described above (International Patent Application Publication Nos. WO 93/12227, WO 92 / 03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible therewith to proliferate, and its ascites The method obtained as follows is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

  In the present invention, as a monoclonal antibody, an antibody gene is cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host, and a recombinant antibody produced using a gene recombination technique can be used ( For example, see Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

  Specifically, mRNA encoding the variable (V) region of an antibody is isolated from a cell that produces the target antibody, for example, a hybridoma. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) etc., and then prepare mRNA by using mRNA Purification Kit (manufactured by Pharmacia). Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

  The cDNA of the antibody V region is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis can be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit or the like. In order to synthesize and amplify cDNA, 5'-Ampli FINDER RACE Kit (Clontech) and 5'-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA ( 1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). The target DNA fragment is purified from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA is confirmed by a known method such as the deoxy method.

  If DNA encoding the V region of the target antibody is obtained, it is ligated with DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector. Alternatively, DNA encoding an antibody V region may be incorporated into an expression vector containing antibody C region DNA.

  In order to produce the antibody used in the present invention, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter, as described later. Next, host cells can be transformed with this expression vector to express the antibody.

  In the present invention, a recombinant antibody artificially modified for the purpose of reducing the heterologous antigenicity to humans, for example, a chimeric antibody or a humanized antibody can be used. These modified antibodies can be produced using known methods.

  A chimeric antibody can be obtained by ligating the DNA encoding the antibody V region obtained as described above with DNA encoding the human antibody C region, incorporating it into an expression vector, introducing it into a host, and producing it (Europe). Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92-19759). Using this known method, a chimeric antibody useful in the present invention can be obtained.

  A humanized antibody is also referred to as a reshaped human antibody or a humanized antibody, and is a non-human mammal such as a mouse antibody complementarity determining region (CDR) grafted to a human antibody complementarity determining region. The general genetic recombination technique is also known (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92-19759).

  Specifically, several oligonucleotides were prepared so that the DNA sequence designed to link the CDR of the mouse antibody and the framework region (FR) of the human antibody had an overlapping portion at the end. It is synthesized from nucleotides by PCR. The obtained DNA is obtained by ligating with the DNA encoding the human antibody C region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication Number EP 239400, International Patent Application Publication Number). See WO 92-19759).

  As the FR of the human antibody to be ligated via CDR, one in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, amino acid in the framework region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

  For chimeric antibodies and humanized antibodies, the human antibody C region is usually used. Examples of the human antibody heavy chain C region include Cγ, Cα, Cμ, Cδ, and Cε. For example, Cγ1, Cγ2, Cγ3, or Cγ4 can be used. Examples of human antibody light chain C region include κ or λ. In addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production.

  The chimeric antibody is composed of a variable region of a non-human mammal-derived antibody and a C region derived from a human antibody, and the humanized antibody is a complementarity determining region of a non-human mammal-derived antibody, a framework region derived from a human antibody, and C These regions are useful as antibodies used in the present invention because of their reduced antigenicity in the human body.

In addition to the above-described methods for obtaining human antibodies, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the variable region of a human antibody can be expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, a suitable expression vector containing the sequence can be prepared and a human antibody can be obtained. These methods are already well known and can be referred to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388. .
The antibody gene constructed as described above can be expressed by a known method.

  The binding activity of the obtained antibody to LIF, LIF receptor, CT-1, CT-1 receptor or gp130 can be measured by methods known to those skilled in the art. In addition, LIF signal inhibitory activity or CT-1 signal inhibitory activity of an antibody can also be measured by methods known to those skilled in the art.

2. The therapeutic agent for heart failure according to the present invention The therapeutic agent for heart failure according to the present invention contains a substance that inhibits differentiation transformation from catecholaminergic neurons to cholinergic neurons as an active ingredient.

  As shown in the Examples of the present application, the inventors used human and animal models in the cardiac sympathetic nervous system of heart failure (especially congestive heart failure), differentiation transformation (CTD) from catecholaminergic neurons to cholinergic neurons. Clarified that is happening. This transformation is mediated by leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1), which are members of the interleukin-6 family, and in particular by its common component, the gp130 signaling pathway. Clarified that it is controlled.

  These facts indicate that inhibition of differentiation transformation from catecholaminergic neurons to cholinergic neurons makes it possible to treat or prevent heart failure (particularly congestive heart failure).

  Differentiation transformation from catecholaminergic neurons to cholinergic neurons can also be viewed as conversion of the neurotransmitter catecholamine to acetylcholine in sympathetic nerves. Therefore, it can be said that the therapeutic agent for heart failure of the present invention contains as an active ingredient a substance that inhibits the conversion of a neurotransmitter released from sympathetic nerves from catecholamine to acetylcholine.

  Differentiation transformation from catecholaminergic neurons to cholinergic neurons is mediated by LIF and CT-1 as described above. Therefore, a substance that inhibits LIF or CT-1 signaling can be used as an active ingredient of the therapeutic agent for heart failure of the present invention.

  A similar effect (therapeutic effect on heart failure) promotes the transformation of cholinergic neurons to catecholaminergic neurons, in other words, the conversion of the neurotransmitter acetylcholine to catecholamine in sympathetic neurons Can also be achieved. Therefore, substances that promote differentiation transformation from cholinergic neurons to catecholaminergic neurons (conversion of neurotransmitter acetylcholine to catecholamine in sympathetic nerves) are also effective as the therapeutic agent for heart failure of the present invention. An example of such a substance is nerve cell growth factor (NGF). NGF may be NGF having a naturally derived sequence, or may be a variant in which one or more amino acids are substituted, deleted, added and / or inserted.

  The therapeutic agent for heart failure of the present invention can be administered orally or parenterally, but is preferably administered parenterally. Specifically, a pulmonary dosage form (for example, a pulmonary dosage form using a device such as a nephriser), a nasal dosage form, a transdermal dosage form (for example, an ointment, a cream), an injection dosage form, etc. are suitable. It is mentioned as a dosage form. Examples of the injection type include intravenous injection such as intravenous drip, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like, which can be administered systemically or locally. Such an administration method can be appropriately selected depending on the age and symptoms of the patient.

  The effective dosage of the therapeutic agent of the present invention can be appropriately determined by those skilled in the art according to the active ingredient to be administered, and is not particularly limited, but is usually 0.001 mg per kg body weight at one time. To 1000mg. Or it is chosen in the range of 0.01-100000 mg / body per patient. The above are general values, and the dosage of each therapeutic agent is appropriately set according to the identification and effect of the active ingredient, and is not limited to these dosages.

  The disease to which the therapeutic agent of the present invention is administered is heart failure, particularly congestive heart failure, and this disease includes those that are combined or accompanied by other diseases.

  The therapeutic agent of the present invention inhibits differentiation transformation from sympathetic nervous system catecholaminergic neurons to cholinergic neurons in heart failure, or promotes differentiation transformation from cholinergic neurons to catecholaminergic neurons. Improves abnormal symptoms of the heart.

The timing of administration of the therapeutic agent of the present invention may be prophylactic or maintenance administration regardless of before or after the occurrence of clinical symptoms of the disease. In that sense, the “therapeutic agent” of the present invention includes a prophylactic agent.
The therapeutic agent for heart failure of the present invention may be used in combination with other therapeutic agents for heart failure.

  The therapeutic agent for heart failure of the present invention can be formulated according to a conventional method (Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) and contains both pharmaceutically acceptable carriers and additives. There may be.

  Examples of such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble Dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), Examples thereof include mannitol, sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.

  The actual additive is selected from the above alone or in appropriate combination depending on the dosage form of the therapeutic agent of the present invention, but is not limited thereto. For example, when used as an injectable preparation, the purified antibody is dissolved in a solvent such as physiological saline, buffer solution, glucose solution and the like, and an adsorption inhibitor such as Tween 80, Tween 20, gelatin, human serum albumin and the like is added thereto. Additions can be used. Alternatively, it may be lyophilized to obtain a dosage form that is reconstituted before use, and as an excipient for lyophilization, for example, sugar alcohols or saccharides such as mannitol and glucose are used. be able to.

  The therapeutic agent for heart failure containing a substance that inhibits differentiation transformation of catecholaminergic neurons into cholinergic neurons of the present invention as an active ingredient inhibits differentiation transformation from catecholaminergic neurons to cholinergic neurons. In other words, it can be rephrased as a method of treating heart failure.

  Further, the therapeutic agent for heart failure containing as an active ingredient a substance that inhibits the conversion of the neurotransmitter catecholamine released from the sympathetic nerve from catecholamine to acetylcholine of the present invention is derived from the neurotransmitter catecholamine released from the sympathetic nerve In other words, it is a method for treating heart failure by inhibiting the conversion to acetylcholine.

  The therapeutic agent for heart failure containing a substance that promotes differentiation transformation from cholinergic neurons to catecholaminergic neurons according to the present invention as an active ingredient is differentiation transformation from cholinergic neurons to catecholaminergic neurons. In other words, it can be rephrased as a method of treating heart failure.

  Furthermore, the therapeutic agent for heart failure containing as an active ingredient a substance that promotes the conversion of the neurotransmitter acetylcholine to catecholamine according to the present invention treats heart failure by promoting the conversion of the neurotransmitter acetylcholine to catecholamine. It can be paraphrased as a method of doing.

3. 3. Screening of therapeutic agents for heart failure of the present invention 3.1 Evaluation by differentiation transformation inhibitory activity The therapeutic agents of the present invention described above are: (a) From a sympathetic nerve (or catecholaminergic neuron) of a test substance to a cholinergic neuron. (B) a therapeutic agent for heart failure based on a substance having an activity to inhibit differentiation transformation of sympathetic nerves (or catecholaminergic neurons) into cholinergic neurons based on the measurement results It can be implemented by selecting as

  The differentiation transformation inhibitory activity of the test substance can be determined, for example, by contacting a nerve cell with the test substance, observing the differentiation transformation from the exchanged nerve to the cholinergic neuron in the presence of the test substance, This can be done by comparison with differentiation transformation in the presence.

3.2 Evaluation by conversion of neurotransmitters The therapeutic agent for heart failure of the present invention measures (a) the activity of inhibiting the conversion of catecholamines from catecholamines to acetylcholine of the neurotransmitter released from the sympathetic nerve of the test substance, (b) Based on the measurement result, it can also be carried out by selecting a substance having an activity of inhibiting the conversion of a neurotransmitter released from sympathetic nerves to acetylcholine as a therapeutic agent for heart failure.

  The activity of inhibiting the conversion of a test substance from catecholamine to acetylcholine is observed, for example, by contacting a test substance with a nerve cell that releases catecholamine and observing the release of catecholamine and / or acetylcholine in the presence of the test substance. This can be done by comparing with the release of catecholamine and / or acetylcholine in the absence of the test substance.

3.3 Evaluation Based on LIF or CT-1 Signal Inhibitory Activity The therapeutic agent for heart failure of the present invention comprises (a) measuring the LIF signal inhibitory activity or CT-1 signal inhibitory activity of a test substance, and (b) the measurement result. Based on the above, it can also be carried out by selecting a substance having LIF signal inhibitory activity or CT-1 signal inhibitory activity as a therapeutic agent for heart failure.

  The test substance used in the screening method of the present invention may be any substance, for example, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non- Peptide compounds, synthetic low molecular compounds, natural compounds, antibodies and the like can be mentioned.

Evaluation by specific markers Differentiation transformation from catecholaminergic neurons to cholinergic neurons can be measured by methods known to those skilled in the art, for example, markers of catecholaminergic neurons such as tyrosine hydroxylase and choline transporter It can be measured using a cholinergic neuron marker such as choline acetyltransferase.

  More specifically, for example, the amount of a neuron that is positive for a catecholaminergic neuron marker is measured using a marker for a catecholaminergic neuron, and at the same time, a cholinergic neuron marker is positive using a marker for a cholinergic neuron. Also measure the amount of neurons. Then, the amount of neurons that are positive for catecholaminergic neuron markers and the amount of neurons that are positive for cholinergic neuron markers are measured again, and the amount of neurons that are positive for catecholaminergic neuron markers decreases, resulting in cholinergic activity. When the amount of neurons that are positive for sex neuron markers is increased, it can be determined that catecholaminergic neurons have been transformed into cholinergic neurons.

Evaluation by electron microscope Differentiation transformation from cholinergic neurons to catecholaminergic neurons can also be measured by electron microscopy. Nerve terminals containing catecholamines are observed as small granular vesicles, whereas nerve terminals containing acetylcholine are observed as small agranular vesicles.

  The neurotransmitter conversion can be measured by detecting catecholamine or acetylcholine and using the expression level as an index, and can also be measured by observing nerve endings with the electron microscope described above. .

  As one embodiment of the screening method of the present invention, for example, stellate ganglion neurons are cultured in a medium containing a culture solution of cardiomyocytes stimulated with angiotensin II together with a test substance. Usually, astrocyte ganglion neurons are promoted to differentiate from catecholaminergic neurons to cholinergic neurons, but the test substance has an activity of inhibiting differentiation transformation from sympathetic to cholinergic neurons. In the case where the neurotransmitter released from the sympathetic nerve has an activity of inhibiting the conversion of catecholamine to acetylcholine, the promotion of differentiation transformation is inhibited, or the neurotransmitter released from the sympathetic nerve Conversion to acetylcholine is inhibited. By comparing in the presence and absence of the test substance, the target test substance can be selected.

  Further, as another aspect of the screening method of the present invention, for example, a test substance is administered to a non-human heart failure model animal such as a Dahl salt-sensitive (DS) rat, and the test substance-administered animal and the test substance non-administered animal are used. By comparing the state of catecholaminergic and cholinergic neurons in rats, or by comparing the state of catecholamine-to-acetylcholine conversion of neurotransmitters released from sympathetic nerves, the test substance is released from catecholaminergic neurons. Whether or not it has an activity of inhibiting differentiation transformation into cholinergic neurons, or whether or not the test substance has the activity of excluding the conversion of the neurotransmitter catecholamine to acetylcholine released from the sympathetic nerve Can be determined.

Evaluation by expression level
The LIF signal inhibitory activity can be evaluated, for example, as the inhibitory activity of the expression of any of LIF, LIF receptor, and its subunit gp130. Inhibition of expression can be evaluated by measuring the amount of each protein or mRNA. Similarly, CT-1 signal inhibitory activity can be evaluated as, for example, the inhibitory activity of the expression of CT-1, CT-1 receptor, or any of its subunits gp130. Inhibition of expression can be evaluated by measuring the amount of each protein or mRNA.

  The amount of protein can be evaluated by an immunological method such as Western blotting, dot blotting, slot blotting, ELISA, or RIA using an antibody specific to the protein of interest.

  Antibodies used for detection can be prepared according to known methods, or commercially available antibodies may be used. The antibody can be obtained by immunizing an animal using an antigen protein or an appropriate fragment selected from the amino acid sequence thereof by an ordinary method, and collecting and purifying the antibody produced in the animal body. Further, according to a known method (for example, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody p.365-367, 1980, Prenum Press, NY) Hybridomas may be established by fusing antibody-producing cells and myeloma cells to be produced, and monoclonal antibodies may be obtained therefrom.

  The antigen polypeptide can be obtained by producing it in a host cell by genetic engineering. Specifically, a vector capable of expressing a gene encoding the protein may be prepared and introduced into a host cell to express the gene.

  The obtained antibody is used for detection by directly labeling it or using the antibody as a primary antibody in cooperation with a labeled secondary antibody that specifically recognizes the primary antibody.

Preferred examples of the type of label include, but are not limited to, an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin. As labeled secondary antibodies (or labeled streptavidin), various types of pre-labeled antibodies (or streptavidin) are commercially available. In the case of RIA, an antibody labeled with a radioisotope such as ¹²⁵ I is used, and the measurement is performed using a liquid scintillation counter or the like.

  When a substrate that develops color is used, it can be detected visually using Western blotting or dot / slot blotting. In the ELISA method, it is preferable to measure and quantify the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader. In addition, by preparing a dilution series of the antigen used for antibody production described above, using this as a standard antigen sample and performing detection simultaneously with other samples, creating a standard curve plotting the standard antigen concentration and measured values, the other It is also possible to quantify the antigen concentration in each sample.

  On the other hand, when a substrate that emits light is used, it can be detected by autoradiography using an X-ray film or an imaging plate or by taking a picture using an instant camera in Western blotting or dot / slot blotting. . Also, quantification using densitometry, a molecular imager Fx system (manufactured by Bio-Rad), etc. is possible. Furthermore, when a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).

  The expression level of LIF, LIF receptor, and gp130 mRNA is determined using RT-PCR, real-time PCR, subtraction, differential display, differential display using primers and probes that can specifically amplify or detect the mRNA. Evaluation can be performed by a hybridization method, a cross hybridization method, or a nucleic acid hybridization method using a solid phase sample such as a gene chip, a cDNA array, and a membrane filter.

Evaluation of signal transduction inhibition The LIF signal or CT-1 signal inhibitory activity of a test substance can be measured by a method known to those skilled in the art, and is not particularly limited.

  For example, it is possible to measure LIF signal or CT-1 signal inhibitory activity using inhibition of binding between LIF and LIF receptor and / or gp130, CT-1 and CT-1 receptor and / or gp130 as an index. It is also possible to measure LIF signal or CT-1 signal inhibitory activity using as an index the physiological activity of cells expressing LIF receptor / gp130 or cells expressing CT-1 receptor / gp130. In addition, LIF signal or CT-1 signal inhibitory activity using as an index the physiological activity of cells expressing a chimeric receptor including the extracellular region of LIF receptor, CT-1 receptor or gp130 and the intracellular region of other receptors Can also be measured.

  For example, a test substance is brought into contact with a cell expressing LIF receptor / gp130 or a cell expressing CT-1 receptor / gp130 in the presence of LIF or CT-1, and the physiological activity of the cell is measured. It is possible to select a substance having LIF signal or CT-1 signal inhibitory activity by selecting a test substance having a change in physiological activity as compared with the case where no contact is made.

  In addition, for example, in the presence of LIF or CT-1, a test substance is applied to a cell expressing a chimeric receptor including the extracellular region of LIF receptor, CT-1 receptor or gp130 and the intracellular region of other receptors. A substance having LIF signal or CT-1 signal inhibitory activity can be obtained by contacting, measuring the physiological activity of the cells, and selecting a test substance having a change in physiological activity compared to the case of not contacting the test substance. It is possible to select.

  In the present invention, “change” usually means an increase (enhancement) or a decrease in physiological activity. In general, it is considered that a substance that increases (enhances) physiological activity is a ligand, whereas a substance that decreases physiological activity is an inhibitor as compared with a case where a test substance is not contacted. By using the above method of the present invention, those skilled in the art can appropriately determine whether a test compound that changes physiological activity is a ligand or an inhibitor in consideration of the type of physiological activity used as an index. Is possible.

  The receptor intracellular region used for the chimeric receptor with the extracellular region of LIF receptor, CT-1 receptor or gp130 is capable of inducing changes in physiological activity when a ligand is bound to the chimeric receptor. There is no particular limitation, and any receptor may be derived. Specific examples of the receptor include a cell membrane receptor, a nuclear receptor, an intracellular receptor, and the like. An example of a preferred receptor is a cell membrane receptor. A cell membrane receptor is a receptor that is expressed on the surface of a cell membrane, and when a ligand binds to an extracellular region, a signal is transmitted into the cell to induce some physiological change. Further, when the intracellular region of the cell membrane receptor is used, it may be the entire intracellular region or a part thereof. When using a part of the intracellular region of the receptor, it is preferable to include a signal transduction region.

  Specific examples of receptors include hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase type receptor family, TNF receptor family, G protein coupled receptor family, Examples include receptors belonging to the receptor family such as GPI anchor type receptor family, tyrosine phosphatase type receptor family, adhesion factor family, hormone receptor family and the like.

  Numerous references exist regarding receptors belonging to these receptor families and their characteristics. For example, Cooke BA., King RJB., Van der Molen HJ. Ed. New Comprehesive Biochemistry Vol. 18B "Hormones and their Actions Part II pp.1-46 (1988) Elsevier Science Publishers BV., New York, USA, Patthy L. (1990) Cell, 61: 13-14., Ullrich A., et al. (1990) Cell, 61: 203 -212., Massagul J. (1992) Cell, 69: 1067-1070., Miyajima A., et al. (1992) Annu. Rev. Immunol., 10: 295-331., Taga T. and Kishimoto T. (1992) FASEB J., 7: 3387-3396., Fantl WI., Et al. (1993) Annu. Rev. Biochem., 62: 453-481., Smith CA., et al. (1994) Cell, 76: 959-962., Flower DR. (1999) Biochim. Biophys. Acta, 1422: 207-234., Supervised by Masayuki Miyasaka, Cellular Engineering Handbook Series "Adhesion Factor Handbook" (1994) Shujunsha, Tokyo, Japan , Etc. Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptor, human or mouse granulocyte colony stimulating factor (G-CSF) receptor, human or mouse thrombopoietin (TPO). ) Receptor, human or mouse insulin receptor, human or mouse Flt-3 ligand receptor, human or mouse platelet derived growth factor (PDGF) receptor, human or mouse interferon (IFN) -α, β receptor, human or Mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human or mouse ciliary neurotrophic factor (CNTF) receptor and the like can be exemplified in the present invention. These receptors can be suitably used. The sequences of these receptors are known (hEPOR: Simon, S. et al. (1990) Blood 76, 31-35 .; mEPOR: D'Andrea, AD. Et al. (1989) Cell 57, 277- 285 .; hG-CSFR: Fukunaga, R. et al. (1990) Proc. Natl. Acad. Sci. USA. 87, 8702-8706 .; mG-CSFR: Fukunaga, R. et al. (1990) Cell 61 , 341-350 .; hTPOR: Vigon, I. et al. (1992) 89, 5640-5644 .; mTPOR: Skoda, RC. Et al. (1993) 12, 2645-2653 .; hInsR: Ullrich, A. et al. (1985) Nature 313, 756-761 .; hFlt-3: Small, D. et al. (1994) Proc. Natl. Acad. Sci. USA. 91, 459-463 .; hPDGFR: Gronwald, RGK Et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 3435-3439 .; hIFNα / βR: Uze, G. et al. (1990) Cell 60, 225-234. And Novick, D. at al. (1994) Cell 77, 391-400.).

  A nuclear receptor is a receptor that binds to a specific DNA sequence by binding to a ligand and induces an increase or decrease in the transcriptional activity of mRNA. The steroid receptor family, retinoid X receptor family, etc. are used. be able to. The steroid receptor family includes glucocorticoid receptors, mineralocorticoid receptors, progesterone receptors, androgen receptors, estrogen receptors. The retinoid X receptor family includes retinoic acid receptors, thyroid hormone receptors, and vitamin D3 receptors. An intracellular receptor means a receptor that is present in a cell and binds to various ligands to induce physiological activity.

  The intracellular region of the chimeric receptor is the G-CSF receptor (eg, mouse G-CSF receptor, human G-CSF receptor) It is possible to use. The mouse G-CSF receptor consists of 813 amino acids and is divided into an extracellular region and an intracellular region by a single transmembrane region (Fukunaga, R. Cell (1990) 61, 341-350). In addition, when the G-CSF receptor gene is expressed in myeloid progenitor cell line FDC-P1 and pro-B cell line Ba / F3 cell, the expressed G-CSF receptor transmits cell proliferation signal. It has also been shown that G-CSF-dependent growth activity is observed. Furthermore, it has been clarified that a region consisting of 76 amino acids in the intracellular region is essential for the transmission of proliferation signals (Fukunaga, R. EMBO J. (1991) 10, 2855-2865). Therefore, a chimeric receptor containing the 76 amino acids as a signal transduction region is prepared and expressed in Ba / F3 cells, whereby the detection index can be cell proliferation activity.

  In the human G-CSF receptor, deletion of the 716th and subsequent amino acids suppresses internalization of the receptor and increases the number of G-CSF receptors expressed on the cell surface. It has been shown that signaling efficiency upon stimulation is significantly enhanced (Melissa G., Blood (1999) 93, 440-446). The deletion region includes a region called box3 containing a sequence assumed to be a motif necessary for internalization, while box1 and box2 necessary for signal transduction are conserved. Therefore, it is easy to increase the signal transduction efficiency when stimulating the G-CSF receptor by deleting the region that does not contain box2, but also contains box3 in the mouse G-CSF receptor. is expected.

  A chimeric receptor preferably includes a transmembrane region. The transmembrane region used for the chimeric receptor is not particularly limited, and may be derived from the receptor used for the extracellular region of the chimeric receptor or may be derived from the receptor used for the intracellular region. Further, it may be derived from a completely different cell membrane receptor.

  In the present invention, the physiological activity is an activity capable of causing a quantitative and / or qualitative change or influence on a living body, tissue, cell, protein, DNA, RNA or the like. Any physiological activity may be used in the present invention. As the physiological activity, cytokine activity, enzyme activity, transcription activity, membrane transport activity, binding activity and the like can be used. Examples of the enzyme activity include proteolytic activity, phosphorylation / dephosphorylation activity, redox activity, transfer activity, nucleic acid degradation activity, and dehydration activity. The binding activity includes, for example, a reaction between an antigen and an antibody, binding and / or activation of cell adhesion factors.

In the present invention, a detection index used for measuring a change in physiological activity can be used as long as a quantitative and / or qualitative change can be measured. For example, a cell-free (cell free assay) index, a cell-based (cell-based assay) index, a tissue-based index, or a biological index can be used. As an index of a cell-free system, enzymatic reaction and quantitative and / or qualitative changes of protein, DNA, and RNA can be used. As the enzyme reaction, for example, amino acid transfer reaction, sugar transfer reaction, dehydration reaction, dehydrogenation reaction, substrate cleavage reaction and the like can be used. Furthermore, protein phosphorylation, dephosphorylation, dimerization, multimerization, degradation, dissociation, and the like, DNA, RNA amplification, cleavage, and extension can be used. For example, phosphorylation of a protein existing downstream in the signal transduction pathway can be used as a detection index. As an indicator of the cell system, changes in cell phenotype, such as quantitative and / or qualitative changes of the produced substance, changes in proliferative activity, changes in morphology, changes in characteristics, etc. can be used. As production substances, secreted proteins, surface antigens, intracellular proteins, mRNA, and the like can be used. Changes in morphology include protrusion formation and / or change in the number of protrusions, change in flatness, change in elongation / aspect ratio, change in cell size, change in internal structure, and deformity / uniformity as a cell population Changes in sex and cell density can be used. These morphological changes can be confirmed by observation under a microscope. As changes in properties, anchorage dependence, cytokine dependence responsiveness, hormone dependence, drug resistance, cell motility, cell migration activity, pulsatility, changes in intracellular substances, and the like can be used. Cell motility includes cell invasion activity and cell migration activity. Examples of changes in intracellular substances include enzyme activity, mRNA levels, intracellular signaling substances such as Ca ²⁺ and cAMP, and intracellular protein quantities. In addition, a change in cell proliferation activity induced by receptor stimulation can be used as an index. As an index of the tissue system, a change in function according to the organization to be used can be used as a detection index. Biological indicators include tissue weight changes, blood system changes such as blood cell number changes, protein content, enzyme activity, electrolysis mass changes, and circulatory system changes such as blood pressure and heart rate changes. Etc. can be used.

  There are no particular limitations on the method for measuring these detection indices, and light emission, color development, fluorescence, radioactivity, fluorescence polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption spectrum, light scattering, fluorescence resonance energy Movement or the like can be used. These measurement methods are well known to those skilled in the art, and can be appropriately selected according to the purpose. For example, the absorption spectrum can be measured with a commonly used photometer, plate reader, etc., emission can be measured with a luminometer, and fluorescence can be measured with a fluorometer. Mass can be measured using a mass spectrometer. Radioactivity is measured using a gamma counter or other measuring device according to the type of radiation, fluorescence polarization is measured by BEACON (Takara Shuzo), surface plasmon resonance signal is measured by BIACORE, time-resolved fluorescence, fluorescence resonance energy transfer is measured by ARVO, etc. it can. Furthermore, a flow cytometer or the like can also be used for measurement. These measurement methods may measure two or more types of detection indices with one measurement method, and if simple, more detections can be made by measuring two or more types simultaneously and / or sequentially. It is also possible to measure an indicator. For example, fluorescence and fluorescence resonance energy transfer can be measured simultaneously with a fluorometer.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the technical scope of this invention is not limited by these Examples.

1. Methods and materials:
1.1 Animals
Dahl salt-sensitive rats (DS rats): 6 weeks old
Dahl salt-tolerant rats (DR rats): 6 weeks old
Wister rats: 6-8 weeks old Rats were all purchased from CLEA Japan and fed a high salt diet (8% NaCl) for 9 weeks.

1.2 Transgenic mice expressing LIF
LIF cDNA is pCALNL5 (Kanegae, Y. et al., Nucleic Acids Res 23, 3816-21 (1995), Betz, UA, et al., J Exp Med 188, 1955-65 (1998)): Prof. Saito, University of Tokyo Sub-cloned into the donor). pCALNL5 contains the chicken β-actin (CAG) promoter, loxP-neo-loxP, and a polyadenylation signal. This construct was microinjected into the pronucleus to produce a transgenic mouse (LIF-loxP mouse) that expresses LIF in the heart. The transgene was confirmed by PCR using the following primers.
Forward Primer: 5'-GAGGATCGTTTCGCATGATT-3 '(SEQ ID NO: 1)
Reverse Primer: 5'-TATTCGGCAAGCAGGCATCG-3 '(SEQ ID NO: 2)

This LIF-loxP mouse was crossed with an α myosin heavy chain (MHC) -Cre mouse to produce a LIF ^αMHCCre mouse. All mice were backcrossed with C567BL / 6.

1.3 gp130 knockout mouse A gp130 knockout mouse was generated by gene targeting.
gp130-floxed mice (gp130 ^{flox / flox} mice: Betz, UA, et al. J Exp Med 188, 1955-65 (1998) ce39) and dopamine β hydroxylase-Cre recombinant transgenic mice (DBH-Cre mice: Rosanna , P., et al. Development 134, 1663-70. (2007)) were provided by Dr. Werner Muller and Dr. Gunther Schutz, respectively.

  CAG-CAT-EGFP transgenic mice (Kawamoto, S., et al., FEBS Lett 470, 263-) having a chicken β-actin (CAG) promoter-chloramphenicol acetyltransferase (CAT)-EGFP as a reporter gene construct 268 (2000)) was provided by Professor Miyazaki of Osaka University.

The DBH-Cre mouse was crossed with a gp130 ^{flox / flox} mouse or a CAG-CAT-EGFP transgenic mouse to create a double transgenic mouse.

1.4 Heart Failure Model Mouse Left ventricular heart failure was created using a chronic transverse aortic stenosis model. Right ventricular heart failure was created using a pulmonary hypertension model with long-term hypoxia (10% O ₂ ).

1.5 Cardiomyocyte conditioned medium
The primary culture of Wistar rat cardiomyocytes was performed according to a previous report (Ieda, M. et al., J Clin Invest 113, 876-84 (2004)). That is, cardiomyocytes were cultured in a medium containing serum for 24 hours, replaced with a new medium not containing serum, and cultured in an conditioned medium containing or not containing angiotensin II (1 × 10 ⁷ M).

1.6 siRNA transfection of LIF and CT-1
LIF siRNA and CT-1 siRNA were purchased from Ambion (Austin, TX). Transfection into cardiomyocytes was performed using Lipofectin RNAiMAX reagent (Invitrogen).

1.7 Culture of exchange nerves
Stellate ganglion neurons were isolated from 2-day old Wistar rats in DMEM (Sigma, St. Louis, MO) containing 5% FBS and nerve growth factor (NGF; 50 ng / ml, Upstate, Charlottesville, VA) In culture. For the first 5 days, cytosine 10 μM 1β-d-arabinofuranoside (Sigma) was added to suppress non-neuronal cell proliferation. After culturing for 2 days, the cells were cultured for 14 days in LIF (Chemicon, Temecula, CA) or cardiomyocyte conditioned medium. The medium was changed every 2-3 days.

1.8 Primary culture of cardiomyocytes Primary culture of neonatal rat cardiomyocytes was performed according to the previous report (cited above). Cardiomyocytes were in vitro 30 minutes in 1x10 ^-7 M angiotensin II, 3 hours in 50 μmol / l hydrogen peroxide, 2 hours in 1 μml / l doxorubicin (DOX), 30 in 10 μmol / l norepinephrine (NE) It was similar to heart failure by exposure to 5 μmol / l ceramide for 15 minutes.

Hypoxic stimulation was applied by culturing cells in a 0% N ₂ /5% CO ₂ /5% O ₂ F atmosphere for 2 hours.

  Mechanical elongation stimulation was applied by culturing cardiomyocytes in a laminin-coated silicon dish stretched to 20%.

1.9 RNA extraction and quantitative PCR
RNA extraction was performed according to a previous report (Ieda, M. et al., J Clin Invest 113, 876-84 (2004)). TaqMan real-time PCR is ABI Prism 7500 sequence detection system (Applied
Biosystems, Foster City, CA). All samples were measured in triplicate. Primer and TaqMan probe (LIF (Rn00573491_g1), CT-1 (Rn00567503_m1), CNTF (Rn00755092_m1), CLC (Rn02133709_s1), BDNF (Rn01484928_m1), NT-3 (Rn00579280_m1), GDNF (Rn005695_h1T (Rn00506029_m1), TH (Rn00562500_m1), BNP (Rn00580641_m1), angiotensinogen (Rn00593114_m1), ACE (Rn00561094_m1)) were purchased from Applied Biosystems. Data were normalized using GAPDH. NGF primers and probes were prepared according to a previous report (Ieda, M. et al., J Clin Invest 113, 876-84 (2004)). mRNA levels were normalized relative to GAPDH mRNA.

1.10 RT-PCR
The above-mentioned primers were used for ChAT44 and VAChT45. Mouse LIF primers having the following sequences were used.
mLIF-F2: 5'-CCTCTAGAGTCCAGCCCATAA-3 '(SEQ ID NO: 3)
mLIF-R2: 5'-CTCTAGAAGGCCTGGACCAC-3 '(SEQ ID NO: 4)

1.11 Western blot analysis Samples were prepared by homogenizing the heart in ice-cold buffer. Immunodetection was performed using an anti-LIF antibody (AB-449-NA; R & D System, Minneapolis, Minn.). After transfer to nitrocellulose membrane, 45-kDa LIF protein was visualized by chemiluminescence detection (SuperSignal West Pico, PIERCE, Rockford, IL).

1.12 Measurement of Norepinephrine Norepinephrine (NE) concentration was measured by HPLC according to a previous report (Ieda, M. et al., J Clin Invest 113, 876-84 (2004)).

1.13 Immunohistochemistry Hearts were perfused with PBS containing 0.4% paraformaldehyde, fixed overnight, embedded in OCT compound, and frozen in liquid nitrogen. Cryosections were stained with anti-TH antibody (Chemicon, Sigma and ImmunoStar, Hudson, Wis.) To detect sympathetic nerves, or CHT (Chemicon) and ChAT (Chemicon) to detect parasympathetic nerves.

  Sections were incubated with secondary antibodies conjugated with Alexa 488, 546, 633 (Molecular Probes, Carlsbad, Calif.), TRITC (DAKO, Glostrup, Denmark), and nuclei were stained with TOTO3 (Molecular Probes). Confocal microscopy was performed using LSM 510 META (Carl Zeiss, Jena, Germany).

  Prior to immunostaining, a portion of the paraffin-embedded section was treated with 10 mM citrate buffer (pH 6) and 20 mM Tris-HCl buffer (pH 9) to recover the antigen, and the signal was transferred to the TSA direct kit ( Visualized by NEN Life Science, Boston, MA). Nerve density was measured with Image J software. For human sympathetic ganglia, cresyl violet staining was performed to determine the total number of neurons.

1.14 BDA antegrade labeling of exchanged nerve fibers Under mechanical ventilation, 10% BDA is injected into the left stellate ganglion via a Hamilton micropipette, and the exchange nerve fibers of the stellate ganglion are labeled. did. Rats were sacrificed 3 days after BDA injection, perfused with PBS containing 0.4% paraformaldehyde, and immunohistochemical analysis was performed.

1.15 Transmission Microscope Observation The heart was fixed with cacodylate buffer containing 0.2% glutaraldehyde and 0.4% paraformaldehyde and embedded in an epoxy resin.

  Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate and observed with a JEOL-1230 transmission electron microscope (JEOL Ltd., Tokyo, Japan). AF64A was prepared from acetylcholine mustard hydrochloride and administered intraperitoneally to rats (150 μmol / kg). AF64A is specifically taken up by choline transporters and degenerated cholinergic nerve fibers. As a control, saline was similarly administered. Three days after administration, the heart was removed and fixed as described above. Samples were immunostained with anti-TH antibody (Chemicon), incubated with secondary antibody conjugated with NANOGOLD-Fab (Nanoprobes, Yaphank, NY) and visualized as described above.

1.16 Preparation of human samples Control samples of left ventricle and stellate ganglion were prepared from 8 anatomical cadaver specimens (mean age, 57 ± 17) without heart disease (Figure 5-2a). Heart failure samples from 8 patients (mean age, 51 ± 13) who died of congestive heart failure due to dilated cardiomyopathy, old myocardial infarction, myocarditis and 3 heart transplant recipients who died of dilated cardiomyopathy Prepared (Figure 5-2b). Samples were quickly fixed in 0.4% paraformaldehyde and embedded in paraffin or OCT compound.

1.17 Statistical analysis Values are given as mean ± standard deviation. Differences between groups were evaluated by a significant difference test using Student's t-test, and a P value <0.05 was considered significant.

2. Results 2.1 Distribution of sympathetic and parasympathetic nerves in rat heart Distribution of tyrosine hydroxylase (TH) and choline transporter (CHT), markers of catecholaminergic and cholinergic neurons, immunostained in the left ventricle The distribution of sympathetic and parasympathetic nerves in the rat heart was examined.

The left ventricle had more TH ⁺ nerves and was distributed more than under the epicardium (Fig. 1a, b). CHT ⁺ neurons less than TH ⁺ nerve, its density was different myocardial layers (Fig. 1a, c). In the epicardium, TH was expressed in densely bundled nerve fibers, but CHT was not expressed (FIG. 1a).

In the stellate ganglion, the majority of nerves were TH ⁺ , and very few co-expressed choline acetyltransferase (ChAT, a cholinergic neuron marker). In contrast, almost all nerves in the cardiac ganglion were ChAT ⁺ and a few were TH ⁺ (Fig. 1-1d). Thus, the distribution of catecholaminergic neurons was prominent in the left ventricle, with the cardiac sympathetic and parasympathetic nervous systems extending from the stellate and cardiac ganglion nerves, respectively.

2.2 Increase in cholinergic neurons in the ventricle of Dahl salt-sensitive rats To see if the distribution of cardiac nerves changes in congestive heart failure (CHF), Dahl salt-sensitive (DS) rats (CHF model) and Dahl salt-tolerant (DR) The function of sympathetic nerve in rats (control) was examined.

  DS rats showed hyperpnea and forced breathing and decreased physical activity. DS rats had significantly lower average body weight and heart weight, and the heart-body weight ratio was 1.7 times that of DR rats at 15 weeks of age (FIGS. 1a-d). In contrast to DR rats, DS rats showed significant left ventricular high expression, high plasma NE concentration, and low heart NE concentration of BNP, a cardiac hypertrophy marker, consistent with the results of congestive heart failure (Fig. 1e-g).

As a result of triple immunostaining, DS rats had fewer catecholaminergic neurons (TH ⁺ ) and more cholinergic neurons (CHT ⁺ ) in the left ventricle than DR rats (FIG. 1h). In the epicardial sympathetic nerve bundle of DS rats, CHT ⁺ nerves were markedly increased and TH ⁺ nerves were markedly reduced compared to DR rats (FIG. 2a). There was also co-expression of CHT in TH ⁺ nerves. In DR rats, most of these fibers expressed TH [TH ⁺ / NF (neurofilament) ⁺ ratio = 84%], and a small part coexpressed CHT (CHT ⁺ / NF ⁺ ratio = 3%). In contrast, DS rats had more CHT ⁺ nerves (CHT ⁺ / NF ⁺ ratio = 21%) and less TH ⁺ nerves (TH ⁺ / NF ⁺ ratio = 39%) (Figure 2-1b, c). At the same time, cholinergic neuron activity was up-regulated in the left ventricle of DS rats, and some cardiac sympathetic fibers had both catecholaminergic and cholinergic neuron characteristics.

2.3 Distribution of cholinergic neurons in rat stellate ganglia with heart failure The expression of specific markers in stellate ganglia was examined. In DR rats, many TH ⁺ neurons, few ChAT ⁺ neurons, and many ChAT ⁺ preneural fibers were observed. In comparison, DS rats were more abundant in ChAT ⁺ neurons, some of which co-expressed TH (FIGS. 2-1d, e). The expression of CHT and vesicular acetylcholine transporter (VAChT) was extremely high in the stellate ganglia of DS rats (1.9-fold and 6.6-fold, respectively) (Figs. 2-1f, g). The shift to cholinergic neurons in this DS rat was stellate ganglion specific, and ChAT expression was not detected in the lumbar sympathetic ganglia in any rat (Figure 3-2a, b). These findings indicate that differentiation transformation of the cardiac sympathetic nervous system into cholinergic neurons occurs not only in the neuron itself but also in peripheral axons.

2.4 Differentiation transformation from catecholaminergic neurons to cholinergic neurons in DS rats To confirm that the cardiac sympathetic nervous system produces a functional change from catecholaminergic neurons to cholinergic neurons The cardiac sympathetic nervous system was antegradely labeled from the stellate ganglion by injecting the neurotracer biotinylated dextranamine (BDA). As a result, sympathetic nerves were labeled with BDA in both stellate ganglia and protruding nerve fibers (Figs. 3-1a and 4-2a). The DR rats left ventricle, although all BDA ⁺ nerve fibers were co-expressed TH, there is BDA ⁺ nerve fibers were labeled with BDA ⁺ nerve fibers and CHT co-expressing TH in DS rats left ventricle (Figure 3-1b).

  In addition, direct observation of the sympathetic nervous system by transmission electron microscopy revealed many small granule vesicles (including SGV and catecholamine) in DR rats, consistent with previous studies. On the other hand, SGV and small granule vesicles (including SAGV and acetylcholine) were observed in nerve endings of DS rats (Fig. 3-1c).

  AF64A is a cholinergic neuron-specific toxin that induces vacuolar degeneration of cholinergic neuron terminals. Therefore, AF64A was injected into rats, and sympathetic nerve endings were analyzed by immunoelectron microscopy using anti-TH antibody. In DR rats, catecholaminergic neuron endings labeled with TH immuno-gold particles remained intact, and vacuolar degeneration was observed only in cholinergic neuron endings marked with SAGV. In DS rats, vacuolar degeneration was also observed in catecholaminergic neuron endings labeled with TH immuno-gold particles, which had both SGV and SAGV (Figure 3-1d, Figure 4-2b, c). ). From the above, it can be said that functional changes to sympathetic nervous system cholinergic neurons occur in DS rats.

2.5 Transduction of neurotransmitter by secretion of cholinergic differentiation factor
The conversion of neurotransmitters in the sympathetic nervous system of DS rats is due to humoral factors, especially IL-6 family cytokines secreted from cardiomyocytes [LIF, CT-1, cardiotrophin-like cytokine (CLC), ciliary body Can be mediated by neurotrophic factor (CNTF)], neurotrophin [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3)], glial cell-derived neurotrophic factor We examined whether it was a thing.

  LIF and CT-1 were significantly higher (approximately 5 times) in the ventricle of DS rats than DR rats, and GDNF was also similar (2 times higher) but expressed between animals with heart failure and controls There was no difference in the amount (Figure 4-1a).

  As an in vitro model of heart failure, various stimuli such as angiotensin II (AngII), hydrogen peroxide, doxorubicin, norepinephrine, ceramide, hypoxia, and mechanical elongation were given to primary cultured cardiomyocytes. LIF and CT-1 mRNA were quantified in congestive heart failure induced in this way. All stimuli up-regulated LIF and CT-1 mRNA, with the strongest effect observed with AngII (Figure 4-1b). Angiotensinogen (AGT) and angiotensin converting enzyme (ACE) are also up-reviewed in the DS rat ventricle, indicating that the renin-angiotensin system is increased in congestive heart failure (CHF) (Figure 4-1a). Based on these findings, cultured cardiomyocytes were treated with AngII to induce a state similar to failing myocardium. AngII significantly increased LIF and CT-1 (8.1 and 4.2 times, respectively), but did not significantly affect other factors (Figure 4-1c).

  Primary cultured stellate ganglion neurons were cultured in the presence of conditioned medium of unstimulated or AngII-stimulated cardiomyocytes (UCM, AngII-CM). AngII-CM significantly up-regulated CHT and VAChT (3.2 and 11.5-fold increase, respectively) and down-regulated TH, but UCM had no effect (Fig. 4-1d). Direct AngII treatment of stellate ganglion neurons did not induce changes in cholinergic related genes. Treatment of stellate ganglion neurons with LIF (10 ng / ml) was the same as treatment with conditioned medium of AngII-stimulated cardiomyocytes, ie up-regulated CHT, ChAT, VAChT and markedly down-regulated TH (Figure 4-1e). All the results were confirmed by immunofluorescence staining (Fig. 4-1f).

  Cardiomyocytes were pretreated with siRNA prior to AngII stimulation, and LIF and / or CT-1 expression was specifically inhibited.The conditioned medium of these cells was 76% of VAChT compared to the medium of untreated cells ( LIF siRNA) and 71% (CT-1 siRNA) (FIG. 4-1g). This result indicates that a combination of cholinergic neuronal differentiation factors derived from hypertrophic cardiomyocytes, mainly LIF and CT-1, mediate the conversion of neurotransmitters in stellate ganglion neurons.

2.6 Transduction of neurotransmitters in the cardiac sympathetic nervous system by heart-specific LIF overexpression
LIF-LoxP mice were created and crossed with αMHC-Cre (heart-specifically expressing Cre) mice (FIG. 5-1a). LIF ^αMHC-Cre double transgenic mice highly expressed LIF specifically in the heart (FIGS. 5-1b and c). Thirteen double transgenic mouse lines were generated, and two lines expressing LIF at different levels were selected (lines 3 and 10) (Fig. 5-1d). Lines 10 (Figure 5-1e) showed cardiac hypertrophy compared to the littermate 8-week-old wild type, but almost no CHT ⁺ nerves. ^Ten LIF ^αMHC-Cre mice showed slight TH ⁺ nerves (78% of wild type) and predominantly high CHT ⁺ nerves (8 times) (Figure 5-1f, g), but in line 3 There were no changes in neurotransmitters and phenotypes. Cardiac NE concentration was decreased in the heart of LIF ^αMHC-Cre mice (line 10) (Figure 5-1h). Consistent with these results, but did not slight ChAT ⁺ nerve Shikami in the stellate ganglion of wild-type mice, the stellate ganglion of ^{LIF αMHC-Cre} mouse seen a large number of ChAT ⁺ nerve, Nakaniwa Some co-expressed TH (Fig. 5-1i, j). These results indicate that heart-specific LIF overexpression induces neurotransmitter conversion and induces cholinergic neuronal differentiation transformation in the cardiac sympathetic nervous system in vivo.

2.7 Conditional targeting of the sympathetic nervous system specific gp130 gene in mice
A gp130-mediated signal induces cholinergic neuronal differentiation transformation of the sympathetic nervous system in congestive heart failure. DBHCre and EGFP-floxed mice were crossed to create a sympathetic nervous system-specific EGFP reporter mouse (EGFP ^DBHCre mouse), and the expression of EGFP in the adrenal gland, stellate ganglion, and sympathetic nervous system was confirmed (Fig. 6-1a). 57% of the stellate ganglion bodies of these mice were marked with EGFP, confirming that this transgenic reporter mouse strain is effective (FIG. 6-1b). Sympathetic nervous system-specific gp130 gene knockout mice were generated by crossing DBHCre and gp130-floxed mice. The resulting Gp130 ^DBHCre mice grew normally and had normal stellate ganglia and left ventricular sympathetic nervous system that stained with TH but not with CHT. This result was consistent with the result of the control gp130flox / flox mouse (FIGS. 6-1c and e).

A model of heart failure and pulmonary hypertension was created as described above. Gp130 ^{flox / flox} mice showed cholinergic neuronal transformation of the cardiac sympathetic nervous system and stellate ganglion as well as the rat congestive heart failure (CHF) model. These changes did not occur in gp130 ^DBHCre mice (FIGS. 6-1c, e).

Quantitative analysis showed that the TH ⁺ / NF ⁺ nerve area ratio in the ventricular sympathetic nervous system of gp130 ^DBHCre mice returned to the control level, but the CHT ⁺ / NF ⁺ nerve area ratio decreased significantly (Figure 6-). 1d). The stellate ganglion ChAT ⁺ / TH ⁺ ratio was significantly reduced in gp130 ^DBHCre mice (FIG. 6-1f). The recovery rate of the ChAT ⁺ / TH ⁺ ratio was consistent with the results in sympathetic nervous system-specific EGFP reporter mice, based on the calculated 57% of stellate ganglion bodies marked with EGFP. These results indicate that sympathetic cholinergic neuronal transformation in congestive heart failure is primarily regulated by the gp130 signaling pathway.

2.8 Transduction of human cardiac sympathetic neurotransmitters in congestive heart failure To see if the above view applies to humans, the phenotype of cardiac sympathetic neurotransmitters in patients with congestive heart failure (Fig. 5-2). Consistent with the experimental results, the control epicardial nerve bundle had the majority of TH ⁺ nerves, but the heart of patients with congestive heart failure had the majority of CHT ⁺ nerves and co-expressed both markers. There were also nerves. The same neurotransmitter phenotype was observed in stellate ganglia (Fig. 6-2), and it was confirmed that differentiation transformation to cholinergic neurons occurred in patients with congestive heart failure.

3. Discussion In both animal models and cardiac tissue of congestive heart failure patients, the left ventricle of congestive heart failure has a marked increase in cholinergic neurons, and the antegrade labeling of cardiac sympathetic nerve fibers causes these CHT ⁺ nerves to It was confirmed to be derived from the sympathetic nervous system. These results indicate that the cardiac sympathetic nervous system potentially has two functions and converts neurotransmitters depending on the environment. Furthermore, we have identified that the mechanism of this process is due to the exogenous angiotensin stimulation of cardiomyocytes secreting many factors that induce the sympathetic nervous system to cholinergic nerves. It was also confirmed that cholinergic differentiation transformation is induced by overexpression of heart-specific LIF and that congestive heart failure-induced cholinergic differentiation can be prevented by targeting the sympathetic nerve-specific gp130 gene.

  The present findings indicate the involvement of differentiation transformation from catecholaminergic neurons to cholinergic neurons as a mechanism of heart failure development. That is, it shows that inhibition of differentiation transformation from catecholaminergic neurons to cholinergic neurons makes it possible to treat or prevent heart failure (particularly congestive heart failure).

  The present invention provides a novel therapeutic agent for heart failure and a screening method thereof. The therapeutic agent of the present invention has a different mechanism of action from conventional therapeutic agents for heart failure, and is useful as a fundamental and prophylactic treatment method for heart failure.

FIG. 1-1 shows the distribution of sympathetic and parasympathetic nervous systems in the rat heart. (a) TH, CHT (green), α-actinin (red), left ventricular immunostaining results (arrows indicate sympathetic nerves on the epicardium), (b) left ventricular TH ⁺ nerve quantitative results (N = 5), (c) CHT ⁺ nerve quantitative analysis results (n = 5), (d) Stellate and cardiac ganglion TH (red), ChAT (green), Toto (blue) immunity As a result of staining, (e) ChAT ⁺ neurons (upper arrow) were surrounded by TH ⁺ neurons, and few TH ⁺ neurons were observed in the cardiac ganglia. * = P <0.01, ns = significant difference, scale bar is (a) 100μm, (d) 50μm FIG. 1-2 shows (a) heart, (b) body weight (n = 5), (c) heart weight (n = 5), (d) heart weight / body weight ratio (n = 5) in DS and DR rats. ), (E) BNP mRNA expression level (n = 5), (f) Serum norepinephrine concentration (n = 5), (g) Left ventricular tissue norepinephrine level (n = 5): * = P <0.01, (h ) Results of triple immunostaining of left ventricle (α-actinin (purple), TH (red), CHT (green)): The area enclosed by the square is high-power photomicrograph, scale bar is 100 μm FIG. 2-1 shows (a) double-stained confocal images with TH (red) and CHT (green), and arrows indicate an increase in CHT ⁺ nerves. The boxed area is high-power photomicrograph, (b, c) left ventricular epicardium TH ⁺ / NF ⁺ and CHT ⁺ / NF ⁺ ratio (n = 5), (d) TH of stellate ganglion (red ) And CHT (green) results of double immunostaining, (e) Quantitative analysis of ChAT ⁺ / TH ⁺ ratio of stellate ganglion (n = 4), (f) Stellate ganglion by RT-PCR Quantitative analysis of VAChT ⁺ expression (n = 4). * = P <0.01, scale bar is 20μm Figure 2-2: (a) Double-stained confocal image of TH (green) and NF (red) in left ventricular epicardium, (b) CHT (green) and NF (red) in left ventricular epicardium By double-stained confocal elephant. Arrows indicate CHT ⁺ nerves in the sympathetic nerve bundle. FIG. 3-1 shows differentiation transformation of cardiac sympathetic nerves in the ventricle of heart failure rats. (a) Right panel shows results of antegrade labeling by BDA. (b) Epicardial sympathetic nerve of DS rat ventricle (left 2 panels are TH co-expressing BDA ⁺ nerve, right 2 panels are CHT co-expressing BDA ⁺ Nerve), (c) Transmission electron micrograph of nerve endings in the left ventricle (black arrow: SGV of sympathetic nerve ending, white arrow: SAGV of parasympathetic nerve ending), (d) Immunoelectron microscope image of left ventricle after AF64A administration . The area enclosed by the square is high-power photomicrograph, scale bar is (c, d) 0.5μm, (b-right cross section) 20μm, (b-left cross section and vertical section) 50μm, (a-right) 100μm, (a-medium) 2mm Fig. 3-2 shows the result of immunostaining of lumbar ganglia of DS rats. (a) TH (red), ChAT (green), Toto (blue), (b) Quantitative analysis results of ChAT ⁺ / TH ⁺ ratio. FIG. 4-1 shows neurotransmitter conversion by cholinergic neuron differentiation factors in cultured sympathetic nerves. (a) Expression analysis results of IL-6 family mRNA (n = 4-6), (b) Angiotensin II (AngII), hydrogen peroxide, doxorubicin, norepinephrine, ceramide, hypoxia, LIF after mechanical elongation stimulation And CT-1 expression analysis results (n = 4), (c) Ang-6 stimulated / unstimulated IL-6 family mRNA expression analysis results (n = 4), (d) AngII stimulated / unstimulated cardiomyocyte conditioned medium Effects on TH, CHT and VAChT expression (n = 3), (e) Effects of LIF stimulation on ChAT, VAChT and GAPDH expression (n = 3), (f) TH (red), ChAT (green) , Immunocytochemical staining of sympathetic nerve with Toto3 (blue), (g) Effect of siRNA on LIF and CT-1 expression. * = P <0.01, ** = P <0.05, ns = significant difference, scale bar is 100 μm. FIG. 4-2 shows the results of antegrade labeling by BDA. (a) BDA detection results in the cardiac plexus (left) and left ventricle (right), (b) Transmission electron microscopic analysis results of nerve endings, (c) Immunoelectron microscopic analysis results after TH-nanogold and AF64A treatment. Scale bars are 0.5 μm (b, c) and 50 μm (a). FIG. 5-1 shows the effect of LIF overexpression on neurotransmitter conversion. (a) LIF ^αMHC-Cre mouse production procedure, (b) LIF mRNA expression analysis results in LIF ^αMHC-Cre mice, (c) Western blot analysis results of LIF in wild type mice and LIF ^αMHC-Cre mice, (d) Results of quantitative analysis of LIF in lines 3 and 10, (e) ^{Hearts of} wild type mice and LIF ^αMHC-Cre mice, (f) Results of double immunostaining of wild type mice and LIF ^αMHC-Cre mice (α actinin (red) , TH / CHT (green)), (g) Norepinephrine levels in wild type and LIF ^αMHC-Cre mice, (i) Results of quantitative analysis of TH ⁺ and CHT ⁺ nerves in stellate ganglia, (h) TH ( Results of immunocytochemical staining of sympathetic nerves with red), ChAT (green), and Toto3 (blue), (j) Quantitative analysis of ChAT ⁺ / TH ⁺ ratio in stellate ganglia of wild-type and LIF ^αMHC-Cre mice result. * = P <0.01, ** = P <0.05, ns = significant difference, scale bar is 50 μm (f), 100 μm (i), 5 mm (e). FIG. 5-2 shows the characteristics of the human sample. (A) Control group, (b) Heart failure patient group, (c) Control group HE staining and Masson trichrome staining result, (d) Heart failure patient group control group HE staining and Masson trichrome staining result, (e) Star shape Results of Nissl staining of ganglia (SG), (f) Quantitative analysis of the number of stellate ganglion neurons (n = 5). Scale bars are 100 μm (c, d) and 200 μm (e). FIG. 6-1 shows inhibition of sympathetic differentiation by gp130 gene knockout. (a) Immunofluorescence staining results (GFP (green), Toto3 (blue), n = 3) of left ventricle (LV), stellate ganglion (SG), adrenal gland (AG) of EGFP ^DBHCre mice, (b) EGFP ^Results of quantitative analysis of EGFP ⁺ cells in ^DBHCre mice (n = 3), (c) gp130 Chronic transverse aortic stenosis (TAC) and hypoxia-induced heart failure in ^DBHCre mice (gp130 knockout) and Gp130 ^{flox / flox} mice (control) , (D) Quantitative analysis of left and right ventricular TH ⁺ / NF ⁺ and CHT ⁺ / NF ⁺ ratio (n = 4), (e) Immunostaining results of gp130 ^DBHCre and Gp130 ^{flox / flox} mice (TH ( (Red), ChAT (green)), (f) Quantitative analysis results of ChAT ⁺ / TH ⁺ ratio (n = 4). * = P <0.01, ** = P <0.05, ns = significant difference, scale bars are 100 μm (a, e), 20 μm (c). FIG. 6-2 shows neurotransmitter conversion in the human sympathetic nervous system. (a) Results of immunostaining in the left ventricle of patients with heart failure (TH (red), CHT (green)), (b) Results of quantitative analysis of TH ⁺ nerve and CHT ⁺ nerve, (c) Immunostaining in the left ventricle of patients with heart failure Results (TH (red), ChAT (green)), (d) Results of quantitative analysis of TH ⁺ nerve and ChAT ⁺ nerve. * = P <0.01, ns = significant difference, scale bar is 50 μm (a), 100 μm (c).

Sequence number 1: Primer Sequence number 2: Primer sequence number 3: Primer sequence number 4: Primer

Claims (13)

  A therapeutic agent for heart failure comprising, as an active ingredient, a substance that inhibits differentiation transformation of catecholaminergic neurons into cholinergic neurons.   A therapeutic agent for heart failure comprising, as an active ingredient, a substance that inhibits conversion of a neurotransmitter released from sympathetic nerves from catecholamine to acetylcholine.   The therapeutic agent for heart failure according to claim 1 or 2, comprising a substance that inhibits leukemia inhibitory factor (LIF) signal as an active ingredient.   The therapeutic agent for heart failure according to claim 3, wherein the substance that inhibits LIF signal is a substance that binds to LIF and inhibits the binding of LIF and LIF receptor.   The therapeutic agent for heart failure according to claim 1 or 2, comprising a substance that inhibits cardiotrophin-1 (CT-1) signal as an active ingredient.   The therapeutic agent for heart failure according to claim 5, wherein the substance that inhibits CT-1 signal binds to CT-1 and inhibits the binding between CT-1 and CT-1 receptor.   A therapeutic agent for heart failure comprising, as an active ingredient, a substance that promotes differentiation transformation from cholinergic neurons to catecholaminergic neurons.   The therapeutic agent for heart failure according to claim 7, wherein the substance that promotes differentiation transformation from cholinergic neurons to catecholaminergic neurons is nerve cell growth factor (NGF).   The therapeutic agent for heart failure according to any one of claims 1 to 8, wherein the heart failure is congestive heart failure. A screening method for a therapeutic agent for heart failure comprising the following steps:
(a) measuring the activity of inhibiting the transformation of a test substance from sympathetic nerve to cholinergic neuron,
(b) A step of selecting, as a therapeutic agent for heart failure, a substance having an activity of inhibiting differentiation transformation of sympathetic nerves to cholinergic neurons based on the measurement results. A screening method for a therapeutic agent for heart failure comprising the following steps:
(a) a step of measuring an activity of inhibiting the conversion of a neurotransmitter released from a sympathetic nerve of a test substance from catecholamine to acetylcholine,
(b) A step of selecting, as a therapeutic agent for heart failure, a substance having an activity of inhibiting the conversion of a neurotransmitter released from sympathetic nerves to acetylcholine based on the measurement result. The screening method according to claim 10 or 11, comprising the following steps:
(a) measuring the LIF signal inhibitory activity of the test substance,
(b) A step of selecting a substance having LIF signal inhibitory activity as a therapeutic agent for heart failure based on the measurement result. The screening method according to claim 10 or 11, comprising the following steps:
(a) measuring the CT-1 signal inhibitory activity of the test substance,
(b) A step of selecting a substance having CT-1 signal inhibitory activity as a therapeutic agent for heart failure based on the measurement result.