JP2005336159A - Novel therapeutic use of pigment epithelium-derived factor which inhibits nadph oxidase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel medical use of a pigment epithelium-derived factor. <P>SOLUTION: A medicinal composition which contains a pigment epithelium-derived factor or a variant thereof as an active ingredient and used for inhibiting NADPH oxidase activity can find possible utility in the therapy and prophylaxis of various diseases including angiopathies. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a pharmaceutical composition or vector for preventing or treating vascular disorders, and in particular, encodes a pharmaceutical composition containing pigment epithelium-derived factor (PEDF) or a variant thereof as an active ingredient, and PEDF or a variant thereof. The present invention relates to a vector containing a nucleic acid.

  Pigment epithelium-derived factor (PEDF) is a glycoprotein belonging to the superfamily of serine protease inhibitors, and was originally purified from the culture supernatant of human retinal pigment epithelial cells as a factor with strong neuronal differentiation activity in human retinoblastoma cells. (Non-Patent Document 1). Recently, PEDF has been shown to inhibit angiogenesis very effectively in cell culture and animal models. PEDF inhibited retinal epithelial cell growth and migration and suppressed ischemia-induced retinal neovascularization (Non-patent Documents 2 and 3).

In the retina, small blood vessels for supplying nutrients and oxygen to the eyeball constitute a network. However, if a hyperglycemic state continues for a long time due to diabetes or the like, these blood vessels are damaged, resulting in increased permeability. As this condition progresses further and the ischemic area expands, it leads to proliferative diabetic retinopathy with new blood vessels. PEDF is present in the vitreous and its levels are reduced in ocular angiogenesis, suggesting that PEDF present in the eye is functionally important in the development of proliferative diabetic retinopathy (Non-patent document 4). However, how PEDF regulates epithelial cell (EC) growth and function is not fully understood.
Atherosclerosis is also an inflammatory fibroproliferative disease associated with EC (Non-Patent Document 5), and its etiology is similar to that of TNF-α and IL-1, which induce thrombotic and inflammatory responses in EC It is considered to be a classic anti-inflammatory cytokine (Non-patent Documents 6 and 7). When exposed to these cytokines, EC undergoes decisive functional changes including IL-6 production, adhesion molecule expression and induction of procoagulant activity (Non-Patent Documents 8-10). Signaling and gene expression of these cytokines are highly dependent on the generation of reactive oxygen species (ROS) in EC (Non-patent Documents 11 and 12). ROS causes oxidative stress that plays a major role in retinopathy and atherosclerosis.
ROS in vascular cells is mainly produced by NADPH oxidase (Non-Patent Document 31), and NADPH oxidase is known to be essential in cell pathophysiological responses, including cell growth and gene expression of adhesion molecules and cytokines. (Non-patent Document 31). In fact, NADPH oxidase is activated by advanced glycation end products (AGE), an aged giant protein derivative that is accelerated in diabetes, thereby correlating oxidative stress with changes in gene expression in EC, diabetic micro and Macrovascular disorders are thought to develop (Non-patent Document 32).

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Yamagishi S, Yonekura H, Yamamoto Y, Katsuno K, Sato F, Mita I, Ooka H, Satozawa N, Kawakami T, Nomura M, Yamamoto H. Advanced glycation end products-driven angiogenesis in vitro.Induction of the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor.J Biol Chem 1997; 272: 8723-8730. Yamagishi S, Fujimori H, Yonekura H, Yamamoto Y, Yamamoto H. Advanced glycation endproducts inhibit prostacyclin production and induce plasminogen activator inhibitor-1 in human microvascular endothelial cells. Diabetologia 1998; 41: 1435-1441. Yamagishi S, Inagaki Y, Okamoto T, Amano S, Koga K, Takeuchi M, Makita Z. Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells.J Biol Chem 2002; 277: 20309-20315. Sulciner DJ, Irani K, Yu ZX, Ferrans VJ, Goldschmidt-Clermont P, Finkel T. 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The present inventors have recently shown that PEDF significantly inhibits AGE-induced pericyte apoptosis and microvascular epithelial cell injury by suppressing ROS generation (Non-patent Documents 15 and 33).
Thus, the present inventors thought that PEDF could prevent the progression of various types of vascular disorders including diabetic vascular complications by blocking the NADPH oxidase-mediated intracellular signaling pathway. According to our research on PEDF, PEDF actually suppresses NADPH oxidase activity, in particular, PEDF suppresses Rac activation in human umbilical vein endothelial cells (hereinafter referred to as HUVEC). It has been shown to inhibit TNF-α-induced NADPH oxidase activity, thereby inhibiting NF-κB activation, IL-6 expression, angiotensin II activity and / or advanced glycation end product (AGE) signaling. Completed the invention.

That is, the present invention provides the following inventions:
(1) a) Pigment epithelium-derived factor;
b) a variant of the factor having a functionally equivalent property to the pigment epithelium-derived factor (a); and
c) a pharmaceutical composition for inhibiting NADPH oxidase activity, comprising an active ingredient selected from the group consisting of a vector comprising a nucleic acid molecule encoding said factor (a) or said variant (b); NADPH oxidase mediated Said pharmaceutical composition that suppresses the production of reactive oxygen species; by inhibiting NADPH oxidase activity, NF-κB activation, IL-6 expression, TNF-α activity, angiotensin II activity, and / or glycation end product signaling Said pharmaceutical composition that inhibits;
In particular,
The pharmaceutical composition of the present invention for preventing or treating a disease caused by NADPH oxidase; more specifically, a disease caused by NADPH oxidase is vascular disorder, NF-κB activation-related disease, IL-6 The pharmaceutical composition selected from the group consisting of expression-related disease, TNF-α activity-related disease, angiotensin II activity-related disease, and advanced glycation end signaling-related disease;
More specifically,
The pharmaceutical composition of the invention, wherein the vascular disorder is selected from the group consisting of vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, and cerebral infarction; NF -κB activation-related diseases include vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, and A pharmaceutical composition of the invention selected from the group consisting of cerebral infarction; IL-6 expression-related disease is coronary event, vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, A pharmaceutical composition of the present invention selected from the group consisting of diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, cerebral infarction, and malignant myeloma; TNF-α activity -Related diseases consist of vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, and cerebral infarction A pharmaceutical composition of the invention selected from the group; angiotensin II activity-related disease is vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, and brain A pharmaceutical composition of the present invention selected from the group consisting of infarcts; a pharmaceutical composition of the present invention for treating diabetic vascular complications associated with the production of advanced glycation end products by inhibiting NADPH oxidase activity; Product communication-a pharmaceutical composition of the invention, wherein the associated disease is diabetic vascular complications associated with the production of advanced glycation end products;
(2) a) Pigment epithelium-derived factor; or
b) a vector for inhibiting NADPH oxidase activity, comprising a nucleic acid encoding a variant of the factor having a functionally equivalent property to pigment epithelium-derived factor (a); inhibiting NADPH oxidase-mediated reactive oxygen species production A vector; inhibiting NADPH oxidase activity to inhibit NF-κB activation, IL-6 expression, TNF-α activity angiotensin II activity and / or terminal glycation product signaling;
In particular,
The vector of the present invention for preventing or treating a disease caused by NADPH oxidase; more specifically, a disease caused by NADPH oxidase is vascular disorder, NF-κB activation-related disease, IL-6 expression The vector selected from the group consisting of a related disease, TNF-α activity-related disease, angiotensin II activity-related disease, and advanced glycation end signaling-related disease;
More specifically,
A vector of the invention wherein the vascular disorder is selected from the group consisting of vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, and cerebral infarction; NF-κB Activation-related diseases include vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, and cerebral infarction A vector of the invention selected from the group consisting of: IL-6 expression-related disease is coronary event, vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complication Vector of the present invention selected from the group consisting of disease, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, cerebral infarction, and malignant myeloma; TNF-α activity-related The disease is from the group consisting of vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, and cerebral infarction The vector of the present invention selected; the group wherein the angiotensin II activity-related disease consists of vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, and cerebral infarction A vector of the present invention selected from: a vector of the present invention for treating diabetic vascular complications associated with the production of glycation end products by inhibiting NADPH oxidase activity; It relates to the vector of the present invention, which is a diabetic vascular complication associated with the production of a terminal glycation product.

(1) Pharmaceutical composition for inhibiting NADPH oxidase activity The present inventors have demonstrated that PEDF inhibits TNF-α-induced NADPH oxidase activity by two different methods (FIG. 2) shown in the Examples described later. Indicated. Although the exact molecular mechanism is not clear, our study suggests that PEDF may inhibit TNF-α-induced NADPH oxidase activity by suppressing Rac activation in HUVEC .

The pharmaceutical composition of the present invention can be used to prevent or treat NADPH oxidase disorders. In the present invention, prevention includes prevention of recurrence. NADPH oxidase mediates the generation of reactive oxygen species (ROS). ROS is a molecule such as hydrogen peroxide, an ion such as a hypochlorite ion or a superoxide anion, a radical such as a hydroxyl radical, and the like, and is involved in sterilization. However, ROS can cause oxidative stress, which plays a major role in retinopathy and atherosclerosis.
Therefore, the present inventors examined the effects of PEDF on reactive oxygen species (ROS) production, NF-κB activation and IL-6 expression in TNF-α-exposed HUVECs. TNF-α significantly increased intracellular ROS production, which was completely suppressed by PEDF or NADPH oxidase inhibitor diphenylene iodonium (hereinafter referred to as DPI). Furthermore, PEDF completely inhibited the increase in NADPH oxidase activity induced by TNF-α. PEDF or the antioxidant N-acetylcysteine significantly inhibited TNF-α-induced NF-κB activation. PEDF inhibited TNF-α-induced IL-6 expression at both the mRNA and protein levels. Furthermore, TNF-α decreased PEDF mRNA levels. Ligand blot analysis revealed that HUVEC has a membrane protein with binding affinity for PEDF. These results indicate that PEDF inhibits TNF-α-induced NF-κB activation and subsequent IL-6 overexpression by suppressing NADPH oxidase-mediated ROS production in HUVEC.

Furthermore, angiotensin II (AngII) markedly induces the activation of NF-κB, a transcription factor sensitive to redox, and then the expression of MCP-1 (Monocyte Chemoattractant protein-1) in HUVEC. Like N-acetylcysteine (hereinafter referred to as NAC), which is an oxidizing agent, it is completely inhibited by PEDF, and the generation of ROS induced by AngII in HUVEC is similar to DPI, which is an NADPH oxidase inhibitor. Found to inhibit.
In addition, advanced glycation end products (AGE) significantly stimulate the activity of intramolecular reactive oxygen species in microvascular endothelial cells, but these are completely suppressed by PEDF, and PEDF is also active in NADPH oxidase activity. Inhibition of NADPH oxidase induced by advanced glycation end products (AGE) as well as increase, and PEDF is significant for cells treated with AGE fraction from hemodialysis diabetic (DM-HD) serum It was revealed that it exhibits antioxidant activity. Furthermore, it showed efficacy against retinal vascular permeability in AGE perfused rats. The series of events described above is referred to as “terminal glycation end product (AGE) information transmission” in the present specification.
From the above findings, the present invention also suppresses NADPH oxidase activity, thereby enabling NF-κB activation, IL-6 expression, TNF-α activity, angiotensin II activity and / or advanced glycation end product (AGE) signaling. Pharmaceutical compositions that inhibit are provided.

The target diseases targeted by the present invention are those caused by NADPH oxidase, such as vascular disorders, NF-κB activation-related diseases, IL-6 expression-related diseases, TNF-α activity-related diseases, Angiotensin II activity-related diseases, and advanced glycation end product (AGE) signaling-related diseases are included.
Vascular disorders include vasculitis, atherosclerosis, vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver Diseases, kidney diseases, cerebral infarction and the like are included.

Examples of NF-κB activation-related diseases include vasculitis, atherosclerosis, acute coronary events, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, Includes kidney disease, and cerebral infarction.
NF-κB, a transcription factor, regulates gene expression of pro-inflammatory cytokines and growth factors and plays a central role in the development of vascular dysfunction and atherosclerosis (Non-patent Document 34). In the present invention, the present inventors have discovered that TNF-α significantly induces IL-6 expression at the mRNA and protein levels through activation of NF-κB. PEDF or antioxidant NAC significantly inhibited TNF-α-induced NF-κB activation and subsequent elevation of IL-6 mRNA expression, so PEDF decreased IL-6 expression through its antioxidant properties It is possible that we have shown a lot of evidence about it as described herein.

  Examples of IL-6 expression-related diseases include coronary events, vasculitis, atherosclerosis, acute coronary events, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver Diseases, kidney disease, cerebral infarction, and malignant myeloma are included. Most of the systemic inflammatory response in atherosclerosis can be explained by the action of IL-6. IL-6 is a major inducer of hepatic synthesis of C-reactive protein (CRP) and elevated serum levels of IL-6 and CRP are associated with poor prognosis in patients with unstable angina (Non-patent documents 35, 36). In addition, several recent studies have shown that CRP may be an independent predictive risk factor for cardiovascular disease in seemingly healthy middle-aged men (37). Therefore, PEDF could have a beneficial effect on the progression of atherosclerosis by weakening IL-6 and CRP production. From the above, PEDF may play an important role in the development and progression of atherosclerosis.

  Examples of TNF-α activity-related diseases include vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney Diseases, and cerebral infarction are included. TNF-α exerts a multifaceted action on a plurality of cell functions through generation of ROS (Non-patent Documents 8, 13, and 14). In fact, the examples shown below show that TNF-α induces ROS generation, NF-κB activation and IL-6 expression. In contrast, the examples shown below also show that these functions elicited by TNF-α were actually suppressed or inhibited by PEDF. Thus, PEDF could be used to prevent or treat TNF-α activity, particularly TNF-α activity-related diseases based on NADPH oxidase activity.

  Angiotensin II activity-related diseases include vasculitis, atherosclerosis, acute coronary events, hypertension, insulin resistance, obesity, diabetic vascular complications, and cerebral infarction. Angiotensin II (AngII) is the dominant effector of the renin-angiotensin system and is known to modulate numerous inflammatory proliferative responses in vascular wall cells and thereby cause arteriosclerosis Yes. The present inventors combined the test results other than the test results of the following Examples, and PEDF inhibited the activation of AngII-induced vascular endothelial cells in vitro, and the mechanism was mediated by NADPH oxidase. We found that this was due to the suppression of ROS generation. This revealed that PEDF has an important preventive and therapeutic effect on the onset and exacerbation of arteriosclerosis. Based on this finding, the present invention further suppresses angiotensin II activity due to the inhibitory action of PEDF on NADPH oxidase activity, and as a result, angiotensin II-derived diseases such as vascular disorders, in particular, cardiovascular diseases such as arteriosclerosis. A prophylactic or therapeutic agent can be provided.

The present invention further provides a pharmaceutical composition for preventing or treating diabetic vascular complications associated with the production of glycation end products (AGE), ie, glycation end products (AGE) signaling-related diseases, by further suppressing NADPH oxidase activity. I will provide a.
Advanced glycation end product (AGE) signaling-related diseases include diabetic vascular complications such as proliferative diabetic retinopathy.

In addition, the present inventors have found in this study that PEDF alone does not affect NADPH oxidase activity, NF-κB promoter activity or IL-6 mRNA level in TNF-α-exposed HUVECs. That is, PEDF does not appear to have a detrimental effect on EC in the basic state. Thus, the compositions and vectors of the present invention can be used to prevent or treat a number of diseases described herein more safely, more conveniently, and without concern about side-effect problems.
Based on this finding, the present invention provides a pharmaceutical composition and / or vector for preventing or treating from a disease caused by NADPH oxidase. In this embodiment, the present invention only prevents or treats a disease caused by NADPH oxidase and does not exhibit an excessive effect.

(2) Active ingredient contained in the pharmaceutical composition of the present invention The active ingredient can be selected from the group consisting of:
a) pigment epithelium-derived factor;
b) a variant of the factor having a functionally equivalent property to the pigment epithelium-derived factor (a); and
c) A vector comprising a nucleic acid molecule encoding the factor (a) or the mutant (b).
(2) -1. Pigment epithelium-derived factor The amino acid sequence and nucleic acid sequence of pigment epithelium-derived factor (PEDF) are described in Proc. Natl. Acad. Sci. USA 90 (4), 1526-1530 (1993). , Registered with Genebank Accession Number M76979. For reference, the amino acid sequence and nucleic acid sequence of human PEDF are shown in SEQ ID NOs: 1 and 2, respectively. According to the present invention, PEDF includes all types of PEDF from mammals such as humans, dogs, cats, cows, and horses, with human-derived PEDF being preferred.
PEDF can be produced by expressing the DNA encoding the protein according to many publications and references such as Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989). In particular, the expression plasmid is constructed by inserting the DNA of the present invention into an appropriate expression vector (eg pBK-CMV). Then, the expression plasmid is introduced into an appropriate host cell to obtain a transformed cell. Examples of host cells include prokaryotes such as E. coli, unicellular eukaryotes such as yeast, and multicellular eukaryotic cells such as insects and animals. Expression plasmids can be introduced into host cells by conventional methods such as the calcium phosphate method, electroporation method, lipofectin method and the like. The desired protein is produced by culturing the transformant in an appropriate medium according to a usual method. The protein thus obtained can be isolated and purified according to standard biochemical techniques.

As used herein, “variants of PEDF functionally equivalent to PEDF” include all types of PEDF variants having properties functionally equivalent to human PEDF. For example, mutants of PEDF are described in US6319687, WO03059248, WO9324529 and the like.
Preferred examples of PEDF variants include amino acid sequences having one or more or several amino acid residue changes that are substitutions, deletions and / or additions to the amino acid sequence of human PEDF, and Variants of PEDF that have functionally equivalent properties to human PEDF are included.
Functionally equivalent to human PEDF means suppression of NADPH oxidase activity equivalent to human PEDF.
Variants of the invention can also be prepared by the recombinant techniques described above.
Whether the mutants prepared as described above have functionally equivalent properties can be proved according to the examples described later in this specification.

A pharmaceutical composition of the invention containing PEDF or a variant thereof can also optionally include a conventional carrier.
A pharmaceutical composition of the invention containing PEDF or a variant thereof can be administered, for example, by intradermal, subcutaneous or intravenous injection. Preferably, such PEDF or variant thereof is administered directly to the site where the lesion is present by injection. The amount of protein in such a formulation will vary depending on the disease being treated, the age and weight of the patient, etc., but is typically administered daily (in one or several divided doses). PEDF or a variant thereof is administered at 0.0001 mg-1000 mg, preferably 0.001 mg-100 mg, more preferably 0.01 mg-10 mg every few days to several months.

(2) -2. Nucleic acid and vector The active ingredient contained in the pharmaceutical composition of the present invention is one that is a substitution, deletion and / or addition to the pigment epithelium-derived factor or the amino acid sequence of the pigment epithelium-derived factor. Alternatively, it may be a vector containing a nucleic acid encoding a variant thereof that contains several amino acid residue changes and has functionally equivalent properties to the pigment epithelium-derived factor.

  As used herein, “nucleic acid” includes DNA and RNA, which may be single-stranded or double-stranded. Nucleic acids can be readily prepared according to typical DNA synthesis or genetic engineering methods, for example as described in standard texts such as “Molecular Cloning”, 2nd ed., Cold Spring Harbor Laboratory Press (1989).

  By administering a nucleic acid incorporated into a vector to a cancer patient, PEDF or a variant thereof is highly expressed in cancer cells. That is, these vectors are useful for gene therapy as are routinely used. Examples of vectors include viruses that incorporate the aforementioned nucleic acids into DNA or RNA viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, poxviruses, polioviruses, or Sindbis viruses and introduce them into cells. Vector is included. Of these methods, those using retrovirus, adenovirus, adeno-associated virus or vaccinia virus are particularly preferred. Specific examples of the vector include pAd / CMV / V5-DEST Gateway Vector (Invitrogen).

The pharmaceutical composition containing the vector of the present invention may also include a conventional carrier, if desired.
A pharmaceutical composition comprising such a vector can be administered, for example, by intradermal administration, subcutaneous administration or intravenous injection. Preferably, such PEDF or variant thereof is administered directly to the site where the lesion is present. The amount of vector in such formulations will vary depending on the disease to be prevented or treated, the age and weight of the patient, etc., but is typically administered daily (in one or several divided doses). The nucleic acid is administered at 0.0001 mg-100 mg, preferably 0.001 mg-10 mg every few days to several months.
The present invention also encompasses the aforementioned vectors themselves.

(3) Binding protein The inventors further studied whether HUVEC has a membrane protein with binding affinity for PEDF.
As mentioned above, inhibition of TNF-α-induced ROS generation by PEDF suggests an antioxidant function of PEDF in TNF-α-exposed HUVECs. However, it must be investigated whether PEDF exerts its effect through interaction with the receptor of TNF-α. Since the present inventors have revealed for the first time that PEDF-specific binding protein exists in the cell membrane of cultured HUVEC (FIG. 6A), the possibility is unlikely. Scatchard analysis of the binding data revealed a single class high affinity receptor (FIG. 6B). Antibodies against peptides in the receptor binding region of PEDF (Non-patent Document 15) completely neutralized the antioxidant properties of PEDF (FIG. 1B). Furthermore, as shown in FIG. 6C, PEDF did not interfere with the binding of TNF-α to its receptor. Taken together, our results suggest that there are PEDF-specific binding proteins involved in the inhibition of TNF-α-induced ROS production in HUVEC. According to Non-Patent Document 30, it was reported that different types of PEDF-binding proteins with Kd values of 1.7-3.6 nM and 80-85 kDa exist on retinoblastoma, and cell type-specific PEDF receptor Suggests that the body may be present.

The invention is further illustrated by the following examples, which are not intended to limit the invention in any way. All values are shown as mean ± SEM. Unless otherwise indicated, one-way ANOVA was followed by Scheffe F test for statistical comparison.
TNF-α, DPI, lucigenin, NADH, NADPH, NAC and pyrrolidine dithiocarbamate (PDTC) used in this Reference Example and Examples were purchased from Sigma (St. Louis, MO). CDP-Star chemiluminescence system and ¹²⁵ I-TNF-α were purchased from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). The IL-6 ELISA system was obtained from Biosource (Camarillo, CA).

  In the examples below, we used HUVEC to examine ROS (reactive oxygen species) production induced by TNF-α. TNF-α exerts pleiotropic effects on many cell functions through the generation of ROS (Non-patent Documents 8, 13, and 14). Therefore, the present inventors first used TNF-α to examine whether and how PEDF can inhibit TNF-α-induced intracellular ROS production in HUVEC. Next, the present inventors examined the effect of PEDF on NF-κB activation and subsequent IL-6 expression in TNF-α-exposed HUVECs.

Reference Example 1 Purification of PEDF protein
PEDF protein was purified as previously reported (Non-patent Document 15). SDS-PAGE analysis of the purified PEDF protein showed a single band with a molecular weight of about 50 kDa, which showed a positive reaction with the anti-human PEDF monoclonal antibody (Non-patent Document 15). A polyclonal antibody against human PEDF was also prepared as previously reported (Non-patent Document 15).

The preparation of PEDF-expression vector and expression vector for PEDF purification is briefly described.
PEDF cDNA was first isolated from a human placenta cDNA library (Clontech, Palo Alto, Calif.) And inserted into a mammalian expression vector pBK-CMV (Stratagene, La Jolla, Calif.) As previously reported (non-patent literature). 35).
Briefly, a gene encoding PEDF was isolated from the cDNA library by PCR under the following conditions:
PCR primers
Fw: 5'-CTCAGTGTGCAGGCTTAGAG-3 '(SEQ ID NO: 3)
Rev: 5'-CCTTCGTGTCCTGTGGAATC-3 '(SEQ ID NO: 4)
x 10 pfu buffer 4 μl
dNTP (2 mM each) 4 μl
Primer (Fw) (10 μM) 4 μl
Primer (Rev) (10 μM) 4 μl
Mold (200 mg)
pfu polymerase (STRATAGENE) 0.5 μl
Distilled water / 40 μl total

PCR conditions (Parkin Elmer 2400)
(Step 1 x 1)
95 ℃ 5min
(Step 2 x 30)
95 ° C 0.5 min
60 ℃ 0.5min
72 ℃ 1.5min
(Step 3 x 1)
72 ℃ 10min
4 ℃ ∞min
After confirming amplification, the PCR product was ligated to the Sma I site of pBluescript II KS. The structure was confirmed by restriction enzyme digestion and sequencing, and the Xba I site was also confirmed to be contained on the 5 ′ side.
The PEDF PCR product cleaved with Xba I and Hind III (Brant) is inserted between the Nhe I (Brant) and Xba I sites of pBK-CMV (STRATAGENE), and the plasmid pBK-CMV-PEDF is the PEDF-expression vector. Got.

Next, an expression vector for purifying PEDF was constructed according to the following procedure.
Oligo DNA for His-Tag,
5'-AATTCCATCATCATCATCATCATTAAT-3 '(SEQ ID NO: 5) and
5′-CTAGATTAATGATGATGATGATGATGG-3 ′ (SEQ ID NO: 6) was synthesized and annealed to each other. In order to remove the PEDF stop codon and insert an Eco RI site at the 3 ′ end, 5′-CGGAATTCGGGGCCCCTGGGGTCC-3 ′ (SEQ ID NO: 7) and the aforementioned Fw primer (SEQ ID NO: 4), and pBK-CMV- as a template. Oligo DNA was synthesized by PCR using PEDF. The amplification product was cleaved with Bgl II and Eco RI, and the cleaved product was ligated to the aforementioned annealed His Tag oligo DNA. The ligated product was inserted into purified pBK-CMV-PEDF cut with Bgl II and Xba I. To insert a translation enhancer whose structure was confirmed by restriction enzyme digestion and sequencing, the enhancer portion isolated from pcDNA4-HisMax (Invitrogen) using Sac I and Xba I was ligated to the same restriction enzyme site of pBluescript II KS. After isolation of the clone, the vector was cleaved at the Nco I site (Brant) and Bam HI site to obtain a vector backbone.

In order to modify the 5 ′ end of PEDF, 5′-GCATGCAGGCCCTGGTGCTACTCC -3 ′ (SEQ ID NO: 8) (Sph I site inserted into PEDF 5 ′ end) and 5′-TTAGGTACCATGGATGTCTGGGCT -3 ′ (SEQ ID NO: 9), And oligo DNA was synthesized using pBK-CMV-PEDF as a template, and the synthesized product was inserted into pGEM-T easy (Promega). Clones were isolated and cut at the Sph I site (Brant) and Bam HI site. The resulting fragment was inserted into the vector backbone described above.
The obtained vector was cleaved at the Sac I site (Brant) and the Bam HI site, and the translation enhancer-PEDF 5 ′ portion was taken out. The fragment containing the translation enhancer-PEDF 5 'part was ligated to 3'His-Tag-containing pBK-CMV-PEDF cleaved at the Nhe I site (Brant) and Bam HI site to obtain an expression vector for purification of PEDF. The resulting vector was confirmed by restriction enzyme digestion and sequencing.

Reference Example 2 Preparation of HUVEC stock
HUVEC (Clonetics, CC-2517) was cultured in EBM medium supplemented with 2% fetal bovine serum, 0.4% bovine brain extract, 10 ng / ml human epidermal growth factor and 1 μg / ml hydrocortisone. In the examples, TNF-α treatment was performed in a medium without epidermal growth factor and hydrocortisone.

Example 1 Effect of PEDF on ROS generation in HUVEC (1)
The inventors first examined whether PEDF can inhibit ROS (reactive oxygen species) production in HUVEC exposed to TNF-α.
HUVECs were treated with 10 ng / ml TNF-α for the time indicated, and then intracellular ROS generation was detected using the fluorescent probe CM-H ₂ DCFDA (Molecular Probes Inc., Eugene, OR) as previously reported. (Non-patent documents 18, 19). As shown in FIG. 1A, ROS production began to rise at 4 hours and reached a maximum at 24 hours; incubation with 10 ng / ml TNF-α for 24 hours increased intracellular ROS production by approximately 1.4-fold. Concentrations of TNF-α higher than 10 ng / ml are toxic to HUVEC.

Treat HUVEC for 24 hours with or without 10 ng / ml TNF-α in the presence or absence of 1 or 10 nM PEDF, 50 nM DPI, or 5 μg / ml anti-PEDF antibody (Abs) Intracellular ROS generation was detected as described above. The ratio of ROS generation is relative to the value of the control without addition, and is shown on the vertical axis. The results are shown in FIG. 1B. PEDF or DPI was found to completely inhibit the increase in ROS production induced by TNF-α.
When a polyclonal antibody against PEDF was added in combination with PEDF, the antioxidant effect of PEDF in TNF-α-exposed HUVEC was completely abolished. 10 nM PEDF does not inhibit superoxide production by the xanthine / xanthine oxidase reaction in a cell-free system. These results suggest that PEDF itself does not have a free radical scavenging action.

Example 2 Effect of PEDF on ROS generation in HUVEC (2)
In Example 1, PEDF was shown to completely inhibit TNF-α-induced increase in ROS production, similar to DPI. However, since pharmacological inhibitors such as DPI are not completely specific for certain target cells, here we investigated the mechanism of PEDF inhibition of ROS production. In order to further investigate that ROS generation in TNF-α-exposed HUVECs is due to NADPH oxidase, we conducted experiments using DN-RacT17N (dominant-negative human Rac-1 mutant) overexpression technology. Went. DN-RacT17 completely blocks the action of GTPase Rac-1 (Non-patent Document 28), which is an essential component of NADPH oxidase.
As previously reported (Non-patent document 17), DN-RacT17N expression vector (provided by Dr. Horiguchi) (Non-patent document 16) or an empty vector was transiently transfected into HUVEC. DN-RacT17N-transfected and mock-transfected HUVEC were treated with 10 ng / ml TNF-α for 24 hours, and ROS was quantitatively analyzed. The ratio of ROS production is relative to the value of mock transfected control cells without TNF-α. As shown in FIG. 1C, overexpression of DN-RacT17N completely inhibited TNF-α-induced increase in ROS production.

We next examined whether TNF-α could induce ROS production through Rac-1 expression and activation in HUVEC.
HUVECs were treated with or without 10 ng / ml TNF-α in the presence or absence of 10 nM PEDF for 4 hours and then analyzed by reverse transcription polymerase chain reaction (RT-PCR). The lower panel shows a quantitative representation of Rac-1 gene induction. Data are normalized by the intensity of the β-actin mRNA-derived signal and contrasted with the control. As shown in FIG. 1D, TNF-α moderately but significantly increased Rac-1 mRNA levels, which were completely suppressed by 10 nM PEDF. Then, Rac activity was measured using Rac Activation Assay Kit (Upstate Biotechnology Inc., Lake Placid, NY). HUVECs were treated for 24 hours with or without 10 ng / ml TNF-α in the presence or absence of 10 nM PEDF. As shown in FIG. 1E, the Rac-GTP pull-down assay showed that TNF-α significantly activated Rac, which was also completely inhibited by PEDF treatment.

  These findings suggest that one of the key components of NADPH oxidase, Rac-1, is directly involved in TNF-α-induced ROS generation and is a molecular target for PEDF. On the other hand, N-ω-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, oxypurinol, a xanthine oxidase inhibitor, or rotenone, a mitochondrial NADPH dehydrogenase inhibitor, induced TNF-α in HUVEC Does not affect sex ROS generation. We have also found that PEDF does not increase apoptotic cell death in TNF-α-exposed HUVECs. These results suggest that the antioxidant effect of PEDF on TNF-α-exposed HUVECs is not simply due to its pro-apoptotic properties.

Example 3 Effect of PEDF on NADPH oxidase activity in HUVEC Next, the inventors examined whether PEDF can actually suppress NADPH oxidase activity.
Treatment of HUVEC with or without 10 ng / ml TNF-α in the presence or absence of 10 nM PEDF for 24 hours, followed by 1 mM EGTA, 150 mM sucrose, 5 μM lucigenin as an electron acceptor, and NADPH oxidase activity was measured by a luminescence assay in a phosphate buffer (50 mM, pH 7.0) containing 100 μM NADPH as a substrate (Non-patent Document 20). As shown in FIG. 2A, TNF-α significantly increased NADPH oxidase activity, which was completely suppressed by 10 nM PEDF. The increase in NADPH oxidase activity induced by TNF-α approximated the increase in ROS production. TNF-α increased NADPH oxidase activity by about 1.3 times. PEDF alone does not affect NADPH oxidase activity in HUVEC.

  HUVECs were treated for 24 hours with or without 10 ng / ml TNF-α in the presence or absence of 10 nM PEDF. The cells were then incubated in phenol red-free Dulbecco's modified Eagle's medium containing 2 μM dihydroethidium. After 30 minutes, the cells were imaged with a confocal laser microscope. As shown in FIG. 2B, cell staining with dihydroethidium also revealed that PEDF inhibited TNF-α-induced NADPH oxidase activity in HUVEC. TNF-α did not increase mRNA levels of p22phox or gp91phox, one of the key components of NADPH oxidase in HUVEC.

Example 4 Effect of PEDF on NF-κB promoter activity in HUVEC
ROS (reactive oxygen species) regulates the expression of numerous genes through the activation of the transcription factor NF-κB (Non-patent Document 29). We therefore investigated whether PEDF can attenuate NF-κB transcriptional activity induced by TNF-α in HUVEC.
A plasmid containing the NF-κB promoter linked upstream of the luciferase reporter gene was provided by Dr. Fujita T (Department of Tumor Cell Biology, Tokyo Metropolitan Institute of Medical Science) (Non-patent Document 21). Treat HUVEC for 4 hours with or without 10 ng / ml TNF-α in the presence or absence of 1 or 10 nM PEDF or the antioxidant 1 mM NAC, then NF-κB as previously reported Luciferase activity was measured (Non-patent Document 17). The ratio of luciferase activity is relative to the control value. As shown in FIG. 3A, TNF-α significantly increased NF-κB promoter activity. PEDF and the antioxidant NAC were found to partially but significantly inhibit TNF-α-induced increase in NF-κB promoter activity.

  In order to measure NF-κB p65 activity, nucleoproteins were extracted and their binding activity to the NF-κB p65 consensus sequence was measured using the TansFactor kit. HUVECs were treated for 4 hours with or without 10 ng / ml TNF-α in the presence or absence of 10 nM PEDF. Nucleoproteins are extracted from cells using the TransFactor extraction kit (BD Biosciences Clontech, Palo Alto, Calif.), And their binding activity to the NF-κB p65 consensus sequence using the TransFactor NF-κB p65 kit (BD Biosciences Clontech). It was measured. As shown in FIG. 3B, TNF-α increased NF-κB p65 activity, which was significantly inhibited by PEDF.

Example 5 Effect of PEDF on IL-6 mRNA and protein levels in HUVEC We next examined whether PEDF could affect IL-6 expression in HUVEC.
HUVEC treated with or without 10 nM PEDF, 1 mM NAC, or 10 μM PDTC (pyrrolidine dithiocarbamate), an inhibitor of NF-κB, with or without 10 ng / ml TNF-α Poly (A) ⁺ RNA was further isolated and analyzed by semi-quantitative RT-PCR as described previously (Non-patent Document 26). The amount of poly (A) ⁺ RNA template for amplification (30 ng) and number of cycles (28 cycles for Rac-1 gene; 35 cycles for IL-6 and PEDF genes; 22 cycles for β-actin gene) Selected in a linearly proceeding quantitative range (determined by plotting signal intensity as a function of template amount and number of cycles) (27). The primer sequences used in semi-quantitative RT-PCR were 5′-ACCCCTTCTGACTGAGCAATATGCC-3 ′ (SEQ ID NO: 10) and 5′-CAATAGGCAAAGCGACTACAGGAGG-3 ′ (SEQ ID NO: 11) per human Rac-1. ), 5′-CCTTCTCCACAAGCGCCTTC-3 ′ (SEQ ID NO: 12) and 5′-GGCAAGTCTCCTCATTGAATC-3 ′ (SEQ ID NO: 13) (Non-Patent Document 23) per human IL-6 mRNA and 5′-TCACAGGCAAACCCATCAAGCTGAC per human PEDF mRNA -3 ′ (SEQ ID NO: 14) and 5′-GCCTTCGTGTCCTGTGGAATCTGCT-3 ′ (SEQ ID NO: 15) (Non-patent Document 24). The internal oligodeoxyribonucleotide probes for detecting human IL-6 and PEDF cDNA were 5'-TCAATTCGTTCTGAAGAGGTGAGTGGCTGT-3 '(SEQ ID NO: 16) and 5'-CGCCCCATCCTCGTTCCACTCAAAGCC-3' (SEQ ID NO: 17), respectively. . The upstream and downstream primer sequences used in semi-quantitative RT-PCR to detect human β-actin mRNA are the same as previously reported (Non-patent Document 25).

  As shown in FIG. 4A, TNF-α significantly increased IL-6 mRNA levels, although partially due to PDTC (pyrrolidine dithiocarbamate), an inhibitor of PEDF, NAC, or NF-κB. Significantly inhibited. PEDF, NAC or PDTC (pyrrolidine dithiocarbamate) alone did not affect IL-6 mRNA levels.

  Thereafter, HUVECs were treated for 24 hours with or without 10 ng / ml TNF-α in the presence or absence of 1 or 10 nM PEDF. IL-6 protein released into the medium was measured using an ELISA kit according to the manufacturer's instructions. PEDF was also found to significantly inhibit IL-6 protein secretion from TNF-α-exposed HUVECs (FIG. 4B).

Example 6 PEDF binding protein in HUVEC We next examined whether HUVEC has a cell surface protein that exhibits binding affinity for PEDF.
Surfactant-soluble cell membrane proteins derived from HUVEC were separated on a 15% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. As previously reported (Non-Patent Document 15), membranes and 30 nM fluorescein-labeled PEDF were incubated in the absence (lane 1) or presence (lane 2) of a 20-fold excess of unlabeled PEDF protein. The resulting immune complex was visualized by CDP-Star chemiluminescence system. As shown in FIG. 6A, a HUVEC membrane protein having a molecular weight of about 60 kDa bound to a fluorescein-labeled PEDF protein. The binding signal was hardly detectable in the presence of a 20-fold excess of unlabeled PEDF protein, confirming the specificity of the binding of fluorescein-labeled PEDF to cultured HUVEC membrane extracts. Furthermore, treatment with 10 ng / ml TNF-α increases PEDF binding to the membrane in HUVEC by about 1.3 fold.

We next analyzed the PEDF binding profile and measured its physicochemical parameters. Radiolabeled PEDF was prepared from purified PEDF protein and ¹²⁵ I as previously reported (Non-patent Document 15). Incubation was carried out with ¹²⁵ I-PEDF protein at a concentration indicating HUVEC in the absence (black circle) or presence (non-specific binding, white circle) of a 20-fold excess of molecular unlabeled PEDF protein. Specific PEDF binding was measured as previously reported (Non-patent Document 15). Specific binding (black squares) was calculated by subtracting nonspecific binding from total binding. All experimental points were shown as an average of 4 cases. As shown in FIG. 6B, Scatchard analysis of the binding data revealed a single class binding site (B _max = 51,000 sites per HUVEC 1 cell) with a dissociation constant (K _d ) of 84.7 nM.

We next examined whether PEDF inhibits TNF-α receptor binding in HUVEC ( ¹²⁵ I-TNF-α binding assay). Incubate HUVEC with 10 ng / ml ¹²⁵ I-TNF-α for 90 min at 4 ° C in the presence or absence of 10 nM PEDF, then lyse the cells with 1 M NaOH and report the radioactivity of the cell lysate as previously reported. (Non-patent document 15). An unpaired t test was performed; p <0.05 was considered significant. As shown in FIG. 6C, PEDF did not affect the binding of TNF-α to the receptor.

Example 7 Anti-atherosclerotic action of PEDF (1) Effect of PEDF on AngII-induced NF-κB promoter activity in HUVEC
HUVEC were treated with 100 nM angiotensin II (AngII) in the presence or absence of 10 nM PEDF for 4 hours, and NF-κB luciferase activity was measured. AngII untreated was also tested.
That is, HUVEC were cultured in EBM medium supplemented with 2% fetal bovine serum (FBS), 0.4% bovine brain extract, 10 ng / mL human epidermal growth factor and 1 μg / mL hydrocortisone. AngII treatment was performed in a medium in which epidermal growth factor and hydrocortisone were removed from the medium. The luciferase assay was measured by the method described in Yamagata S et al., J. Cardiovasc. Pharmacol. 2004; 43: 724-730, using a plasmid in which the NF-κB promoter was connected upstream of the luciferase reporter gene. It was.
The results are shown in FIG. As is apparent from FIG. 7, AngII significantly increased NF-κB promoter activity, while PEDF completely suppressed the action.

(2) Effect of PEDF on AngII-induced MCP-1 mRNA and protein level increase in HUVEC
MCP-1 is one of the NF-κB target genes and induces chemotaxis to monocytes in vitro and in vivo. Therefore, we examined how PEDF affects the expression of MCP-1 in HUVEC. To test the effect on MCP-1 mRNA, Poly (A) ⁺ RNA isolated from HUVEC was treated with 100 nM AngII for 4 hours in the presence or absence of 10 nM PEDF, RT-PCR Measured at AngII untreated was also tested. The RT-PCR method was performed by the method described in Yamabeishi S et al. Diabetologia. 1998; 41: 1435-1441. To test the effect on MCP-1 protein, HUVEC was treated with 100 nM AngII in the presence or absence of 10 nM PEDF for 24 hours, and MCP-1 protein produced in the medium was analyzed by ELIZA method. It was measured. AngII untreated was also tested.
The results are shown in FIG. As shown in FIGS. 8A and B, AngII significantly increased MCP-1 expression at the mRNA and protein levels, which was completely inhibited by PEDF.

(3) Effect of PEDF on AngII-induced intramolecular ROS generation in HUVEC
ROS is known to modulate the expression of genes that induce arteriosclerosis through transcriptional activation of NF-κB (see, eg, Cir. Res. 1994; 74: 1141-1148 by Griendling KK et al.). On the other hand, it has been found that NAC, an antioxidant, acts on NF-κB activation and MCP-1 expression in HUVEC exposed to AngII. From these, ROS generated by AngII may be a molecular target of PEDF. Therefore, the effect of PEDF on intramolecular ROS generation in HUVEC was investigated. First, the effect of AngII on ROS generation was examined. In the experiment, HUVEC was treated with 100 nM AngII for the time shown in FIG. 9A, and the produced ROS was quantitatively analyzed using a fluorescent probe CM-H ₂ DCFDA (Molecular Probe Inc., USA) ( The detection method is described in, for example, Yamazaki S. et al., J. Biol. Chem. 2001; 276: 25096-25100). As shown in FIG. 9A, ROS begins to occur after 4 hours with Ang II and is maximal at 24 hours. In 24-hour culture with 100 nM AngII, 1.4-fold ROS generation was observed. Therefore, the effect of PEDF on the occurrence of ROS was investigated. HUVEC treated with 1 nM or 10 nM PEDF, 10 nM PEDF with 50 μg / mL anti-PEDF antibody, and 100 nM AngII in the presence or absence of 50 nM DPI for 24 hours ROS was quantified in the same manner as described above. As a result, as shown in FIG. 9B, PEDF suppressed the increase of ROS induced by AngII in a concentration-dependent manner, and was completely suppressed by 10 nM PEDF. In the system where anti-PEDF antibody (for example, J. Mol. Cell. Cardiol. 2004; 37: 497-506 by Yamagishi S. et al.) Coexisted, the inhibitory action was neutralized. It can be seen that PEDF acts specifically. Moreover, since DPI known as a NADPH oxidase inhibitor suppressed ROS generation, the molecular action mechanism of PEDF is considered to be suppression of NADPH oxidase.

(4) Effect of PEDF on AngII-induced NADPH oxidase activity in HUVEC The above examples suggested that PEDF inhibited NADPH oxidase activity and acted on AngII. This was confirmed by this experiment. did.
HUVECs were treated with 100 nM Ang II in the presence or absence of 1 nM or 10 nM PEDF, 10 nM PEDF and 5 μg / mL anti-PEDF antibody for 24 hours to prepare cells in a homogeneous solution 50 mM phosphoric acid containing NADPH (100 μM) as substrate, suspended in buffer (20 mM Hepes, pH 7.0, 100 mM potassium chloride, 1 mM EDTA containing protease inhibitor cocktail) NADPH was measured by phosphorescence analysis in buffer (containing 7.0, 1 mM EDTA, 150 nM sucrose and 5 μM lucigenin as electron acceptor) (eg, Circ by Griendling KK Res. 1994; 74: 1141-1148).
The results are shown in FIG. As is clear from FIG. 10, PEDF suppressed the concentration of the NADPH oxidase activity induced by AngII in a concentration-dependent manner. On the other hand, when the anti-PEDF antibody was coexisted, the inhibitory effect was neutralized.

Example 8 Application to diabetic vascular complications associated with the production of advanced glycation end products (AGE) by inhibiting NADPH oxidase activity
(1) Production of intracellular reactive oxygen species in microvascular endothelial cells and effects on NADPH oxidase activity
(a) Antisense DNA (10 μM) for human adult skin microvascular endothelial cells (hereinafter referred to as vascular endothelial cells) to 1M or 10M PEDF, 50M DPI or advanced glycation end product (AGE) receptor (RAGE) mRNA Was treated with AGE-treated bovine serum albumin (AGE-BSA) or non-glycated bovine serum albumin at a predetermined concentration in the presence or absence of sucrose for 24 hours, and then reactive oxygen species (ROS) were quantitatively analyzed. Here, AGE-BSA was prepared by the method described in Non-Patent Document 15. Specifically, bovine serum albumin (50 mg / ml) was prepared by culturing under aseptic conditions with 0.1 MD-glyceraldehyde in 0.2 M sodium phosphate buffer (pH 7.4) for 7 days. Unreacted reducing sugar was removed by dialysis against phosphate buffered saline. Non-glycated bovine serum albumin as a control was prepared by incubation under the same conditions except that no reducing sugar was present. It was confirmed that the obtained preparation did not contain endotoxin (equipment used: Endospecy ES-20S, Seikagaku Corporation). Antisense DNA against RAGE mRNA was prepared according to the method described in FEBS Lett. 1996, 384: 103-106 by Yamagishi, S. et al. Intracellular generation of reactive oxygen species is performed using a fluorescent probe (CM-H2DCFDA, Molecular Probes Inc.) according to the method described in Kidney Int., 2003, 63: 464-473 by Yamagishi, S. et al. It was measured. The result is shown in FIG. 11 (a). In the figure, * indicates that the value treated with 100 μg / ml of AGE-BSA is significant at P <0.01. BSA indicates the result of treatment with non-glycated bovine serum albumin, and AGEs indicates the result of treatment with AGE-treated bovine serum albumin. As is clear from this figure, PEDF showed an inhibitory action superior to DPI on ROS production from serum albumin modified with AGE.

(b) Vascular endothelial cells were treated with 100 μg / ml AGE-BSA or non-glycated bovine serum albumin in the presence or absence of 10 nM PEDF for 24 hours, and NADPH oxidase activity was measured by the luminescence method. That is, the cells were suspended in a buffer solution (20 mM Hepes, pH 7.0, 100 mM potassium chloride, 1 mM EDTA containing a protease inhibitor cocktail), and the cell homogenate was prepared according to the method described in Non-Patent Document 20, 1 mM EDTA, 150 mM sucrose. Measurement was performed by a luminescence method in a 50 mM phosphate buffer (pH 7.0) containing 5 μM lucigenin as an electron acceptor and 100 μM NADPH as a substrate. The results are shown in FIG. 11 (b). As is clear from this figure, PEDF showed an excellent NADPH oxidase inhibitory action in serum albumin-treated cells modified with AGE.
Human adult skin microvascular endothelial cells (Clonetics Corp.), the vascular endothelial cells used in this experiment (a) and (b), are 5% fetal bovine serum, 0.4% bovine brain extract, 10 ng / ml human epithelial growth. The cells were cultured in eosin methylene blue medium supplemented with factors and 1 μg / ml hydrocortisone. AGE treatment was performed in a medium excluding human epidermal growth factor and hydrocortisone.

(2) Effect on serum AGE fractionation in microvascular endothelial cells AGE fraction was obtained from hemodialysis diabetic (DM-HD) serum or healthy human (Normal) serum with informed consent by a conventional method, and the fractionation Were used to treat microvascular endothelial cells in the presence or absence of 10 nMPEDF for 24 hours. The results of quantitative analysis of the production of superoxide, which is a reactive oxygen species, are shown in FIG. Here, superoxide generation is performed by culturing the cells obtained by the above treatment in phenol red-free Dulbecco's modified Eagle's medium (DMEM) containing 3 μM dihydroethidium, and measuring the fluorescence intensity after 30 minutes. Were imaged and quantified with a laser scanning confocal microscope. As is clear from this figure, PEDF significantly suppressed the production of reactive oxygen species from cells treated with the AGE fraction from hemodialysis diabetic patient (DM-HD) serum.

(3) Effects of intravenous administration on retinal vascular permeability in AGE perfused rats
According to the method of Adamis, AP et al. (Invest. Ophthalmol. Vis. Sci., 2000, 44: 2155-2162), the effect of PEDF on blood retinal barrier (hereinafter referred to as BRB) damage was examined. That is, SD rats were anesthetized and then intravenously injected with FITC-conjugated dextran (4.4 kDa, Sigma). After 10-15 minutes, blood samples were taken and each rat perfused with phosphate buffered saline. After perfusion, the retina was collected, weighed and homogenized to extract FITC-conjugated dextran. BRB damage was calculated by the formula of [(a) / (b)] / [(c) x (d)]. Here, (a) is the amount of FITC-conjugated dextran (μg) in the retina, (b) is the retinal weight (g), (c) is the FITC-conjugated dextran concentration in plasma (μg / mL), (d ) Indicates the circulation time (h). As shown in FIG. 13, in the AGE-BSA treatment group, the PEDF administration group exhibited a significant effect compared to the non-administration group.

  Example 8 shows that PEDF suppresses intramolecular reactive oxygen species in microvascular endothelial cells caused by terminal glycation products (AGE), inhibits NADPH oxidase activity induced by terminal glycation products (AGE), and PEDF. Showed significant activity against cells treated with AGE fraction from hemodialysis diabetic (DM-HD) serum. Furthermore, it showed efficacy against retinal vascular permeability in AGE perfused rats.

DISCUSSION In the present invention, we inhibit PEDF by restoring TNF-α-induced NF-κB activation and IL-6 expression in HUVEC from NADPH oxidase-mediated ROS (reactive oxygen species) production, Based on the following evidence: [1] TNF-α significantly stimulates intracellular ROS (reactive oxygen species) production in HUVEC, which is PEDF, NADPH oxidase inhibitor DPI (see Example 1), Or completely suppressed by overexpression of DN-RacT17N (dominant-negative human Rac-1 mutant) (see Example 2);
[2] PEDF completely suppressed TNF-α-induced increase in NADPH oxidase activity (see Example 3); and
[3] PEDF or antioxidant NAC significantly inhibited TNF-α-induced NF-κB activation and IL-6 expression (see Examples 4 and 5). Example 7 showed that PEDF significantly suppressed the NADPH oxidase activity induced by AngII in a concentration-dependent manner. Moreover, from Example 8, it was shown that PEDF could prevent or treat diabetic vascular complications associated with the production of advanced glycation end products (AGE) by suppressing NADPH oxidase activity.

It was interesting that PEDF reduced TNF-α-induced ROS production below control levels at 10 nM, while not affecting ROS production in the absence of TNF-α. The inventors do not understand the exact mechanism, but can be explained as follows. To examine the effect of TNF-α on PEDF gene expression in HUVEC, poly (A) ⁺ RNA was isolated from HUVEC treated with or without 10 ng / ml TNF-α and analyzed by RT-PCR did. As shown in FIG. 5, TNF-α was found to decrease PEDF mRNA levels in HUVEC. Therefore, we speculate that a decrease in PEDF mRNA may conversely increase its functional binding protein, thereby enhancing the inhibitory effect of externally administered PEDF on ROS production. doing. TNF-α treatment increased PEDF binding to the membrane in HUVECs by approximately 1.3-fold, which supports our speculation. The mechanism of PEDF mRNA reduction induced by TNF-α is not clear from our study. The present inventors have recently discovered that PEDF mRNA levels are suppressed under oxidative stress conditions in both cultured ECs and pericytes, so that TNF-α-induced ROS generation is effective in suppressing PEDF mRNA levels. It may be involved. Whatever the mechanism, the decrease in PEDF expression by TNF-α will further exacerbate oxidative stress-induced cell damage.

The effect of PEDF on intracellular ROS generation in HUVEC is shown. FIG. 1A is a graph showing the effect of TNF-α on intracellular ROS generation in HUVEC: *, P <0.01 (compared to values without TNF-α). FIG. 1B is a graph showing the effect of PEDF, DPI, or anti-PEDF antibody (Abs) on intracellular ROS generation in HUVEC: *, P <0.01 (compared to values of TNF-α alone). FIG. 1C is a graph showing DN-RacT17N versus ROS production in HUVEC: *, P <0.01 (compared to values of mock-transfected cells treated with TNF-α). FIG. 1D is a graph showing the effect of PEDF on TNF-α induced Rac-1 mRNA levels. FIG. 1E is a graph showing the effect of PEDF on TNF-α-induced Rac activity: *, P <0.01 (compared to the value of TNF-α alone). It is a graph which shows the effect of PEDF with respect to NADPH oxidase activity in HUVEC. FIG. 2A is a graph showing the effect of PEDF on TNF-α-induced NADPH oxidase activity: *, P <0.01 (compared to the value of TNF-α alone). FIG. 2B shows a typical photomicrograph of cells under a confocal microscope. FIG. 2 is a graph showing the effect of PEDF on NF-κB activity in HUVEC: *, P <0.05 (compared to the value of TNF-α alone). FIG. 3A is a graph showing that PEDF and NAC suppress TNF-α-induced increase in NF-κB promoter activity. FIG. 3B is a graph showing that TNF-α increases NF-κB p65 activity, which is significantly inhibited by PEDF: *, P <0.05 (compared to the value of TNF-α alone). Figure 6 shows the effect of PEDF on IL-6 mRNA and protein levels in HUVEC. FIG. 4A is a graph showing quantitatively the induction of IL-6 gene. Data were normalized by the intensity of the β-actin mRNA-derived signal and compared to control values: *, P <0.05; **, P <0.01 (compared to TNF-α alone values). FIG. 4B is a graph quantitatively showing the induction of IL-6 protein: *, P <0.01 (compared to the value of TNF-α alone). The effect of TNF-α on PEDF gene expression in HUVEC is shown. The lower panel shows quantitative induction of PEDF gene. Data were normalized by the intensity of the β-actin mRNA-derived signal and compared to the control value. An unpaired t test was performed; p <0.05 was judged to be significant: *, P <0.01 (compared to control cell values). The binding of purified PEDF protein (A and B) and TNF-α (C) to HUVEC is shown. FIG. 6A is a ligand blot with fluorescein-labeled PEDF. FIG. 6B is a graph showing a PEDF binding profile. FIG. 6C is a graph showing the effect of PEDF on TNF-α binding in HUVEC. FIG. 5 is a graph showing the effect of PEDF on angiotensin II (AngII) -induced NF-κB activity in HUVEC: * indicates significant at P <0.05 versus AngII alone treatment group. FIG. 6 is a graph showing the effect of PEDF on AngII-induced MCP-1 mRNA and protein level elevation in HUVEC: * indicates significant at P <0.01 vs. AngII alone treatment group. FIG. 8A is a graph showing the effect of PEDF on MCP-1 mRNA, and FIG. 8B is a graph showing the effect of PEDF on MCP-1 protein. It is a graph which shows the effect of PEDF with respect to AngII induction intramolecular ROS generation | occurrence | production in HUVEC. FIG. 9A shows that ROS is generated by AngII (* indicates that it is significant at P <0.01 with respect to the AngII untreated group), and FIG. 9B shows that PEDF suppresses ROS generation. (* And ** marks indicate significance at P <0.05 and P <0.01, respectively, compared to the values at AngII). It is a graph which shows the effect of PEDF with respect to the AngII induction NADPH oxidase activity in HUVEC (* and ** mark show that it is significant at P <0.05 and P <0.01, respectively, compared with the value in AngII). It is a graph which shows the effect | action of PEDF with respect to the production | generation of the intracellular reactive oxygen species in a microvascular endothelial cell, and NADPH oxidase activity (* is P <0.01 with respect to an AGE-BSA treatment group (it is written as AGEs in a figure). Is significant). It is a graph which shows the effect with respect to the serum AGE fraction in a microvascular endothelial cell (* shows that the PEDF administration group is significant at P <0.01 with respect to DM-HD group). It is a graph which shows the effect by intravenous administration with respect to retinal vascular permeability in AGE perfusion rat (* shows that PEDF administration group is significant at P <0.01 compared with non-administration group).

Claims (18)

a) pigment epithelium-derived factor;
b) a variant of the factor having a functionally equivalent property to the pigment epithelium-derived factor (a); and
c) A pharmaceutical composition for inhibiting NADPH oxidase activity, comprising an active ingredient selected from the group consisting of a vector comprising a nucleic acid molecule encoding the factor (a) or the mutant (b).   The pharmaceutical composition according to claim 1, which suppresses the production of NADPH oxidase-mediated reactive oxygen species.   The pharmaceutical composition according to claim 1 or 2, which inhibits NF-κB activation, IL-6 expression, TNF-α activity, angiotensin II activity, and / or terminal glycation end signaling by suppressing NADPH oxidase activity. Stuff.   The pharmaceutical composition according to any one of claims 1 to 3, for preventing or treating a disease caused by NADPH oxidase.   Diseases caused by NADPH oxidase are vascular disorders, NF-κB activation-related diseases, IL-6 expression-related diseases, TNF-α activity-related diseases, angiotensin II activity-related diseases, and terminal glycation product signaling- 5. A pharmaceutical composition according to claim 4 selected from the group consisting of related diseases.   6. The pharmaceutical composition of claim 5, wherein the vascular disorder is selected from the group consisting of vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, and cerebral infarction. .   NF-κB activation-related diseases include vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, 6. The pharmaceutical composition according to claim 5, which is selected from the group consisting of and cerebral infarction.   IL-6 expression-related diseases include coronary events, vasculitis, atherosclerosis, acute coronary events, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney 6. The pharmaceutical composition according to claim 5, selected from the group consisting of disease, cerebral infarction, and malignant myeloma.   TNF-α activity-related diseases include vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, cancer, inflammation, rheumatoid arthritis, liver disease, kidney disease, and 6. A pharmaceutical composition according to claim 5, selected from the group consisting of cerebral infarction.   6. The angiotensin II activity-related disease is selected from the group consisting of vasculitis, atherosclerosis, acute coronary event, hypertension, insulin resistance, obesity, diabetic vascular complications, and cerebral infarction. Pharmaceutical composition. The pharmaceutical composition according to claim 1 for treating diabetic vascular complications associated with the production of glycation end products by inhibiting NADPH oxidase activity.   6. The pharmaceutical composition according to claim 5, wherein the terminal glycation product information transmission-related disease is diabetic vascular complication associated with terminal glycation product generation. a) pigment epithelium-derived factor; or
b) A vector for suppressing NADPH oxidase activity, comprising a nucleic acid encoding a variant of the factor having a property equivalent to that of the pigment epithelium-derived factor (a).   14. The vector of claim 13, which inhibits NADPH oxidase-mediated reactive oxygen species production.   The vector according to claim 12 or 14, which inhibits NADPH oxidase activity to inhibit NF-κB activation, IL-6 expression, TNF-α activity angiotensin II activity and / or terminal glycation product signal transduction.   To prevent or treat vascular disorders, NF-κB activation-related diseases, IL-6 expression-related diseases, TNF-α activity-related diseases, angiotensin II activity-related diseases, and terminal glycation end product signaling-related diseases The vector according to any one of claims 13 to 15. 14. The vector according to claim 13 for treating diabetic vascular complications associated with the production of advanced glycation end products by inhibiting NADPH oxidase activity. The vector according to claim 16, wherein the glycation end product signal transduction-related disease is diabetic vascular complication associated with end glycation end product generation.

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