JP2007528355A - Compositions and methods for inhibiting vascular wall inflammation and neointimal hyperplasia - Google Patents

Abstract

Compositions and methods are provided that inhibit vascular wall inflammation and / or neointimal hyperplasia by gene therapy using a soluble Flt-1 (sFlt-1) gene. VEGF has an essential role in the development of neointimal hyperplasia by causing inflammation. By transferring the sFlt-1 gene to the site of vascular injury, Flt-1-mediated VEGF signaling is blocked, thereby inhibiting early inflammation and late neointimal hyperplasia. The present invention is useful for inhibiting or treating vessel wall inflammation and / or neointimal hyperplasia in patients at risk for post-coronary angioplasty restenosis, atherosclerosis, arteriosclerosis, or edema It is.

Description

  The present invention inhibits neointimal hyperplasia using gene therapy, more specifically, a gene encoding a soluble fragment of fms-like tyrosine kinase-1 (Flt-1). Or relates to compositions and methods of treatment.

  Neointimal hyperplasia (NIH) is a major cause of restenosis after coronary angioplasty (Libby P, Ganz P, “Restenosis revisited--new targets, new therapies.”, N. Engl. Med., 1997, 337: 418-9; and Topol EJ, Serruys PW, "Frontiers in interventional cardiology", Circulation, 1998, 98: 1802-20). Vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1: fms-like tyrosine kinase 1 receptor (Flt-1), VEGFR-2: endothelial type 2 receptor (Flk-1)) are atheromatous arteries It is up-regulated in vascular inflammation and proliferative disorders such as sclerosis and restenosis (Shibata M, Suzuki H, Nakatani M, Koba S, Geshi E, Katagiri T, Takeyama Y, “The involvement of vascular endothelial growth factor and flt-1 in the process of neointimal proliferation in pig coronary arteries following stent implantation. ", Histochem. Cell Biol., 2001, 116: 471-81; Ruef J, Hu ZY, Yin LY, Wu Y, Hanson SR , Kelly AB, Harker LA, Rao GN, Runge MS, Patterson C, "Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis", Circ. Res., 1997, 81: 24-33; Chen YX, Nakashima Y, Tanaka K, Shiraishi S, Nakagawa K, Sueishi K, “Immunohistochemical e xpression of vascular endothelial growth factor / vascular permeability factor in atherosclerotic intimas of human coronary arteries. ", Arterioscler Thromb Vasc. Biol., 1999, 19: 131-9; and Inoue M, Itoh H, Ueda M, Naruko T, Kojima A , Komatsu R, Doi K, Ogawa Y, Tamura N, Takaya K, Igaki T, Yamashita J, Chun TH, Masatsugu K, Becker AE, Nakao K, “Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions ： possible pathophysiological significance of VEGF in progression of atheroscleosis. ", Circulation, 1998, 98: 2108-16). VEGF is thought to protect the arteries from such disorders, primarily by inducing endothelial regeneration through the endothelial type 2 receptor Flk-1 and improving endothelial function, and VEGF gene transfer or its protein Administration induces endothelial regeneration after endothelial injury and decreases NIH (Baumgartner I, Isner JM, “Somatic gene therapy in the cardiovascular system.”, Annu. Rev. Physiol., 2001, 63: 427-50 ). However, there is still considerable controversy over the role of VEGF in the development of NIH after injury (Isner JM, “Still more debate over VEGF.”, Nat. Med., 2001, 7: 639-41; and Ware JA. “Too many vessels? Not enough? The wrong kind? The VEGF debate continues.”, Nat. Med., 2001, 7: 403-4). Evidence is being suggested to suggest that VEGF causes or accelerates the development of atherosclerosis or NIH after injury. VEGF, through the VEGF receptor Flt-1, migrates and activates monocytes (Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D, “Migration of human monocytes in response to vascular endothelial growth factor. (VEGF) is mediated via the VEGF receptor Flt-1. ", Blood, 1996, 87: 3336-43), adhesion molecules (Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY," Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. '', J. Biol. Chem., 2001 276: 7614-20), or monocyte chemoattractant protein-1 (MCP-1) (Marumo T, Schini-Kerth VB, Busse R, “Vascular endothelial growth factor activates nuclear factor-kappa B and induces monocyte chemoattractant protein- 1 in bovine retinal endothelial cells. ", Diabetes 1999, 48: 1131-7). In addition, administration of VEGF protein to hypercholesterolemic animals induces monocyte infiltration and activation and enhances atherogenesis (Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR , Dake MD, “Vascular endothelial growth factor enhances atherosclerotic plaque progression.”, Nat. Med., 2001, 7: 425-9). In addition, VEGF may promote vascular smooth muscle cell migration through Flt-1 (Grosskreutz CL, Anand-Apte B, Duplaa C, Quinn TP, Terman BI, Zetter B, D'Amore PA, “ Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro. ", Microvasc. Res., 1999, 58: 128-36; and Ishida A, Murray J, Saito Y, Kanthou C, Benzakour O, Shibuya M, Wijelath ES, “Expression of vascular endothelial growth factor receptors in smooth muscle cells”, J. Cell Physiol., 2001, 188: 359-68).

  Various functions of Flt-1 have been reported as well. Flt-1 in monocytes is involved in chemical migration (Barleon B et al., Supra) and Flt-1 in smooth muscle cells is involved in migration (Ishida A et al., Supra, and Wang H, Keiser JA, “Vascular endothelial growth. factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1. ", Circ. Res., 1998, 83: 832-40). Flt-1 acts as an important mediator of chemotaxis through VCAM-1, ICAM-1, and MCP-1 (Barleon B et al., Supra, Kim I et al., Supra, and Marumo T et al., Supra). Luttun et al. Reported that treatment with anti-Flt-1 antibodies attenuated the development of experimental tumor angiogenesis, arthritis, and atherosclerosis (Luttun A, Tjwa M, Moons L, Wu Y , Angelillo-Scherrer A, Liao F, Nagy JA, Hooper A, Priller J, De Klerck B, Compernolle V, Daci E, Bohlen P, Dewerchin M, Herbert JM, Fava R, Matthys P, Carmeliet G, Collen D, Dvorak HF, Hicklin DJ, Carmeliet P, “Revascularizaion of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis, and atherosclerosis by anti-Flt-1.”, Nat. Med., 2002, 8: 831-40.

  One reason for the disagreement regarding the role of VEGF may be due to the fact that there are no selective VEGF inhibitors examined. The only known endogenous VEGF inhibitor is the soluble form of Flt-1 (sFlt-1), this isoform is expressed primarily by vascular endothelial cells, by directly sequestering VEGF, and dominant negative inhibition It can inhibit VEGF activity by acting as an agent (Kendall RL, Wang G, Thomas KA, “Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem. Biophys. Res. Commun., 1996, 226: 324-8). Similarly, sFlt-1 probably binds to VEGF, but also binds to and blocks the ectodomain of membrane-bound Flt-1, so sFlt-1 has vasosuppressive properties due to antagonist activity against VEGF (Kendall RL & Thomas KA, “Inhibition of vascular endothelial cell growth factor activity by an inherently encoded soluble receptor.”, Proc. Natl. Acad. Sci. USA, 1993, November 15, 90 ( 22): 10705-9; Goldman CK, Kendall RL, Cabrera G, Soroceanu L, Heike Y, Gillespie GY, Siegal GP, Mao X, Bett AJ, Huckle WR, Thomas KA, Curiel DT, “Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate. ", Proc. Natl. Acad. Sci. USA, 1998, 95: 8795-800, International Publication No. 94/21679).

  WO 98/13071 discloses a gene therapy methodology for inhibiting primary tumor growth and metastasis by introgressing a nucleotide sequence encoding a soluble form of the VEGF tyrosine kinase receptor into a mammalian host is doing.

  It is an object of the present invention to clarify the role of VEGF in the development of NIH and to provide compositions and methods that inhibit vascular wall inflammation and / or NIH formation. The inventors have already demonstrated that transfection of the sFlt-1 gene into muscle effectively and specifically blocks VEGF signaling, thus eliminating VEGF activity in vivo (Zhao Q, Egashira K , Inoue S, Usui M, Kitamoto S, Ni W, Ishibashi M, Hiasa Ki K, Ichiki T, Shibuya M, Takeshita A, `` Vascular endothelial growth factor is necessary in the development of arteriosclerosis by recruiting / activating monocytes in a rat model of long-term inhibition of nitric oxide synthesis. "Circulation 2002, 105: 1110-5, and Goldman CK et al., supra). Subsequently, the inventors investigated the role of VEGF in the pathogenesis of NIH after cuff-induced periarterial inflammation in hypercholesterolemic mice. This cuff model has the advantage that cuff placement in the presence of hypercholesterolemia induces reproducible site-controlled NIH and remodeling, and cuff-induced damage induces vascular inflammation, Selected for the reason that it induces neointimal damage similar to the restenosis and atherosclerotic lesions observed in (Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh VW, van Bockel JH, Quax PH, “Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE * 3 Leiden transgenic mice.”, Circ. Res., 2000, 87: 248-53; and von der Thusen JH, van Berkel TJ, Biessen EA, "Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.", Circulation, 2001, 103: 1164-70).

  The inventors have discovered that blockade of VEGF by sFlt-1 gene transfer reduces early inflammation and proliferative changes, thus reducing the onset of NIH. Perivascular inflammation has a major role in the pathogenesis of cuff-induced NIH (Egashira K, Zhao Q, Kataoka C, Ohtani K, Usui M, Charo IF, Nishida K, Inoue S, Katoh M, Ichiki T, Takeshita A , "Importance of monocyte chemoattractant protein-1 pathway in neointimal hyperplasia after periarterial injury in mice and monkeys", Circ. Res., 2002, 90: 1167-72, and Wu L, Iwai M, Nakagami H, Li Z, Chen R , Suzuki J, Akishita M, de Gasparo M, Horiuchi M, `` Roles of angiotensin II type 2 receptor stimulation associated with selective angiotensin II type 1 receptor blockade with valsartan in the improvement of inflammation-induced vascular injury. '', Circulation, 2001, 104: 2716-21). Thus, increased expression and vascular inflammation and proliferation mediated by VEGF activity are essential in the pathogenesis of NIH after cuff-induced perivascular injury.

  VEGF is usually thought to be an endothelial cell-specific growth factor and is thought to reduce vascular disease by inducing endothelial growth and regeneration primarily through the endothelial type 2 receptor Flk-1 ( Baumgartner I et al., Supra). However, recent evidence suggests that functional VEGF receptors are expressed in damaged arterial walls of cells other than endothelial cells. Thus, the relative effect of Flt-1 versus Flk-1 mediated action may depend on the relative expression of Flt-1 and Flk-1 in target cells. We herein demonstrate that Flt-1 was increased in injured monocytes and intimal smooth muscle cells at an early stage and in neointimal and intimal smooth muscle cells at a later stage. To do. Increased Flk-1 expression was observed only in the late stage. Taking into account the previously reported Flt-1 function (Barleon B et al., Supra; Ishida A et al., Supra; Wang YH et al., Supra; Kim I et al., Supra; and Marumo T et al., Supra) , Cause inflammation and migration of vascular smooth muscle cells through Flt-1 mediated signals, and thus may cause NIH after cuff-induced periarterial injury.

  Evidence is being suggested to suggest that hematopoietic stem cells in the bone marrow are recruited after vascular injury and differentiate into neointimal cells. Sata et al. Found that a significant proportion of neointimal and medial cells were smooth in vascular injury in models of restenosis after angioplasty, transplant-related arteriosclerosis, and hyperlipidemia-induced atherosclerosis. Bone marrow-derived progenitor cells that differentiated into myocytes and endothelial cells were reported (Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R, “Hematopoietic stem cells differentiated into vascular cells that participate in the pathogenesis of atherosclerosis”, Nat. Med., 2002, 8: 403-9). Flt-1 is an important mediator of stem cell mobilization and migration (Luttun A, Tjwa M, Moons L, Wu Y, Angellilo-Scherrer A, Liao F, Nagy JA, Hooper A, Priller J, De Klerck B, Compernolle V, Daci E, Bohlen P, Dewerchin M, Herbert JM, Fava R, Matthys P, Carmeliet G, Collen D, Dvorak HF, Hicklin DJ, Carmeliet P., `` Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. ", Nat. Med., 2002, 8: 831-40. We have demonstrated herein that cells in the neointima or media are rarely expressed markers of bone marrow origin in bone marrow transplanted mice after cuff placement, which It shows that bone marrow-derived cells are hardly involved in neointimal formation in the model.

  In order to understand the mechanism of VEGF-mediated inflammation after cuff placement, we evaluated the gene expression of various inflammatory genes. sFlt-1 gene transfer reduced the increase in gene expression of inflammatory cytokines, adhesion molecules, chemokines, and chemokine receptors (FIG. 5). These data are consistent with previous reports showing that VEGF induces adhesion molecules (VCAM-1 and ICAM-1) or MCP-1 in endothelial cells in vitro (Kim I et al., Supra, and Marumo T Et al., Above). The essential role of these pro-inflammatory molecules in the development of neointimal hyperplasia after arterial injury has been reported (Egashira K et al., Usui M, Egashira K, Ohtani K, Kataoka C, Ishibashi M, Hiasa KI, Katoh M, Zhao Q, Kitamoto S, Takeshita A., `` Anti-monocyte chemoattractant protein-1 gene therapy inhibits restenotic changes (neointimal hyperplasia) after balloon injury in rats and monkeys. '', Faseb J., 2002, Mori E , Komori K, Yamaoka T, Tanii M, Kataoka C, Takeshita A, Usui M, Egashira K, Sugimachi K., `` Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointimal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic rabbits. ", Circulation, 2002, 105: 2905-10, and Oguchi S, Dimayuga P, Zhu J, Chyu KY, Yano J, Shah PK, Nilsson J, Cercek B," Monoclonal antibody against vascular cell adhesion molecule- 1 inhibits neointimal formation after periadventitial carot id artery injury in genetically hypercholesterolemic mice. ", Arterioscler Thromb. Vasc. Biol., 2000, 20: 1729-36). sFlt-1 gene transfer reduces the increase in VEGF and Flt-1 gene expression, indicating that VEGF is intracellular in arterial walls with diseases like smooth muscle cells, endothelial cells, and damaging monocytes It was shown that the activity is regulated by the mechanism of autocrine loop. Positive feedback for VEGF is supported by previous studies that showed monocyte enhancement of VEGF production by Flt-1 stimulation (Bottomley MJ, Webb NJ, Watson CJ, Holt L, Bukhari M, Denton J, Freemont AJ, Brenchley PE., “Placenta growth factor (PlGF) induces vascular endothelial growth factor (VEGF) secretion from mononuclear cells and is co-expressed with VEGF in synovial fluid.”, Clin. Exp. Immunol., 2000, 119: 182-8). Thus, sFlt-1 gene transfer reduced cuff-induced NIH primarily by suppressing inflammation (monocyte recruitment and activation).

  Taken together, VEGF and its receptor signals appear to be essential for the development of early inflammation as well as delayed NIH after cuff-induced perivascular cuff injury. The data presented herein supports the notion that VEGF acts as a pro-inflammatory and pro-arteriosclerotic factor after cuff-induced periarterial injury.

The present invention provides the following (1) to (30):
(1) A composition comprising a nucleic acid encoding soluble Flt-1 (sFlt-1) and a pharmaceutically acceptable carrier, wherein the nucleic acid inhibits inflammation of the blood vessel wall and / or neointimal hyperplasia Or a composition that expresses an effective amount of sFlt-1 for treatment;
(2) The composition according to (1), wherein the nucleic acid is inserted into a vector;
(3) The composition according to (2), wherein the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Sendai virus envelope (HVJ-E) vector;
(4) The composition according to (2), wherein the vector is a eukaryotic cell expression plasmid;
(5) The composition according to any one of (1) to (4), wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
(6) The nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 having one or more amino acid substitutions, deletions, additions, and / or insertions, and the polypeptide is SEQ ID NO: The composition according to any one of (1) to (5), which is functionally equivalent to a polypeptide comprising the amino acid sequence of 2 and has at least 65% identity;
(7) Any one of (1) to (6), wherein the amount effective to inhibit vascular wall inflammation and / or neointimal hyperplasia is about 0.0001 mg to 100 mg / day / patient. A composition according to claim 1;
(8) The composition according to any one of (1) to (7), which is administered intramuscularly to a patient;
(9) The composition according to any one of (1) to (8), which is administered to a patient at risk of restenosis after coronary angioplasty, atherosclerosis, arteriosclerosis, or edema;
(10) The composition according to (9), wherein the patient is a hypercholesterolemia patient;
(11) Use of a nucleic acid encoding soluble Flt-1 (sFlt-1) for the manufacture of a pharmaceutical composition that inhibits or treats vascular wall inflammation and / or neointimal hyperplasia;
(12) The use according to (11), wherein the nucleic acid is inserted into a vector;
(13) The use according to (12), wherein the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Sendai virus envelope (HVJ-E) vector;
(14) The use according to (12), wherein the vector is a eukaryotic cell expression plasmid;
(15) The use according to any one of (11) to (14), wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
(16) The nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 having one or more amino acid substitutions, deletions, additions, and / or insertions, and the polypeptide is SEQ ID NO: The use according to any one of (11) to (15), which is functionally equivalent to a polypeptide comprising the amino acid sequence of 2 and has at least 65% identity;
(17) The use according to any one of (11) to (16), wherein the composition is administered at a dose of about 0.0001 mg to 100 mg / day / patient.
(18) The use according to any one of (11) to (17), wherein the composition is administered intramuscularly to a patient;
(19) The composition according to any one of (11) to (18), wherein the composition is administered to a patient having a risk of restenosis after coronary angioplasty, atherosclerosis, arteriosclerosis, or edema. Use of;
(20) The use according to (19), wherein the patient is a hypercholesterolemia patient;
(21) A method of inhibiting or treating vascular wall inflammation and / or neointimal hyperplasia comprising the step of administering a nucleic acid encoding soluble Flt-1 (sFlt-1) to a patient in need thereof;
(22) The method according to (21), wherein the nucleic acid is inserted into a vector;
(23) The method according to (22), wherein the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Sendai virus envelope (HVJ-E) vector;
(24) The method according to (22), wherein the vector is a eukaryotic cell expression plasmid;
(25) The method according to any one of (21) to (24), wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
(26) The nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 having one or more amino acid substitutions, deletions, additions, and / or insertions, and the polypeptide has the SEQ ID NO: The method according to any one of (21) to (25), which is functionally equivalent to a polypeptide comprising the amino acid sequence of 2 and has at least 65% identity;
(27) The method according to any one of (21) to (26), wherein the nucleic acid is administered at a dose of about 0.0001 mg to 100 mg / day / patient;
(28) The method according to any one of (21) to (27), wherein the nucleic acid is administered intramuscularly;
(29) The method according to any one of (21) to (28), wherein the patient has risk factors for post-coronary angioplasty restenosis, atherosclerosis, arteriosclerosis, or edema; (30) The method according to (29), wherein the patient is a hypercholesterolemia patient.

Polynucleotides As used herein, a “soluble Flt-1 (sFlt-1) gene” refers to a polynucleotide or nucleic acid that encodes and expresses an sFlt-1 protein. Such a polynucleotide or nucleic acid may be DNA or RNA. It can be obtained by isolation or synthesis from natural materials.

  As used herein, an “isolated polynucleotide or nucleic acid” is separated from its original environment (eg, the natural environment if it is naturally occurring), and thus from its natural state. A “artificially” altered polynucleotide or nucleic acid.

  Thus, for example, this term is naturally occurring in the genomic DNA of a naturally occurring organism that does not include coding sequences that naturally flank the nucleic acid (ie, the sequences present at the 5 ′ and 3 ′ ends of the nucleic acid). (B) a nucleic acid incorporated into the vector or prokaryotic or eukaryotic genomic DNA such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c ) Individual molecules such as cDNA, genomic fragments, fragments produced by polymerase chain reaction (PCR), or restriction fragments; and (d) hybrid genes, ie recombinant nucleotides that are part of a gene encoding a fusion protein Spans an array. Nucleic acids present in random, uncharacterized mixtures of different DNA molecules, transfected cells, or cell clones, such as those occurring in DNA libraries such as cDNA libraries or genomic DNA libraries, are particularly Excluded from this definition.

  Naturally occurring nucleic acids may be derived from mammals including mice, rats, and humans. A known human sFlt-1 gene as shown in SEQ ID NO: 1 (Kendall RL et al., 1993, supra, GenBank accession number U01134), and a mouse sFlt-1 gene as shown in SEQ ID NO: 3 (GenBank accession) Session number D88690) can be used. The sFlt-1 gene used in the present invention can be synthesized based on its known sequence. For example, sFlt-1 cDNA can be cloned by performing an RT-PCR reaction on mRNA derived from a suitable source using a portion of the suitable DNA as a PCR primer. Such cloning can be easily performed by those skilled in the art according to references such as Maniatis T. et al., “Molecular Cloning”, 2nd edition, Cold Spring Harbor Laboratory Press (1989).

  As long as the polypeptide expressed from the sFlt-1 gene is functionally equivalent to sFlt-1, the sFlt-1 gene may have partial deletions, substitutions, or insertions of one or more nucleic acids. Or it may have other nucleotide sequences ligated to it at its 5 ′ end and / or 3 ′ end. As used herein, “sFlt-1 activity” means an activity that binds to VEGF but does not cause signal transduction.

  As used herein, the term “functionally equivalent” means that the test polypeptide retains a biologically important activity characteristic of sFlt-1. Biologically important activities of sFlt-1 include VEGF inhibitory activity that inhibits inflammation and migration of vascular smooth muscle cells. Therefore, in the present invention, as long as the obtained protein retains sFlt-1 activity, one or more amino acids are substituted, deleted, inserted and / or added. SEQ ID NO: 1 A polynucleotide comprising a nucleotide sequence encoding a protein having Furthermore, in the present invention, as long as the obtained polynucleotide encodes a protein functionally equivalent to sFlt-1, it hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1. Polynucleotides to be included. The determination of sFlt-1 was determined by the VEGF binding assay as described in Duan, DS, R. et al. (1991), J. Biol. Chem., 266: 413-418, and WO 94/21679. Can be performed by methods well known to those skilled in the art, such as the mitogen inhibition assay described in.

  The polynucleotide of the present invention can be obtained by methods well known to those skilled in the art. Examples of such methods include site-directed mutagenesis (Kramer W and Fritz, HJ (1987), Methods in Enzymol., 154: 350-367), hybridization techniques (EM Southern, J. Mol. Biol. , 1975, 98: 503-517) and polymerase chain reaction (PCR) technology (RK Saiki et al., Science 1985, 230: 1350-1354; RK Saiki et al., Science, 1988, 239: 487-491). More specifically, those skilled in the art generally use the polynucleotide shown in SEQ ID NO: 1 or a part thereof as a probe, or an oligo that specifically hybridizes with the polynucleotide shown in SEQ ID NO: 1. Using the nucleotide as a primer, a polynucleotide having high homology with the polynucleotide shown in SEQ ID NO: 1 can be isolated from other animals. Furthermore, polynucleotides that can be isolated by hybridization techniques or PCR techniques, and polynucleotides that hybridize to the polynucleotide shown in SEQ ID NO: 1 are also included in the polynucleotides of the present invention. Examples of such polynucleotides include those disclosed in WO 94/21679.

  The hybridization reaction for isolating the above polynucleotide is preferably carried out under stringent conditions. Hybridization may be performed using a buffer that allows the formation of a hybridization complex between nucleic acid sequences containing several mismatches. At high stringency, the hybridization complex is considered to remain stable only if the nucleic acid molecules are almost completely complementary. Many factors determine the stringency of hybridization, including the G + C content of the cDNA, salt concentration, and temperature. For example, stringency may be increased by decreasing the salt concentration or by increasing the hybridization temperature. The temperature conditions for hybridization and washing greatly affect stringency and can be adjusted using the melting temperature (Tm). Tm varies depending on the component ratio of hybridizing base pair nucleotides and on the composition of the hybridization solution (concentration of salt, formamide, and sodium dodecyl sulfate). In solutions used for some membrane-based hybridization, the reaction occurs at lower temperatures when organic solvents such as formamide are added. Thus, when considering the relevant parameters, one of ordinary skill in the art can select the appropriate conditions to obtain the appropriate stringency based on experience or experimentation.

  Examples of stringent hybridization conditions include conditions containing 65 ° C., 2 × SSC, 0.1% SDS, and conditions having stringency equivalent to this condition. In general, the higher the temperature, the higher the homology of the two strands that hybridize at equilibrium. A polynucleotide isolated under conditions of high stringency as described above is expected to encode a polypeptide having a higher homology at the amino acid level with the amino acid sequence shown in SEQ ID NO: 2. In this sense, “high homology” means at least 65% or more of the entire amino acid sequence, more preferably 70% or more, even more preferably 80%, even more preferably 90% or more, And most preferably means 95% or more identity.

Polypeptides The sFlt-1 protein encoded by the above nucleic acid includes human sFlt-1 shown in SEQ ID NO: 2, mouse sFlt-1 shown in SEQ ID NO: 4, and variants thereof. The variant is preferably encoded by a nucleotide sequence having at least 65% identity with the human sFlt-1 gene. More preferably, the variant is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% with the nucleotide sequence of the human sFlt-1 gene , Or higher identity. For example, if the isolated polynucleotide of the invention, eg, SEQ ID NO: 1, is longer or equivalent in length to the prior art sequence, a comparison is made for the full length of the sequence of the invention. Alternatively, if the isolated polynucleotide of the present invention is shorter than the prior art sequence, compare for a segment of the prior art sequence that is the same length as the sequence of the present invention (excluding any loops necessary to calculate homology) I do.

  Determination of percent identity between two sequences is determined by Karlin and Altschul (S. Karlin and SF Altschul, Proc. Natl. Acad. Sci. USA, 1990, 87: 2264-2268; S. Karlin and SF Altschul, Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-5877). Any conventional mathematical algorithm can be used. The BLAST algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (S.F. Altschul et al., J. Mol. Biol., 1990, 215: 403). When analyzing nucleotide sequences according to BLASTN, suitable parameters include, for example, score = 100, word length = 12. On the other hand, suitable parameters for amino acid sequence analysis by BLASTX include, for example, score = 50 and word length = 3. Gapped BLAST can be used as described in Altschul et al. (1997), Nucleic Acids Res., 25: 3389 to obtain alignments containing gaps for comparison purposes. Alternatively, iterative search can be performed to detect the distance relationship between molecules using PSI-Blast. When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters for the respective programs (eg, XBLAST and NBLAST) are preferably used. However, those skilled in the art can easily adjust the parameters to suit a particular purpose. Specific techniques for such analysis are known in the art (see, eg, the BLAST website of the National Center for Biotechnology Information on the web worldwide, www.ncbi.nlm.nih.gov ). Another example of a mathematical algorithm that can be used to compare sequences is the algorithm of Myers and Miller (1988), CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

  Polypeptides having amino acid sequences modified by deleting, adding, and / or substituting one or more amino acid residues of a particular amino acid sequence are known to retain their original biological activity (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984), 81: 5562-5666, Zoller, MJ & Smith, M., Nucleic Acids Research (1982), 10: 6487-6500; Wang, A Science 224: 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79: 6409-6413).

  The number of amino acids mutated by substitution, deletion, addition, and / or insertion is not particularly limited. Usually this is 10% or less of the total number of amino acid residues, preferably 5% or less, and more preferably 1% or less.

  Amino acid substitutions may be made at one or more predicted, preferably non-essential amino acid residues. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein (eg, the sequence shown in SEQ ID NO: 2) without altering biological activity, but the “essential” amino acid residue Groups are necessary for biological activity. The amino acid is preferably replaced with a different amino acid that preserves the side chain properties of the amino acid. Thus, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a chemically similar side chain. Groups of amino acid residues having similar side chains have been defined in the art. These groups include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, Tyrosine, cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), and aromatic side chains (eg , Tyrosine, phenylalanine, tryptophan, histidine).

Vectors The sFlt-1 gene of the present invention may be incorporated into various vectors. Any vector can be used as long as the vector allows in vivo expression of the gene. Examples of vectors include plasmids, liposomes, and viral vectors.

  As a plasmid vector, a eukaryotic plasmid containing pCAGGS (Gene 108: 193-200 (1991)), pBK-CMV, pcDNA3.1, and pZeoSV (Invitrogen, Stratagene) is preferably used. In such an expression vector, the gene of the present invention can be operably linked to a promoter / enhancer element. Promoter / enhancer elements may be selected to optimize for in vivo expression of the gene. The promoter may be inducible or constitutive and may optionally be tissue specific. Promoters isolated from viral genomes that grow in mammalian cells may be used, such as the vaccinia virus 7.5K, SV40, HSV, adenovirus MLP, MMTV, LTR, and CMV promoters.

  Alternatively, the sFlt-1 gene of the present invention can be encapsulated in any known liposome composed of a lipid bilayer such as an electrostatic liposome. Liposomes containing the sFlt-1 gene of the present invention can be fused to viruses such as inactivated Sendai virus (Hemagglutinating virus of Japan; HVJ). HVJ-liposomes have a very high fusion activity with respect to cell membranes compared to normal liposomes. In particular, the Z strain (available from ATCC) is preferred as the HVJ strain, but other HVJ strains (eg, ATCC VR-907 and ATCC VR-105) may be used similarly.

  Viral vectors can be used as well. The viral vector can be a DNA virus or an RNA virus. Examples of viral vectors include detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus envelope (HVJ-E, Sendai virus), SV40, and Human immunodeficiency virus (HIV) is included. Among the above viral vectors, adenovirus infection efficiency is known to be considerably higher than other viral vectors. In this respect, preferably an adenovirus vector system can be used.

  The above-mentioned vectors and liposomes can be prepared according to known methods ("Supplement of Experimental Medicine, Basic Technology in gene Therapy", Yodosha (1996); "Experimental Supplementary Experimental Medicine, Experimental Methods in Gene Introduction and Expression Analysis ”, Yodosha (1997);“ Handbook for Development and Research of Gene Therapy ” "Nippon Gene Therapy Society, NTS (1999), J. Clin. Invest. 93: 1458-1464 (1994); Am. J. Physiol. 271: R1212-1220 (1996), etc.).

Gene therapy substance The vector having the sFlt-1 gene of the present invention can be prepared in an appropriate gene therapy substance. The term “gene therapy substance” as used herein means a pharmaceutical composition used as a dosage form for gene therapy. The composition may vary depending on the dosage regimen (eg, liquid) described below. For example, the injection may be prepared by dissolving the gene in an appropriate solvent (buffer solution such as PBS, physiological saline, sterilized water, etc.). Next, the injection solution may be sterilized by filtration with a filter, if necessary, and filled into an aseptic container. A normal carrier or the like may be added to the injection. Liposomes such as HVJ-liposomes may be in the form of suspensions, frozen preparations, centrifugal concentrated frozen preparations and the like.

The gene therapy substance of the present invention is a vector having the sFlt-1 gene so that sFlt-1 is expressed in an amount effective to inhibit or treat inflammation of the blood vessel wall or to inhibit the formation of NIH including. Such inhibitory effects can be determined as described in the examples below. For example, the inflammation-inhibiting effect can be determined by measuring the number of Mac3-positive monocytes (see Example 2, Example 3 and Example 4, FIG. 3E). NIH formation inhibition can be determined by measuring intimal area, intima / media ratio, and percent stenosis (see Example 4, Figure 3B, Figure 3C and Figure 3D) ). The expression level of VEGF, Flt-1, CCR1, IL-6, CCR2, MCP-1, Flt-1, CXCR2, eotaxin, VCAM-1, or ICAM-1 is also an indicator of inflammation and NIH formation inhibition Can be used (see Example 3 and Example 5). With respect to VEGF levels, VEGF ₁₈₈ and VEGF ₁₆₄ or their corresponding isoforms should be determined, excluding VEGF ₁₂₁ or its corresponding isoform. Expression levels can be measured for mRNA or protein levels using known methods such as Northern blotting and Western blotting (eg, Maniatis, T. et al., Supra). Alternatively, NIH can be measured by cardiovascular angiography (CAG) or intravascular ultrasound (IVUS). By comparing the data obtained by these methods with the data obtained for normal NIH-free sites, the NIH formation inhibitory effect can be determined.

Gene Transfer Methods The gene therapy agents of the present invention may be introduced into target cells or patient tissues by in vivo or ex vivo methods. In vivo methods allow the direct introduction of gene therapy substances into the body. In the ex vivo method, a specific cell is collected from a human, a gene therapy substance is introduced into the cell, and the obtained cell is then returned to the body (Nikkei Science April 1994, pages 20-24) Monthly Yakuji 36 (1): 23-48 (1994); Supplement to Experimental Medicine 12 (15) (1994); Handbook for Development and Research of Gene Therapy ), NTS (1999)). According to the present invention, in vivo methods are preferred.

  Examples of methods for transferring genes into cells include lipofection method, calcium phosphate coprecipitation method, DEAE-dextran method, direct DNA introduction method using micro glass tube, and the like.

  Examples of methods for transferring genes to tissues include internal liposome method, electrostatic liposome method, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), receptor-mediated gene transfer, particle gun method , Naked DNA method, introduction method with positively charged polymer.

  Electroporation may be applied after gene transfer to enhance transgene expression. Microinjection or viral vectors can be used as well for efficient gene transfer or transgene expression.

  Appropriate methods and sites for appropriate administration for the disease or condition to be treated are selected for the gene therapy of the present invention. The gene therapy substance of the present invention can be administered parenterally, preferably intramuscularly, at the site of vascular injury.

  The dose of the substance of the present invention varies depending on the age, sex, and symptoms of the patient, but the sFlt-1 gene is about 0.0001 mg to about 100 mg / day / adult patient dose, preferably about 0.001 to about It can be administered at a dose of 10 mg / day / adult patient. When the HVJ-liposome type is used, the gene of the present invention can be administered in the range of about 1 to about 4000 μg, preferably about 10 to about 400 μg / adult patient.

  The therapeutic agents of the invention may be administered once every few days, once every few weeks, or once a week. The number of doses should be selected according to the patient's symptoms. Multiple doses are suitable depending on the purpose of the treatment.

  The gene therapy of the present invention is effective to inhibit vascular wall inflammation and / or NIH formation. Vascular wall inflammation and NIH formation are symptoms observed after vascular injury caused by post-coronary angioplasty restenosis, atherosclerosis, or arteriosclerosis. The gene therapy of the present invention can be applied to patients at risk for these diseases. Risk factors for these diseases include hypercholesterolemia.

  All publications cited herein are hereby incorporated by reference for a more detailed description of the state of the art to which this invention belongs.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail with reference to the following examples. However, it should be noted that the present invention is not limited in any way by these examples.

General Methods The following methods are general for all of the following examples.

Expression vector
A 3.3 kb mouse sFlt-1 gene (GenBank accession number D88690; nucleotide and amino acid sequences shown in SEQ ID NOs: 3 and 4, respectively) was obtained from a mouse lung cDNA library (Kondo K, Hiratsuka S, Subbalakshmi E, Matsushime H, Shibuya M, `` Genomic organization of the flt-1 gene encoding for vascular endothelial growth factor (VEGF) receptor-1 suggests an intimate evolutionary relationship between the 7-Ig and the 5-Ig tyrosine kinase receptors. '', Gene, 1998, 208: 297-305), cloned into the multicloning site, BamH1 (5 ′) and Not1 (3 ′) sites of eukaryotic expression vector plasmid cDNA3 (Invitrogen). In this plasmid, gene expression is controlled by the cytomegalovirus immediate early enhancer / promoter.

Experimental animals
Apolipoprotein E knockout mice (8-10 weeks old) with a genetic background of C57BL / 6J were purchased from Jackson Laboratory (Bar Harbor, ME) and given a standard commercial diet. It was. Cuff placement and gene transfer were performed as previously described (Zhao Q et al., Supra; and Egashira K et al., Supra). A non-stenotic polyethylene cuff (1.5-mm ng; PE20, 0.38-mm inner diameter, 1.09-mm outer diameter) was loosely placed around the left femoral artery. Either empty plasmid or sFlt-1 plasmid (300 μg / 100 μl PBS) was injected into the right thigh muscle immediately after and 10 days later using a 27 gauge needle. In order to enhance the expression of the transgene, these animals were electroporated at the injection site immediately after injection. Using an electric pulse generator CUY21 (BTX), 100 V 50 ms electric pulses were applied 6 times.

Cuff placement was also performed in wild-type mice with a C57BL / 6J genetic background that was replaced by the bone marrow of ROSA26 mice whose bone marrow expressed β-galactosidase (LacZ) broadly (Sata M et al., Supra). Wild type mice that were lethally irradiated were administered 1 × 10 ⁶ bone marrow cells from ROSA26 mice. Four weeks after the bone marrow transplant, the cuff was placed around the left femoral artery. The femoral artery was removed and stained with X-gal solution for 7 hours before further fixation in 4% paraformaldehyde.

Histopathology and immunohistochemistry Mice were anesthetized with pentobarbital, femoral arteries were harvested, fixed overnight in 3.7% formaldehyde phosphate buffered saline, and embedded in paraffin (Egashira K et al., Supra). ). Serial sections (thickness 5 μm) spanning the entire length of the cuffed femoral artery were used for histological analysis. Frozen sections were prepared from 2 mice for each condition. All sections were routinely stained with hematoxylin-eosin (HE) or van Gieson. Monocytes / macrophages were detected using Mac-3 (PharMingen) staining. Vascular proliferation was detected using proliferating cell nuclear antigen (Santa Cruz Biotech, CA). Endothelial cells were labeled with an antibody against von Willebrand factor (vWF; Sigma Chemical Co., St. Louis, MO). Indirect immunofluorescence double staining with matched primary antibody and fluorescein-conjugated secondary antibody was used for staining for co-localization with VEGF receptor in smooth muscle cells or monocytes: rabbit anti-mouse Flt-1 (Santa Cruz Biotech), rabbit anti-mouse Flk-1 (Santa Cruz Biotech), rat anti-mouse Mac-3, anti-smooth muscle actin (α-SMA) (Boehringer Mannheim), anti-rabbit IgG conjugated to FITC or rhodamine, and FITC Or anti-rat IgG conjugated to rhodamine (Santa Cruz Biotech).

Quantification of intimal damage in sections of cuffed femoral artery To quantify intimal damage, 10 equidistant sections were examined in all mice. Using image analysis software, the total cross-sectional area of the media between the outer elastic layer and the inner elastic layer was measured; the total cross-sectional area of the intima between the monolayer of endothelial cells and the inner elastic layer was measured .

Reverse transcription polymerase chain reaction and RNase protection assay
RNA was prepared from the collected samples (n = 5-7 for each group) using TRIzol reagent (Gibco-BRL). First strand DNA was synthesized using reverse transcriptase with random hexamers from 1 μg of total RNA in a reaction volume of 20 μl according to the manufacturer's protocol (GeneAmp RNA PCR kit; Perkin-Elmer). 25% of the polymerase chain reaction (PCR) was used to amplify 10% of the resulting reverse transcriptase (RT) product. Primers used for amplification of VEGF were 5′-GGA TCC ATG AAC TTT CTG CT-3 ′ (SEQ ID NO: 5) and 5′-GAA TTC ACC GCC TCG GCT TGT C-3 ′ (SEQ ID NO: 6). And the expected sizes for the three VEGF isoforms (VEGF 188, 164 and 121, respectively) were 654 bp, 582 bp and 450 bp. Primers for internal control β-actin are 5′-ATG GAT GAC GAT ATC GCT-3 ′ (SEQ ID NO: 7) and 5′-ATG AGG TAG TCT GCT AGG T-3 ′ (SEQ ID NO: 8) The expected product was 550 bp. PCR products were separated by 2% agarose gel electrophoresis, visualized with ethidium bromide, photographed and analyzed by scanning density measurement.

  The RNase protection assay was performed using two custom template sets and 5 μg total RNA according to the manufacturer's protocol (PharMingen). After RNase digestion, the protected probe was resolved on a denaturing polyacrylamide gel and quantified using the BASS-3000 system (Fujifilm). The value of each hybridized probe was normalized to the values of L32 and GAPDH, internal controls included within each template set.

Statistical analysis Data are expressed as mean ± SE. Statistical analysis on differences was compared by analysis of variance. Post hoc analysis was performed using Bonferroni correction for multiple comparison tests. P values less than 0.05 were considered statistically significant.

Results Example 1: Plasma Lipid Levels Total cholesterol, triacylglycerol, and high density lipoprotein-cholesterol levels in plasma were determined by a commercial kit (Wako Pure Chemicals, Osaka, Japan). There were no statistically significant differences in serum total cholesterol and triacylglycerol levels in the three groups, control group, empty plasmid group and sFlt-1. Total cholesterol and triacylglycerol levels were 503 ± 11 mg / dL and 38 ± 6 mg / dL in the control group, 512 ± 16 mg / dL and 40 ± 5 mg / dL in the empty plasmid group, and the sFlt-1 group And 497 ± 10 mg / dL and 39 ± 3 mg / dL.

Example 2: In vivo plug assay The in vivo Matrigel plug assay was used to determine the effect of sFlt-1 gene transfer on VEGF activity (Zhao Q et al., Supra; and Egashira K et al., Supra). Matrigel matrix alone (300 μl) or mixed with recombinant VEGF protein (100 ng / ml) was injected subcutaneously into the flank of C57BL / 6J mice. The matrigel was then removed 7 or 14 days after injection and examined for angiogenesis and inflammation by histopathological analysis.

Seven days after cuff placement, Matrigel plugs containing recombinant VEGF protein showed a significant angiogenic response (CD31 positive cells / mm ² , 380 ± 29 compared to Matrigel alone (8 ± 3 and 5 ± 2 respectively). ) And inflammatory reaction (Mac-3 positive cells / mm ² ; 87 ± 10 cells / mm ² ) were observed. Soluble Flt-1 gene transfer suppressed both the angiogenic response to VEGF (11 ± 5 / mm ² ) and the inflammatory response (6 ± 3 / mm ² ) to levels similar to Matrigel plugs without VEGF However, it was not suppressed when an empty plasmid was injected.

Example 3: Increased VEGF mRNA expression and immunoreactivity mRNA levels of the two VEGF isoforms (188 and 164) were significantly increased after cuff placement, but were undetectably low in control intact arteries (Figure 1A, Figure 1B). Peak expression was observed on day 7. VEGF121 mRNA was undetectable before and after cuff placement.

  In immunohistochemical staining, VEGF increased in the vicinity of inflammatory lesions in the intima and adventitia on day 7 and in three layers of cells in the cuff indwelling artery on day 21 compared to weak staining in control arteries (Figure 1C). Endothelial layers detected by vWF staining were preserved before and after cuff placement (Figure 1C).

  Flt-1 was undetectable except for the endothelial layer in control intact arteries, but increased dramatically in intima, media and adventitia at 7 and 21 days after cuff placement (Figure 1C). VEGFR-2 (Flk-1) did not increase on day 7, but increased on day 21. To identify the location of the VEGF receptor, immunofluorescence double staining was performed (Figure 2). On day 7, α-SM actin positive cells in the media and neointima expressed very little Flk-1, but they expressed Flt-1 (FIG. 2A). Mac-3 positive cells were recruited to neointima, media and outer membranes that express Flt-1 but not Flk-1. Similarly, some α-SM actin positive cells in the outer membrane (probably outer membrane myofibroblasts) expressed Flt-1. On day 21, many α-SM actin positive cells in the neointima and media expressed both VEGF receptors (FIG. 2B).

Example 4: Time course of development of neointimal hyperplasia Mice were sacrificed on days 1, 3, 7, 14, and 21. As published (Lardenoye JH et al., Supra; Egashira K et al., Supra; Wu L et al., Supra; and Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL, “Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. ", J. Clin. Invest., 1998, 101: 1225-32), most of which are Mac3- within 7 days after cuff placement. Mononuclear leukocytes, positive monocytes, were mobilized to the outer membrane, media and intima (Fig. 2). Seven days later, neointimal injury developed and thickened over time (FIG. 3A). Monocyte infiltration decreased spontaneously, and α-SM actin-positive cells appeared mainly in the neointima. On day 21, she developed significant NIH with stenosis of the lumen. Cells in the neointimal were mainly composed of α-SM actin positive cells.

  In order to determine whether bone marrow-derived progenitor cells are involved in the formation of neointimal, we used mice transplanted with bone marrow expressing β-galactosidase. Normal and cuff indwelling artery β-galactosidase (LacZ) was stained 21 days after cuff placement. LacZ-positive cells were hardly observed in the neointima or media (Fig. 4). Some mononuclear leukocytes recruited to the outer membrane were positive for LacZ.

Example 5: Soluble Flt-1 gene transfer reduces cuff-induced neointimal hyperplasia
sFlt-1 transfected mice had significantly less inflammation (Mac-3 positive cells) and proliferation (PCNA index) compared to day 7 empty plasmid transfected mice (FIG. 3E). Transfer of the sFlt-1 gene significantly reduced NIH (increased neointimal area, intima / media ratio, and luminal stricture) 21 days after cuff placement (Figure 3A, Figure 3B, Figure 3C and Figure 3D).

  A series of inflammatory cytokines, chemokines, and chemokine receptor gene expression was examined by RNase protection assay 7 days after cuff placement (FIG. 5). Gene expression was upregulated after cuff placement. sFlt-1 gene transfer did not affect the gene expression of RANTES, MIP-1α, TGF-β, MIP-2, but CCR1, IL-6, CCR2, MCP-1, Flt-1, Increased or decreased gene expression of CXCR2, eotaxin, VCAM-1, ICAM-1, and VEGF.

Industrial Applicability The present invention may have significant clinical significance. Blocking VEGF by sFlt-1 gene transfer can be an attractive anti-VEGF therapy for inflammatory vascular disease and other inflammatory disorders. In particular, the gene therapy is useful for inhibiting the development of NIH after vascular injury caused by restenosis, atherosclerosis, arteriosclerosis, or edema after coronary angioplasty. Thus, the compositions and methods of the present invention can be applied to patients at risk for these diseases, including hypercholesterolemia patients.

VEGF expression in the femoral artery indwelling the cuff. A, A photograph showing changes in VEGF mRNA level over time. Expression of arterial VEGF and β-actin mRNA after cuff placement. mRNA levels were evaluated at the indicated times. This is a representative assay from 5 different experiments. Density analysis of data in B and A. The expression of VEGF mRNA in each sample was normalized by β-actin mRNA expression in the same sample. N = 5 for each bar. * P <0.01 versus intact control artery. C, immunohistochemically stained for VEGF, VEGF receptor 1 (Flt-1), VEGF receptor 2 (Flk-1), or vWF on day 7 or 21 after cuff placement, A photograph showing a cross section of the aorta intact or with a cuff placed. Bar indicates 50 μm. Shown are immunofluorescent staining of VEGF receptor, monocytes, and α-SM actin in the femoral artery indwelling the cuff. A, 7 days after cuff placement, Flt-1 (VEGF-R1, green) and α-SM actin (red), Flk-1 (VEGF-R2, green) and α-SM actin (red), and Mac Photomicrograph of cuffed femoral artery stained with -3 (green) and Flt-1 (VEGF-R1, red). Bar indicates 10 μm. B, Cuff instillation femoral artery stained with Flt-1 (VEGF-R1) and α-SM actin and Flk-1 (VEGF-R2) and α-SM actin in the cuff instillation femoral artery 21 days after cuff placement Photomicrograph. Single fluorescence-positive cells stained green or red, whereas double positive cells stained yellow. The scale bar indicates 10 μm. The histopathology of the femoral artery with a cuff placed is shown. A, Photograph showing changes over time in cuff damage-induced NIH and the effect of sFlt-1 gene transfer. Micrographs of cross sections of control (intact) and cuff indwelling arteries stained with van Gieson Elastica (vGE) on days 3, 7 and 21. The scale bar indicates 100 μm. Effects of sFlt-1 gene transfer on B, C and D, neointimal thickening (B), intima / media ratio (C), stenosis (%) (D) 21 days after cuff placement. E, Effect of sFlt-1 gene transfer on changes in inflammation and proliferation 7 days after cuff placement. * P <0.01 vs control and sFlt-1 group. The involvement of bone marrow-derived cells in the development of neointimal after cuff placement is shown. A photomicrograph of a cross section stained with LacZ 21 days after cuff placement in bone marrow transplanted mice. There were no LacZ positive cells in the neointima or media. LacZ-positive cells were present only in the outer membrane (arrow). This is a representative sample from 6 animals. Scale bar indicates 50 μm. Chemokines and chemokine receptors (MCP-1, CCR2, Lantes, CCR1, MIP-1α, CXCR2, Eotaxin, MIP-2), adhesion molecules (ICAM-1, VCAM-1), cytokines (IL- 6 is a photograph showing the effect of sFlt-1 gene transfer on TGF-β), VEGF, and Flt-1. Data are expressed as the ratio of each mRNA to the corresponding GAPDH mRNA. * P <0.01 vs control site; † P <0.01 vs empty plasmid group; NS is not significant. N = 5-6.

Claims (30)

  A composition comprising a nucleic acid encoding soluble Flt-1 (sFlt-1) and a pharmaceutically acceptable carrier, wherein the nucleic acid inhibits or treats vascular wall inflammation and / or neointimal hyperplasia A composition that expresses an effective amount of sFlt-1.   2. The composition of claim 1, wherein the nucleic acid is inserted into a vector.   3. The composition of claim 2, wherein the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Sendai virus envelope (HVJ-E) vector.   The composition according to claim 2, wherein the vector is a eukaryotic cell expression plasmid.   The composition according to any one of claims 1 to 4, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.   The nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 having one or more amino acid substitutions, deletions, additions, and / or insertions, wherein the polypeptide is set forth in SEQ ID NO: 2. 6. The composition according to any one of claims 1 to 5, which is functionally equivalent to a polypeptide comprising the amino acid sequence of and has at least 65% identity.   7. The composition of any one of claims 1-6, wherein the amount effective to inhibit vascular wall inflammation and / or neointimal hyperplasia is about 0.0001 mg to 100 mg / day / patient. .   The composition according to any one of claims 1 to 7, which is intramuscularly administered to a patient.   9. The composition of any one of claims 1-8, wherein the composition is administered to a patient at risk for post-coronary angioplasty restenosis, atherosclerosis, arteriosclerosis, or edema.   10. The composition according to claim 9, wherein the patient is a hypercholesterolemia patient.   Use of a nucleic acid encoding soluble Flt-1 (sFlt-1) for the manufacture of a pharmaceutical composition for inhibiting or treating vascular wall inflammation and / or neointimal hyperplasia.   12. Use according to claim 11, wherein the nucleic acid is inserted into a vector.   13. Use according to claim 12, wherein the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Sendai virus envelope (HVJ-E) vector.   13. Use according to claim 12, wherein the vector is a eukaryotic cell expression plasmid.   15. Use according to any one of claims 11 to 14, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.   The nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 having one or more amino acid substitutions, deletions, additions, and / or insertions, wherein the polypeptide is set forth in SEQ ID NO: 2. 16. Use according to any one of claims 11 to 15, which is functionally equivalent to a polypeptide comprising the amino acid sequence of and has at least 65% identity.   17. Use according to any one of claims 11 to 16, wherein the composition is administered at a dose of about 0.0001 mg to 100 mg / day / patient.   18. Use according to any one of claims 11 to 17, wherein the composition is administered intramuscularly to the patient.   19. Use according to any one of claims 11 to 18, wherein the composition is administered to a patient at risk for post-coronary angioplasty restenosis, atherosclerosis, arteriosclerosis or edema.   20. Use according to claim 19, wherein the patient is a hypercholesterolemia patient.   A method of inhibiting or treating vascular wall inflammation and / or neointimal hyperplasia comprising the step of administering a nucleic acid encoding soluble Flt-1 (sFlt-1) to a patient in need thereof.   The method of claim 21, wherein the nucleic acid is inserted into a vector.   23. The method of claim 22, wherein the vector is selected from the group consisting of a plasmid, an adenovirus vector, and a Sendai virus envelope (HVJ-E) vector.   23. The method of claim 22, wherein the vector is a eukaryotic cell expression plasmid.   25. The method according to any one of claims 21 to 24, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.   The nucleic acid encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 having one or more amino acid substitutions, deletions, additions, and / or insertions, wherein the polypeptide is set forth in SEQ ID NO: 2. 26. The method of any one of claims 21-25, wherein the method is functionally equivalent to a polypeptide comprising the amino acid sequence of and has at least 65% identity.   27. The method of any one of claims 21-26, wherein the nucleic acid is administered at a dose of about 0.0001 mg to 100 mg / day / patient.   28. A method according to any one of claims 21 to 27, wherein the nucleic acid is administered intramuscularly.   29. The method of any one of claims 21-28, wherein the patient has risk factors for post-coronary angioplasty restenosis, atherosclerosis, arteriosclerosis, or edema.   30. The method of claim 29, wherein the patient is a hypercholesterolemia patient.

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