WO2006084007A2

WO2006084007A2 - Novel gene underlying atherosclerosis locus i

Info

Publication number: WO2006084007A2
Application number: PCT/US2006/003645
Authority: WO
Inventors: Alan R. Tall; Carrie Welch; Sara Bretschger Seidelmann
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2005-02-01
Filing date: 2006-02-01
Publication date: 2006-08-10
Anticipated expiration: 2007-08-01
Also published as: WO2006084007A3

Abstract

This invention provides an isolated nucleic acid encoding Adamtsl-1 protein and related compositions. This invention further provides a method of inhibiting expression of Adamtsl-1 in a cell. This invention further provides a method for inhibiting the onset of atherosclerosis in a subject and a method of treating atherosclerosis in a subject. This invention further provides transgenic animals. Finally, this invention provides for a method of screening for possible candidate agents for treating a human afflicted with atherosclerosis.

Description

NOVEL GENE UNDERLYING ATHEROSCLEROSIS LOCUS I

This application claims benefit of U . S . Provisional Application No . 60/648 , 837 , filed February 1 , 2005 , the contents of which are hereby incorporated by reference .

This invention was made with support under United States Government Grant No . HL54591 from the National Institutes of Health . Accordingly, the United States Government has certain rights in the subj ect invention .

Throughout this application, various publications are referenced . Full citations for these publications may be found immediately preceding the claims . The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art as of the date of the invention described and claimed herein .

Background of the Invention

Atherosclerosis, a progressive disease of the large arteries , is the primary cause of coronary heart disease and stroke . While environmental factors , including smoking, diet , and exercise, contribute to atherosclerosis susceptibility, an estimated 40-60% of variance is due to genetic factors [ 1 ] . However, the genes contributing to inter-individual variation in susceptibility are largely unknown [2 ] . One method of studying the genetics of complex diseases is through animal models . Several atherosclerosis loci have been mapped in the murine model [ 3-7 ] , but none of the underlying genes have been identified by positional cloning .

Summary of the Invention

This invention provides an isolated nucleic acid encoding Adamtsl-1 protein, an isolated Adamtsl-1 protein, an antibody that specifically binds to Adamtsl-1 protein and a vector comprising a nucleic acid encoding Adamtsl-1. This invention also provides a cell comprising a vector, which vector comprises a nucleic acid encoding Adamtsl-1.

This invention further provides a method for inhibiting expression of Adamtsl-1 in a cell wherein Adamtsl-1 expression would otherwise occur comprising contacting the cell with an agent that inhibits expression of Adamtsl-1 in the cell .

This invention further provides a method for inhibiting the onset of atherosclerosis in a subj ect at risk for becoming afflicted therewith, comprising administering to the subj ect a prophylactically effective amount of an agent that inhibits the expression of Adamtsl-1 in the subj ect' s cells , thereby inhibiting the onset of atherosclerosis in the subj ect .

This invention further provides a method of treating a subj ect afflicted with atherosclerosis comprising administering to the subj ect a therapeutically effective amount of an agent that inhibits the expression of Adamtsl- 1 in the subj ect' s cells , thereby treating the subj ect .

This invention further provides a transgenic animal whose somatic cells are characterized by a genotype comprising ( i ) an LDL-receptor knockout and (ii) a polymorphism for Adamtsl-1, wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions .

This invention further provides a transgenic animal whose somatic cells are characterized by a genotype comprising ( i ) an Apo-E knockout and (ii ) a polymorphism for Adamtsl- 1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions .

This invention further provides a transgenic animal whose somatic cells are characterized by a genotype comprising a polymorphism for Adamtsl-1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions .

This invention further provides a method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises : (a) administering the agent to a transgenic animal whose somatic cells are characterized by a genotype comprising

( i ) an LDL-receptor knockout and (ii) a polymorphism for

Adamtsl-1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ; (b) after a sufficient period of time, determining the severity of atherosclerotic lesions present in the transgenic animal ; and (c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions determined in step (b) is less than the severity of lesions present in the transgenic animal to which no agent was administered .

This invention further provides a method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis, which comprises : (a) administering the agent to a transgenic animal whose somatic cells are characterized by the genotype comprising (i ) an Apo-E knockout and (ii) a polymorphism for Adamtsl- 1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ; (b) after a sufficient period of time, determining the severity of atherosclerotic lesions present in the transgenic animal; and (c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions determined in step (b) is less than the severity of lesions present in the transgenic animal to which no agent was administered .

Finally, this invention provides a method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises : (a) administering the agent to a transgenic animal whose somatic cells are characterized by the genotype comprising a polymorphism for Adamtsl-1, wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and the transgenic animal has atherosclerotic lesions ; (b) after a sufficient period of time , determining the severity of atherosclerotic lesions present in the transgenic animal ; and (c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions determined in step (b) is less than the severity of lesions determined in the transgenic animal to which no agent was administered .

Brief Description of the Figures

Figure 1

Accelerated atherosclerotic lesion development in Athsql congenic mice . (a) Genetic map of mouse chromosome 4 showing the Athsql region . (b) B6-Ldlr^~/~ mice carrying one (b/m, 43-81 cM) or two (m/m, 43-54 cM) copies of the MOLF- derived Athsql interval exhibit increasing aortic root lesion area with increasing gene copy number compared to non-congenic littermate controls (b/b) after six-week WTD feeding . (c-k) Atherosclerotic lesions from the proximal aorta of B6-Ldlr^~/~ mice carrying zero, one or two copies of the MOLF-derived Athsql susceptibility locus and fed a Western-type diet for six weeks . (c-e ) H&E stain; ( f-h) Verhoeff' s stain for elastin (black) ; ( i-k) Masson' s trichrome stain for collagen (blue) . (c, f, i ) Typical focal fatty streak lesions of Athsql b/b non-congenic littermate controls . (d, g, j ) More advanced fatty- fibrous lesions of Athsql b/m heterozygotes . Some of the lesions have elastin- and collagen-containing fibrous caps

( arrows ) and large necrotic cores (NC) . (e, h, k)

Dramatically-advanced fibrous lesions of Athsql m/m homozygotes . The lesion area is nearly continuous around the vessel wall with a thick fibrous cap (closed arrow) , abundant intraplaque connective tissue (dotted arrow) , and large necrotic cores (NC) . (c, d, f, g, i, j ) IOOX magnification; (e , h, k) 4OX magnification .

Figure 2 Genetic and physical mapping of Athsql . (a) Localization to a <3.4 Mb interval of chromosome 4 in a set of overlapping congenic strains fed a WTD for twelve weeks . Filled boxes indicate B6 alleles and open boxes indicate

MOLF alleles . Positions of genetic markers are given in megabases (Mb) from the centromere as taken from Ensembl .

(b) Further localization to a <1.2 Mb interval using mice fed a WTD for six weeks . Lesion area is in μmVsection x

ICT⁴ ± SEM and necrotic core area is in μmVsection x ICT³ +

SEM. NS, not significant . ND, not determined. (c)

Reduction of the Λi±sgl-containing interval to a 0.5 Mb interval by SNP analysis . Filled bars represent sub- congenic intervals not conferring lesion susceptibility and the hatched bar represents extension of the sub-congenic interval conferring susceptibility. The recombination region is indicated with dotted lines showing that only Adamtsll and the unknown testis-specific EST Tex30 are within the smallest congenic interval associated with lesion susceptibility.

Figure 3

Expression of Adamtsll and Tex30. (a) Tissue expression pattern of Adamtsll and (b) Tex30 in a mouse cDNA panel normalized for housekeeping genes . Lanes ( 1 ) heart , (2 ) brain, ( 3) spleen, ( 4 ) lung, ( 5 ) liver, ( 6) skeletal muscle, (7 ) kidney, ( 8 ) testis , ( 9) 7-day embryo, ( 10) 11- day embryo, ( 11) 15-day embryo, ( 12 ) 17-day embryo . (c) Presence of Adamtsll and absence of Tex30 in cDNA derived from atherosclerotic aorta following six-week WTD feeding . The acidic ribosomal phosphoprotein PO (Arbp/36B4) housekeeping gene serves as a positive control . Similar results were observed in multiple mouse aortas . (d) Relative expression of Adamtsll in thioglycollate-elicited peritoneal macrophages from Athsql b/m and Athsql m/m congenics versus Athsql b/b controls using quantitative PCR . (e) Relative expression of Adamtsll , CD68, Mac2_r CD36, and Ahca 7 in atherosclerotic aorta following six-week WTD feeding in Athsql m/m (n=8 ) congenics versus Athsql b/b (n=5 ) controls using quantitative PCR . Data were normalized to the Arbp housekeeping gene .

Figure 4

Predicted domain structure of ADAMTSLl and related murine proteins .

Figure 5

Increased abundance of versican in Athsql congenic atherosclerotic plaques . Atherosclerotic lesions stained for versican (brown) from the proximal aorta of B6-JJdlr^~'/~ mice carrying zero or two copies of the MOLF-derived Athsql susceptibility locus and fed a Western-type diet for (a-b) 3.5 weeks ; (c-d) 6 weeks ; (e-f) 12 weeks . (a, c , e) Absence of staining for versican in lesions of Athsql b/b non-congenic littermate controls . (b, d, f) Accumulation of versican in lesions of Athsql m/m homozygotes , most dramatic at the 6 week time point . Intense staining was observed in the media (M) underlying atherosclerotic plaques and to a lesser extent near the surface and within cellular regions of the plaque (Pl) . 400X magnification .

Figure 6

Human Adamtsl-1 nucleotide (SEQ . ID . NO : 1) and amino acid sequence ( SEQ ID . N0 : 2 ) . Figure 7

(a) B6 ( SEQ . ID . NO : 3 ) and MOLF ( SEQ . ID . NO : 5 ) Adamtsl-1 nucleotide alignment . (b) Bβ (SEQ. ID. NO : 4 ) and MOLF ( SEQ . ID . NO : 6) amino acid alignment .

Figure 8

Effect of the Athsql locus on lesion development in a uniform genetic background.

Figure 9

ADAMTSLl protein expression . A. Full-length ADAMTSLl (250 kDa) is increased in aortic extracts from B6-Ldlr-/- congenic mice (m/m) compared to non-congenic controls (b/b) . In contrast, no difference was observed in the levels of the known splice variant , punctin ( 67 kDa) . The 75 kDa band corresponds to an unknown species present in aortic extracts but not macrophages . Briefly, 50 μg of protein were loaded on a 6% SDS-PAGE gel . Following overnight transfer, blots were incubated overnight with a polyclonal antibody raised against recombinant punctin at 1 : 250 dilution . B . Full-length ADAMTSLl (250 kDa) is secreted by macrophages . Thioglycollate-elicited peritoneal macrophages were collected from BG-LdIr-/- mice and cultured for 24 hours in DMEM plus 0.1% FBS . Excess albumin was removed from the media by immunoprecipitation using an anti-BSA agarose matrix (Sigma) . Western blotting was performed as for A. For both A and B, similar results were observed in three independent sets of samples .

Figure 10

Quantification of versican staining in atherosclerotic lesions derived from Athsql congenic or non-congenic littermates following six or twelve weeks of Western diet (WTD) feeding . B, B6-Ld2r-/- mice homozygous for B6 alleles at the congenic interval ; M, Bβ-Ldlr-/- mice homozygous for MOLF alleles . Morphometric analysis was performed using ImagePro Plus (NIH) .

Figure 11

Intact versican is increased in aortic homogenates from

Athsql congenic mice carrying copies of the MOLF-derived susceptibility allele . Proximal aortas were collected from 6-week WTD-fed mice . Two aortas were pooled for each homogenate . Immunoblots were incubated with a polyclonal antibody raised against a fusion protein of the β GAG domain of the core mouse protein . 97 , 99A is a pool from non-congenic controls and 15 , 16Α from congenics . Similar results were observed in^' three independent sets of samples .

Detailed Description of the Invention

Terms

"Adamtsl-1" is used herein to mean a disintegrin-like and metalloprotease with thromospondin-like repeats-1 like-1. "Adamtsl-1" and "Adamtsll" are used synonymously.

'"Administering" an agent can be effected or performed using any of the various methods and delivery systems known to those skilled in the art . The administering can be performed, for example, intravenously, orally, nasally, via the cerebrospinal fluid, via implant, transmucosally, transdermally, intramuscularly, and subcutaneously. The following delivery systems, which employ a number of routinely used pharmaceutically acceptable carriers , are only representative of the many embodiments envisioned for administering compositions according to the instant methods .

Inj ectable drug delivery systems include solutions (e . g. , water or buffer) , suspensions , gels, microspheres and polymeric inj ectables , and can comprise excipients such as solubility-altering agents (e . g . , ethanol , propylene glycol and sucrose ) and polymers (e . g . , polycaprylactones and PLGA' s ) . Implantable systems include rods and discs , and can contain excipients such as PLGA and polycaprylactone .

Oral delivery systems include tablets and capsules . These can contain excipients such as binders (e . g . , hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch) , diluents (e . g . , lactose and other sugars , starch, dicalcium phosphate and cellulosic materials ) , disintegrating agents (e . g . , starch polymers and cellulosic materials) and lubricating agents (e . g . , stearates and talc) .

Transmucosal delivery systems include patches , tablets , suppositories , pessaries , gels and creams , and can contain excipients such as solubilizers and enhancers ( e . g . , propylene glycol , bile salts and amino acids) , and other vehicles (e . g . , polyethylene glycol , fatty acid esters and derivatives , and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid) .

Dermal delivery systems include , for example, aqueous and nonaqueous gels , creams , multiple emulsions , microemulsions , liposomes , ointments , aqueous and nonaqueous solutions , lotions , aerosols , hydrocarbon bases and powders, and can contain excipients such as solubilizers , permeation enhancers ( e . g . , fatty acids , fatty acid esters , fatty alcohols and amino acids ) , and hydrophilic polymers (e . g . , polycarbophil and polyvinylpyrolidone) . In one embodiment , the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer .

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents ( e . g . , gums , zanthans , cellulosics and sugars ) , humectants ( e . g . , sorbitol ) , solubilizers ( e . g . , ethanol , water, PEG and propylene glycol ) , surfactants (e . g . , sodium lauryl sulfate , Spans , Tweens , and cetyl pyridine) , preservatives and antioxidants ( e . g . , parabens, vitamins E and C, and ascorbic acid) , anti-caking agents, coating agents , and chelating agents ( e . g . , EDTA) .

^{^}"Agent ' ' shall mean any chemical entity, including, without limitation, a glycomer, a protein, an antibody, a lectin, a nucleic acid, a small molecule , and any combination thereof .

"Antibody" shall include, by way of example, both naturally occurring and non-naturally occurring antibodies . Specifically, this term includes polyclonal and monoclonal antibodies , and antigen-binding fragments (e . g . , Fab fragments ) thereof . Furthermore, this term includes chimeric antibodies (e . g . , humanized antibodies ) and wholly synthetic antibodies, and antigen-binding fragments thereof .

"Antisense nucleic acid" shall mean any nucleic acid which, when introduced into a cell (directly or via expression of another nucleic acid directly introduced into the cell ) , specifically hybridizes to at least a portion of an mRNA in the cell encoding a protein ( i . e . , target protein) whose expression is to be inhibited, and thereby inhibits the target protein ' s expression .

"Bacterial cell" shall mean any bacterial cell . One example of a bacterial cell is E. coll .

"Candidate agent for treating a human afflicted with atherosclerosis" shall mean an agent which has shown some potential for treating a human afflicted with atherosclerosis ( e . g . , via the instant assays ) , and which is suitable for further animal tests and/or human tests wherein its efficacy for treating atherosclerosis is determined .

"DNAzyme" shall mean a catalytic nucleic acid that is DNA or whose catalytic component is DNA, and which specifically recognizes and cleaves a distinct target nucleic acid sequence, which can be either DNA or RNA. Each DNAzyme has a catalytic component (also referred to as a "catalytic domain" ) and a target sequence-binding component consisting of two binding domains, one on either side of the catalytic domain .

"High cholesterol diet" includes , but is not limited to, a diet containing 21% (wt/wt) butterfat and 0.15% (wt/wt) cholesterol (Harlan Teklad Adjusted Calories TD 88137 , Madison WI) .

"Inhibiting" the onset of a disorder shall mean either lessening the likelihood of the disorder ' s onset , or preventing the onset of the disorder entirely . In the preferred embodiment , inhibiting the onset of a disorder means preventing its onset entirely .

"" Isolated protein ¹¹ (e . g . , Adamtsl- 1) shall mean a protein separated, in whole or in part , from other proteins . An isolated protein includes , for example, isolated protein obtained from cells , and isolated protein produced by chemical and/or recombinant synthesis . In one embodiment , an isolated protein (e . g . , Adamtsl-1) is free of other protein . In another embodiment , an isolated protein constitutes at least half of the protein in a protein sample .

"" Isolated nucleic acid ' ¹ (e . g . , Adamtsl- 1-encoding nucleic acid) shall mean the nucleic acid free , in whole or in part , from nucleic acid encoding other proteins . In one embodiment , isolated nucleic acid means the nucleic acid free of any other nucleic acid. A nucleic acid in a vector is considered isolated, where the nucleic acid is not naturally present in the vector. Isolated nucleic acid can be, for example , obtained from cells , or produced by chemical and/or recombinant synthesis .

"Mammalian cell" shall mean any mammalian cell . Mammalian cells include , without limitation, cells which are normal , abnormal and transformed, and are exemplified by neurons , epithelial cells , muscle cells , blood cells , immune cells , stem cells , osteocytes , endothelial cells and blast cells .

"Nucleic acid" shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof . The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and ϋ, as well as derivatives thereof . Derivatives of these bases are well known in the art , and are exemplified in PCR Systems , Reagents and Consumables ( Perkin Elmer Catalogue 1996-1997 , Roche Molecular Systems , Inc . , Branchburg, New Jersey, USA) .

Examples of a "polymorphism for Adamtsl-1" include , but are not limited to the polymorphisms described in Table 1.

"Polypeptide" and "protein" are used interchangeably herein, and each means a polymer of amino acid residues . The amino acid residues can be naturally occurring or chemical analogues thereof . Polypeptides and proteins can also include modifications such as glycosylation, lipid attachment , sulfation, hydroxylation, and ADP-ribosylation . " Prophylactically effective amount" means an amount sufficient to prevent , or reduce the likelihood of, the onset of a disorder or a complication associated with a disorder in a subj ect .

"Ribozyme" shall mean a catalytic nucleic acid molecule which is RNA or whose catalytic component is RNA, and which specifically recognizes and cleaves a distinct target nucleic acid sequence, which can be either DNA or RNA. Each ribozyme has a catalytic component (also referred to as a "catalytic domain" ) and a target sequence-binding component consisting of two binding domains, one on either side of the catalytic domain .

The "severity of atherosclerotic lesions" in a subj ect correlates with, and can be determined by comparing factors including, without limitation : atherosclerotic lesion area, necrotic core area, coloration, total number of lesions in the subj ect or an area and fibrous cap thickness .

"siRNA" shall mean small interfering ribonucleic acid. Methods of designing and producing . siRNA to decrease the expression of a target protein are well known in the art .

"Subj ect" shall mean any animal, such as a human, non-human primate, mouse, rat , guinea pig or rabbit .

"Therapeutically effective amount" means an amount sufficient to treat a subj ect afflicted with a disorder or a complication associated with a disorder . The therapeutically effective amount will vary with the subj ect being treated, the condition to be treated, the agent delivered and the route of delivery. A person of ordinary skill in the art can perform routine titration experiments to determine such an amount . Depending upon the agent delivered, the therapeutically effective amount of agent can be delivered continuously, such as by continuous pump, or at periodic intervals ( for example, on one or more separate occasions ) . Desired time intervals of multiple amounts of a particular agent can be determined without undue experimentation by one skilled in the art .

'"Treating" a disorder shall mean slowing, stopping or reversing the disorder' s progression . In the preferred embodiment, treating a disorder means reversing the disorder' s progression, ideally to the point of eliminating the disorder itself .

"Vector" shall mean a nucleic acid encoding a nucleic acid of interest and/or a protein of interest, which nucleic acid, when placed in a cell, permits the expression of the nucleic acid or protein of interest . For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine- Dalgarno sequence and the start codon AUG . Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II , a downstream polyadenylation signal, the start codon AUG and a termination codon for detachment of the ribosome . Such vectors may be obtained commercially or assembled from the sequences described methods well-known in the art . Embodiments of the Invention

This invention provides an isolated nucleic acid encoding Adamtsl-1 protein . In the preferred embodiment, the Adamtsl-1 protein is human Adamtsl-1 protein . In another embodiment, the Adamtsl-1 protein is mouse Adamtsl-1 protein . In another embodiment , the nucleic acid encodes a protein having a sequence comprising the amino acid sequence set forth in SEQ . ID . NO : 2. In another embodiment , the nucleic acid encodes a protein having a sequence comprising the amino acid sequence set forth in SEQ . ID . NO : 4 and SEQ. ID. NO: 6. In another embodiment, the nucleic acid is DNA or RNA. In another embodiment , the DNA is cDNA.

This invention further -provides an isolated Adamtsl-1 protein . In the preferred embodiment, the isolated Adamtsl-1 protein is human Adamtsl-1 protein . In another embodiment, the isolated Adamtsl-1 protein is mouse Adamtsl-1 protein . In another embodiment, the isolated Adamtsl-1 protein has a sequence comprising the amino acid sequence set forth in SEQ . ID . NO : 2. In another embodiment , the isolated Adamtsl-1 protein has a sequence comprising the amino acid sequence set forth in SEQ. ID. NO : 4 and SEQ . ID . NO : 6.

This invention further provides an antibody that specifically binds to Adamtsl-1 protein . This invention further provides a vector comprising a nucleic acid encoding Adamtsl-1. In one embodiment, the vector is adapted for expression of the nucleic acid in a cell and comprises regulatory elements necessary for the expression of the nucleic acid in the cell operatively linked to the nucleic acid so as to permit expression thereof .

This invention further provides a cell comprising a vector, which vector comprises a nucleic acid encoding Adamtsl-1 protein . In one embodiment, the cell is bacterial, amphibian, yeast , fungal , insect or mammalian .

This invention further provides a method for inhibiting expression of Adamtsl-1 in a cell wherein Adamtsl-1 would otherwise occur comprising contacting the cell with an agent that inhibits expression of Adamtsl-1 in the cell .

In one embodiment, the cell is a macrophage cell . In another embodiment, the agent is selected from the group consisting of an antisense nucleic acid, siRNA, a DNAzyme and a ribozyme .

This invention further provides a method for inhibiting the onset of atherosclerosis in a subj ect at risk for becoming afflicted therewith, comprising administering to the subj ect a prophylactically effective amount of an agent that inhibits the expression of Adamtsl-1 in the subj ect' s cells , thereby inhibiting the onset of atherosclerosis in the subject . In one embodiment, the cell is a macrophage cell . In another embodiment, the agent is selected from the group consisting of an antisense nucleic acid, siRNA, a DNAzyme , a ribozyme and an antibody that specifically binds to Adamtsl-1.

This invention further provides method of treating a subj ect afflicted with atherosclerosis comprising administering to the subj ect a therapeutically effective amount of an agent that inhibits the expression of Adamtsl- 1 in the subj ect' s cells , thereby treating the subj ect . In one embodiment , the cell is a macrophage cell . In another embodiment, the agent is selected from the group consisting of an antisense nucleic acid, siRNA, a DNAzyme, a ribozyme and an antibody that specifically binds to Adamtsl-1.

This invention further provides a transgenic animal whose somatic cells are characterized by a genotype comprising (i) an LDL-receptor knockout and (ii) a polymorphism for Adamtsl-1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions . In one embodiment , the animal is a mouse . In another embodiment, the mouse is fed a high-cholesterol diet .

This invention further provides a transgenic animal whose somatic cells are characterized by a genotype comprising

( i ) an Apo-E knockout and (ii) a polymorphism for Adamtsl- 1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions . In one embodiment, the animal is a mouse . In another embodiment, the mouse is fed a high-cholesterol diet .

This invention further provides a transgenic animal whose somatic cells are characterized by a genotype comprising a polymorphism for Adamtsl-1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions . In one embodiment , the animal is a mouse . In another embodiment, the mouse is fed a high- cholesterol diet . In the instant transgenic animals , in one embodiment, the Adamtsl-1 polymorphism is MOLF .

This invention further provides a method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises : (a) administering the agent to a transgenic animal whose somatic cells are characterized by a genotype comprising (i ) an LDL-receptor knockout and (ii) a polymorphism for Adamtsl-1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ; (b) after a sufficient period of time , determining the severity of atherosclerotic lesions present in the transgenic animal; and (c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions determined in step (b) is less than the severity of lesions present in the transgenic animal to which no agent was administered . In one embodiment, the transgenic animal is a mouse . In another embodiment, the transgenic mouse is fed a high-cholesterol diet .

This invention further provides a method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises : (a) administering the agent to a transgenic animal whose somatic cells are characterized by a genotype comprising (i) an Apo-E knockout and (ii ) a polymorphism for Adamtsl- 1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ; (b) after a sufficient period of time, determining the severity of atherosclerotic lesions present in the transgenic animal ; and ( c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions present in step (b) is less than the severity of lesions present in the transgenic animal to which no agent was administered. In one embodiment , the transgenic animal is a mouse . In another embodiment , the transgenic mouse is fed a high-cholesterol diet .

Finally, this invention provides a method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises : (a) administering the agent to a transgenic animal whose somatic cells are characterized by a genotype comprising a polymorphism for Adamtsl-1, wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ; (b) after a sufficient period of time, determining the severity of atherosclerotic lesions present in the transgenic animal; and ( c) comparing the severity of atherosclerotic lesions determined in step

(b ) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions determined in step (b) is less than the severity of lesions present in the transgenic animal to which no agent was administered . In one embodiment , the transgenic animal is a mouse . In another embodiment , the transgenic mouse is fed a high-cholesterol diet .

In the instant methods , in one embodiment , the Adamtsl-1 polymorphism is MOLF .

This invention is illustrated in the Experimental Details section which follows . This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to limit in any way the invention as set forth in the claims which follow thereafter .

Experimental Details

Introduction

Atherosclerosis is genetically complex and the genes contributing to inter-individual variation in susceptibility are largely unknown . Although several atherosclerosis loci have been mapped in the murine model, none of the underlying genes has been identified by positional cloning . A locus affecting atherosclerotic lesion area, independent of plasma lipid levels , was previously mapped in a cross between strains MOLF/Ei (MOLF) and 057BLZSJ (Be) -LdIr^"7" [ I] . Atherosclerosis susceptibility QTLl {Athsql ) was identified on chromosome 4 with susceptibility derived from the MOLF strain . In the current study, B6-Ldlr congenic mice carrying the MOLF- derived Athsql interval exhibited greatly accelerated lesion development in a gene dosage-dependent manner . Sub- congenic strain analysis reduced the Athsql interval to 0.5 Mb containing two candidate genes . Genetic sequence and expression analysis indicate that extracellular matrix protein Adamtsl-1 (a. disintegrin-like a.nd metalloprotease with thromoj3pondin-like repeats-1 like-1 ) likely underlies Athsql . The proposed function of ADAMTS like proteins is regulation of matrix remodeling through direct interaction with ADAMTS proteins and modulation of protease activity. This study demonstrates the feasibility of using complex trait analysis to positionally identify an atherosclerosis susceptibility gene and suggests that variants of Adamtsl-1 have a maj or impact on early lesion development . Materials and Methods

Mice

MOLF/Ei (MOLF) and B6.129Sl-Ldlr^tmlHer (B6~Ldlr^v~) were purchased from The Jackson Laboratory (Bar Harbor, Maine) . MOLF females were mated with B6-LdIr^"Λmales to produce F₁ mice . Female Fis were backcrossed to B6-LdIr^~/"males to produce N2 mice homozygous for the LdIr knockout allele . The Athsql b/m congenic strain, heterozygous for MOLF and B6 alleles at the Athsql susceptibility locus , was created through successive backcrossing to the Bβ-Ldlr^~/~ strain . At each generation, the mice were typed for four microsatellite markers representing the proximal, medial , and distal portion of the introgressed interval . In order to speed up the breeding process , animals were screened for -100 markers to select for animals that inherited more of the Bβ-Ldlr^"7" background than others . By the N7 generation, all animals were 100% B6-Ldlr^~/~ background with the exception of one sub-congenic line which was ^99.22% Bβ-Ldlr^"7" and 0.78% MOLF with no MOLF alleles on chromosome 6 {Athsq2 [4 ] ) . Athsql b/m mice carry an approximately 38 centiMorgan (cM) donor interval between D4Mltl 85 (at 43 cM distal to the centromere as listed in the Mouse Genome Database , www . informatics . j ax . org/ ) and D4M±t42 (at 81 cM) . The Athsql m/m congenic strain, homozygous for MOLF alleles at the Athsql susceptibility locus , was created through intercrossing Athsql b/m mice and selecting for those carrying 2 MOLF alleles at the locus . Due to difficulty in breeding mice which carried 2 copies of the distal part of the interval, six-week WTD-fed Athsql m/m congenic mice carry only an interval from D4Mi tl85 to D4Mitl20 (at 54 cM) .

All congenic mice were weaned onto standard laboratory chow ( PicoLab Rodent 20 , #5053 ) at 21 days of age and switched to a Western-type diet at 8-12 weeks of age . The Western diet contained 21% (wt/wt ) butterfat and 0.15% (wt/wt) cholesterol (Harlan Teklad Adjusted Calories TD 88137 , Madison, WI ) . Mice were sacrificed after six weeks or three months of Western diet feeding . The breeding colony was produced and maintained in a specific pathogen- free environment . All mice were given ad libitum access to food and water and maintained on a standard 12-h light-dark cycle throughout the study . All experimental protocols were approved by the Institutional Animal Care and Research Advisory Committee .

DNA Extraction and LdIr Genotyping

DNA was extracted from tail tips by a quick alkaline lysis protocol as previously described [ 4 ] . Primer sequences and a protocol ^' for LdIr genotyping were obtained from http : //www . j ax . org/resources/documents/imr/protocols/Ldlr K 0. html ( 8 /5/1998 ) .

Atherosclerotic Lesion Measurements

Anesthetized mice were killed by cervical dislocation . The hearts were perfused with 0.9% NaCl alone (for frozen sections ) or 0.9% NaCl followed by 10% formalin ( for paraffin sections ) , and the aortic root was dissected and fixed in 10% formalin . Sequential, 10-micron (frozen) or 6-micron (paraffin) thick cross-sections were stained with oil red O ( frozen) or hematoxylin and eosin (H&E) (paraffin) . Every tenth section, for a total of six sections , was quantified by video microscopy with Image Pro 3.0 Plus software (Media Cybernetics ) and an average lesion size was determined for each mouse . Necrotic core area was determined from H&E stained sections as the lesion acellular area . Cap thickness was determined from Verhoeff' s stained sections using a scoring system based on numbers of elastic layers as described previously [ 8 ] . Frozen sections were used for all twelve-week WTD data and paraffin sections for six-week data .

Sequence analysis

RNA was isolated from mouse tissues . First-strand cDNA was synthesized with oligo (dT) ^12"18 primer using Superscript™ II RNase H^" reverse transcriptase ( Invitrogen) according to the manufacturer' s protocol . Direct bi-directional sequencing was performed on PCR products using an ABI Genetic Analyzer ' 3100 (Applied Biosystems ) . MOLF gene sequences were analyzed using Sequencher version 3.1 and compared to B6 and published sequence (Celera and Ensembl) . Gel electrophoresis was used to confirm the size difference caused by 5769 del45nt in the MOLF strain . A fragment from the 3 ' UTR of Adamtsll was amplified by PCR, using the following primers : 5 ' -primer ( 5 ' -

GGACAGCAAGGAGTTTATAACACATA-S ' ) ( SEQ . ID . NO : 7 ) ; 3 ' -primer (5' - TTTCTTGGTCTTCCCACACC-S ' ) (SEQ. ID. NO: 8 ) and DNA fragments were analyzed on a 3% agarose gel ( 124bp and 169bp for MOLF and B6 respectively) . Tissue Expression Analysis

RT-PCR was performed using Mouse Multiple Tissue cDNA Panel

I according to the manufacturer' s instructions (Clontech) or cDNA prepared from proximal aorta homogenates .

Amplification of Tex30 was carried out with the following primers : 5' -primer ( 5' - AAATGTTACCGGTGACAT-3' ) ( SEQ . ID .

NO: 9) ; 3' -primer (5' - TATTTCTCTGCCTCCTTA-3' ) (SEQ . ID.

NO : 10 ) and Adamtsll with the following primers : 5' -primer ( 5 ' - CTCCCACCACCTGCATGA-3' ) (SEQ. ID . N0 : ll ) ; 3' ~primer

( 5 ' -CCTTGAGGTCCTCCACTGAG-S ' ) (SEQ . ID . NO : 12 ) . The expected fragment sizes were 441bp for Tex30 and 408bp for

Adamtsll .

Quanti ta tive PCR

Peritoneal macrophages were harvested 4 days after thioglycollate inj ection, centrifuged at 1000 rpm, and plated with DMEM+10%FBS . Cells were washed 3x with PBS and total RNA was extracted with the use of RNeasy mini kit (QIAGEN) according to the manufacturer' s instructions . First strand cDNA synthesis was carried out as described above . Adamtsll expression was determined with Assays-on- Demand Gene Expression product Mm.00553186 (Applied Biosystems ) according to manufacturer' s instruction using the Stratagene Mx4000 Multiplex Quantitative PCR System. Assays were performed in duplicate and normalized to the acidic ribosomal phosphoprotein PO {Arbp/36B4) . Primer and probe sequences for Arbp were as follows : forward, 5 ' - AGATGCAGCAGATCCGCAT-S ' ; reverse, 5 ' -GTTCTTGCCCATCAGCACC-3 ' ; and probe , 5 ' -JOE-CGCTCCGAGGGAAGGCCG-3 ' . Statistical Analysis

ANOVA or Mann Whitney (if unequal variance) was performed using STATVIEW 5.0 (Abacus Concepts, Inc . ) .

Table IA . Athsql candidate sequence variants identified in MOLF relative to the B6 mouse strain .

Results

A locus affecting atherosclerotic lesion area, independent of traditional risk factors, was previously mapped in a cross between strains MOLF/Ei (MOLF) and C57BL/6J (B6) -LdIr^' ^/- [ 4 ] . Atherosclerosis susceptibility QTLl {Athsql ) was identified on chromosome 4 with susceptibility derived from the MOLF strain . To confirm the Athsql susceptibility locus and test its amenability to fine mapping, congenic mice carrying one or two copies of MOLF chromosome 4 between D4Mitl85 ( 43 cM) and D4Mitl20 ( 54 cM) or D4Mit42 ( 81 cM) on a B6~Ldlr^~/~ background were created ( Fig . Ia) . After six weeks of Western-type diet (WTD) feeding, mean atherosclerotic lesion area was significantly greater in Athsql b/m heterozygotes ( 93 , 000 ± 9 , 000 μm²/section, mean ± SEM) and Athsql m/m homozygotes ( 220 , 000 ± 35, 000 μm²/section) compared to non-congenic Athsql b/b controls (53 , 000 + 5 , 000 μm²/section; p<0.0003 and p<0.0001, respectively) ( Fig . Ib) . The difference in lesion areas was independent of plasma cholesterol levels (data not shown) . Although the effect of Athsql was female-specific in the original cross [ 4 ] , the locus affected lesion development in both sexes in the uniform genetic background ( Fig . 8 ) .

While non-congenic littermate controls exhibited small, focal, fatty streak lesions (Fig . Ic, f, and i) , Athsql b/m heterozygotes developed both fatty streak lesions and more advanced fatty-fibrous lesions characterized by fibrous cap and necrotic core formation ( Fig . Id, g, and j ) . In stark contrast to Athsql b/b controls , Athsql m/m homozygotes exhibited advanced fibrous lesions , often covering the entire circumference of the vessel wall ( Fig . Ie, h, and k) . Mean necrotic core area was significantly greater in congenic mice ( 10 , 000 ± 4 , 000 μm²/section for Athsql b/m and 32 , 000 ± 11 , 000 μm²/section for Athsql m/m) compared to non- congenic controls ( 300 ± 1200 μm²/section; p<0.04 and p<0.0006 , respectively) . The most advanced lesions from control mice exhibited thin ( single elastic layer) fibrous caps while advanced lesions from Athsql b/m and Athsql m/m mice exhibited intermediate (two to four elastic layers ) or thick (greater than four elastic layers ) fibrous caps

(trend for heterozygotes versus controls and p<0.0002 for homozygotes versus controls ) [ 8 ] . Thus , the congenic mice exhibited greatly accelerated atherosclerotic lesion development in a gene dosage-dependent manner .

For fine mapping, nine congenic/sub-congenic strains carrying varying lengths of the MOLF-derived chromosome 4 interval were generated (Fig 2 ) . Atherosclerotic lesion development was compared with non-congenic littermate controls to determine whether or not a particular strain carried the Athsql variant . Using the twelve-week WTD feeding protocol employed in the original mapping study [4] , no difference in lesion area was observed between homozygous congenics carrying a region extending from D4Mitl 85 to D4Mit42 and those carrying a shorter region from D4M±tl85 to D4Mitl20 ( Fig . 2a) . Furthermore, analysis of four sub-congenic strains , heterozygous for MOLF alleles , confirmed this observation and narrowed the Athsql containing interval to a <3.4 Mb region between D4M±tl52 and Adfp-rs ( Fig . 2a) . While this region lies proximal to the original linkage peak, sub-congenic strain analysis strongly supports the presence of a single gene affecting atherosclerosis susceptibility localizing to this sub- region . However, the possibility that the effect of a second, distally located gene was lost in the uniform genetic background cannot be ruled out . Following a pilot study, a six-week feeding regimen was used for further mapping studies . Comparison of two additional heterozygous sub-congenic strains to non-congenic littermates reduced the interval to a <1.2 Mb region between D4Mit302 and Adfp- rs ( Fig . 2b ) . To narrow the region, single nucleotide polymorphisms ( SNPs ) were identified between the B6 and MOLF strains by comparative sequence analysis . Sequencing of SNPs in critical sub-congenics narrowed Athsql to an approximate 0.5 Mb region between SNPs Adamtsl-479 and Rraga-22dellnt (Fig . 2c) . This interval included only two expressed sequences : Adamtsll and an unknown EST (Celera Tex30 ; RIKEN 4930500009 ) which was designated as Tex30.

To identify candidate sequence variant ( s ) underlying Athsql , the 5 ' UTR, coding sequence, and 3' UTR of Tex30 and Adamtsll derived from the MOLF strain were sequenced and compared those sequences to B6. Two non-conservative amino acid variants for Tex30 and three variants for Adamtsll were detected (Table IB) .

Table IB . Athsql candidate sequence variants identified in MOLF relative to the B6 mouse strain .

In addition, multiple deletion/insertion polymorphisms were detected in the ^' 3' UTR of Adamtsll in MOLF versus B6 (Table IB) . Fragments spanning each of the two deletions were amplified and sequenced from MOLF genomic DNA, ruling out the possibility that these polymorphisms represented splice variants as opposed to deletions . Next , the tissue expression patterns of Tex30 and Adamtsll were compared by RT-PCR. Adamtsll was widely expressed in adult as well as embryonic tissues ( Fig . 3a) . In contrast, Tex30 was expressed in a highly testis-specific manner ( Fig . 3b) . Adamtsll, but not Tex30_r mRNA was also detected in atherosclerotic lesions ( Fig . 3c) . The testis specific expression of Tex30 is supported by data in the corresponding GenBank UniGene Cluster (Mm.56451 ) where cDNA sources included only testis and round spermatids . Because Tex30 is not expressed in tissues relevant to atherogenesis , it is unlikely to be the gene underlying Athsql . Therefore, further investigation focused on Adamtsll .

Using a probe-based quantitative PCR assay, a significant induction of Adamtsll mRNA was observed in lesion- containing aorta compared to unaffected vessels (32-fold, p=0.005 ) . Adamtsll levels were approximately 4-fold {Athsql b/rn) and 5.5-fold {Athsql m/m) higher in isolated peritoneal macrophages derived from congenics versus controls ( Fig . 4d, p<0.006 and p<0.001 , respectively) . In addition, Athsql m/m mice showed greater expression of Adamtsll mRNA in lesion-containing aorta compared to Athsql b/b mice ( Fig . 3e, p=0.001 ) while the levels of other genes expressed by macrophages were not different ( Fig . 3e) . Although functional promoter variants between MOLF and B6 have been excluded by sub-congenic SNP analysis ( Fig . 2c) , the altered expression level of Adamtsll may be due to an intronic regulatory variant or an effect of one or more of the 3 ' UTR polymorphisms on transcript stability. These data suggest that increased expression of Adamtsll in lesional macrophages may contribute to accelerated atherogenesis .

Discussion

Adamtsll is an extracellular matrix (ECM) glycoprotein that is a member of a non-enzymatic sub-family related to the Adamts metalloprotease family . The complete coding sequence of Adamtsll has not been previously published . However, an N-terminal fragment termed punctin has been described [ 9] . A 7.2 kb transcript was sequenced, encoding 30 exons , which predicts a protein with structural homology to other family members , including ADAMTSL3 [ 10 ] and papilin [ 11 ] ( Fig . 4 ) . The cDNA sequence has been deposited in Genbank. A conserved cassette of domains

(papilin cassette ) at the 5 ' end of the ADAMTS-liJce molecules is homologous to a non-catalytic cassette of domains at the 3 ' end of ADAMTS molecules ( Fig . 4 ) .

Papilin, which has been described in some detail in Drosophila, is an ECM glycoprotein secreted by macrophage- like phagocytes that binds ADAMTS protease and possibly modulates protease activity and local matrix composition

[11-13] . Homology between ADAMTSLl and papilin likely reflects conservation of function between the two proteins and suggests that ADAMTSLl may also modulate the activity of ADAMTS metalloproteases [ 9] .

Currently, the role of ADAMTS proteases in atherogenesis is unknown but they have been implicated in the processing of procollagen [ 14 ] , cleavage of proteoglycans [ 15-19] , inflammation [20 ] , and cell migration [21 , 22 ] . Specifically, cleavage of versican, a proteoglycan implicated in atherogenesis [23 , 24 ] , can occur via multiple ADAMTS protease family members [ 16] . Thus , lesions derived from congenic and non-congenic mice were stained using a specific versican antibody . Athsql congenic mice exhibited a dramatic accumulation of versican in lesions while non-congenic littermates exhibited virtually no accumulation ( Fig . 5 ) . These data are in agreement with a previous report that versican is nearly absent from early through advanced lesions of B6-Ldlr^~/~ and B6~apoE^~/~ mice [25] . In contrast , versican is prominent in human lesions and has been proposed to increase atherosclerosis by a variety of mechanisms [23 , 24 ] . Versican accumulation in Athsql congenic mice was most prominent at the six-week diet time point when differences in lesion development were most pronounced between congenics and controls . Staining for collagen, hyaluronan, and total proteoglycans showed no differences in comparable lesions derived from the two strains (data not shown) , suggesting a specific role for versican . Hence, increased ADAMTSLl activity may inhibit one or more ADAMTS family members responsible for proteolysis of aortic versican, providing a local niche for intimal expansion and lesion progression .

While a number of atherosclerosis susceptibility loci have been mapped in the mouse [3-7 ] , none of the underlying genes have been previously identified by positional cloning . In this study, genetic exclusion narrowed the

Athsql interval to two genes and gene expression patterns were used to identify the underlying gene . Although the precise molecular actions of Adamtsll in atherogenesis remains to be elucidated, this study provides a more rigorous method of complex gene identification than candidate gene selection [26] .

References

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17. Russell, D . L . , K. M. H . Doyle, S . A. Ochsner, J. D . Sandy, and J. S . Richards . Processing and Localization of ADAMTS-I and Proteolytic Cleavage of Versican during Cumulus Matrix Expansion and Ovulation . J. Biol . Chem . 2003 ; 278 : 42330-42339.

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19. Abbaszade, I . , R . Q . Liu, F . Yang, S .A. Rosenfeld, O . H . Ross , J. R. Link, D . M. Ellis, M. D. Tortorella, M. A. Pratta, and a . Hollis et . Cloning and characterization of ADAMTSIl , an aggrecanase from the ADAMTS family . The Journal Of Biological Chemistry. 1999 ; 274 : 23443- 23450. 20. Kuno, K. , N . Kanada, E . Nakashima, F. Fuj iki , F. Ichimura, and K . Matsushima . Molecular cloning of a gene encoding a new type of metalloproteinase- disintegrin family protein with thrombospondin motifs as an inflammation associated gene . Journal of Biological Chemistry. 1997 ; 272 : 556-562.

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25. Kunj athoor, V . V . , D . S . Chiu, K. D . O ' Brien, and R. C . LeBoeuf . Accumulation of Biglycan and Perlecan, but Not Versican, in Lesions of Murine Models of Atherosclerosis . Arterioscler Thromb Vase Biol . 2002 ; 22 : 462-468. 26. Glazier, A. M. , J. H . Nadeau, and T . J. Aitman . Finding Genes That Underlie Complex Traits . Science. 2002 ; 298 : 2345-2349.

10

15

?.S

30

Claims

What is claimed is :

1. An isolated nucleic acid encoding Adamtsl-1 protein .

2. The isolated nucleic acid of claim 1 , wherein the Adamtsl-1 protein is human Adamtsl-1 protein .

3. The isolated nucleic acid of claim 1 , wherein the Adamtsl-1 protein is mouse Adamtsl-1 protein .

4. The isolated nucleic acid of claim 2 , wherein the nucleic acid encodes a protein having a sequence comprising the amino acid sequence set forth in SEQ . ID . N0 : 2.

5. The isolated nucleic acid of claim 3 , wherein the nucleic acid encodes a protein having a sequence comprising the amino acid sequence set forth in SEQ . ID . N0 : 4.

6. The isolated nucleic acid of claim 1 , wherein the nucleic acid is DNA or RNA.

7. The isolated nucleic acid of claim 6, wherein the DNA is cDNA.

8. An isolated Adamtsl-1 protein .

9. The isolated Adamtsl-1 protein of claim 8 , wherein the Adamtsl-1 protein is human Adamtsl-1 protein .

10. The isolated Adamtsl-1 protein of claim 8 , wherein the Adamtsl-1 protein is mouse Adamtsl-1 protein .

11. The isolated Adamtsl-1 protein of claim 9, wherein the protein has a sequence comprising the amino acid sequence set forth in SEQ . ID. NO : 2.

12. The isolated Adamtsl-1 protein of claim 10 , wherein the protein has a sequence comprising the amino acid sequence set forth in SEQ. ID. NO: 4.

13. An antibody which specifically binds to Adamtsl-1 protein .

14. A vector comprising a nucleic acid encoding Adamtsl-1 protein .

15. The vector of claim 14 , wherein the vector is adapted for expression of the nucleic acid in a cell and comprises regulatory elements necessary for the expression of the nucleic acid in the cell operatively linked to the nucleic acid so as to permit expression thereof .

16. A cell comprising a vector, which vector comprises a nucleic acid encoding Adamtsl-1 protein .

17. The cell of claim 16, wherein the cell is a bacterial , amphibian, yeast, fungal, insect, or mammalian cell .

18. A method for inhibiting expression of Adamtsl-1 in a cell wherein Adamtsl-1 expression would otherwise occur comprising contacting the cell with an agent that inhibits expression of Adamtsl-1 in the cell .

19. The method of claim 18 , wherein the cell is a macrophage cell .

20. The method of claim 18 , wherein the agent is selected from the group consisting of an antisense nucleic acid, siRNA, a DNAzyme and a ribozyme .

21. A method for inhibiting the onset of atherosclerosis in a subj ect at risk for becoming afflicted therewith, comprising administering to the subject a prophylactically effective amount of an agent that inhibits the expression of Adamtsl-1 in the subj ect ' s cells , thereby inhibiting the onset of atherosclerosis in the subj ect .

22. The method of claim 21 , wherein the cells are macrophage cells .

23. The method of claim 21, wherein the agent is selected from the group consisting of an antisense nucleic acid, siRNA, a DNAzyme, a ribozyme and an antibody that specifically binds to Adamtsl-1.

24. A method of treating a subj ect afflicted with atherosclerosis comprising administering to the subj ect a therapeutically effective amount of an agent that inhibits the expression of Adamtsl-1 in the subj ect' s cells , thereby treating the subj ect .

25. The method of claim 24 , wherein the cells are macrophage cells .

26. The method of claim 24 , wherein the agent is selected from the group consisting of an antisense ■ nucleic acid, siRNA, a DNAzyme, a ribozyme and an antibody that specifically binds to Adamtsl-1.

27. A transgenic animal whose somatic cells are characterized by a genotype comprising (i) an LDL- receptor knockout and (ii) a polymorphism for Adamtsl- 1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions .

28. The transgenic animal of claim 27 , wherein the animal is a mouse .

29. The transgenic animal of claim 28 , wherein the transgenic mouse is fed a high-cholesterol diet .

30. A transgenic animal whose somatic cells are characterized by a genotype comprising (i ) an Apo-E knockout and (ii ) a polymorphism for Adamtsl-1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions .

31. The transgenic animal of claim 30 , wherein the animal is a mouse .

32. The transgenic animal of claim 31 , wherein the transgenic mouse is fed a high-cholesterol diet .

33. A transgenic animal whose somatic cells are characterized by a genotype comprising a polymorphism for Adamtsl-1, wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions .

34. The transgenic animal of claim 33 , wherein the animal is a mouse .

35. The transgenic animal of claim 34 , wherein the transgenic mouse is fed a high-cholesterol diet .

36. A method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises :

( a) administering the agent to a transgenic animal whose somatic cells are characterized by a genotype comprising (i ) an LDL-receptor knockout and ( ii) a polymorphism for Adamtsl-1 , wherein the polymorphism for

Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ;

(b) after a sufficient period of time , determining the severity of atherosclerotic lesions present in the transgenic animal; and

(c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions determined in step

(b) is less than the severity of lesions present in the transgenic animal to which no agent was administered.

37. The method of claim 36, wherein the transgenic animal is a mouse .

38. The method of claim 37 , wherein the transgenic mouse is fed a high-cholesterol diet .

39. A method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises :

(a) administering the agent to a transgenic animal whose somatic cells are characterized by a genotype comprising (i ) an Apo-E knockout and (ii) a polymorphism for Adamtsl-1 , wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ;

(b) after a sufficient period of time, determining the severity of atherosclerotic lesions present in the transgenic animal; and

(c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions present in step (b) is less than the severity of lesions present in the transgenic animal to which no agent was administered.

40. The method of claim 39 , wherein the transgenic animal is a mouse .

41. The method of claim 40 , wherein the transgenic mouse is fed a high-cholesterol diet .

42. A method for determining whether an agent is a candidate agent for treating a human afflicted with atherosclerosis , which comprises :

(a) administering the agent to a transgenic animal whose somatic cells are characterized by a genotype comprising a polymorphism for Adamtsl- 1, wherein the polymorphism for Adamtsl-1 predisposes the animal to developing atherosclerotic lesions , and wherein the transgenic animal has atherosclerotic lesions ; (b) after a sufficient period of time, determining the severity of atherosclerotic lesions present in the transgenic animal ; and

(c) comparing the severity of atherosclerotic lesions determined in step (b) with the severity of atherosclerotic lesions present in the same type of transgenic animal to which no agent was administered, whereby the agent is determined to be a candidate agent for treating a human afflicted with atherosclerosis if the severity of atherosclerotic lesions determined in step (b) is less than the severity of lesions present in the transgenic animal to which no agent was administered.

43. The method of claim 42 , wherein the transgenic animal is a mouse .

44. The method of claim 43, wherein the transgenic mouse is fed a high-cholesterol diet .