ATHEROSCLEROSIS-ASSOCIATED GENES

Abstract

The invention provides novel atherosclerosis-associated polynucleotides and polypeptides encoded by those genes. The invention also provides expression vectors, host cells, and antibodies. The invention also provides methods for screening or purifying ligands and diagnosing, treating or preventing atherosclerosis.

Description

FIELD OF THE INVENTION

[0001] The invention relates to 34 atherosclerosis-associated polynucleotides identified by their co-expression with known atherosclerosis genes and their corresponding gene products. The invention also relates to the use of these biomolecules in diagnosis, prognosis, prevention, treatment, and evaluation of therapies for diseases associated with atherosclerosis.

BACKGROUND OF THE INVENTION

[0002] Atherosclerosis is a disorder characterized by cellular changes in the arterial intima and the formation of arterial plaques containing intra- and extracellular deposits of lipids. The resultant thickening of artery walls and the narrowing of the arterial lumen is the underlying pathologic condition in most cases of coronary artery disease, aortic aneurysm, peripheral vascular disease, and stroke. A cascade of molecules is involved in the cellular morphogenesis, proliferation, and cellular migration which results in an atherosclerotic lesion (Libby et al. (1997) Int. J. Cardiol. 62 (S2): 23-29).

[0003] A healthy artery consists of three layers. The vascular intima, lined by a monolayer of endothelial cells in contact with the blood, contains smooth muscle cells in extracellular matrix. An internal elastic lamina forms the border between the intima and the tunica media. The media contains layers of smooth muscle cells surrounded by a collagen and elastin-rich extracellular matrix. An external elastic lamina forms the border between the media and the adventitia. The adventitia contains nerves and some mast cells and is the origin of the vasa vasorum which supplies blood to the outer layers of the tunica media.

[0004] Initiation of an atherosclerotic lesion often occurs following vascular endothelial cell injury as a result of hypertension, diabetes mellitus, hyperlipidemia, fluctuating shear stress, smoking, or transplant rejection. The injury results in the local release of nitric oxide and superoxide anions which react to form cytodestructive peroxynitrite radicals, causing injury to the endothelium and myocytes of the intima. This cellular injury leads to the expression of a variety of molecules that produce local and systemic effects. The initial cellular response to injury includes the release of mediators of inflammation such as cytokines, complement components, prostaglandins, and downstream transcription factors. These molecules promote monocyte infiltration of the vascular intima and lead to the upregulation of adhesion molecules which encourage attachment of the monocytes to the damaged endothelial cells. Additionally, components of the extracellular matrix including collagens, fibrinogens, and matrix Gla protein are induced and provide sites for monocyte attachment. Annexins, plasminogen activator inhibitor 1, and nitric oxide synthases are triggered to counteract these effects.

[0005] Monocytes that infiltrate the lesion accumulate modified low density lipoprotein lipid through scavenger receptors such as CD36 and macrophage scavenger receptor type I. The abundance of modified lipids is a factor in atherogenesis and is influenced by modifying enzymes such as lipoprotein lipase, carboxyl ester lipase, serum amyloid P component, LDL-receptor related protein, microsomal triglyceride transfer protein, and serum esterases such as paraoxonase. Lipid metabolism is governed by cholesterol biosynthesis enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A synthase, and products of the apolipoprotein genes. Modified lipid stabilization and accumulation is aided by perilipin and alpha-2-macroglobulin.

[0006] As monocytes accumulate in the lesion, they can rupture and release free cholesterol, cytokines, and procoagulants into the surrounding environment. This process leads to the development of a plaque which consists of a mass of lipid-engorged monocytes and a lipid-rich necrotic core covered by a fibrous cap. The gradual progression of plaque growth is punctuated by thrombus formation which leads to clinical symptoms such as unstable angina, myocardial infarction, or stroke. Thrombus formation is initiated by episodic plaque rupture which exposes flowing blood to tissue factors, which induce coagulation, and collagen, which activates platelets. After initiation of the atherosclerotic lesion, enzymes that degrade extracellular matrix components such as matrix metalloproteinases and cathepsin K are up-regulated, and their inhibitors are down-regulated. This results in destabilization of the atherosclerotic lesion and subsequent complications including myocardial infarction, angina, and stroke. Further arterial occlusion and infiltration increase with the expression of coagulation factors and down-regulation of their inhibitors, antithrombin III, and lipoprotein-associated coagulation inhibitor.

[0007] Smooth muscle cells build up in the arterial media and constitute one of the principal cell types in atherosclerotic and restenotic lesions. They show a high degree of plasticity and are able to shift between a differentiated, contractile phenotype and a less differentiated, synthetic phenotype. This modulation occurs as a response to factors secreted from cells at the site of vascular injury and results in structural reorganization with a loss of myofilaments and the formation of an extensive endoplasmic reticulum and a large Golgi complex. Genes encoding secreted protein, acidic and rich in cysteine (SPARC) and endothelin-1 contribute to these changes. At the same time, the expression of cytoskeletal proteins such as calponin, myosin, desmin, and other gene products in the cells is altered. As a result, the smooth muscle cells lose their contractility and become able to migrate from the media to the intima, to proliferate, and to secrete extracellular matrix components which contribute to arterial intimal thickening.

[0008] The initiation and progression of atherosclerotic lesion development requires the interplay of various molecular pathways Many genes that participate in these processes are known, and some of them have been shown to have a direct role in atherosclerosis pathogenesis by animal model experiments, in vitro assays, and epidemiological studies (Krettek et al. (1997) Arterioscler. Thromb. Vasc. Biol. 17(11):2897-2903; Fisher et al. (1997) Atherosclerosis 135(2):145-159; Shih et al. (1998) Circulation 95(120:2684-2693; and Bocan et al. (1998) Atherosclerosis 139(1):21-30).

[0009] The present invention satisfies a need in the art by providing new compositions that are useful for diagnosis, prognosis, treatment, prevention, and evaluation of therapies for diseases associated with atherosclerosis. We have implemented a method for analyzing gene expression patterns and have identified 34 atherosclerosis-associated polynucleotides through their co-expression with 66 known atherosclerosis-associated genes.

SUMMARY OF THE INVENTION

[0010] The invention provides for a substantially purified polynucleotide comprising a gene that is coexpressed with one or more known atherosclerosis-associated genes in a biological sample. Known atherosclerosis-associated genes include and encode human 22 kDa smooth muscle protein, calponin, desmin, smooth muscle myosin heavy chain, alpha tropomyosin, human tissue inhibitor of metalloproteinase 3, human tissue inhibitor of metalloproteinase-2, human tissue inhibitor of metalloproteinase-4, pro alpha 1(I) collagen, collagen alpha-2 type I, collagen alpha-6 type I, procollagen alpha 2(V), collagen VI alpha-2, type VI collagen alpha3, pro-alpha-1 type 3 collagen, pro-alpha-1 (V) collagen, collagenase type IV/matrix metalloproteinase 9/gelatinase B, matrix Gla protein, cathepsin K, fibrinogen beta chain gene, fibrinogen gamma chain gene, pre-pro-von Willebrand factor, coagulation factor II/prothrombin, coagulation factor XII, coagulation factor VII, platelet endothelial cell adhesion molecule, lipoprotein-associated coagulation inhibitor, antithrombin III variant, plasminogen activator inhibitor-1, lipoprotein lipase, alpha-2-macroglobulin, apolipoprotein AI, apolipoprotein AII, apolipoprotein B-100, lipoprotein apoCII, pre-apolipoprotein CIII, apolipoprotein apo C-IV, macrophage scavenger receptor type I, human antigen CD36 gene, serum amyloid P component, carboxyl ester lipase gene, paraoxonase 1, paraoxonase 2, paraoxonase 3, LDL-receptor related protein, hepatic triglyceride lipase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, very low density lipoprotein receptor, microsomal triglyceride transfer protein, perilipin, endothelin-1, endothelin receptor A, interleukin 6, interleukin 1, complement protein C8 alpha, complement component C9, prostaglandin D2 synthase, annexin II/lipocortinlI, annexin I/lipocortin, prostaglandin-endoperoxide synthase 2, insulin-like growth factor binding protein-1, secreted protein, acidic and rich in cysteine, human NF-kappa-B transcription factor, angiotensinogen, nitric oxide synthase 3, and nitric oxide synthase 2.

[0011] The invention also provides a substantially purified polynucleotide comprising a gene that is coexpressed with one or more known atherosclerosis-associated genes in a plurality of samples. In one aspect, the polynucleotide comprises a polynucleotide sequence selected from (a) a polynucleotide encoding a peptide selected from SEQ ID NOs: 1-34; (b) a polynucleotide sequence complementary to the polynucleotide sequence of (a) or (b); and (c) a probe comprising at least 18 sequential nucleotides of the polynucleotide sequence of (a) or (b). The invention further provides a pharmaceutical composition comprising a polynucleotide and a pharmaceutical carrier.

[0012] The invention additionally provides methods for using a polynucleotide. One method uses the polynucleotide to screen a library of molecules or compounds to identify at least one ligand which specifically binds the polynucleotide and comprises combining the polynucleotide with a library of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the polynucleotide. In this first method, the library is selected from DNA molecules, RNA molecules, PNAs, mimetics, and proteins; and the ligand identified using the method may be used to modulate the activity of the polynucleotide. A second method uses the polynucleotide to purify a ligand which specifically binds the polynucleotide and comprises combining the polynucleotide with a sample under conditions to allow specific binding, detecting specific binding between the polynucleotide and a ligand, recovering the bound polynucleotide, and separating the polynucleotide from the ligand, thereby obtaining purified ligand. A third method uses the polynucleotide to diagnose a disease or condition associated with the altered expression of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a plurality of biological samples and comprises hybridizing a polynucleotide to a sample under conditions to form one or more hybridization complexes, detecting the hybridization complexes, and comparing the levels of the hybridization complexes with the level of hybridization complexes in a non-diseased sample, wherein the altered level of hybridization complexes compared with the level of hybridization complexes of a non-diseased sample indicates the presence of the disease or condition. A fourth method uses the polynucleotide to produce a polypeptide and comprises culturing a host cell containing an expression vector containing the polynucleotide under conditions for expression of the polypeptide and recovering the polypeptide from cell culture.

[0013] The invention provides a substantially purified polypeptide comprising the product of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a plurality of samples. The invention also provides a polypeptide comprising a polypeptide sequence selected from (a) the polypeptides encoded by SEQ ID NOs: 1-34; and b) an oligopeptide sequence comprising at least 6 sequential amino acids of the polypeptide sequence of a). The further provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 35. The invention still further provides a pharmaceutical composition comprising a polypeptide and a pharmaceutical carrier.

[0014] The invention additionally provides methods for using a polypeptide. One method uses the polypeptide to screen a library of molecules or compounds to identify at least one ligand which specifically binds the polypeptide and comprises combining the polypeptide with the library of molecules or compounds under conditions to allow specific binding and detecting specific binding between the polypeptide and ligand, thereby identifying a ligand which specifically binds the polypeptide. In this method, the library is selected from DNA molecules, RNA molecules, PNAs, mimetics, polypeptides, agonists, antagonists, and antibodies; and the ligand identified using the method is used to modulate the activity of the polypeptide. A second method uses the polypeptide to purify a ligand from a sample and comprises combining the polypeptide with a sample under conditions to allow specific binding, detecting specific binding between the polypeptide and a ligand, recovering the bound polypeptide, and separating the polypeptide from the ligand, thereby obtaining purified ligand. A third method uses the polypeptide to treat or to prevent a disease associated with the altered expression of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need and comprises administering to the subject in need the pharmaceutical composition containing the polypeptide in an amount effective for treating or preventing the disease.

[0015] The invention provides an antibody or Fab comprising an antigen binding site, wherein the antigen binding site specifically binds to the polypeptide. The invention also provides a method for treating or preventing a disease associated with the altered expression of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need, the method comprising the step of administering to the subject in need the antibody or the Fab in an amount effective for treating or preventing the disease. The invention further provides an immunoconjugate comprising the antigen binding site of the antibody or Fab joined to a therapeutic agent. The invention additionally provides a method for treating or preventing a disease associated with the altered expression of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need, the method comprising the step of administering to the subject in need the immunoconjugate in an amount effective for treating or preventing the disease.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0016] The Sequence Listing provides exemplary atherosclerosis-associated gene sequences including polynucleotide sequences SEQ ID NOs: 1-34 and the polypeptide sequence, SEQ ID NO:35. Each sequence is identified by a sequence identification number (SEQ ID NO).

DESCRIPTION OF THE INVENTION

[0017] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

[0018] Definitions

[0019] “Atherosclerosis-associated gene” refers to a gene or polynucleotide that exhibits a statistically significant coexpression pattern with known atherosclerosis-associated genes which are useful in the diagnosis, treatment, prognosis, or prevention of atherosclerosis.

[0020] “Known atherosclerosis-associated gene” refers to a sequence which has been previously identified as useful in the diagnosis, treatment, prognosis, or prevention of atherosclerosis and includes polynucleotides encoding human 22 kDa smooth muscle protein, calponin, desmin, smooth muscle myosin heavy chain, alpha tropomyosin, human tissue inhibitor of metalloproteinase 3, human tissue inhibitor of metalloproteinase-2, human tissue inhibitor of metalloproteinase-4, pro alpha 1(I) collagen, collagen alpha-2 type I, collagen alpha-6 type I, procollagen alpha 2(V), collagen VI alpha-2, type VI collagen alpha3, pro-alpha-1 type 3 collagen, pro-alpha-1(V) collagen, collagenase type IV/matrix metalloproteinase 9/gelatinase B, matrix Gla protein, cathepsin K, fibrinogen beta chain gene, fibrinogen gamma chain gene, pre-pro-von Willebrand factor, coagulation factor II/prothrombin, coagulation factor XII, coagulation factor VII, platelet endothelial cell adhesion molecule, lipoprotein-associated coagulation inhibitor, antithrombin III variant, plasminogen activator inhibitor-1, lipoprotein lipase, alpha-2-macroglobulin, apolipoprotein AI, apolipoprotein AII, apolipoprotein B-100, lipoprotein apoCII, pre-apolipoprotein CIII, apolipoprotein apo C-IV, macrophage scavenger receptor type I, human antigen CD36 gene, serum amyloid P component, carboxyl ester lipase gene, paraoxonase 1, paraoxonase 2, paraoxonase 3, LDL-receptor related protein, hepatic triglyceride lipase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, very low density lipoprotein receptor, microsomal triglyceride transfer protein, perilipin, endothelin-1, endothelin receptor A, interleukin 6, interleukin 1, complement protein C8 alpha, complement component C9, prostaglandin D2 synthase, annexin II/lipocortinII, annexin I/lipocortin, prostaglandin-endoperoxide synthase 2, insulin-like growth factor binding protein-1, secreted protein, acidic and rich in cysteine, human NF-kappa-B transcription factor, angiotensinogen, nitric oxide synthase 3, and nitric oxide synthase 2. Typically, this means that the known gene is expressed at higher levels (i.e., has more abundant transcripts) in atherosclerotic lesions than in normal or non-diseased arterial intima or any other tissue.

[0021] “Ligand” refers to any molecule, agent, or compound which will bind specifically to a complementary site on a polynucleotide or polypeptide. Such ligands stabilize or modulate the activity of polynucleotides or polypeptides of the invention. For example, ligands are libraries of inorganic and organic molecules or compounds such as nucleic acids, proteins, peptides, carbohydrates, fats, and lipids.

[0022] “NSEQ” refers generally to a polynucleotide sequence of the present invention, including SEQ ID NO: 1-34. “PSEQ” refers generally to a polypeptide sequence of the present invention, including SEQ ID NO:35.

[0023] A “fragment” refers to a nucleic acid sequence that is preferably at least 20 nucleotides in length, more preferably 40 nucleotides, and most preferably 60 nucleotides in length, and encompasses, for example, fragments consisting of 1-50, 51-400, 401-4000, 4001-12,000 nucleotides, and the like, of SEQ ID NO: 1-34.

[0024] “Gene” refers to the partial or complete coding sequence of a gene including 5′ or 3′ untranslated regions. The gene may be in a sense or antisense (complementary) orientation.

[0025] “Polynucleotide” refers to a nucleic acid, nucleic acid sequence, oligonucleotide, nucleotide, or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein or other materials to perform a particular activity or form a useful composition. “Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligomer, element, and probe.

[0026] “Polypeptide” refers to an amino acid, amino acid sequence, oligopeptide, peptide, or protein or portions thereof whether naturally occurring or synthetic.

[0027] A “portion” refers to peptide sequence which is preferably at least 5 to about 15 amino acids in length, most preferably at least 10 amino acids long, and which retains some biological or immunological activity of, for example, a portion of SEQ ID NO: 35.

[0028] “Sample” is used in its broadest sense. A sample containing nucleic acids may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; and the like.

[0029] “Substantially purified” refers to a nucleic acid or an amino acid sequence that is removed from its natural environment and that is isolated or separated, and is at least about 60% free, preferably about 75% free, and most preferably about 90% free, from other components with which it is naturally present.

[0030] “Substrate” refers to any rigid or semi-rigid support to which polynucleotides or polypeptides are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.

[0031] A “variant” refers to a polynucleotide or polypeptide whose sequence diverges from SEQ ID NO: 1-35. Polynucleotide sequence divergence may result from mutational changes such as deletions, additions, and substitutions of one or more nucleotides; it may also be introduced to accommodate differences in codon usage. Each of these types of changes may occur alone, or in combination, one or more times in a given sequence.

[0032] The Invention

[0033] The present invention encompasses a method for identifying biomolecules that are associated with a specific disease, regulatory pathway, subcellular compartment, cell type, tissue type, or species. In particular, the method identifies polynucleotides useful in diagnosis, prognosis, treatment, prevention, and evaluation of therapies for diseases associated with atherosclerosis including, but not limited to, stroke, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, coronary artery disease, angina pectoris, peripheral vascular disease, intermittent claudication, renal artery stenosis, and hypertension.

[0034] The method entails first identifying polynucleotides that are expressed in a plurality of cDNA libraries. The identified polynucleotides include genes of known or unknown function which are expressed in a specific disease process, subcellular compartment, cell type, tissue type, or species. The expression patterns of the genes with known function are compared with those of genes with unknown function to determine whether a specified coexpression probability threshold is met. Through this comparison, a subset of the polynucleotides having a high coexpression probability with the known genes can be identified. The high coexpression probability correlates with a particular coexpression probability threshold which is preferably less than 0.001 and more preferably less than 0.00001.

[0035] The polynucleotides originate from cDNA libraries derived from a variety of sources including, but not limited to, eukaryotes such as human, mouse, rat, dog, monkey, plant, and yeast; prokaryotes such as bacteria; and viruses. These polynucleotides can also be selected from a variety of sequence types including, but not limited to, expressed sequence tags (ESTs), assembled polynucleotide sequences, full length gene coding regions, promoters, introns, enhancers, 5′ untranslated regions, and 3′ untranslated regions. To have statistically significant analytical results, the polynucleotides need to be expressed in at least three cDNA libraries.

[0036] The cDNA libraries used in the coexpression analysis of the present invention can be obtained from adrenal gland, biliary tract, bladder, blood cells, blood vessels, bone marrow, brain, bronchus, cartilage, chromaffin system, colon, connective tissue, cultured cells, embryonic stem cells, endocrine glands, epithelium, esophagus, fetus, ganglia, heart, hypothalamus, immune system, intestine, islets of Langerhans, kidney, larynx, liver, lung, lymph, muscles, neurons, ovary, pancreas, penis, peripheral nervous system, phagocytes, pituitary, placenta, pleurus, prostate, salivary glands, seminal vesicles, skeleton, spleen, stomach, testis, thymus, tongue, ureter, uterus, and the like. The number of cDNA libraries selected can range from as few as 3 to greater than 10,000. Preferably, the number of the cDNA libraries is greater than 500.

[0037] In a preferred embodiment, genes are assembled from related sequences, such as assembled sequence fragments derived from a single transcript. Assembly of the sequences can be performed using sequences of various types including, but not limited to, ESTs, extensions, or shotgun sequences. In a most preferred embodiment, the polynucleotide sequences are derived from human sequences that have been assembled using the algorithm disclosed in “Database and System for Storing, Comparing and Displaying Related Biomolecular Sequence Information”, Lincoln et al. Serial No: 60/079,469, filed Mar. 26, 1998, incorporated herein by reference.

[0038] Experimentally, differential expression of the polynucleotides can be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational difference analysis, and transcript imaging. Additionally, differential expression can be assessed by microarray technology. These methods may be used alone or in combination.

[0039] Known atherosclerosis-associated genes are selected based on the use of these genes as diagnostic or prognostic markers or as therapeutic targets.

[0040] The procedure for identifying novel genes that exhibit a statistically significant coexpression pattern with known atherosclerosis-associated genes is as follows. First, the presence or absence of a gene in a cDNA library is defined: a gene is present in a cDNA library when at least one cDNA fragment corresponding to that gene is detected in a cDNA sample taken from the library, and a gene is absent from a library when no corresponding cDNA fragment is detected in the sample.

[0041] Second, the significance of gene coexpression is evaluated using a probability method to measure a due-to-chance probability of the coexpression. The probability method can be the Fisher exact test, the chi-squared test, or the kappa test. These tests and examples of their applications are well known in the art and can be found in standard statistics texts (Agresti (1990) Categorical Data Analysis, John Wiley & Sons, New York N.Y.; Rice (1988) Mathematical Statistics and Data Analysis, Duxbury Press, Pacific Grove Calif.). A Bonferroni correction (Rice, supra, p. 384) can also be applied in combination with one of the probability methods for correcting statistical results of one gene versus multiple other genes. In a preferred embodiment, the due-to-chance probability is measured by a Fisher exact test, and the threshold of the due-to-chance probability is set preferably to less than 0.001, more preferably to less than 0.00001.

[0042] To determine whether two genes, A and B, have similar coexpression patterns, occurrence data vectors can be generated as illustrated in Table 1. The presence of a gene occurring at least once in a library is indicated by a one, and its absence from the library, by a zero. 1 TABLE 1 Occurrence data for genes A and B Library 1 Library 2 Library 3 . . . Library N gene A 1 1 0 . . . 0 gene B 1 0 1 . . . 0

[0043] For a given pair of genes, the occurrence data in Table 1 can be summarized in a 2×2 contingency table. 2 TABLE 2 Contingency table for co-occurrences of genes A and B Gene A present Gene A absent Total Gene B present 8  2 10 Gene B absent 2 18 20 Total 10  20 30

[0044] Table 2 presents co-occurrence data for gene A and gene B in a total of 30 libraries. Both gene A and gene B occur 10 times in the libraries. Table 2 summarizes and presents: 1) the number of times gene A and B are both present in a library; 2) the number of times gene A and B are both absent in a library; 3) the number of times gene A is present, and gene B is absent; and 4) the number of times gene B is present, and gene A is absent. The upper left entry is the number of times the two genes co-occur in a library, and the middle right entry is the number of times neither gene occurs in a library. The off diagonal entries are the number of times one gene occurs, and the other does not. Both A and B are present eight times and absent 18 times. Gene A is present, and gene B is absent, two times; and gene B is present, and gene A is absent, two times. The probability (“p-value”) that the above association occurs due to chance as calculated using a Fisher exact test is 0.0003. Associations are generally considered significant if a p-value is less than 0.01 (Agresti, supra; Rice, supra).

[0045] This method of estimating the probability for coexpression of two genes makes several assumptions. The method assumes that the libraries are independent and are identically sampled. However, in practical situations, the selected cDNA libraries are not entirely independent, because more than one library may be obtained from a single subject or tissue. Nor are they entirely identically sampled, because different numbers of cDNAs may be sequenced from each library. The number of cDNAs sequenced typically ranges from 5,000 to 10,000 cDNAs per library. In addition, because a Fisher exact coexpression probability is calculated for each gene versus 45,233 other assembled genes, a Bonferroni correction for multiple statistical tests is used.

[0046] The present invention identifies 34 novel atherosclerosis-associated polynucleotides that exhibit strong association with genes known to be specific to atherosclerosis. The results presented in Table 4 show that the expression of the 34 novel atherosclerosis-associated polynucleotides has direct or indirect association with the expression of known atherosclerosis-associated genes. Therefore, the novel atherosclerosis-associated polynucleotides can potentially be used in diagnosis, treatment, prognosis, or prevention of diseases associated with atherosclerosis or in the evaluation of therapies for atherosclerosis. Further, the gene products of the 34 novel atherosclerosis-associated polynucleotides are either potential therapeutics or targets of therapeutics against atherosclerosis.

[0047] Therefore, in one embodiment, the present invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO: 1-34. These 34 polynucleotides are shown by the method of the present invention to have strong coexpression association with known atherosclerosis-associated genes and with each other. The invention also encompasses a variant of the polynucleotide sequence, its complement, or 18 consecutive nucleotides of a sequence provided in the above described sequences. Variant polynucleotide sequences typically have at least about 75%, more preferably at least about 85%, and most preferably at least about 95% polynucleotide sequence identity to NSEQ.

[0048] NSEQ or the encoded PSEQ may be used to search against the GenBank primate (pri), rodent (rod), mammalian (mam), vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt, BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25:217-221), PFAM, and other databases that contain previously identified and annotated motifs, sequences, and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5:35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol. 215:403-410), BLOCKS (Henikoff and Henikoff (1991) Nucleic Acids Res. 19:6565-6572), Hidden Markov Models (HMM; Eddy (1996) Cur. Opin. Str. Biol. 6:361-365; Sonnhammer et al. (1997) Proteins 28:405-420), and the like, can be used to manipulate and analyze nucleotide and amino acid sequences. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., p 856-853).

[0049] Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to SEQ ID NO: 1-34, and fragments thereof under stringent conditions. Stringent conditions can be defined by salt concentration, temperature, and other chemicals and conditions well known in the art. Conditions can be selected, for example, by varying the concentrations of salt in the prehybridization, hybridization, and wash solutions or by varying the hybridization and wash temperatures. With some substrates, the temperature can be decreased by adding formamide to the prehybridization and hybridization solutions.

[0050] Hybridization can be performed at low stringency, with buffers such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60° C., which permits complex formation between two nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45° C. (medium stringency) or 68° C. (high stringency), to maintain hybridization of only those complexes that contain completely complementary sequences. Background signals can be reduced by the use of detergents such as SDS, Sarcosyl, or Triton X-100, and/or a blocking agent, such as salmon sperm DNA. Hybridization methods are described in detail in Ausubel (supra, units 2.8-2.11, 3.18-3.19 and 4-6-4.9) and Sambrook et al. (1989; Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.)

[0051] NSEQ can be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences such as promoters and other regulatory elements. (See, e.g., Dieffenbach and Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.). Additionally, one may use an XL-PCR kit (PE Biosystems, Foster City Calif.), nested primers, and commercially available cDNA libraries (Life Technologies, Rockville Md.) or genomic libraries (Clontech, Palo Alto Calif.) to extend the sequence. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth Minn.) or another program, to be about 18 to 30 nucleotides in length, to have a GC content of about 50%, and to form a hybridization complex at temperatures of about 68° C. to 72° C.

[0052] In another aspect of the invention, NSEQ can be cloned in recombinant DNA molecules that direct the expression of PSEQ, or structural or functional portions thereof, in host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express the polypeptide encoded by NSEQ. The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

[0053] In order to express a biologically active polypeptide, NSEQ, or derivatives thereof, may be inserted into an expression vector, i.e., a vector which contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5′ and 3′ untranslated regions. Methods which are well known to those skilled in the art may be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, supra; and Ausubel, supra).

[0054] A variety of expression vector/host cell systems may be utilized to express NSEQ. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus vectors; plant cell systems transformed with viral or bacterial expression vectors; or animal cell systems. For long term production of recombinant proteins in mammalian systems, stable expression in cell lines is preferred. For example, NSEQ can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable or visible marker gene on the same or on a separate vector. The invention is not to be limited by the vector or host cell employed.

[0055] In general, host cells that contain NSEQ and that express PSEQ may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or amino acid sequences. Immunological methods for detecting and measuring the expression of PSEQ using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).

[0056] Host cells transformed with NSEQ may be cultured under conditions for the expression and recovery of the polypeptide from cell culture. The polypeptide produced by a transgenic cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing NSEQ may be designed to contain signal sequences which direct secretion of the polypeptide through a prokaryotic or eukaryotic cell membrane.

[0057] In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the polypeptide may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Bethesda Md.) and may be chosen to ensure the correct modification and processing of the expressed polypeptide.

[0058] In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences are ligated to a heterologous sequence resulting in translation of a fusion polypeptide containing heterologous polypeptide moieties in any of the aforementioned host systems. Such heterologous polypeptide moieties facilitate purification of fusion polypeptides using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase, maltose binding protein, thioredoxin, calmodulin binding peptide, 6-His, FLAG, c-myc, hemaglutinin, and monoclonal antibody epitopes.

[0059] In another embodiment, the nucleic acid sequences are synthesized, in whole or in part, using chemical or enzymatic methods well known in the art (Caruthers et al. (1980) Nucl. Acids Res. Symp. Ser. 215-233; Ausubel, supra). For example, peptide synthesis can be performed using various solid-phase techniques (Roberge et al. (1995) Science 269:202-204), and machines such as the ABI 431A Peptide synthesizer (PE Biosystems) can be used to automate synthesis. If desired, the amino acid sequence may be altered during synthesis and/or combined with sequences from other proteins to produce a variant protein.

[0060] In another embodiment, the invention entails a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO: 35 and fragments thereof.

[0061] Screening, Diagnostics and Therapeutics

[0062] The polynucleotide sequences can be used in diagnosis, prognosis, treatment, prevention, and selection and evaluation of therapies for atherosclerosis including, but not limited to, stroke, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, coronary artery disease, angina pectoris, peripheral vascular disease, intermittent claudication, renal artery stenosis, and hypertension.

[0063] The polynucleotide sequences may be used to screen a library of molecules for specific binding affinity. The assay can be used to screen a library of DNA molecules, RNA molecules, PNAs, peptides, ribozymes, antibodies, agonists, antagonists, immunoglobulins, inhibitors, proteins including transcription factors, enhancers, repressors, and drugs and the like which regulate the activity of the polynucleotide sequence in the biological system. The assay involves providing a library of molecules, combining the polynucleotide sequence or a fragment thereof with the library of molecules under conditions suitable to allow specific binding, and detecting specific binding to identify at least one molecule which specifically binds the polynucleotide sequence.

[0064] Similarly the polypeptide or a portion thereof may be used to screen libraries of molecules in any of a variety of screening assays. The portion of the polypeptide employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. Specific binding between the polypeptide and molecule may be measured. The assay can be used to screen a library of DNA molecules, RNA molecules, PNAs, peptides, mimetics, ribozymes, antibodies, agonists, antagonists, immunoglobulins, inhibitors, peptides, polypeptides, drugs and the like, which specifically bind the polypeptide. One method for high throughput screening using very small assay volumes and very small amounts of test compound is described in Burbaum et al. U.S. Pat. No. 5,876,946, incorporated herein by reference, which screens large numbers of molecules for enzyme inhibition or receptor binding.

[0065] In one preferred embodiment, the polynucleotide sequences are used for diagnostic purposes to determine the absence, presence, and excess expression of the polypeptide. The polynucleotides may be at least 18 nucleotides long and consist of complementary RNA and DNA molecules, branched nucleic acids, and/or peptide nucleic acids (PNAs). In one alternative, the polynucleotides are used to detect and quantify gene expression in samples in which expression of NSEQ is correlated with disease. In another alternative, NSEQ can be used to detect genetic polymorphisms associated with a disease. These polymorphisms may be detected in the transcript cDNA.

[0066] The specificity of the probe is determined by whether it is made from a unique region, a regulatory region, or from a conserved motif. Both probe specificity and the stringency of diagnostic hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring, exactly complementary sequences, allelic variants, or related sequences. Probes designed to detect related sequences should preferably have at least 75% sequence identity to any of the polynucleotides encoding PSEQ.

[0067] Methods for producing hybridization probes include the cloning of nucleic acid sequences into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by adding RNA polymerases and labeled nucleotides. Hybridization probes may incorporate nucleotides labeled by a variety of reporter groups including, but not limited to, radionuclides such as 32P or 35S, enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, fluorescent labels, and the like. The labeled polynucleotide sequences may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; and in microarrays utilizing samples from subjects to detect altered PSEQ expression.

[0068] NSEQ can be labeled by standard methods and added to a sample from a subject under conditions for the formation and detection of hybridization complexes. After incubation the sample is washed, and the signal associated with hybrid complex formation is quantitated and compared with a standard value. Standard values are derived from any control sample, typically one that is free of the suspect disease. If the amount of signal in the subject sample is altered in comparison to the standard value, then the presence of altered levels of expression in the sample indicates the presence of the disease. Qualitative and quantitative methods for comparing the hybridization complexes formed in subject samples with previously established standards are well known in the art.

[0069] Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual subject. Once the presence of disease is established and a treatment protocol is initiated, hybridization or amplification assays can be repeated on a regular basis to determine if the level of expression in the subject begins to approximate that which is observed in a healthy subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to many years.

[0070] The polynucleotides may be used for the diagnosis of a variety of diseases associated with atherosclerosis. These include, but are not limited to, stroke, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, coronary artery disease, angina pectoris, peripheral vascular disease, intermittent claudication, renal artery stenosis, and hypertension.

[0071] The polynucleotides may also be used as targets in a microarray. The microarray can be used to monitor the expression patterns of large numbers of genes simultaneously and to identify splice variants, mutations, and polymorphisms. Information derived from analyses of the expression patterns may be used to determine gene function, to understand the genetic basis of a disease, to diagnose a disease, and to develop and monitor the activities of therapeutic agents used to treat a disease. Microarrays may also be used to detect genetic diversity, single nucleotide polymorphisms which may characterize a particular population, at the genome level.

[0072] In yet another alternative, polynucleotides may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data as described in Heinz-Ulrich et al. (In: Meyers, supra, pp. 965-968).

[0073] In another embodiment, antibodies or Fabs comprising an antigen binding site that specifically binds PSEQ may be used for the diagnosis of diseases characterized by the over-or-under expression of PSEQ. A variety of protocols for measuring PSEQ, including ELISAs, RIAs, and FACS, are well known in the art and provide a basis for diagnosing altered or abnormal levels of expression. Standard values for PSEQ expression are established by combining samples taken from healthy subjects, preferably human, with antibody to PSEQ under conditions for complex formation The amount of complex formation may be quantitated by various methods, preferably by photometric means. Quantities of PSEQ expressed in disease samples are compared with standard values. Deviation between standard and subject values establishes the parameters for diagnosing or monitoring disease. Alternatively, one may use competitive drug screening assays in which neutralizing antibodies capable of binding PSEQ specifically compete with a test compound for binding the polypeptide. Antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PSEQ. In one aspect, the anti-PSEQ antibodies of the present invention can be used for treatment or monitoring therapeutic treatment for atherosclerosis.

[0074] In another aspect, the NSEQ, or its complement, may be used therapeutically for the purpose of expressing mRNA and polypeptide, or conversely to block transcription or translation of the mRNA. Expression vectors may be constructed using elements from retroviruses, adenoviruses, herpes or vaccinia viruses, or bacterial plasmids, and the like. These vectors may be used for delivery of nucleotide sequences to a particular target organ, tissue, or cell population. Methods well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences or their complements. (See, e.g., Maulik et al. (1997) Molecular Biotechnology, Therapeutic Applications and Strategies, Wiley-Liss, New York N.Y.) Alternatively, NSEQ, or its complement, may be used for somatic cell or stem cell gene therapy. Vectors may be introduced in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors are introduced into stem cells taken from the subject, and the resulting transgenic cells are clonally propagated for autologous transplant back into that same subject. Delivery of NSEQ by transfection, liposome injections, or polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman et al. (1997) Nature Biotechnology 15:462-466.) Additionally, endogenous NSEQ expression may be inactivated using homologous recombination methods which insert an inactive gene sequence into the coding region or other targeted region of NSEQ. (See, e.g. Thomas et al. (1987) Cell 51: 503-512.)

[0075] Vectors containing NSEQ can be transformed into a cell or tissue to express a missing polypeptide or to replace a nonfunctional polypeptide. Similarly a vector constructed to express the complement of NSEQ can be transformed into a cell to downregulate the overexpression of PSEQ. Complementary or antisense sequences may consist of an oligonucleotide derived from the transcription initiation site; nucleotides between about positions −10 and +10 from the ATG are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco N.Y., pp. 163-177.)

[0076] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the cleavage of mRNA and decrease the levels of particular mRNAs, such as those comprising the polynucleotide sequences of the invention. (See, e.g., Rossi (1994) Current Biology 4: 469-471.) Ribozymes may cleave mRNA at specific cleavage sites. Alternatively, ribozymes may cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The construction and production of ribozymes is well known in the art and is described in Meyers (supra).

[0077] RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′ O-methyl rather than phosphodiester linkages within the backbone of the molecule. Alternatively, nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases, may be included.

[0078] Further, an antagonist, or an antibody that binds specifically to PSEQ may be administered to a subject to treat or prevent atherosclerosis. The antagonist, antibody, or fragment may be used directly to inhibit the activity of the polypeptide or indirectly to deliver a therapeutic agent to cells or tissues which express the PSEQ. An immunoconjugate comprising a PSEQ binding site of the antibody or the antagonist and a therapeutic agent may be administered to a subject in need to treat or prevent disease. The therapeutic agent may be a cytotoxic agent selected from a group including, but not limited to, abrin, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudomonas exotoxin A and 40, radioisotopes, and glucocorticoid.

[0079] Antibodies to PSEQ may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, such as those which inhibit dimer formation, are especially preferred for therapeutic use. Monoclonal antibodies to PSEQ may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma, the human B-cell hybridoma, and the EBV-hybridoma techniques. In addition, techniques developed for the production of chimeric antibodies can be used. (See, e.g., Pound (1998) Immunochemical Protocols, Methods Mol. Biol. Vol. 80). Alternatively, techniques described for the production of single chain antibodies may be employed. Fabs which contain specific binding sites for PSEQ may also be generated. Various immunoassays may be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.

[0080] Yet further, an agonist of PSEQ may be administered to a subject to treat or prevent a disease associated with decreased expression, longevity or activity of PSEQ.

[0081] An additional aspect of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic applications discussed above. Such pharmaceutical compositions may consist of PSEQ or antibodies, mimetics, agonists, antagonists, or inhibitors of the polypeptide. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a subject alone or in combination with other agents, drugs, or hormones.

[0082] The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0083] In addition to the active ingredients, these pharmaceutical compositions may contain pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton Pa.).

[0084] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0085] A therapeutically effective dose refers to that amount of active ingredient which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating and contrasting the ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population) statistics. Any of the therapeutic compositions described above may be applied to any subject in need of such therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

EXAMPLES

[0086] It is to be understood that this invention is not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the invention. The described embodiments are provided to illustrate the invention and are not intended to limit the scope of the invention which is limited only by the appended claims.

I cDNA Library Construction

[0087] The cDNA library SMCCNOS01 was selected as an example to demonstrate the construction of cDNA libraries from which the polynucleotides associated with known atherosclerosis-associated genes were derived. The SMCCNOS01 subtracted coronary artery smooth muscle cell library was constructed using 7.56 ×106 clones from the SMCCNOT02 library and was subjected to two rounds of subtraction hybridization for 48 hours with 6.12 ×106 clones from SMCCNOT01. The SMCCNOT02 library was constructed using RNA isolated from coronary artery smooth muscle cells removed from a 3-year-old Caucasian male. The cells were treated for 20 hours with TNF&agr; and IL-1&bgr; at 10 ng/ml each. The SMCCNOT01 was constructed using RNA isolated from untreated coronary artery smooth muscle cells from the same donor. Subtractive hybridization conditions were based on the methodologies of Swaroop et al. (1991; Nucleic Acids Res. 19:1954) and Bonaldo et al. (1996; Genome Research 6:791).

[0088] For both cDNA libraries, SMCCNOT01 and SMCCNOT02, the frozen coronary artery smooth muscle cells (50-100 mg) were homogenized in GTC buffer (4.0 M guanidine thiocyanate, 0.1 M Tris-HCl pH 7.5, 1% 2-mercaptoethanol). Two volumes of binding buffer(0.4 M LiCl, 0.1 M Tris-HCl pH 7.5, 0.02 M EDTA) were added, and the resulting mixture was vortexed at 13,000 rpm. The supernatant was removed and combined with Oligo(dT)25 bound streptavidin particles (MPG). After rotation at room temperature, the mRNA-Oligo(dT)25 bound streptavidin particles were separated from the supernatant, washed twice with hybridization buffer I (0.1 5 M NaCl, 0.01 M Tris-HCl pH8.0, 1 mM EDTA, 0.1% lauryl sarcosinate) using magnetic separation at each step to remove the supernatant from the particles. Bound mRNA was eluted from the particles with release solution and heated to 65° C. The supernatant containing eluted mRNA was magnetically separated from the particles and used to construct the cDNA libraries.

[0089] The RNA was used for cDNA synthesis and construction of the cDNA library according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia Biotech, Piscataway N.J.), and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (Incyte Pharmaceuticals, Palo Alto, Calif.). Recombinant plasmids were transformed into DH5&agr; competent cells or ELECTROMAX cells (Life Technologies).

II Isolation and Sequencing of cDNA Clones

[0090] Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Qiagen, Valencia Calif.). The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cells were cultured for 19 hours and then lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml distilled water, and samples were transferred to a 96-well block for storage at 4° C.

[0091] The cDNAs were prepared using a MICROLAB 2200 System (Hamilton, Reno Nev.) in combination with the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.). cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol. Biol. 94:441f) using ABI PRISM 377 (PE Biosystems) or MEGABACE 1000 sequencing systems (Amersham Pharmacia Biotech).

[0092] Most of the sequences disclosed herein were sequenced using standard ABI protocols and kits (PE Biosystems) at solution volumes of 0.25x -1.0x concentrations. In the alternative, some of the sequences disclosed herein were sequenced using solutions and dyes from Amersham Pharmacia Biotech.

III Selection, Assembly, and Characterization of Sequences

[0093] The sequences used for co-expression analysis were assembled from EST sequences, 5′ and 3′ longread sequences, and full length coding sequences. Selected assembled sequences were expressed in at least three cDNA libraries.

[0094] The assembly process is described as follows. EST sequence chromatograms were processed and verified. Quality scores were obtained using PHRED (Ewing et al. (1998) Genome Res. 8:175-185; Ewing and Green (1998) Genome Res. 8:186-194), and edited sequences were loaded into a relational database management system (RDBMS). The sequences were clustered using BLAST with a product score of 50. All clusters of two or more sequences created a bin which represents one transcribed gene.

[0095] Assembly of the component sequences within each bin was performed using a modification of Phrap, a publicly available program for assembling DNA fragments (Green, P. University of Washington, Seattle Wash.). Bins that showed 82% identity from a local pair-wise alignment between any of the consensus sequences were merged.

[0096] Bins were annotated by screening the consensus sequence in each bin against public databases, such as GBpri and GenPept from NCBI. The annotation process involved a FASTn screen against the GBpri database in GenBank. Those hits with a percent identity of greater than or equal to 75% and an alignment length of greater than or equal to 100 base pairs were recorded as homolog hits. The residual unannotated sequences were screened by FASTx against GenPept. Those hits with an E value of less than or equal to 10−8 were recorded as homolog hits.

[0097] Sequences were then reclustered using BLASTn and Cross-Match, a program for rapid amino acid and nucleic acid sequence comparison and database search (Green, supra), sequentially. Any BLAST alignment between a sequence and a consensus sequence with a score greater than 150 was realigned using cross-match. The sequence was added to the bin whose consensus sequence gave the highest Smith-Waterman score (Smith et al. (1992) Protein Engineering 5:35-51) amongst local alignments with at least 82% identity. Non-matching sequences were moved into new bins, and assembly processes were repeated.

IV Coexpression Analyses of Atherosclerosis-Associated Genes

[0098] Sixty-six known atherosclerosis-associated genes were selected to identify novel genes that are closely associated with atherosclerosis. The known atherosclerosis-associated genes which were examined in this analysis and brief descriptions of their functions are listed in Table 3. 3 TABLE 3 Descriptions of Known Atherosclerosis-Associated Genes GENE DESCRIPTION AND REFERENCES Human 22kDa Smooth muscle cell-specific gene which is down-regulated during smooth muscle smooth muscle cell dedifferentiation as part of atherogenic process (Sobue et al. (1998) Horm. protein (SM22) Res. 50 Suppl. 2:15-24; Sobue et al. (1999) Mol. Cell. Biochem. 190(1-2): 105- 18) calponin (CNN1) Calponin is smooth muscle-specific and may mediate smooth muscle contractility through it's binding of the amino-terminal end of the myosin regulatory light chain. Involved in phenotypic modulation of smooth muscle cells, a feature of atherosclerosis (Szymanski et al. (1999) Biochemistry 38(12): 3778-84) desmin Contractile component of myofibrils in differentiated smooth muscle cells. (DES) Regarded as a marker for smooth muscle cells (Shi et al. (1997) Circulation 95(12): 2684-93) smooth muscle Contractile component of myofibrils in differentiated smooth muscle cells. myosin heavy Regarded as a marker for smooth muscle cells (Sobue et al. (1999) Mol. Cell. chain (MYH11) Biochem. 190(1-2): 105-18) alpha Contractile component of myofibrils in differentiated smooth muscle cells (Sobue tropomyosin et al. (1999) Mol. Cell. Biochem. 190(1-2): 105-18; Kashiwada et al. (1997) J. (TPM1) Biol. Chem. 272(24): 15396-404) Human tissue TIMPs control the activity of matrix metalloproteinases and are important in local inhibitor of matrix remodeling of vasculature. Atheroma extracts shown to have 5× higher metallo- TIMP3 expression levels than non-atherosclerotic tissue. Abundant TIMP1, 2, 3 proteinase 3 expression noted in plaque macrophages and smooth muscle cells. PDGF and (TIMP3) TGFbeta augment TIMP3 expression. TIMP3 possible important role in plaque stability (Fabunmi et al. (1998) Circ. Res. 83(3): 270-8) Human tissue TIMPs control the activity of matrix metalloproteinases and are important in local inhibitor of matrix remodeling of vasculature. Abundant TIMP1, 2, 3 expression noted in metallo- plaque macrophages and smooth muscle cells. Expression of TIMP2 is greatly proteinase-2 increased during neointima formation in organ cultures of human saphenous vein (TIMP-2) (Kranzhofer et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19(2): 255-65) Human tissue TIMPs control the activity of matrix metalloproteinases and are important in local inhibitor of matrix remodeling of vasculature (Greene et al. (1996) J. Biol. Chem. 271(48): metallo- 30375-80) proteinase-4 (TIMP4) pro alpha 1(I) Member of family of fibrous structural proteins. Most abundant structural collagen component of the extracellular matrix. Secreted as procollagen and converted to (COL1A1) collagen by matrix metalloproteinases. Collagens are important in atherosclerosis for promoting platelet aggregation and for providing sites for platelet adhesion to the vessel wall (Wen et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19(3): 519- 24) collagen alpha-2 see COL1A1 above type I (COL1A2) COL6A1 see COL1A1 above procollagen see COL1A1 above alpha 2(V) (COL5A2) collagen VI see COL1A1 above alpha-2 (COL6A2) type VI collagen see COL1A1 above alpha3 (COL6A3) pro-alpha-1 type see COL1A1 above 3 collagen (COL3A1) pro-alpha-1 (V) collagen see COL1A1 above (COL3A1 collagenase type Contributes to the degradation of vascular wall/smooth muscle cells associated IV/matrix with local matrix remodeling. Expression of metalloproteinases controlled by metallo- tissue inhibitors of metalloproteinases (TIMPs). Balance between MMP and proteinase TIMP expression becomes distorted during onset and progression of 9/gelatinase B atherosclerosis. MMP9 localized to lesional macrophages, along with MMP-1, (MMP9) MMP-2, MMP-3. Rabbit aortic macrophage foam cells express immunoreactive MMP-9 (Moreau et al. (1999) Circulation 99(3): 420-426; Zaltsman et al. (1997) Atherosclerosis 130(1-2): 61-70) matrix Gla Role in active calcification of vascular smooth muscle cells, suggested by protein (MGP) expression study on VSMC in vitro differentiation study. Calcifying phenotype associated with high MGP levels. MGP knockout mice develop to term, but die up to 2 months after birth due to extensive calcification of the arteries, causing blood vessel rupture (Luo et al. (1997) Nature 386(6620): 78-81; Mori et al. (1998) FEBS Lett. 433(1-2): 19-22) cathepsin K Nonmetalloenzyme, potent elastase present in advanced atherosclerotic plaques. (CTSK) Contributes to the breakdown of components of vascular extracellular matrix, reducing tensile strength, increasing plaque vulnerability (Sukhova et al. (1998) J. Clin. Invest. 102(3): 576-83) fibrinogen beta Component of fibrin in the extracellular matrix. Fibrin deposition is an integral chain gene part of advanced atherosclerotic lesion development. Variation at the beta (FGB) fibrinogen locus associated with peripheral atherosclerosis (Sueishi et al. (1998) Semin. Thromb. Hemost. 24(3): 255-260; Fowkes et al. (1992) Lancet 339 (8795): 693-696) fibrinogen beta Participant in adhesion and aggregation of platelets which occurs through binding chain gene of platelet receptors. FGG carries the main binding site for the platelet receptor (FGG) binding. Mutations in FGG associated with clotting defects and thrombotic tendency. Fibrin deposition is an integral part of advanced atherosclerotic lesion development (Sueishi et al. (1998) Semin. Thromb. Hemost. 24 (3): 255-60; Cote et al. (1998) Blood 92(7): 2195-2212) pre-pro-von Blood glycoprotein involved in normal hemostasis. Mediates adhesion of platelets Willebrand to sites of vascular damage. Also acts as a cofactor in factor VIII activity in blood factor (VWF) coagulation. Increased levels of VWF are found in atherosclerosis and in several of its major risk factors, including hypercholesterolemia, diabetes, obesity, hypertension. Levels serve as a predictor of adverse clinical outcome following vascular surgery, possibly as an indicator of thrombus formation (Sadler, J. E. (1998) Annu Rev Biochem 67: 395-424; Blann et al. (1994) Eur. J. Vasc. Surg. 8(1): 10-15; Kessler et al. (1998) Diabetes Metab. 24(4): 327-36; Folsom et al. (1997) Circulation 96(4): 1102-1108) coagulation Central role in blood hemostasis by regulating platelet aggregation and blood factor II/ coagulation. Converts fibrinogen to fibrin in the final stage of clotting cascade. prothrombin (F2) Promotes cellular chemotaxis and proliferation, extracellular matrix turnover and release of inflammatory cytokines (Goldsack et al. (1998) Int. J. Biochem. Cell. Biol. 30(6): 641-646) Coagualtion Activation of blood coagulation is an important part of post-vascular injury with factor XII (F12) initiation of atherosclerotic lesion formation and contributes to thrombosis in advanced stage atherosclerosis (Sueishi et al. (1998) Semin. Thromb. Hemost. 24(3): 255-260) coagulation Central role in coagulation, influences plasma triglyceride levels, a risk factor in factor VII(F7) atherosclerosis. Epidemiological studies have linked F7 with cardiovascular risk/atherothrombotic tendency (Ghaddar et al. (1998) Circulation 98(25): 2815- 2821; Koenig, W. (1998) Eur. Heart J. 19 Suppl C: C39-43; Folsom et al. (1997) Circulation 96(4): 1102-1108) platelet Signalling molecule in the migration of cells as part of the pathophysiology of endothelial cell vascular occulsive diseases such as atherosclerosis. Analysis of adhesion endothelial/monocyte co-cultures indicates oxidative stress induces molecule transendothelial migration of monocytes as a result of phosphorylation of (PECAM-1) PECAM-1 (Rattan et al. (1997) Am. J. Physiol. 273(3 Pt 1): E453-61) lipoprotein- Natural anticoagulant, inhibits factor VII/tissue factor complexes. Role in associated regulating coagulation in atherosclerotic plaques. Circulates in association with coagulation plasma lipoproteins VLDL, HDL and LDL. In situ expression studies indicate inhibitor (LACI) TFPI is expressed in adventitial layer of large arteries, and in atherosclerotic vessels is expressed by macrophages in focal areas throughout the plaque (Drew et al. (1997) Lab. Invest. 77(4): 291-298; Sandset, P.M. (1996) Haemostasis 26 Suppl 4: 154-165) antithrombin III ATIII is the sole blood component through which heparin exerts its anti variant (AT3) coagulation effect. Deficiency in ATIII causes recurrent venous thrombosis and pulmonary embolism and can be inherited in autosomal dominant fashion (Hultin et al. (1988) Thromb. Haemost. 59(3): 468-73; Lane et al. (1996) Blood Rev. 10(2): 59-74) plasminogen Major physiological inhibitor of fibrinolysis. Plasma levels correlate with activator incidence of MI and venous thrombosis. Both adipocytes and endothelial cells inhibitor-1 produced PAI, possibly under the control of PPARG, as demonstrated using (PAI-1) recombinant PPARG expression constructs in endothelial cell lines. Increased expression of PAI observed in coronary heart disease. 4G polymorphism in promotor causes increased PAI expression associated with MI in some studies (Eriksson et al. (1995) Proc. Natl. Acad. Sci. USA 92(6): 1851-5; Marx et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19(3): 546-551) lipoprotein lipase Hydrolises triglyceride in chylomicrons and therefore regulates metabolism of (LPL) circulating lipoproteins. Appears to have an atherogenic effect on the arterial wall due to its ability to alter the properties of LDL. Increased activity of LPL is found in atherosclerotic arteries when compared to normal. Expressed by macrophages in atherosclerotic lesions. Mutations in LPL responsible for familial hypercholesterolemia and premature atherosclerosis (Fisher. et al. (1997) Atherosclerosis 135(2): 145-159; Goldberg, I. J. (1996) J. Lipid Res. 37(4): 693- 707; Gerdes et al. (1997) Circulation 96(3): 733-740) alpha-2- Foam cell formation - retains LDL cholesterol in the lipid core of atherosclerotic macroglobulin plaque (Llorente et al. (1998). Rev. Esp. Cardiol. 51(8): 633-641) (A2M) apolipoprotein Participates in reverse cholesterol transport from tissues to the liver. Promotes AI (APOA1) cholesterol efflux from tissues and acts as a cofactor for lecithin cholesterol acyltransferase (LCAT). Mutations in ApoA1 and of ApoAI/CIII/AIV gene cluster assoc with atherosclerosis. Transgenic mice expressing high plasma APOAI levels are protected from fatty streak development with a high atherogenic diet (Gordon et al. (1989) Circulation 79(1): 8-15; Rubin et al. (1991) Nature 353(6341): 265-7; Karathanasis et al. (1987) Proc. Natl. Acad. Sci. USA 84(20): 7198-7202) apolipoprotein Major component of HDL. Appears to have an opposite effect to that of APOAI, AII (APOA2) though exact function unknown. APOAII may have ability to convert HDL from an anti- to a pro-inflammatory particle, with paraoxonase having a role in this transformation process. Plasma APOAII levels significantly associated with plasma free fatty acid levels. Transgenic mice expressing varying levels of APOAII show increased atherosclerotic lesions than wt when fed an atherogenic diet. Possible interaction between diet/genotype and atherogenic potential (Escola-Gil et al. (1998) J. Lipid Res. 39(2): 457-462; Warden et al. (1993) Proc. Natl. Acad. Sci. USA 90(22): 10886-10890) apolipoprotein B- Main apolipoprotein of chylomicrons and low density lipoproteins. Mutations in 100 (APOB) APOB100 underly familial defective apolipoprotein B-100 in which patients suffer from premature atherosclerosis. Mutations result in defect in binding of LDL to LDL receptor, and accumulation of plasma LDL. High-expressing APOB transgenic mice exibit elevated VLDL-LDL cholesterol and atherogenic lesions (Callow et al. (1995) J Clin Invest 96(3): 1639-1646; Brasaemle et al. (1997) J. Biol. Chem. 272(14): 9378-9387) lipoprotein Role in lipoprotein metabolism. Cofactor in the activity of lipoprotein lipase the apoCII (APOC2) enzyme that hydrolyzes triglycerides in plasma and transfers the fatty acids to tissues. Mutations in APOC2 responsible for hyperlipoproteinemia 1B. similar to lipoprotein lipase deficiency (Cox et al. (1978) N. Engl. J. Med. 299(26): 1421- 1424; Arimoto et al. (1998) J. Lipid Res. 39(1): 143-151) pre- Inhibits lipoprotein lipase and hepatic lipase, decreases uptake of lymph apolipoprotein chylomicrons by hepatic cells. APOA3 possibly delays breakdown of triglyceride CIII (APOC3) rich particles. SstI RFLP in apoCIII is associated with plasma triglyceride and apoCIII levels and hyperlipidemic phenotypes (Henderson et al. (1987) Hum. Genet. 75(1): 62-65) apolipoprotein APOC4 is a lipid-binding protein that has the potential to alter lipid metabolism. apoC-IV Human APOC4 transgenic mice are hypertriglyceridaemic compared to normal (APOC4) controls 9Allan et al. (1996) J. Lipid Res. 37(7): 1510-1518) macrophage Mediates binding, internalisation and processing of negatively-charged scavenger macromolecules. Implicated in the pathological deposition of cholesterol in receptor type I arterial walls during atherogenesis (Han et al. (1998) Hum. Mol. Genet. 7(6): (MSR1) 1039-1046) Human antigen Acts as a scavenger receptor for oxidised LDL. Transient regulation under control CD36 gene of M-CSF during monocyte-macrophage differentiation increases foam cell (CD36) accumulation, Possible role in atherogenesis: increased M-CSF levels detected in atherosclerotic lesions in rabbits and humans. Huh et al. (1996) Blood 87(5): 2020-2028; Aitman, et al. (1999) Nat. Genet. 21(1): 76-83) serum amyloid P Plasma glycoprotein expressed in atherosclerotic lesions. Interacts with component lipoproteins in specific manner (Li et al. (1995) Arterioscler. Thromb. Vasc. Biol. (SAP) 15(2): 252-257; Li et al. (1998) Biochem. Biophys. Res. Commun. 244(1): 249- 252) carboxyl ester CEL gene expression increases in presence of oxidised and native LDL in vitro. It lipase gene is expressed in the vessel wall and in aortic extracts - may interact with cholesterol (CEL) to modulate progression of atherosclerosis (Li et al. (1998) Biochem. J. 329(Pt 3): 675-679) paraoxonase 1 Serum esterase exclusively associated with high-density lipoproteins; it might (PON1) confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. PON1 gln192-to-arg polymorphism associated with CAD. Association between PON1 genetic variation and plasma LDL, HDL and non-HDL and apoB levels in genetically isolated Alberta Hutterite population. When fed on a high-fat, high-cholesterol diet, PON1-null mice were more susceptible to atherosclerosis than wild-type (Serrato et al. (1995) J. Clin. Invest. 96(6): 3005-3008; Boright et al. (1998) Atherosclerosis 139(1): 131-136; Shih et al. (1998) Nature 394(6690): 284-287) paraoxonase 2 Serum esterase exclusively associated with high-density lipoproteins; it might (PON2) confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. Common polymorphism at codon 311 (cys-ser) in PON2 associated with CHD alone and synergistically with the 192 polymorphism in PON1 in Asian Indians. Association between genetic variation in PON2 and plasma cholesterol and apolipoprotein A1 in genetically isolated Alberta Hutterite population (Sanghera et al. (1998) Am. J. Hum. Genet. 62(1): 36-44; Boright et al. (1998) Atherosclerosis 139(1): 131-136) paraoxonase 3 Serum esterase exclusively associated with high-density lipoproteins; it might (PON3) confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. Other members PON2, 3 associated with CHD and cholesterol levels (Laplaud et al. (1998) Clin. Chem. Lab. Med. 36(7): 431-441) LDL-receptor Possible important role in atherosclerotic lesion development. Abundant related protein expression of mRNA and protein found in vascular smooth muscle cells and (LRP1) macrophages of early and advanced atherosclerotic lesions. Receptor for uptake of ApoE-containing lipoprotein particles (Beisiegel. et al. (1989) Nature 341(6238): 162-164; Hiltunen et al. (1998) Atherosclerosis 137 Suppl: S81-88) hepatic Hepatic lipase is involved in cholesterol efflux. Downstream of cholesterol ester triglyceride transfer protein in pathway: acts on triglyceride-rich HDL to promote formation of lipase (HTGL) smaller HDL particles - effectors of cellular cholesterol efflux Fan et al. (1998) J. Atheroscler. Thromb. 5(1): 41-45; Santamarina-Fojo et al. (1998) Curr. Opin. Lipidol. 9(3): 211-219) 3-hydroxy-3- Catalyses rate limiting step in cholesterol biosynthesis as well as being involved in methylglutaryl other systems (eg. primordial germ cell migration). Expression of HMG CoA coenzyme A reductase is regulated by oxysterols via sterol-regulatory element in the promotor, synthase as is found in APOE. Target for cholesterol-lowering therapies: prevastatin, (HMGCR) “statins” (Bocan et al. (1998) Atherosclerosis 139(1): 21-30; Farnier et al. (1998) Am. J. Cardiol. 82(4B): 3J-10J) very low density Role in triglyceride metabolism. Marked induction of VLDLR expression lipoprotein observed in fatty streaks and plaques in rabbit atherosclerosis models (Hiltunen et receptor al. (1998) Circulation 97(11): 1079-1086) (VLDLR) Microsomal Catalyses transport of triglyceride, cholesterol ester and phospholipid between triglyceride phospholipid surfaces. Mutations cause abetalipoproteinemia. Linkage found transfer protein between MTP genotype and plasma triglyceride levels in a quantitative sib-pair (MTP) analysis of female dizygotic twins. Inhibitors of MTP normalise atherogenic lipoprotein profiles in an atherosclerotic rabbit model (Wetterau et al. (1992) Science 258(5084): 999-1001; Austin et al. (1998) Am. J. Hum. Genet. 62(2): 406-419; Wetterau et al. (1998) Science 282(5389): 751-754) perilipin (PLIN) Lipid storage droplets of steroidogenic cells are surrounded by perilipins, family of phosphorylated proteins encoded by a single gene, detected in adipocytes and steroidogenic cells. Possible role in lipid metabolism (Brasaemle et al. (1997) J. Biol. Chem. 272(14): 9378-9387) endothelin-1 Secretion of EDN1 coincides with the location of native and oxidised low density (EDN1) lipoproteins and occurs in a specific fashion suggesting that EDN1 may be involved in pathophysiological processes such as atherogenesis. Quantitative and qualitative immunohistochemical analysis of anti EDN1 antibodies in the wall layers of human arteries ex vivo suggest that EDN1 is normally expressed exclusively in endothelial cells. However, in cases of coronary artery disease and atherosclerosis, EDN1 expression is enhanced and can be found in the tunica media and vascular smooth muscle cells. Analysis of recombinant EDN1 expression in vitro suggests it influences vascular smooth muscle cell proliferation. Potent vasoconstriction properties(Unoki et al. (1999) Cell Tissue Res. 295(1): 89-99; Rossi et al. (1999) Circulation 99(9): 1147-1155; Yoshizumi et al. (1998) Br. J. Pharmacol. 125(5): 1019-1027; Alberts et al. (1994) J. Biol. Chem.269(13): 10112-10118) endothelin Mediates action of endothelin1 on vascular smooth muscle migration, proliferation receptor A and monocyte/endothelial cell interaction during initiation and progression of (EDNRA) atherosclerotic lesion development (Kohno et al. (1998)J. Cardiovasc. Pharmacol. 31 Suppl 1: S84-9; Alberts et al. (1994) J. Biol Chem 269(13): 10112-10118) interleukin 6 Inflammatory cytokine present in arterial atherosclerotic wall which is upregulated (IL6) by platelets to stimulate smooth muscle cell growth. Increased expression of IL6 in atherosclerotic aortas of APOE knockout vs aortas from aged-matched controls. Secretion levels of IL6 is positively associated with increased lesion surface area in APOE aortic tissue samples (Sukovich, et al. (1998) Arterioscler. Thromb. Vasc. Biol. 18(9): 1498-1505; Loppnow, et al. (1998) Blood 91(1): 134-141) interleukin 1 May contribute to regulation of local pathogenesis in the vessel wall by activation (IL1) of the cytokine regulatory network. IL-1 antagonist inhibits platelet-induced cytokine production of smooth muscle cells (Loppnow et al. (1998) Blood 91(1): 134-141) complement Complement activation of C8 shown to be an initial event in atherogenesis protein C8 alpha (Torzewski et al. (1996) Arterioscler. Thromb. Vasc. Biol. 16(5): 673-677) (C8A) complement Complement activation of C9 shown to be an initial event in atherogenesis component C9 (Torzewski et al. (1996) Arterioscler. Thromb. Vasc. Biol. 16(5): 673-677) (C9) Prostaglandin D2 Catalyses conversion of PGH2 to PGD2, a prostaglandin important in smooth synthase muscle contraction/relaxation and potent inhibitor of platelet aggregation. (PTGDS) Northern analysis shows strong specific expression in heart. Immunocytochemical localisation to myocardial and atrio endocardial cells, and accumulates in end-stage atherosclerotic plaques. High plasma levels detected in severe angina patients (Eguchi et al. (1997) Proc. Natl. Acad. Sci. USA 94(26): 14689-14694) Annexin Inhibits phospholipase A2 activity and hence the production of arachidonic acid, II/lipocortinII the precursor of the inflammatory mediators prostaglandins and leukotrienes. (ANX2) ANX2 is an important anti-inflammatory molecule. Independently binds plasminogen and t-PA and therefore suspected of having a role in atherogenesis. Binding of plasminogen to ANX2 is specifically inhibited by the excess atherogenic Lp(a) (Hajjar et el. (1998) J. Investig. Med. 46(8): 364-369) Annexin Inhibits phospholipase A2 activity and hence the production of arachidonic acid, I/lipocortin the precursor of the inflammatory mediators prostaglandins and leukotrienes. (ANX1) ANXI is an important anti-inflammatory molecule (Wallner, et al. (1986) Nature 320(6057): 77-81) Prostaglandin- Major mechanism for the regulation of prostaglandin synthesis. Arachidonic acid endoperoxide pathway. Role in inflammation and endothelial cell migration/angiogenesis. Synthase 2 Regulated enzyme - major mediator of inflammation. Antiinflammatory (PTGS2) glucocorticoids are potent inhibitors of this cyclooxygenase. Over expression of PTGS2 in vitro in rabbit epithelial cells causes increased adhesion to extracellular matrix proteins and inhibition of apoptosis, hallmarks of atherosclerotic plaque formation (Morham et al. (1995) Cell 83(3): 473-482; O'Banion et al. (1992) Proc. Natl. Acad. Sci. USA 89(11): 4888-4892; Tsujii et al. (1995) Cell 83(3): 493-501) insulin-like A study of 218 individuals indicates free IGFBP1 levels are associated with high growth factor HDL cholesterol and more favourable cardiovascular outcome. The binding protein-1 IGF1/IGFBP1 system found to be associated with cardiovascular risk and (IGFBP-1) atherosclerosis (Janssen et al. (1998) Arterioscler. Thromb. Vasc. Biol. 18(2): 277-282) Secreted protein, Extracellular glycoprotein secreted by endothelial cells which has a suspected role acidic and rich in in calcification of atherosclerotic plaques. Interacts with PDGF-B containing cysteine dimers and inhibits binding to its receptors. Expression of SPARC and PDGF is (SPARC) minimal in most adult tissues, but is enhanced following injury and advanced atherosclerotic lesions. Selective expression of SPARC causes rounding of adherent endothelial cells and influences extravasation of macromolecules (Raines et al. (1992) Proc. Natl. Acad Sci U.S.A. 89(4): 1281-1285; Goldblum et al. (1994) Proc. Natl. Acad. Sci. USA 9 1(8): 3448-3452) Human NF- Activated NF kappa B occurs in atherosclerotic lesions, and regulates the kappa-B expression of gene important in recruitment of monocytes and inflammatory transcription response. Responsible for cytokine production by smooth muscle cells during factor (NFkB) atherogenesis (Navab et al. (1995) Am. J. Cardiol. 76(9): 18C-23C; Hernandez- Presa et al. (1998) Am J Pathol 153(6): 1825-1837; Thurberg et al. (1998) Curr. Opin. Lipidol. 9(5): 387-396; Brand et al. (1997) Arterioscler. Thromb. Vasc. Biol. 17(10): 1901-1909) angiotensinogen Concentration of angiotensinogen influences the renin-angiotensin system (RAS). (AGT) Hypertensive mice carrying renin and angiotensinogen transgenes found to have higher total cholesterol levels on an atherogenic diet than their wt counterparts, and atherogenic lesions were 4× larger in surface area. Suggests hypertension induced by activated RAS is important atherogenic factor (Sugiyama et al. (1997) Lab. Invest. 76(6): 835-842) Nitric Oxide Mediates basal vasodilation. Regulates the production of nitric oxide, an Synthase 3 important signal transduction component and scavenger of reactive oxygen (NOS3) species. Activity of NOS3 appears to be a factor in endothelin/endothelin receptor B mediated endothelial cell migration and angiogenesis. Polymorphism associated with smoking dependent coronary artery disease (Goligorsky et al. (1999) Clin. Exp. Pharmacol. Physiol. 26(3): 269-271; Stroes et al. (1998) J. Cardiovasc. Pharmacol. 32 Suppl 3: S14-21; Sobue, et al. (1998) Horm. Res. 50 Suppl 2:15- 24) Nitric Oxide Mediates basal vasodilation. Regulates the production of nitric oxide, an Synthase 2 important signal transduction component and scavenger of reactive oxygen (NOS2) species. NOS2, known as inducible NOS is expressed in most cells only after induction by immunologic and inflammatory stimuli, and is upregulated in pathological conditions such as atherosclerosis (Dusting et al. (1998) Clin. Expt. Pharmacol. Phisiol. Suppl. 25: S34-41)

[0099] From a total of 45,233 assembled gene sequences, 34 novel genes were identified, SEQ ID NOs: 1-34, that show strong association with 66 known atherosclerosis-associated genes. Initially, the degree of association was measured by probability values using a cutoff p value less than 0.00001. The sequences were further examined to ensure that the genes that passed the probability test had strong association with known atherosclerosis-associated genes. Details of the co-expression patterns for the 66 known and 34 novel atherosclerosis-associated polynucleotides are presented in Table 4. The entries in Table 4 are the negative log of the p-value (−log p) for the coexpression of the two genes. The novel atherosclerosis-associated polynucleotides identified are listed in the table by their SEQ ID NOs numbers, and the known genes, by their names or the abbreviations shown in Table 3. 4 TABLE 4 Co-expression of 34 novel genes with 66 known atherosclerosis genes. (−log p) SM22 CNN1 DES MYH11 TPM1 TIMP3 TIMP2 TIMP4 COL1A1 COL1A2 COL6A1 COL5A2 SEQ ID NO:1 0 0 1 0 0 0 1 0 1 0 0 0 SEQ ID NO:2 3 1 1 2 4 4 5 0 1 3 2 2 SEQ ID NO:3 0 0 0 0 1 1 2 1 1 0 1 1 SEQ ID NO:4 1 1 1 1 0 0 0 1 0 1 1 1 SEQ ID NO:5 0 1 0 0 1 0 0 1 0 1 0 1 SEQ ID NO:6 1 0 1 0 1 0 0 0 1 1 0 1 SEQ ID NO:7 1 0 1 0 0 0 0 1 1 1 1 0 SEQ ID NO:8 9 5 8 7 6 4 7 0 9 12 13 3 SEQ ID NO:9 14 6 5 10 10 8 10 2 4 7 9 4 SEQ ID NO:10 8 5 5 5 9 6 3 0 4 5 5 2 SEQ ID NO:11 2 1 1 2 1 2 4 2 1 0 2 1 SEQ ID NO:12 4 1 1 3 4 5 5 2 1 4 2 1 SEQ ID NO:13 3 4 4 5 2 0 1 0 1 1 1 2 SEQ ID NO:14 4 3 0 3 2 3 2 7 4 3 3 3 SEQ ID NO:15 0 1 1 0 1 2 1 0 1 1 1 1 SEQ ID NO:16 9 11 12 8 11 7 4 1 4 7 9 2 SEQ ID NO:17 2 2 2 1 4 2 3 2 6 5 6 9 SEQ ID NO:18 0 0 1 0 1 0 0 0 0 0 0 0 SEQ ID NO:19 27 17 14 14 15 11 12 3 7 13 17 2 SEQ ID NO:20 20 13 9 12 12 19 19 4 22 27 27 15 SEQ ID NO:21 3 2 0 1 4 7 4 4 1 3 3 0 SEQ ID NO:22 11 6 3 6 6 10 3 2 5 6 7 3 SEQ ID NO:23 1 0 0 0 0 1 1 0 1 1 2 1 SEQ ID NO:24 10 8 6 7 7 8 5 5 7 8 12 3 SEQ ID NO:25 3 0 1 0 1 3 2 3 2 4 2 4 SEQ ID NO:26 1 0 1 0 0 1 0 0 1 0 1 0 SEQ ID NO:27 0 1 1 0 0 0 0 1 1 0 0 0 SEQ ID NO:28 2 2 0 1 1 1 1 1 1 0 0 0 SEQ ID NO:29 1 1 1 1 1 1 1 0 0 1 0 1 SEQ ID NO:30 1 1 1 1 2 4 1 1 1 0 1 3 SEQ ID NO:31 2 1 1 0 1 5 2 0 7 8 6 11 SEQ ID NO:32 0 1 0 1 0 1 0 0 1 0 1 0 SEQ ID NO:33 0 0 1 0 1 0 0 0 1 2 0 0 SEQ ID NO:34 30 48 44 44 30 15 10 3 11 17 24 3 COL6A2 COL6A3 COL3A1 COL5A1 MMP9 MGP CTSK FGB FGG VWF F2 F12 F7 SEQ ID NO:1 0 0 0 0 0 0 0 4 3 1 5 1 0 SEQ ID NO:2 2 4 3 4 4 5 3 1 1 6 0 0 1 SEQ ID NO:3 1 0 0 1 0 1 1 4 4 0 6 6 2 SEQ ID NO:4 0 1 1 1 0 0 1 7 4 0 3 1 0 SEQ ID NO:5 0 0 0 0 0 0 0 2 2 0 4 1 4 SEQ ID NO:6 1 0 0 1 1 0 1 4 6 0 4 2 6 SEQ ID NO:7 1 0 1 0 1 0 0 7 5 0 4 2 2 SEQ ID NO:8 14 11 12 4 8 10 13 1 2 3 0 1 0 SEQ ID NO:9 13 5 6 8 11 14 6 1 2 12 1 0 0 SEQ ID NO:10 7 5 6 5 5 10 1 2 3 1 1 2 1 SEQ ID NO:11 1 0 1 0 0 1 0 1 0 1 1 1 0 SEQ ID NO:12 6 4 4 5 4 6 1 0 1 10 0 0 0 SEQ ID NO:13 2 2 2 2 1 1 0 0 1 0 1 0 1 SEQ ID NO:14 4 4 3 1 2 4 1 0 1 5 0 0 0 SEQ ID NO:15 1 1 0 1 2 1 2 1 2 1 2 2 4 SEQ ID NO:16 9 11 10 7 5 11 3 0 1 5 0 0 1 SEQ ID NO:17 8 4 6 7 6 1 3 1 1 1 1 2 2 SEQ ID NO:18 0 0 1 0 0 1 1 4 4 1 4 3 1 SEQ ID NO:19 25 12 11 6 10 21 6 3 1 7 1 0 0 SEQ ID NO:20 33 24 34 15 27 20 18 0 2 8 1 0 0 SEQ ID NO:21 2 6 3 1 3 9 1 0 1 7 0 0 1 SEQ ID NO:22 7 10 6 2 8 14 5 0 6 5 0 1 1 SEQ ID NO:23 1 0 1 0 1 0 1 7 7 0 7 5 0 SEQ ID NO:24 9 11 10 3 11 11 6 1 0 8 1 0 0 SEQ ID NO:25 2 5 5 3 4 2 1 0 0 4 0 0 1 SEQ ID NO:26 0 1 0 0 1 1 0 0 0 0 0 0 0 SEQ ID NO:27 0 0 0 1 0 0 0 6 4 0 7 4 2 SEQ ID NO:28 1 0 0 0 0 1 1 12 10 0 9 7 1 SEQ ID NO:29 I I 1 1 0 1 0 6 4 0 4 0 0 SEQ ID NO:30 1 2 1 1 2 1 1 4 5 1 7 5 2 SEQ ID NO:31 8 9 10 7 9 3 7 1 2 3 1 2 4 SEQ ID NO:32 1 0 1 0 0 0 0 7 4 0 6 4 2 SEQ ID NO:33 0 0 1 0 1 1 0 8 5 1 8 4 1 SEQ ID NO:34 26 18 17 13 14 20 6 1 1 8 0 1 2 PECAM1 LAC1 AT3 PA11 LPL A2M APOA1 APOA2 APOB APOC2 APOC3 APOC4 MSR1 CD36 SEQ ID NO:1 0 1 5 1 0 1 3 4 2 2 5 4 0 0 SEQ ID NO:2 7 4 0 3 1 4 0 0 0 0 0 2 3 SEQ ID NO:3 0 1 8 1 1 1 5 4 3 3 5 7 0 0 SEQ ID NO:4 0 0 4 0 1 2 4 5 4 4 6 2 0 1 SEQ ID NO:5 1 3 1 2 0 0 5 4 3 5 2 0 0 0 SEQ ID NO:6 0 3 6 1 1 1 10 6 5 6 4 0 0 1 SEQ ID NO:7 1 2 5 1 1 1 6 5 6 3 6 3 0 0 SEQ ID NO:8 2 1 0 0 4 9 1 0 0 1 0 0 4 2 SEQ ID NO:9 6 3 1 2 5 7 0 1 1 0 1 0 7 3 SEQ ID NO:10 4 6 3 5 3 8 1 1 2 0 2 1 2 7 SEQ ID NO:11 0 1 0 0 3 3 0 0 0 2 0 0 1 1 SEQ ID NO:12 8 7 0 3 5 6 1 1 1 2 0 0 5 6 SEQ ID NO:13 0 0 0 1 1 1 11 0 10 2 4 0 0 0 SEQ ID NO:14 3 0 0 1 4 4 0 0 0 0 0 0 9 5 SEQ ID NO:15 1 1 2 1 1 0 3 1 2 2 1 0 0 2 SEQ ID NO:16 2 1 1 0 3 6 2 0 1 0 0 0 4 3 SEQ ID NO:17 0 1 0 5 1 1 3 2 1 2 1 0 1 2 SEQ ID NO:18 0 1 5 2 1 1 4 5 5 5 3 1 0 0 SEQ ID NO:19 5 2 1 4 1 13 1 1 1 0 1 0 10 1 SEQ ID NO:20 7 1 0 4 3 12 1 0 0 1 1 0 9 5 SEQ ID NO:21 5 3 0 2 6 7 0 0 0 1 0 0 5 9 SEQ ID NO:22 4 4 0 2 6 10 1 0 1 0 0 0 8 5 SEQ ID NO:23 1 1 8 1 1 3 5 8 4 6 8 9 1 1 SEQ ID NO:24 2 1 0 0 12 10 1 1 0 1 0 0 9 14 SEQ ID NO:25 4 1 0 1 8 5 0 1 0 1 1 0 5 11 SEQ ID NO:26 0 0 0 0 0 0 0 0 0 0 0 0 1 0 SEQ ID NO:27 1 1 8 2 0 1 5 7 6 4 7 3 0 0 SEQ ID NO:28 0 3 13 1 0 2 10 16 10 8 14 7 1 1 SEQ ID NO:29 1 2 5 0 0 1 5 6 5 3 4 2 0 0 SEQ ID NO:30 4 2 9 3 0 2 6 8 7 6 4 3 1 1 SEQ ID NO:31 3 2 1 1 2 2 3 2 1 1 0 0 1 2 SEQ ID NO:32 0 1 6 2 0 1 6 7 7 4 8 1 0 1 SEQ ID NO:33 0 2 7 3 0 2 8 10 10 7 8 4 0 0 SEQ ID NO:34 2 0 0 1 2 20 1 0 3 0 0 0 10 4 SAP CEL PON1 PON2 PON3 LRP1 HTGL HMGCR VLDLR MTP PLIN EDN1 EDNRA IL6 SEQ ID NO:1 6 0 5 1 2 1 0 3 0 1 0 0 1 0 SEQ ID NO:2 0 1 1 6 1 2 1 0 6 0 1 4 3 0 SEQ ID NO:3 8 1 5 2 2 0 2 2 1 1 0 0 0 1 SEQ ID NO:4 3 0 2 0 1 0 1 1 0 0 0 0 0 1 SEQ ID NO:5 1 1 1 1 2 0 2 1 0 1 1 1 2 0 SEQ ID NO:6 2 3 5 3 8 1 6 4 2 3 2 2 2 0 SEQ ID NO:7 4 1 2 0 1 0 1 1 0 3 0 1 0 0 SEQ ID NO:8 0 2 1 1 1 8 1 2 2 1 6 1 2 0 SEQ ID NO:9 0 2 1 4 0 8 0 1 3 0 11 2 3 1 SEQ ID NO:10 2 0 1 4 5 2 1 5 1 0 2 3 1 6 SEQ ID NO:11 1 0 1 5 0 5 1 0 4 0 1 1 0 1 SEQ ID NO:12 0 1 0 7 1 4 1 2 3 0 6 4 4 0 SEQ ID NO:13 1 3 1 1 1 1 1 5 1 9 0 0 1 0 SEQ ID NO:14 0 0 0 1 0 3 0 0 1 0 15 1 0 3 SEQ ID NO:15 3 6 6 2 7 1 3 2 2 2 4 2 2 0 SEQ ID NO:16 0 3 1 3 1 4 1 3 4 0 3 1 4 1 SEQ ID NO:17 1 3 2 3 2 2 2 2 3 1 2 2 7 2 SEQ ID NO:18 2 1 2 2 1 0 3 1 1 1 1 1 0 0 SEQ ID NO:19 1 0 0 2 1 8 1 1 2 0 1 0 1 2 SEQ ID NO:20 0 1 1 2 2 17 0 1 1 0 7 0 4 4 SEQ ID NO:21 0 1 0 1 0 2 0 0 2 0 6 0 0 2 SEQ ID NO:22 1 1 1 2 3 3 1 2 1 0 8 0 1 2 SEQ ID NO:23 11 0 5 1 2 1 0 4 0 0 0 1 0 0 SEQ ID NO:24 0 0 0 0 0 6 0 0 2 0 19 0 1 2 SEQ ID NO:25 1 1 0 1 0 2 1 2 2 0 8 3 2 1 SEQ ID NO:26 0 0 0 0 1 0 0 0 0 0 0 0 0 2 SEQ ID NO:27 9 1 6 1 5 0 1 3 0 2 0 0 1 0 SEQ ID NO:28 11 0 7 3 2 0 3 3 0 1 0 1 0 0 SEQ ID NO:29 1 0 1 1 1 0 2 1 0 1 0 0 0 0 SEQ ID NO:30 7 0 4 2 2 1 3 4 1 2 1 1 1 0 SEQ ID NO:31 1 1 0 1 1 3 2 2 2 0 3 1 6 1 SEQ ID NO:32 7 1 4 1 3 0 1 4 0 2 1 0 1 0 SEQ ID NO:33 5 1 3 1 1 0 2 2 0 2 0 0 0 0 SEQ ID NO:34 0 1 1 1 1 10 2 6 3 3 3 0 4 0 IL1 C8A C9 PTGDS ANX2 ANX1 PTGS2 IGFBP1 SPARC NFkB AGT NOS3 NOS2 SEQ ID NO:1 0 3 4 0 2 0 1 4 1 0 2 1 0 SEQ ID NO:2 0 0 0 2 3 4 0 2 8 5 0 4 1 SEQ ID NO:3 0 5 4 0 1 0 1 4 1 1 4 0 1 SEQ ID NO:4 0 3 4 0 1 0 1 2 1 0 3 0 0 SEQ ID NO:5 0 1 0 1 0 0 0 5 0 1 2 2 0 SEQ ID NO:6 0 4 0 0 0 0 1 8 0 0 5 6 1 SEQ ID NO:7 0 4 3 1 0 0 0 5 1 1 2 0 0 SEQ ID NO:8 2 0 0 4 6 3 1 1 9 5 1 2 2 SEQ ID NO:9 0 0 1 7 6 4 0 0 11 6 3 2 0 SEQ ID NO:10 1 5 2 1 11 7 6 2 7 2 1 1 0 SEQ ID NO:11 1 0 0 23 1 1 0 1 4 2 15 0 0 SEQ ID NO:12 0 1 0 3 3 2 1 1 7 3 5 6 1 SEQ ID NO:13 0 1 0 0 1 1 0 1 0 1 1 1 1 SEQ ID NO:14 0 0 0 2 2 1 0 0 2 3 1 1 0 SEQ ID NO:15 0 3 0 0 0 2 0 4 1 0 2 4 1 SEQ ID NO:16 0 0 0 3 6 3 1 1 7 3 0 1 0 SEQ ID NO:17 2 1 0 1 1 3 1 4 4 4 2 3 0 SEQ ID NO:18 0 6 1 0 0 0 0 4 1 0 5 1 0 SEQ ID NO:19 1 1 0 12 7 9 3 1 14 6 2 0 1 SEQ ID NO:20 0 0 0 6 12 9 0 0 22 8 0 1 1 SEQ ID NO:21 1 0 0 2 3 1 1 1 6 2 1 0 1 SEQ ID NO:22 0 1 1 6 3 10 1 1 6 2 0 2 1 SEQ ID NO:23 1 5 11 1 0 1 1 3 0 0 3 0 0 SEQ ID NO:24 0 0 0 4 4 2 1 0 7 3 1 0 0 SEQ ID NO:25 1 1 0 0 2 2 0 1 3 5 1 1 0 SEQ ID NO:26 0 0 0 1 0 0 2 0 0 0 0 0 5 SEQ ID NO:27 0 9 8 0 1 1 1 6 0 0 3 2 0 SEQ ID NO:28 0 5 8 2 0 0 0 10 1 0 8 0 0 SEQ ID NO:29 0 1 2 1 2 1 0 5 2 0 3 0 0 SEQ ID NO:30 1 7 6 2 1 3 1 6 1 3 5 0 0 SEQ ID NO:31 0 2 0 1 3 2 0 1 6 4 1 2 0 SEQ ID NO:32 0 7 6 0 0 0 1 7 1 0 4 2 0 SEQ ID NO:33 0 7 4 2 1 1 0 6 1 0 5 0 1 SEQ ID NO:34 0 0 0 11 9 6 1 1 9 3 0 3 1

V Novel Genes Associated with Atherosclerosis

[0100] Using the co-expression analysis method, 34 novel atherosclerosis-associated polynucleotides were identified, SEQ ID NOs: 1-34, that exhibit strong association, or co-expression, with 66 known atherosclerosis-associated genes.

[0101] Polynucleotides comprising the consensus sequences of SEQ ID NO: 1-34 of the present invention were first identified from Incyte bins and assembled as described in Example III. BLAST and other motif searches were performed for SEQ ID NOs: 1-34 according to Example VI. The full length and 5′-complete sequences were translated and sequence identity was sought with known sequences.

[0102] SEQ ID NO: 35 of the present invention was encoded by the nucleic acids of SEQ ID NO: 11. SEQ ID NO: 35 has 366 amino acids which are encoded by SEQ ID NO: 11. Motif analyses of SEQ ID NO: 35 shows one potential cAMP- and cGMP-dependent protein kinase phosphorylation sites at residue S343, two potential casein kinase II phosphorylation sites at residues S179 and T351, and four potential protein kinase C phosphorylation sites at residues T29, S85, T269, and T324. Additionally, SEQ ID NO: 35 contains a potential sugar transport protein signature sequence from residues L201 to S217.

VI Homology Searching for Atherosclerosis-Associated Polynucleotides and Polypeptides

[0103] The polynucleotide sequences, SEQ ID NO: 1-34, and polypeptide sequence, SEQ ID NO: 35, were queried against databases derived from sources such as GenBank and SwissProt. These databases, which contain previously identified and annotated sequences, were searched for regions of similarity using BLAST (Altschul, supra). BLAST searched for matches and reported only those that satisfied the probability thresholds of 10−25 or less for nucleotide sequences and 10−8 or less for polypeptide sequences.

[0104] The polypeptide sequence was also analyzed for known motif patterns using MOTIFS, SPSCAN, BLIMPS, and HMM-based protocols. MOTIFS (Genetics Computer Group, Madison Wis.) searches polypeptide sequences for patterns that match those defined in the Prosite Dictionary of Protein Sites and Patterns (Bairoch, supra) and displays the patterns found and their corresponding literature abstracts. SPSCAN (Genetics Computer Group) searches for potential signal peptide sequences using a weighted matrix method (Nielsen et al. (1997) Prot. Eng. 10: 1-6). Hits with a score of 5 or greater were considered. BLIMPS uses a weighted matrix analysis algorithm to search for sequence similarity between the polypeptide sequences and those contained in BLOCKS, a database consisting of short amino acid segments, or blocks of 3-60 amino acids in length, compiled from the PROSITE database (Henikoff; supra; Bairoch, supra), and those in PRINTS, a protein fingerprint database based on non-redundant sequences obtained from sources such as SwissProt, GenBank, PIR, and NRL-3D (Attwood et al. (1997) J. Chem. Inf. Comput. Sci. 37:417-424). For the purposes of the present invention, the BLIMPS searches reported matches with a cutoff score of 1000 or greater and a cutoff probability value of 1.0×10−3. HMM-based protocols were based on a probabilistic approach and searched for consensus primary structures of gene families in the protein sequences (Eddy, supra; Sonnhammer, supra). More than 500 known protein families with cutoff scores ranging from 10 to 50 bits were selected for use in this invention.

VII Labeling of Probes and Hybridization Analyses

[0105] Substrate Preparation

[0106] Nucleic acids are isolated from a biological source and applied to a substrate for standard hybridization protocols by one of the following methods. A mixture of target nucleic acids, a restriction digest of genomic DNA, is fractionated by electrophoresis through an 0.7% agarose gel in 1xTAE [Tris-acetate-ethylenediamine tetraacetic acid (EDTA)] running buffer and transferred to a nylon membrane by capillary transfer using 20x saline sodium citrate (SSC). Alternatively, the target nucleic acids are individually ligated to a vector and inserted into bacterial host cells to form a library. Target nucleic acids are arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on bacterial growth medium, LB agar containing carbenicillin, and incubated at 37° C. for 16 hours. Bacterial colonies are denatured, neutralized, and digested with proteinase K. Nylon membranes are exposed to UV irradiation in a STRATALINKER UV-crosslinker (Stratagene) to cross-link DNA to the membrane.

[0107] In the second method, target nucleic acids are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. Amplified target nucleic acids are purified using SEPHACRYL-400 beads (Amersham Pharnacia Biotech). Purified target nucleic acids are robotically arrayed onto a glass microscope slide (Corning Science Products, Corning N.Y.). The slide is previously coated with 0.05% aminopropyl silane (Sigma-Aldrich, St. Louis Mo.) and cured at 110° C. The arrayed glass slide (microarray) is exposed to UV irradiation in a STRATALINKER UV-crosslinker (Stratagene).

[0108] Probe Preparation

[0109] cDNA probes are made from mRNA templates. Five micrograms of mRNA is mixed with 1 &mgr;g random primer (Life Technologies), incubated at 70° C. for 10 minutes, and lyophilized. The lyophilized sample is resuspended in 50 &mgr;l of 1x first strand buffer (cDNA Synthesis systems; Life Technologies) containing a dNTP mix, [&agr;-32P]dCTP, dithiothreitol, and MMLV reverse transcriptase (Stratagene), and incubated at 42° C. for 1-2 hours. After incubation, the probe is diluted with 42 &mgr;l dH2O, heated to 95° C. for 3 minutes, and cooled on ice. mRNA in the probe is removed by alkaline degradation. The probe is neutralized, and degraded mRNA and unincorporated nucleotides are removed using a PROBEQUANT G-50 microcolumn (Amersham Pharmacia Biotech). Probes can be labeled with fluorescent markers, Cy3-dCTP or Cy5-dCTP (Amersharn Pharmacia Biotech), in place of the radionucleotide, [32P]dCTP.

[0110] Hybridization

[0111] Hybridization is carried out at 65° C. in a hybridization buffer containing 0.5 M sodium phosphate (pH 7.2), 7% SDS, and 1 mM EDTA. After the substrate is incubated in hybridization buffer at 65° C. for at least 2 hours, the buffer is replaced with 10 ml of fresh buffer containing the probes. After incubation at 65° C. for 18 hours, the hybridization buffer is removed, and the substrate is washed sequentially under increasingly stringent conditions, up to 40 mM sodium phosphate, 1% SDS, 1 mM EDTA at 65° C. To detect signal produced by a radiolabeled probe hybridized on a membrane, the substrate is exposed to a PHOSPHORIMAGER cassette (Amersham Pharmacia Biotech), and the image is analyzed using IMAGEQUANT data analysis software (Amersham Pharmacia Biotech). To detect signals produced by a fluorescent probe hybridized on a microarray, the substrate is examined by confocal laser microscopy, and images are collected and analyzed using GEMTOOLS gene expression analysis software (Incyte Pharmaceuticals).

VIII Complementary Polynucleotides

[0112] Molecules complementary to the polynucleotide, or a fragment thereof, are used to detect, decrease, or inhibit gene expression. Although use of oligonucleotides comprising from about 18 to about 60 base pairs is described, the same procedure is used with larger or smaller fragments or their derivatives (PNAs). Oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and SEQ ID NO: 1-34 or fragments thereof To inhibit transcription by preventing promoter binding, a complementary oligonucleotide is designed to bind to the most unique 5′ sequence, most preferably about 10 nucleotides before the initiation codon of the open reading frame. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the mRNA encoding the polypeptide.

IX Production of Specific Antibodies

[0113] The polypeptides encoded by SEQ ID NO: 1-34, or portions thereof, substantially purified using polyacrylamide gel electrophoresis or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols as described in Pound (1998; Immunochemical Protocols, Methods Mol. Biol. Vol. 80).

[0114] Alternatively, the amino acid sequence is analyzed using LASERGENE software (DNASTAR, Madison Wis.) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. Typically, oligopeptides 15 residues in length are synthesized using an ABI431A Peptide synthesizer (PE Biosystems) using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH, Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, supra) to increase immunogenicity. Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

X Screening Molecules for Specific Binding with the Polynucleotide or Polypeptide

[0115] The polynucleotide, or fragments thereof, or the polypeptide, or portions thereof, are labeled with 32P-dCTP, Cy3-dCTP, or Cy5-dCTP (Amersham Pharmacia Biotech), or with BIODIPY or FITC (Molecular Probes, Eugene OR), respectively. Libraries of candidate molecules previously arranged on a substrate are incubated in the presence of labeled polynucleotide or polypeptide. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the binding molecule is identified. Data obtained using different concentrations of the polynucleotide or polypeptide are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.

Claims

1. A substantially purified polynucleotide comprising a gene that is coexpressed with one or more known atherosclerosis-associated genes in a plurality of samples.

2. The polynucleotide of claim 1, comprising a polynucleotide sequence selected from

(a) a polynucleotide encoding a peptide selected from SEQ ID NOs: 1-34;

(b) a polynucleotide sequence complementary to the polynucleotide sequence of (a) or (b);

(c) a probe comprising at least 18 sequential nucleotides of the polynucleotide sequence of (a) or (b).

3. A pharmaceutical composition comprising one of the polynucleotides of claim 2 and a pharmaceutical carrier.

4. A method of using a polynucleotide to screen a library of molecules or compounds to identify at least one ligand which specifically binds the polynucleotide, the method comprising:

(a) combining the polynucleotide of claim 1 with a library of molecules or compounds under conditions to allow specific binding, and

(b) detecting specific binding, thereby identifying a ligand which specifically binds the polynucleotide.

5. The method of claim 4 wherein the library is selected from DNA molecules, RNA molecules, PNAs, mimetics, and proteins.

6. A ligand identified by the method of claim 4 which modulates the activity of the polynucleotide.

7. A method of using a polynucleotide of to purify a ligand which specifically binds the polynucleotide, the method comprising:

(a) combining the polynucleotide of claim 1 with a sample under conditions to allow specific binding,

(b) detecting specific binding between the polynucleotide and a ligand,

(c) recovering the bound polynucleotide, and

(d) separating the polynucleotide from the ligand, thereby obtaining purified ligand.

8. A method for diagnosing a disease or condition associated with the altered expression of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a plurality of biological samples, the method comprising the steps of:

(a) hybridizing a polynucleotide of claim 2 to a sample under conditions effective to form one or more hybridization complexes;

(b) detecting the hybridization complexes; and

(c) comparing the levels of the hybridization complexes with the level of hybridization complexes in a non-diseased sample, wherein the altered level of hybridization complexes compared with the level of hybridization complexes of a non-diseased sample indicates the presence of the disease or condition.

9. An expression vector comprising the polynucleotide of claim 2.

10. A host cell comprising the expression vector of claim 9.

11. A method for producing the polypeptide, the method comprising:

(a) culturing the host cell of claim 10 under conditions for expression of the polypeptide,

(b) recovering the polypeptide from cell culture.

12. A substantially purified polypeptide comprising the product of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a plurality of samples.

13. The polypeptide of claim 12, comprising a polypeptide sequence selected from

(a) the polypeptides encoded by SEQ ID NOs: 1-34; and

(b) an oligopeptide sequence comprising at least 6 sequential amino acids of the polypeptide sequence of a).

14. The polypeptide comprising the amino acid sequence of SEQ ID NO: 35.

15. A pharmaceutical composition comprising a polypeptide of claim 12 and a pharmaceutical carrier.

16. A method for using a polypeptide to screen a library of molecules or compounds to identify at least one ligand which specifically binds the polypeptide, the method comprising:

(a) combining the polypeptide of claim 12 with the library of molecules or compounds under conditions to allow specific binding, and

(b) detecting specific binding between the polypeptide and ligand, thereby identifying a ligand which specifically binds the polypeptide.

17. The method of claim 16 wherein the library is selected from DNA molecules, RNA molecules, PNAs, mimetics, proteins, agonists, antagonists, and antibodies.

18. A ligand identified by the method of claim 16 which modulates the activity of the polypeptide.

19. A method of using the polypeptide to purify a ligand from a sample, the method comprising:

(a) combining the polypeptide of claim 12 with a sample under conditions to allow specific binding,

(b) detecting specific binding between the polypeptide and a ligand,

(c) recovering the bound polypeptide, and

(d) separating the polypeptide from the ligand, thereby obtaining purified ligand.

20. A method for treating or preventing a disease associated with the altered expression of a gene that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need, the method comprising the step of administering to the subject in need the pharmaceutical composition of claim 15 in an amount effective for treating or preventing the disease.