Identification of genes involved in restenosis and in atherosclerosis

I. BACKGROUND OF THE INVENTION

[0001] Coronary artery disease is a disease that is endemic in Western society. In this disease the arteries that supply blood to the heart muscle become narrowed by deposits of fatty, fibrotic, or calcified material on the inside of the artery. The build up of these deposits is called atherosclerosis. Atherosclerosis reduces the blood flow to the heart, which starves the heart muscle of oxygen, leading to either/or angina pectoris (chest pain), myocardial infarction (heart attack), and congestive heart failure.

[0002] One common treatment to clear arteries blocked by atherosclerosis is balloon angioplasty, more formally referred to as percutaneous transluminal coronary angioplasty (PTCA). This treatment involves opening up a blocked artery by inserting and inflating a small balloon, which compresses and rearranges the blocking plaque against the arterial wall. After deflation and removal of the balloon, the arterial lumen is enlarged, thereby improving blood flow. About one million angioplasty procedures are performed each year.

[0003] In a significant number of angioplasty patients the treated artery narrows again within six months of the procedure in a process called restenosis. Restenosis begins soon after angioplasty, wherein the increased size of the vascular lumen (the open channel inside the artery) becomes gradually occluded by the proliferation of smooth muscle cells. Approximately 20 to 30% of all angioplasty patients experience restenosis to the extent that they must undergo repeated angioplasty or even coronary bypass surgery.

[0004] Restenosis has a complex pathology, triggered by the stretch-induced injury of the vessel walls during balloon inflation This stimulates smooth muscle cell migration and proliferation, and thereby leads to neointimal accumulation (which constitutes the restenotic lesion). Additional processes contributing to restenosis include inflammation and accumulation of extracellular matrix. Remodeling of the vessel wall, leading to narrowing of the vessel, is a critically important component of restenosis. However, this is totally eliminated by the implacement of a stent at the site of angioplasty, which prevents the vessel from remodeling. Stenting has become almost routine, being performed in many centers in over 70% of all angioplasty procedures. Restenosis also occurs in the arteries supplying the legs when these vessels are narrowed by atherosclerosis and are treated by angioplasty.

[0005] Currently, restenosis is diagnosed by visualizing the narrowed vessel through the injection of radioopaque dye into the vessel being examined and performing a cineangiogram (angiography). Angiography is an expensive invasive technique that requires radiation and special instruments to visualize and interpret the results. Typically, angioplasty is considered successful, not by the maintenance of the post-operative increase in the vascular lumen, but merely if the post-operative diameter of the vessel narrows less than 50% within 6-8 months of the procedure.

[0006] While several factors appear to be related to the occurrence of restenosis, including diabetes, the number of times the procedure has been performed, or the placement of a stent in the vessel, there presently are no reliable predictive indicators for the large majority of patients as to whether or not a given patient is at high risk for the development of restenosis. If a reliable risk profile were available, it would importantly influence how the patient were treated. Some patients deemed to be at very high risk for restenosis might be offered bypass surgery. Others might forego angioplasty and treated very aggressively with medical management. In still others brachytherapy (intravascular radiation) might be added to the usual angioplasty, a procedure normally reserved for patients who are now identified as being at high risk of restenosis using a rather blunt assessment—they already have had multiple episodes of restenosis. It is apparent, therefore, that new and improved methods for detecting and treating restenosis are greatly to be desired.

[0007] Finally, it is commonly appreciated that restenosis shares, with atherosclerosis, many common and overlapping processes and mechanisms. One of the key differences in these two conditions is the speed at which functionally important narrowing of the involved artery develops. Hence, restenosis can be used as an efficient model to understand many of the mechanisms responsible for atherosclerosis.

II. SUMMARY OF THE INVENTION

[0008] It is therefore an object of this invention to provide methods for predicting the risk of the development of restenosis.

[0009] It is a further object of this invention to provide methods of treating restenosis and of reducing its recurrence.

[0010] In accomplishing these objects there is provided a method for the detection of restenosis in a mammal, comprising assaying the level of expression of at least three genes in a sample obtained from the mammal. The presence of restenosis is indicated either by increased expression of at least three, five, ten, twenty, or fifty genes in the sample, or by decreased expression of at least three, five, ten, twenty, or fifty genes in the sample. The presence of restenosis may also be indicated by the altered (raised or lowered) expression of at least three, five, ten, twenty, or fifty genes in the sample. The genes may be selected from the group of genes listed in Table 1.

[0011] The increased gene expression of a gene may be at least two fold higher, four fold higher, or ten fold higher, than a reference level. The decreased expression of a gene may be at least one-half or at least one-tenth a reference level.

[0012] The altered expression of a gene, when increased, may be at least two fold higher than a reference level of that gene and when decreased, may be one-half the level of that gene when compared to a reference level.

[0013] In each case, the said reference level may be the level in healthy (non-stenotic) vascular tissue. Alternatively, the reference level may be determined from pre-stenotic levels. The vascular tissue may be vascular arterial tissue and/or vascular venous tissue. The sample also may be blood and/or lymph.

[0014] In other embodiments, the method of assay is genetic microarray, quantitative PCR, and/or by assay of the level of protein expression in a sample. When protein expression is measured, one or more of the proteins may be soluble proteins. The level of protein expressions may be determined by ELISA.

[0015] In accordance with another object of the invention there is provided a method of inhibiting restenosis comprising administering to a patient suffering from restenosis a composition that inhibits smooth muscle cell proliferation or neointimal hyperplasia, where the composition modifies expression of at least one gene listed in Table 1. The composition may induce the expression of a gene or gene transcript that ameliorates effects of restenosis. The composition may inhibit genes that promote smooth muscle cell proliferation or neointimal hyperplasia. The composition may comprise an antisense oligonucleotide and/or an oligonucleotide that binds to mRNA to form a triplex.

[0016] In one embodiment, the composition inhibits the activity of at least one protein that promotes smooth muscle cell proliferation or neointimal hyperplasia. In another embodiment, the composition comprises an antibody that binds to a protein that promotes smooth muscle cell proliferation or neointimal hyperplasia. The composition may comprise a human antibody, and/or a soluble protein receptor. In another embodiment, the composition comprises a protein that is administered to supplement the loss of a protein down-regulated during the course of restenosis.

[0017] In a further embodiment, detection is carried out using a kit suitable for performing PCR, where the kit comprises primers specific for the amplification of DNA or RNA sequences identified by the genes in Table 1.

[0018] In accordance with another object of the invention, there is provided a method to estimate the risk of developing restenosis or of atherosclerosis in an individual, comprising detecting the presence of biologically important polymorphisms in at least three, five, ten, twenty, or fifty genes in a sample obtained from the individual. The genes may be selected from the group of genes listed in Table 1. The sample may comprise, lymph, venous or arterial blood, and/or vascular tissue of the individual. The vascular tissue may be vascular arterial tissue.

[0019] In one embodiment the polymorphisms are detected using a genetic microarray. In another embodiment the polymorphisms are detected using quantitative PCR.

[0020] In accordance with another object of the invention, there is provided a kit for carrying out any of the methods described above.

[0021] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Table 1 lists the genes whose expression was detectably altered during the development of restenosis.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention provides new and improved methods for prediction, prevention, and treatment of restenosis and of atherosclerosis. Those genes that have altered expression levels during the healing response to acute vascular injury, and therefore during restenosis and during atherosclerosis, have been identified, and the changes in gene expression have been quantified. The relative changes in gene expression at different time points during the restenosis process have been measured, and these measurements allow additional insight into the progress and development of restenosis. Moreover, by measuring changes in gene expression, the risk of restenosis (or atherosclerosis) can be determined.

[0024] Because differential expression of genes is involved in the healing response to vascular injury, changes in the degree of expression, or in the length of time during which they are differentially expressed, lead to abnormal patterns of healing. In the context of injury to the vessel wall (either acute as in restenosis or chronic as in atherosclerosis), the excessive healing response contributes to the development of either restenosis or atherosclerosis. Changes in the degree of gene expression, or in the length of time during which the genes are differentially expressed, are caused by polymorphisms either in the gene or in the regulatory components of the gene. This invention, therefore, identifies those genes in which polymorphisms can convey susceptibility to the development of either restenosis or atherosclerosis.

[0025] The identification of genes that are involved in the healing response to acute vascular injury allows those genes having changed degree or duration of expression, caused in part by polymorphisms of the gene, to be used as targets to identify genetic abnormalities conveying altered risk of restenosis or atherosclerosis. Identification of polymorphisms associated with increased risk allows prediction of the risk for restenosis development in patients prior to the performance of the angioplasty procedure, This pre-procedure risk prediction will importantly influence how the patient is treated. Some patients deemed to be at very high risk for restenosis might be offered bypass surgery. Others might forego angioplasty and be treated aggressively with medical management. In still others brachytherapy (intravascular radiation) might be added to the usual angioplasty, a procedure normally reserved for patients who are now identified as being at high risk of restenosis using a rather blunt assessment—they already have had multiple episodes of restenosis. Accordingly, the present invention provides new and improved methods for predicting risk of restenosis.

[0026] Moreover, identification of the genes that are activated during the healing response to acute vascular injury provides new methods for preventing, ameliorating, or treating the disease by targeted inhibition of the expression of a suitable set or subset of those genes. In addition, the invention permits the monitoring of the effectiveness of restenosis treatment by measuring the changes in gene expression that occur during treatment.

[0027] Furthermore, restenosis shares, with atherosclerosis, many common and overlapping processes and mechanisms. Therefore, many of the genes differentially expressed during the healing response to acute vascular injury are the same genes differentially expressed during chronic vascular injury leading to atherosclerosis. The invention therefore also allows risk profiling of individuals for the development of atherosclerosis prior to the actual development of clinically significant atherosclerosis; i.e. prior to the development of detectable or significant narrowing of the relevant cardiac artery or peripheral arteries. This information therefore allows prophylactic intervention to prevent atherosclerosis, and prompt detection to allow delay or amelioration of the disease process.

[0028] The invention also allows the identification of genes to be analyzed for polymorphisms that predispose to atherosclerosis risk. Because different polymorphisms play a role in the development of atherosclerosis in different patients, the invention allows identification of specific abnormalities that may be characteristic to a specific patient. The invention therefore allows for greater specificity of treatment. A regime that may be efficacious in one patient with a specific polymorphism profile may not be effective in a second patient with a different polymorphism profile. Such a profiling also allows treatment to be individualized so that unnecessary side effects of a treatment strategy that would not be effective for a specific patient can be avoided.

[0029] Specifically, approximately two hundred genes are identified whose expression changes during the course of the healing response to acute vascular injury—the intrinsic process leading to restenosis.

[0030] Since the differential expression of these genes is involved in the healing response to vascular injury, changes in the degree of expression, or in the length of time during which they are differentially expressed, could lead to abnormal patterns of healing. Analogous to a keloid scar, in which a genetic precondition leads to excessive fibrous tissue developing on the skin in response to cutaneous injury, in the context of injury to the vessel wall (either acute as in restenosis or chronic as in atherosclerosis), the excessive healing response can contribute to the development of restenosis.

[0031] Changes in the degree of gene expression, or in the length of time during which the genes are differentially expressed, can be caused by polymorphisms in the gene or in the regulatory components of the gene. Such polymorphisms, conveying an increased risk of disease development, have already been identified for several genes associated with several diseases. This invention, therefore, identifies those genes in which polymorphisms can convey susceptibility to the development of restenosis. Subsequent reference, therefore, to prediction of restenosis (or atherosclerosis-see below), relate to polymorphisms of the genes identified by this invention, or of their regulatory units.

[0032] Restenosis may be predicted by identifying polymorphisms of at least three genes whose expression is up-regulated during the healing response to acute vascular injury. Identification of polymorphisms of at least three of those genes down-regulated during the healing response to acute vascular injury is predictive of restenosis. In addition, identification of polymorphisms of at least three genes, some of which are up-regulated and some of which are down-regulated, is predictive of restenosis. Further, the expression of some genes is altered during the course of the healing response to acute vascular injury.

[0033] The change in expression of certain of the identified genes is predictive, not just of the risk for restenosis itself, but is diagnostic of the stage of development of the disease. By identifying almost 200 genes whose expression changes during the healing response to acute vascular injury and therefore during the development of restenosis, the inventors recognize that analysis of greater numbers of polymorphisms of those genes leads to a greater ability to predict the development of restenosis, to determine the probability of its development, and to predict its ultimate severity. In view of the importance that the identified genes may play in the etiology of restenosis, an ability to manipulate the expression of those genes may be efficacious in the treatment of restenosis. Methods to treat restenosis may include gene therapy to increase the expression of genes down-regulated during the disease. Treatment may also include methods to decrease the expression of genes up-regulated during restenosis. Treatment to decrease gene expression may include, but is not limited to, the expression of anti-sense mRNA, triplex formation or inhibition by co-expression.

[0034] Identification of genes involved in the development of restenosis also makes possible an identification of proteins that may effect the development of restenosis. Identification of such proteins makes possible the use of methods to affect their expression or alter their metabolism. Methods to alter the effect of expressed proteins include, but are not limited to, the use of specific antibodies or antibody fragments that bind the identified proteins, specific receptors that bind the identified protein, or other ligands or small molecules that inhibit the identified protein from affecting its physiological target and exerting its metabolic and biologic effects. In addition, those proteins that are down-regulated during the course of restenosis may be supplemented exogenously to ameliorate their decreased synthesis.

[0035] The identification of genes involved in the development of restenosis makes possible the prophylactic use of methods to affect gene expression or protein function, and such methods may be used to treat individuals at risk for the development of restenosis.

[0036] Different polymorphisms may play a role in the development of restenosis in different patients. Accordingly, the present invention makes possible an identification of specific abnormalities that are characteristic of a specific patient, which allows for greater specificity of treatment. A regime that may be efficacious in one patient with a specific polymorphism profile may not be effective in a second patient with a different polymorphism profile. Such a profiling also allows treatment to be individualized so that unnecessary side effects of a treatment strategy that would not be effective for a specific patient can be avoided.

[0037] Finally, restenosis shares, with atherosclerosis, many common and overlapping processes and mechanisms. One of the key differences in these two conditions is the speed at which functionally important narrowing of the involved artery develops. Hence, restenosis can be used as an efficient model to understand many of the mechanisms responsible for atherosclerosis. Accordingly, each of the methods disclosed herein may be used to predict the occurrence of atherosclerosis as well as restenosis. Similarly, the methods of treatment disclosed herein may be used to treat, prevent, and/or ameliorate the symptoms of atherosclerosis as well as restenosis

[0038] Elucidation of Changes in Gene Expression in Restenosis

[0039] The present inventors have identified the genes that undergo changes in expression during the healing response to acute vascular injury, and therefore during the process of restenosis. Those genes are listed in Table 1. The inventors have carried out this analysis using nucleic acid array analysis of rat cardiac tissue as described in more detail below.

[0040] The rat is a widely accepted model for the human for vascular studies, and results obtained in the rat are considered highly predictive of results in humans. Accordingly, it is expected that the changes in gene expression in humans during the healing response to acute vascular injury will be similar to or essentially the same as those observed in the rat. Exaggerated changes in the degree of expression in these genes, or in the length of time during which the genes are differentially expressed, will predispose to restenosis. Such exaggerated changes are usually caused by polymorphisms in the gene or in the regulatory components of the gene, and therefore the rat genes identified as being differentially regulated during the healing response to acute vascular injury will be homologous to the human genes in which such polymorphisms will be found to convey susceptibility to restenosis. Moreover, both rat and human homologues are known for each of the genes described in Table 1, demonstrating further that the results obtained in the rat studies will be highly predictive of results obtained in humans.

[0041] Because restenosis shares many of the processes and mechanisms as atherosclerosis, and since both result from vascular injury, then the genes identified in the rat model of the healing response to acute vascular injury will also be the genes whose abnormal expression will predispose to atherosclerosis. The specific abnormalities are determined by identifying polymorphisms of these genes that are associated with atherosclerosis. Such genes also serve as the target for therapeutic interventions—those genes upregulated during the healing response to acute vascular injury can be targeted by therapy designed to decrease gene expression or function of the proteins encoded by these genes; those genes down-regulated during the healing response to acute vascular injury can be targeted by therapy designed to increase gene expression or function of the proteins encoded by these genes.

[0042] Changes in gene expression in the rat carotid artery during experimentally induced acute vascular injury have been studied, a model commonly accepted as a reasonable animal model simulating restenosis as it occurs in humans. Sample and control rat carotid artery tissues were obtained, RNA was prepared from the tissues, labeled cRNA generated from it and analyzed using an Affymetrix GeneChip® Rat Genome U34A Set. Sample and control tissues were compared and those genes that experienced significant changes in gene expression were identified. For the purposes of this study, a two fold increase or decrease in gene expression was deemed significant, although the skilled worker will recognize that under certain circumstances smaller changes in gene expression may also be significant. Corresponding human genes for each of the genes determined to have a significant change in expression were identified.

[0043] Now that an essentially complete set of genes that undergo changes in expression in the healing response to acute vascular injury has been identified, it is possible to predict the risk of restenosis and/or atherosclerosis developing by studying the changes of a smaller subset of those genes. Thus, although about 200 genes have been shown to have altered expression in restenosis, it is possible to reliably predict the risk of restenosis by analyzing a subset of these genes that contains as few as three members. In other embodiments, at least five, ten, fifteen, twenty or fifty genes may be studied or, if desired, all or most of the genes listed in Table 1 can be studied. Moreover, these genes can also be analyzed for polymophisms associated with restenosis and atherosclerosis. All of the genes can be analyzed intially, but reliable predictions can be made by analyzing a subset of these genes that contains as few as three members. In other embodiments, at least five, ten, fifteen, twenty or fifty genes may be studied or, if desired, all or most of the genes listed in Table 1 can be studied, for example, using sequencing, short tandem repeat association studies, single nucleotide polymorphism association studies, etc. In each case, however, it generally is more convenient to study gene expression or polymorphisms in a smaller subset of the genes.

[0044] By measuring changes in expression of a set of genes (or by identification of polymorphisms influencing expression of sets of genes), rather than of a single gene, the present invention provides increased statistical confidence that the changes observed are predictive of the risk of developing restenosis or atherosclerosis—ie, provides reliable risk profiling of an individual. Thus, a change in expression of a single gene, or a single gene polymorphism, may not increase susceptibility to disease sufficiently to cross the threshold for disease development. On the other hand, coordinated changes in expression of multiple specified genes, due the presence of multiple polymorphisms, is much more likely increase the risk of restenosis or of atherosclerosis. This is analogous to the situation of an individual have only one risk factor predisposing to atherosclerosis (elevated cholesterol). Risk is increased markedly as the number of risk factors increase (elevated cholesterol plus hypertension, obesity, smoking, diabetes, etc).

[0045] By assaying gene expression and/or the presence of polymorphisms that influence expression of these genes it is possible to predict the risk of restenosis development prior to performing the angioplasty procedure, and predict the risk of atherosclerosis development prior to the development of clinically detectable atherosclerosis. Such early prediction provides the clinician with opportunities to slow or halt the restenosis or atherosclerosis Moreover, the invention provides new compositions that can be used to inhibit, slow, or prevent restenosis and atherosclerosis.

[0046] Dysregulation of Multiple Genes that Increase Susceptibility to Restenosis or to Atherosclerosis

[0047] Gene polymorphisms that lead to biologically important exaggerated changes in the expression of genes that are differentially expressed during the course of the healing response to acute vascular injury, and which thereby predispose to restenosis or atherosclerosis, can be measured directly in patient samples. These samples comprise DNA that is most conveniently obtained from peripheral blood. The present inventors used nucleic acid array methods to identify the complete set of genes that exhibit significantly changed expression during the course of the healing response to acute vascular injury. However, other methods for measuring changes in gene expression are well known in the art. For example, levels of proteins can be measured in tissue sample isolates using quantitative immunoassays such as the ELISA. Kits for measuring levels of many proteins using ELISA methods are commercially available from suppliers such as R&D Systems (Minneapolis, Minn.) and ELISA methods also can be developed using well known techniques. See for example Antibodies: A Laboratory Manual (Harlow and Lane Eds. Cold Spring Harbor Press). Antibodies for use in such ELISA methods either are commercially available or may be prepared using well known methods.

[0048] Other methods of quantitative analysis of multiple proteins include, for example, proteomics technologies such as isotope coded affinity tag reagents, MALDI TOF/TOF tandem mass spectrometry, and 2D-gel/mass spectrometry technologies. These technologies are commercially available from, for example, Large Scale Proteomics Inc. (Germantown Md.) and Oxford Glycosystems (Oxford UK).

[0049] Alternatively, quantitative mRNA amplification methods, such as quantitative RT-PCR, can be used to measure changes in gene expression at the message level. Systems for carrying out these methods also are commercially available, for example the TaqMan system (Roche Molecular System, Alameda, Calif.) and the Light Cycler system (Roche Diagnostics, Indianapolis, Ind.). Methods for devising appropriate primers for use in RT-PCR and related methods are well known in the art. In particular, a number of software packages are commercially available for devising PCR primer sequences.

[0050] Nucleic acid arrays offer are a particularly attractive method for studying the expression of multiple genes. In particular, arrays provide a method of simultaneously assaying expression of a large number of genes. Such methods are now well known in the art and commercial systems are available from, for example, Affymetrix (Santa Clara, Calif.), Incyte (Palo Alto, Calif.), Research Genetics (Huntsville, Ala.) and Agilent (Palo Alto, Calif.). See also U.S. Pat. Nos. 5,445,934, 5,700,637, 6,080,585, 6,261,776 which are hereby incorporated by reference in their entirety.

[0051] Changes in the degree of gene expression, or in the length of time during which the genes are differentially expressed, can be caused by polymorphisms in the gene or in the regulatory components of the gene. Such polymorphisms, conveying an increased risk of disease development, have already been identified for genes associated with several diseases. The present invention, therefore, identifies those genes in which polymorphisms can convey susceptibility to the development of restenosis or atherosclerosis.

[0052] To study a set of genes having altered expression in restenosis using nucleic acid arrays, samples of total RNA or mRNA are obtained from cardiac tissue, and analyzed using methods that are well known in the art. Thus, for example, samples of suitable cardiac tissue, such as carotid artery, can be obtained by biopsy. Total RNA can be obtained using commercially available kits, such as Triazol reagent (Invitrogen, Carlsbad, Calif.) and mRNA can be obtained from this sample by chromatography on oligo(dT) cellulose. The RNA is reverse transcribed and the resulting cDNA subjected to an amplification step. In one embodiment, the amplification is a linear RNA amplification method such as that described in U.S. Pat. Nos. 5,716,785 and 5,891,636, which are hereby incorporated by reference in their entirety. Detailed instructions for preparing amplified RNA are available, for example, in the manufacturer's directions for preparing samples for assay using the Affymetrix GeneChip system.

[0053] Once suitable nucleic acid samples have been obtained, the gene expression profiles are determined using the nucleic acid arrays according to the manufacturer's instructions. For every gene probe on the array this provides a quantitative gene expression level in the sample. The expression level for each gene can then be compared to a baseline value to determine whether expression has been altered. Thus, the gene expression level of genes in tissue under study can be compared to reference levels of those genes in healthy tissue where restenosis is not occurring. Preferably, those reference levels are obtained from the same, although it is possible to use reference levels from different subjects. In such cases it is preferred to use reference levels from subjects that resemble the test subject as closely as possible, for example in demographic criteria such as age, gender, ethnicity, etc.

[0054] Although it is possible to measure absolute gene expression levels, it often is more convenient to measure relative gene expression levels. Thus, levels of expression of a particular gene on the array are compared to a reference gene on the same array whose expression is known to be unaffected in restenosis, for example, a gene not shown in Table 1. This provides an internal control mechanism for the array and reduces any differences in results that are due to variability in the array, assay conditions, etc.

[0055] In each case, the level of gene expression is compared to a suitable baseline level of expression. The baseline level of expression can be the level found in healthy vascular tissue, the level assayed prior to angioplasty, a global concentration assayed from a pool of healthy individuals or some other objective baseline.

[0056] Methods for identifying polymorphisms in genes are well known in the art. See, for example, U.S. Pat. Nos. 6,235,480 and 6,268,146, which are hereby incorporated by reference. Once polymorphisms are identified, methods for detecting specific polymorphisms in a gene using nucleic acid arrays are also well known in the art

[0057] Thus, in one embodiment, the invention provides methods where the expression of at least three genes selected from the genes shown in Table 1 are assayed. The genes can be selected in combinations such that (i) increased expression of all three genes indicates restenosis; (ii) decreased expression of all three genes indicates restenosis; (iii) decreased expression of some gene(s) combined with increased expression of the remaining selected genes indicates restenosis, or (iv) decreased expression of some genes and the increased expression of other genes at the beginning or shortly after angioplasty followed by an increase in the expression of those down-regulated genes and a decrease in the expression of genes initially up-regulated is indicative of the development of restenosis. In other embodiments of the invention the expression of at least five genes or at least about five genes is assayed to determine the development of restenosis. In yet further embodiments the number of genes assayed is ten. In yet other embodiments the number of genes assayed is 20 or at least about 20. In still yet other embodiments the number of genes assayed is 50 or at least about 50. Regardless of the number of genes in the subset of analyzed genes, the expression profile satisfies the criteria to diagnose the disease set out above when (i) the expression of some genes is increased throughout the course of the disease; (ii) the expression of some genes is decreased throughout the course of the disease; (iii) expression of some of the genes are increased while others are decreased, or (iv) the expression of some genes is altered during the development of the disease.

[0058] The invention also provides methods where the presence of at least three gene polymorphisms, selected from the genes shown in Table 1, are assayed. The aggregate number of polymorphisms can then provide an estimate of risk of restenosis or atherosclerosis. The more biologically significant polymorphisms are present, the greater the risk. As more polymorphisms of the genes listed in Table 1 are identified, even more powerful risk profiling will be possible. Thus, in other embodiments of the invention the expression of at least five genes or at least about five genes is assayed to determine the risk of developing restenosis or atherosclerosis. In yet further embodiments the number of genes assayed is ten. In yet other embodiments the number of genes assayed is 20 or at least about 20. In still yet other embodiments the number of genes assayed is 50 or at least about 50.

[0059] Regardless of the number of genes in the subset of analyzed genes, the polymorphism profile satisfies the criteria to determine the risk of developing restenosis or atherosclerosis set out above when the aggregate number of polymorphisms in the genes listed in Table 1 that exaggerate gene expression in biologically significant ways and that thereby predispose to the development of restenosis or atherosclerosis. The skilled artisan will recognize that, due to the heterogeneous nature of restenosis and of atherosclerosis, not all individuals with restenosis or atherosclerosis will exhibit altered expression of every last one of the genes listed in Table 1. Thus, it is possible that one, a few, or many genes will not exhibit significantly altered expression (and therefore will contain no biologically important polymorphisms), and that different individuals will exhibit different combinations of polymorphisms; yet, the coordinated changes induced by the polymorphisms in the expression of the totality of genes are highly predictive of the presence of development of restenosis and of atherosclerosis.

[0060] In general, where the expression of only a relatively small number of genes is studied, changes in expression in most or all of the genes must be observed to provide a reliable diagnosis of restenosis or atherosclerosis. For example, where only three genes are measured, all three genes must show relevant changes in expression to permit a reliable diagnosis of restenosis or atherosclerosis. Where five genes are studied, changes in at least four genes typically will provide a reliable diagnosis. Where ten genes are measured, a reliable diagnosis is obtained where changes in at least seven genes are observed. Where more than 10 genes are measured, changes in 90%, 80%, 70%, 60% or 50% of the measured genes are predictive of restenosis or atherosclerosis. As these percentages decrease, the reliability of the diagnosis also decreases, but the skilled worker will recognize that when a coordinated change in expression of 20 or 30 genes of the genes listed in Table 1 is observed this is highly predictive of the presence of restenosis. In general, as the number of genes increases, it is possible to provide a reliable diagnosis by observing coordinated changes in expression in a relatively smaller subset of the genes studied.

[0061] In general, where biologically important polymorphisms (leading to a predisposition to restenosis or of atherosclerosis) of only a relatively small number of genes is studied, polymorphisms in most or all of the genes must be observed to provide a reliable estimate of risk of developing restenosis or of atherosclerosis. For example, where only three genes are measured, all three genes must show relevant polymorphisms to permit a reliable estimate of risk of developing restenosis or of atherosclerosis. Where five relevant polymorphisms or ten polymorphisms are identified, the greater the number of such polymorphisms an individual has the greater the estimated risk of developing restenosis or of atherosclerosis. The skilled worker will recognize that when a coordinated change in expression of 20 or 30 genes of the genes listed in Table 1 is observed (as a result biologically important polymorphisms) this is highly predictive of the risk of developing restenosis or of atherosclerosis.

[0062] Tissues Sampled to Determine Altered Gene Expression and the Presence of Polymorphisms that Cause Biologically Important Alterations in Relevant Gene Expression

[0063] Although any sample containing nucleic acid would be appropriate for this purpose, the simplest tissue to sample is peripheral venous or arterial blood. However, tissue may be used, such as vascular tissue, in particular arterial vascular tissue or venous vascular tissue.

[0064] Significance of Altered Gene Expression

[0065] The terms increased expression, decreased expression or altered expression mean at least a two fold difference or at least about a two fold difference in the expression of the identified gene when compared to the expression of that gene in a control or non-restenotic animal. The change in gene expression may be at least four fold higher or at least about four fold higher than the reference level. In yet other embodiments the change in gene expression is at least ten fold higher or at least about ten fold higher than the reference level. Because some of the genes identified are down-regulated the term decreased expression means those genes that have at least a two-fold decrease or at least a about two-fold decrease in expression compared to control values. In other embodiments the term decreased expression means those genes that have at least a ten-fold decrease or at least about a ten-fold decrease in expression when compared to reference values.

[0066] Determination of Reference Level

[0067] The reference level used in the methods of the present invention is the level of gene expression in relatively healthy vascular tissue. This may mean the level of gene expression in pre-stenotic tissue, or it may mean the level of gene expression prior to angioplasty. The reference level may be determined from global values assayed from healthy individuals.

[0068] Methods of Studying Gene Expression and Polymorphisms of the Genes Listed in Table 1

[0069] Gene expression may be studied at the nucleic acid (RNA) level or the protein level. While each cell nucleus carries a complete set of genes only those genes expressed in each cell are transcribed into mRNA which is then translated into proteins. Consequently, gene expression is tissue or even cell specific. Generally, it is thought that the greater the number of RNA molecules transcribed the greater the number of protein molecules translated from them and, accordingly, the results obtained using RNA or protein analysis should be the same, at least in terms of relative changes in levels of gene expression. An analysis of gene expression may therefore be directed at the quantity of a particular mRNA transcript or the amount of protein translated from it.

[0070] Polymorphisms can be identified by several methods including sequencing, short tandem repeat association studies, single nucleotide polymorphism association studies, etc. These methods are well-known in the art.

[0071] Gene expression can also be studied at the protein level. While each cell nucleus carries a complete set of genes only those genes expressed in each cell are transcribed into mRNA which is then translated into proteins. Consequently, gene expression is tissue or even cell specific. Generally, it is thought that the greater the number of RNA molecules transcribed the greater the number of protein molecules translated from them and, accordingly, the results obtained using protein analysis should be the same, at least in terms of relative changes in levels of gene expression. An analysis of gene expression may therefore be directed at the quantity of a particular mRNA transcript or the amount of protein translated from it. However, although gene polymorphisms are detected reliably with tissue derived from any source, including peripheral blood, assay of the mRNA or protein encoded by any of the genes listed in Table 1 to determine relevant changes in the level of gene expression is critically dependent on tissue sampled. While some idea of altered gene expression occurring at the site of developing restenosis or of atherosclerosis can be obtained from sampling and testing peripheral blood, much more reliable estimates of altered gene expression would be obtained from sampling the actually artery developing restenosis or of atherosclerosis.

[0072] RNA Expression

[0073] Methods of isolating RNA from tissue are well known in the art. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (Third Edition) Cold Spring Harbor Press, 2001. Commercial reagents also are available for isolating RNA.

[0074] Briefly, for example, cells or tissue are lysed and the lysed cells centrifuged to remove the nuclear pellet. The supernatant is then recovered and the nucleic acid extracted using phenol/chloroform extraction followed by ethanol precipitation. This provides total RNA, which can be quantified by measurement of optical density at 260-280 nM.

[0075] mRNA can be isolated from total RNA by exploiting the “PolyA” tail of mRNA by use of several commercially available kits. QIAGEN mRNA Midi kit (Cat. No. 70042); Promega PolyATtract® mRNA Isolation Systems (Cat. No. Z5200). The QIAGEN kit provides a spin column using Oligotex Resin designed for the isolation of poly A mRNA and yields essentially pure mRNA from total RNA within 30 minutes. The Promega system uses a biotinylated oligo dT probe to hybridize to the mRNA poly A tail and requires about 45 minutes to isolate pure mRNA.

[0076] mRNA can also be isolated by using the cesium chloride cushion gradient method. Briefly the flash frozen tissue if homogenized in Guanethedium isothiocyanate, layered over a cushion of cesium chloride and ultracentrifuged for 24 hours to obtain the total RNA.

[0077] Genetic Microarray Analysis

[0078] Microarray technology is an extremely powerful method for assaying the expression of multiple genes in a single sample of mRNA. For example, Gene Chip® technology commercially available from Affymetrix Inc. (Santa Clara, Calif.) uses a chip that is that is plated with probes for over thousands of known genes and expressed sequence tags (ESTs). Biotinylated cRNA (linearly amplified RNA) is prepared and hybridized to the probes on the chip. Complementary sequences are then visualized and the intensity of the signal is commensurate with the number of copies of mRNA expressed by the gene.

[0079] Quantitative PCR

[0080] Quantitative PCR (qPCR) employs the co-amplification of a target sequence with serial dilutions of a reference template. By interpolating the product of the target amplification with that a curve derived from the reference dilutions an estimate of the concentration of the target sequence may be made. Quantitative reverse transcription PCR (RTPCR) may be carried out on mRNA using kits and methods that are commercially available from, for example, Applied BioSystems (Foster City, Calif.) and Stratagene (La Jolla, Calif.) See also Kochanowsi, Quantitative PCR Protocols” Humana Press, 1999. For example, total RNA may be reverse transcribed using random hexamers and the TaqMan Reverse Transcription Reagents Kit (Perkin Elmer) following the manufacturer's protocols. The cDNA is amplified using TaqMan PCR master mix containing AmpErase UNG dNTP, AmpliTaq Gold, primers and TaqMan probe according to the manufacture's protocols. The TaqMan probe is target-gene sequence specific and is labeled with a fluorescent reporter (FAM) at the 5′ end and a quencher (e.g. TAMRA) at the 3′ end. Standard curves for both endogenous control and the target gene may be constructed and the comparison of the ration of CT (threshold cycle number) of target gene to control in treated and untreated cells is determined. This technique has been widely used to characterize gene expression.

[0081] Protein Expression

[0082] Gene expression may also be studied at the protein level. Target tissue is first isolated and then total protein is extracted by well known methods. Quantitative analysis is achieved, for example, using ELISA methods employing a pair of antibodies specific to the target protein.

[0083] A subset of the proteins listed in Table 1 are soluble or secreted. In such instances the proteins may be found in the blood, plasma or lymph and an analysis of those proteins may be afforded by any of those methods described for the analysis of proteins in such tissues. This provides a minimally invasive means of obtaining patient samples for estimate of risk of developing restenosis or of atherosclerosis. Methods for identifying secreted proteins are known in the art.

[0084] Treatment of Restenosis

[0085] The identification of the set of genes having altered expression during the healing response to acute vascular injury, provides new opportunities to treat restenosis or atherosclerosis. Identification of genes up-regulated during the healing response to acute vascular injury affords the ability to use methods to negatively affect their transcription or translation. Similarly, the identification of genes that are down-regulated during the healing response to acute vascular injury affords the ability to positively affect their expression. Finally, the determination of the proteins encoded by these genes allows for the use of appropriate methods to ameliorate or potentiate the protein activities, which thereby could influence the development of restenosis or atherosclerosis.

[0086] Methods of Enhancing Gene Expression

[0087] For genes that exhibit decreased expression during the the healing response to acute vascular injury, it is possible to ameliorate or prevent restenosis or atherosclerosis by enhancing expression of one or more of these genes. Gene transcription may be deliberately modified in a number of ways. For example, exogenous copies of a gene may be inserted into the genome of cells in vascular tissue by genomic transduction via homologous recombination. While expression by genomic transduction is relatively stable it also is of low efficiency. An alternative method is transient transduction where the gene is inserted within a vector allowing for its transcription independent of the genomic allele making use of a vector specific promoter. While transient transduction generally has a higher expression the gene is maintained for a much shorter period of time, although use of episomal vector containing a eukaryotic origin of transcription provides for greater persistence of the vector. Yet another method is transfection with naked DNA. However, this method generally results in very low expression and the DNA appears to be rapidly degraded.

[0088] Methods of Inhibiting of Gene Expression

[0089] The present invention also affords an ability to negatively affect the expression of genes that are up-regulated during the healing response to acute vascular injury. Methods for down regulating genes are well known. It has been shown that antisense RNA introduced into a cell will bind to a complementary mRNA and thus inhibit the translation of that molecule. In a similar manner, antisense single stranded cDNA may be introduced into a cell with the same result. Further, co-suppression of genes by homologous transgenes may be effected because the ectopically integrated sequences impair the expression of the endogenous genes (Cogoni et al. Antonie van Leeuwenhoek, 1994; 65(3):205-9), and may also result in the transcription of antisense RNA (Hamada and Spanu, Mol. Gen Genet 1998). Methods of using short interfering RNA (RNAi) to specifically inhibit gene expression in eukaryotic cells have recently been described. See Tuschl et al., Nature 411:494-498 (2001).

[0090] In addition, stable triple-helical structures can be formed by bonding of oligodeoxyribonucleotides (ODNs) to polypurine tracts of double stranded DNA. (See, for example, Rininsland, Proc. Nat'l Acad. Sci. USA 94:5854-5859 (1997). Triplex formation can inhibit DNA replication by inhibition of transcription of elongation and is a very stable molecule.

[0091] Methods to Inhibit the Activity of Specific Proteins

[0092] When a specific protein has been implicated in the restenotic or atherosclerotic pathway its activity can be altered by several methods. First, specific antibodies may be used to bind the protein thereby blocking its activity. Such antibodies may be obtained through the use of conventional hybridoma technology or may be isolated from libraries commercially available from Dyax (Cambridge, Mass.), MorphoSys (Martinsried, Germany), Biosite (San Diego, Calif.) and Cambridge Antibody Technology (Cambridge, UK). In addition, proteins usually exert their cellular effects by ligating to cellular receptors. Identification of the receptors to which proteins, which are implicated by the current invention as contributing to restenosis or atherosclerosis, bind will allow the design of specific ligand antagonists that block pathways mediating the effects leading to the development of restenosis or atherosclerosis.

[0093] The identification of genes that are down regulated during the the healing response to acute vascular injury leads to the ability to identify their protein products. Down-regulated proteins may then be supplemented, thereby ameliorating the effect of their decreased synthesis.

[0094] The methods of the present invention may be used prophylactically to prevent the development of restenosis or atherosclerosis in at risk individuals.

[0095] The present invention also provides kits having chips containing the DNA of the biologically important polymorphisms for the genes identified in Table 1. Such chips permit the rapid detection of the polymorphisms, providing a convenient means for the rapid detection of those individuals at high or at low risk of developing restenosis or of atherosclerosis. The detection of specific polymorphisms in specific patients will allow highly specific and individualized treatment strategies to be devised for each patient to prevent or attenuate restenosis and or atherosclerosis.

[0096] The present invention, thus generally described, will be understood more readily by reference to the following example, which is provided by way of illustration and is not intended to be limiting of the present invention.

EXAMPLE

Microarray Analysis of the Restenotic Carotid Artery

[0097] Isolation of RNA

[0098] Rats were divided into two groups. One group was treated with carotid angioplasty and the control group was treated by sham surgery. Rat carotids after surgery and sham surgery were collected and flash frozen. Pooled carotids (30-50 mg) were crushed into powder using a mortar and pestle (collected with liquid nitrogen) and then homogenized in 2.5 ml of guanidinium isothiocyanate. Total RNA was extracted using ultracentrifugation on cesium chloride cushion gradient for 24 hours at 4° C. See Sambrook et al supra.

[0099] Target Preparation and DNA Microarray Hybridizations

[0100] For the first strand cDNA synthesis reaction, 5.0-8.0 μg of total RNA was incubated at 70° C. for 10 minutes with T7-(dT) 24 primer, then placed on ice. For the temperature adjustment step, 5×first stand cDNA buffer, 0.1 M DTT, and 10 mM dNTP mix was added and the reaction incubated for 1 hour at 42° C. SSII reverse transcriptase was added, and the reaction incubated for 1 hour at 42° C. With the first strand synthesis completed, 5×second strand reaction buffer, 10 mM dATP, dCTP, dGTP, dTTP, DNA Ligase, DNA Polymerase I, and RNaseH were added to the reaction tube. Samples were then incubated at 16°. Following the addition of 0.5M EDTA, cDNA was cleaned using phase lock gels-phenol/chloroform extraction, followed by ethanol precipitation.

[0101] Synthesis of Biotin-Labeled cRNA (In Vitro Transcription)

[0102] The synthesis of biotin-labeled cRNA was completed using the ENZO BioArray RNA transcript labeling kit from (ENZO Biochem, Inc., New York, N.Y.) according to the manufacturers protocol. To set up the reaction 1 μg of cDNA, 10×HY reaction buffer, 10×Biotin labeled ribonucleotides, 10×DTT, 10×RNase inhibitor mix and 20×T7 RNA polymerase were incubated at 37° C. for 4-5 hours. RNeasy spin columns from QIAGEN were used to purify the labeled RNA, followed by ethanol precipitation and quantification.

[0103] Fragmentation of cRNA for Target Preparation

[0104] 5×fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM Mg)Ac) was added to the cRNA. Samples were incubated at 94° C. for 35 minutes, then placed on ice. Fragmented cRNA was stored at −70° C.

[0105] Target Hybridization

[0106] Hybridization cocktail was prepared as follows: fragmented cRNA (15 μg adjusted), control oligonucleotide B2 (Affymetrix), 20×eukaryotic hybridization controls (Affymetrix), herring sperm DNA, acetylated BSA, and 2×hybridization buffer (Affymetrix) were combined, and heated to 99° C. for five minutes. Hybridization cocktail was then centrifuged at maximum speed for five minutes to remove any insoluble materials from the mixture. Following centrifugation, cocktail was heated at 45° C. for five minutes. The clarified hybridization cocktail was then added to the Affymetrix U34A probe array cartridge that had been pre-wet with 1×hybridization buffer. The probe array was then placed in a 45° C. rotisserie box oven set at 60 rpm and hybridized for 16 hours.

[0107] Washing, Staining and Scanning Probe Arrays

[0108] The GeneChip® Fluidics Station 400 was used to wash and stain the array. This instrument was run using GeneChip® software. Briefly, arrays were washed for 10 cycles with non-stringent wash buffer at 25° C., followed by 4 cycles of washing with stringent wash buffer at 50° C. The array was then stained for 10 minutes with Phycoerythrin-streptavidin at 25° C. The array was then washed for 10 cycles with non-stringent wash buffer at 25° C. The probe array was the stained again with phycoerythrin-streptavidin for 10 minutes at 25° C., and then washed for 15 cycles with non-stringent wash buffer at 30° C. Hybridization signals are detected by placing the probe array in an HP Gene Array™ Scanner, which operated using GeneChip® software.

[0109] Data Analysis

[0110] Data analysis was performed using GeneChip® software (version 3.3) using the manufacturer's instructions. Lockhart, D. J. et al., Nat. Biotechnol. 14:1675-80 (1996). Briefly, each gene was represented and queried by 1-3 probe sets on the chip. Each probe set comprises 16 perfect match (PM) and 16 mismatch (MM) 25 nucleotide base probes, The mismatch has a single base change in the middle of the 25 base pair probe. The hybridization signal from the PM and the MM probes were compared and this allowed for a measure of signal intensity that is specific and eliminated the nonspecific cross hybridization from the data of the two control chips. Intensity differences as well as ratios of intensity of each probe pair are used to make a “present” or “absent” call. The controls were used as baseline and the experimental GeneChip® assay values compared to the base line to derive four matrixes which were used to determine the difference calls that indicate whether the transcription level of a particular gene is changed.

[0111] Iterative comparisons were performed using a spreadsheet analysis (Microsoft Excel). Each experimental data set at a particular time point (n=2) and the difference in expression between the controls and experimental was determined for each gene. Genes with a consistent difference call across all four pairwise comparisons were extracted for further analysis.

[0112] GeneSpring® Analysis

[0113] The data from each GeneChip® assay was fed into the GeneSpring® software and clustering of genes based on their temporal expression profile was analyzed. Correlation coefficients of 0.97 or greater were taken as a cutoff to create gene-clusters with significant expression homology.

[0114] This application claims priority from U.S. application Ser. No. 60/326,210, the specification of which is incorporated by reference in its entirety.

1RAT GENES IN RESTENOSIS: 3 HOUR DOWN REGULATEDABCD1Rat Accession #Locus linkDESCRIPTIONHuman locusE23AF022136_atGJA5GJA5 gap junction protein, alphaLocusID: 270240kD (connexin 40)4J00738_s_atCALD1CALD1: caldesmon 1LocusID: 80056K01934mRNA#2_atTHRSPTHRSP: thyroid hormoneLocusID: 7069responsive SPOT14 (rat)homolog7rc_AI237731_s atLPLLPL: lipoprotein lipaseLocusID: 40238L03294_atLPLLPL: lipoprotein lipaseLocusID: 40239L03294_g_atLPLLPL: lipoprotein lipaseLocusID: 402310L46791_atCES3CES3 carboxylesterase 3 (brain)LocusID: 234911112A04674cds_s_at7350UCP1: uncoupling protein 1LocusID: 7350(mitochondrial, proton carrier)13X03894_atUCP: BROWN ADIPOSELocusID: 7350TISSUE UNCOUPLING4q31PROTEINTHERMOGENIN1415M22400_atGPG3GPC3: glypican 3LocusID: 271916U66470_atCGR11CGR11: cell growth regulatoryLocusID: 10669with EF-hand domain17U95178_s_at1601DAB2: disabled (Drosophila)LocusID: 1601homolog 2 (mitogen-responsivephosphoprotein)18J03179_atDBPDBP: D site of albumin promoterLocusID: 1628(albumin D-box) binding protein19J03179_g_atDBPDBP: D site of albumin promoterLocusID: 1628(albumin D-box) binding protein20M31837_atIGFBP3IGFBP3: insulin-like growth factorLocusID: 3486binding protein 321M64711_atEDN1EDN1: endothelin 1LocusID: 190622M80367_at2633GBP1: guanylate binding proteinLocusID: 26331, interferon-inducible, 67kD23D89730_at24J03624_atGALGAL: galaninLocusID: 258625U18314_g_at7112TMPO: thymopoietinLocusID: 711226U45965_atGRO3 ONCOGENELocusID: 2921 4q2127AF009329_atDEC2DEC2: bHLH protein DEC2LocusID: 7936528M10934_s_atRBP4RBP4: retinol-binding protein 4,LocusID: 5950plasma2930Z22607_atBMP4BMP4: bone morphogeneticLocusID: 652protein 431X58830_atBMP6BMP6: bone morphogeneticLocusID: 654protein 6B32U92564_g_at*FINGER PROTEINLocusID: Rn 94188 5q3433D49494UTR#1_g_atGDF10GDF10: growth differentiationLocusID: 2662factor 103435M84719_atFMO1FMO1: flavin containingLocusID: 2326monooxygenase 136rc_AA859837_atGDAGDA: guanine deaminaseLocusID: 961537M80633_at38rc_AI172247_atXDHXDH: xanthene dehydrogenaseLocusID: 749839rc_AI232374_g_atH1F0H1F0: H1 histone family, memberLocusID: 3005040rc_AA965261_atH2AFYH2AFY: H2A histone family,LocusID: 9555member Y4142X71127_atC1QBC1QB: complement componentLocusID: 7131, q suboomponent, betapolypeptide43M92059_s_at1675DF: D component of complementLocusID: 1675(adipsin)44L03201_atCTSSCTSS: cathepsin SLocusID: 15204546J03637_atAldh3a1Aldh3: Aldehyde dehydrogenaseLocusID: 25375class 347M15327_atADH6ADH6: alcohol dehydrogenase 6LocusID: 130(class V)48X72792cds_s_at126ADH1C: alcohol dehydrogenaseLocusID: 1261C (class I), gamma polypeptide49M27434_s_at50X68101_at51rc_AA799666_at52rc_AA874875_at53rc_AA891069_at1198CLK3: CDC-like kinase 3LocusID: 119854rc_AA893244_at55rc_AA894089_s_at56rc_AI639246_at5758596061626364*Rat gene no apparent Human Homologue

[0115]

2

RAT GENES IN RESTENOSIS: 3 HOUR UP REGULATED GENES AND HUMAN HOMOLOGUES 3

A
B
C
D

1
Rat Accession #
Locus link
DESCRIPTION
Human Locus
E

2

3
AA848563_s_at
3303
HSPA1A: heat shock 70kD protein
LocusID: 3303

1A

4
Z27118cds_s_at

5
rc_AA818604_s_at

HSPA1L: heat shock 70kD protein-
LocusID: 3305
14q24 1

like 1

6
S74351_s_at
1843
DUSP1: dual specificity
LocusID: 1843

7
X68041cds_s_at
SOD3
SOD3: superoxide dismutase 3,
LocusID: 6649

extracellular

8

9

10
rc_AA800784_at
3491
CYR61: cysteine-rich, angiogenic
LocusID: 3491

inducer, 61

11
L05489_at
DTR
DTR: diphtheria toxin receptor
LocusID: 1839

(heparin-binding epidermal growth

factor-like growth factor)

12
rc_AA858520_at
Fst
*Fst: Follistatin
LocusID: 24373
MGC14128

hypothetical protein

MGC 14128; LocusID

84985

13
rc_AA858520_g_at
Fst
*Fst: Follistatin
LocusID: 24373

14
M19651_at
FOSL1
FOSL1 FOS-like antigen-1
LocusID: 8061

15
U19866_at

16
X13167cds_s_at
4774
NFIA: nuclear factor I/A
LocusID: 4774

17
Z54212_at
EMP1
EMP1: epithelial membrane protein
LocusID: 2012

1

18

19

20
M61875_s_at

21
AB015432_s_at
8140
SLC7A5: solute carrier family 7
LocusID: 8140

(cationic amino acid transporter, y+

system), member 5

22

23

24
AF004811_at
MSN
MSN: moesin
LocusID: 4478

25
AF028784mRNA#1_s_at
GFAP
GFAP: glial fibrillary acidic protein
LocusID: 2670

26
AJ002556_s_at
29457
*Mtap6: microtubule-associated
LocusID: 29457

protein 6

27
rc_AA799773_at
2318
FLNC: filamin C, gamma (actin-
LocusID: 2318

binding protein-280)

28
rc_AA799773_g_at
2318
FLNC: filamin C, gamma (actin-
LocusID: 2318

binding protein-280)

29

30

31
rc_A1029183_s_at
2697
GJA1: gap junction protein, alpha 1,
LocusID: 2697

43kD (connexin 43)

32
L12407_at
DBH
DBH: dopamine beta-hydroxylase
LocusID: 1621

(dopamine beta-monooxygenase)

33
M74494_g_at
Atp1a1
Atp1a1: ATPase, Na+K+
LocusID: 24211

transporting, alpha 1 polypeptide

34

35

36
X13722_at

37

38

39
X71898_at
PLAUR
PLAUR: plasminogen activator,
LocusID: 5329

urokinase receptor

40

41

42
rc_AA892813_s_at
MBLL
MBLL: C3H-type zinc finger protein,
LocusID: 10150

similar to D melanogaster

muscleblind B protein

43
rc_AA894318_at

44
S56464mRNA_g_at

45
X74S6Scds_at
PTB
PTB: polypyrimidine tract binding
LocusID: 5725

protein (heterogeneous nuclear

ribonucleoprotein I)

46
X62875mRNA_g_at

47
AF015953_at
406
ARNTL: aryl hydrocarbon receptor
LocusID: 406

nuclear translocator-like

49
rc_AI639343_at

50
rc_H31747_s_at

51
rc_AA800708_at

52
rc_AA875405_at

55

56

58

*Rat gene no apparent Human Holomogue

[0116]

3

RAT GENES IN RESTENOSIS: 3 DAY DOWN REGULATED AND HUMAN HOMOLOGUES

1
A

E Locus
F
G

2
RAT ACCESSION NO
B
C
D
Link
DESCRIPTION
HUMAN LOCUS

3
M17526_g_at
RR

2775
50664
RATBPGTPC: GTP binding
LocusID: 50664

protein

4
M60921_at
601597

BTG2
BTG2: BTG family, member 2
LocusID: 7832

5
M60921_g_at

6
rc_AA944156_s_at
601597

GENE2: BTG2
Gene map locus 1q32

7
rc_AA925717_at
605276
Reddy E P

9625
AATK: apoptosis-associated
LocusID: 9625

8
M62752_at
Wang E
Leffers H
602959
24799
Stnl: Stalin-like protein**RAT
LocusID: 24799

9
rc_H31144_g_at
PMID,
Greene
http:/www.n

8497321

cbi.nlm.nih.g

ov/LocusLin

k/LocRpt.cgi

?l=10188

10
rC_AI103238_at

604325

PROTEIN PHOSPHATASE 2,
LocusID: 5521, Gene

REGULATORY SUBUNIT B,
map locus 5q31-33

BETA, PPP2R2B

11
Y13275_at
Zoller M

7103
TM4SF3: transmembrane 4
LocusID: 7103

12

13

14
rc_AI71462_s_at

600074

CD24: ANTIGEN, CD24
LocusID: 934, Gene

map locus 6q21

15
U49062_at
Hirokawa K
Y

CD24: ANTIGEN, CD24

16
U49062_g_at

Altevogt P

17
M63282_at
Taub R
S

none: rat leucine zipper

18
X58830_at
BMP6
Wooley D E
112266

BMP6: bone morphogenetic
LocusID: 654

protein 6

19
rc_H31479_at

20
rc_AA859882_s_at

21

22

23
L00088expanded_cds#2_at

24
rc_AI639532_at
Leiden J M

25
rc_AI136540_at
Ginard B

26
M73701_at

191043

SKELETAL MUSCLE
LocusID: 7136 Gene

map locus 11p15 5

27
rc_AI639096_at
NM 004533

28
X00975_at
Levy Z

29
X00975_at
Levy Z

30
X02412_at
Ginard B

31
S68736_at
Rotkind M

Lanfranchi

32
rc_AA799471_at
G

33
rc_AA851223_at
172430

Enolase 1
LocusID: 2023, Gene

map locus 1pter-

p36.13

34
M75148_at

35
M10140_at

123310

CREATINE KINASE,
LocusID: 1158, Gene

MUSCLE TYPE, CKM
map locus 19q13

36
L24897_s_at
Ryan A F

37
L10669_g_at

232600

GLYCOGEN STORAGE
LocustID: 5837, Gene

DISEASE V
map locus 11q12-

q13 2

38
L10669_at

39
K02423cds_s_at

40
J00692_at
Nedel U.

41
M63656_s_at

103870

ALDOLASE C, FRUCTOSE-
LocusID: 230, Gene

BISPHOSPHATE, ALDOC
map locus 17cen-q12

42

43

44
U14398_at
TC
600103

SYNAPTOTAGMIN 4
LocusID: 6860, Gene

map locus 18q12 3

45
U14398_g_at

46
U16802_at
604667
9289400

Ca2+ de[emdemt actovator
LocustID: 8616, Gene

protein for secretion, CADPS
map locus 3p23-cen

47
rc_AI175539_at
168890

PARVALBUMIN, PVALB
LocusID: 5816, Gene

map locus 22q12-

q13 t

48
M99223_at

49
M26161_at
North R A

50
AF055477_at
D

51
rc_AI177026 at
182310
JX

477
ATP1A2: ATPase, Na+/K+
LocustID: 477

transporting, alpha 2 (+)

polypeptide

52

53

54
A04674cds_s_at

113730

UNCOUPLING PROTEIN 1:
LocusID: 7350, Gene

UCP1
map locus 4q31

55
rc_AI044900_s_at
B
A

56
L46791_at
Grogan

WM

57
D43623_g_at

Terada H.

58
J02585_at

59

60

61
AF019974_at
Metz

62
AF031878_at
Parysek L M

63
M85214_at
Matsuda I

64
U17254_g_at
Milbrandt J
Olsson T

65
U88958_at

Theill L E

66
L21192_at

162060

GROWTH ASSOCIATED
LocusID: 2596, Gene

PROTEIN 43, GAP43
map locus 3q13 1-

q13 2

67
AF031880_at
Saarma M

68

69

70
X79321_at

71
K03242_at
*159440

MYELIN PROTEIN ZERO,
LocusID: 4359, Gene

MPZ
map locus 1q22

72
M73049_at

605336

INTERNEXIN, ALPHA, INA
LocusID: 9118, 10cen-

q26 11

73
X52817cds_s_at

74
X90475cds_at

75
D10699_at

76
D10699_g_at
HL
191342

UBIQUITIN CARBOXYL-
LocusID: 7345, Gene

TERMINAL ESTERASE L1,
map locus 4p14

UCHL1

77
rc_AA957930_s_at

78
rc_AI227608_s_at
Ginzburg I
*157140

MICROTUBULE-ASSOCIAT-
LocusID: 4137, Gene

ED PROTEIN TAU, MAPT
map locus 17q21 1

79
rc_AI044508_s—l at
FE

80
U03414_s_at
Sutcliffe J G
Kihara I

81
Z12152_at
Brophy P J
PJ

82
X86789_at

AM

83
rc_0AA875659_s_at
605336

INTERNEXIN, ALPHA, INA,
LocusID: 9118

NEUROFILAMENT PROTEIN

5

84
Z29649_at
Brophy P J
Lupski J R

85
M72711_at
G

86
M24852_at

87
rc_AI072770_st

88

89

90
rc_AI171644_s_at
B
*602009

CYTOCHROME c
LocusID: 1339

OXIDASE, SUBUNIT VIa,

POLYPEPTIDE 2, COX6A2

91
U78977_at

92
M37942exon#2-3_s
Holmes E W

93

94

95
M27925_at
Greengard

P

96
rc_AI145494_g_at

97
rc_AI145494_at
P

98
L25633_g_at
Eipper B A
Eipper B A

99
L31621_s_at
Patrick J

100
U17604_at
Seki N
*600865

RETICULON 1, RTN1
LocusID: 6252, Gene

map locus 4q21-22

101
S50879_g_at

102
rc_AI043796_s_at
193001

SLC18A2
LocusID: 6571, Gene

map locus 10q25

103
U02983_at

104
X06655 at
313475
TC

SYNAPTOPHYSIN, SYP
LocusID: 6855, Gene

map locus Xp11 23-

p11 22

105
L00603_at
Hoffman

B J, Sabban

106
L12407_at
E L

107
U25967_at

602753

ARISTALESS HOMEO BOX,
LocusID: 401, Gene

DROSOPHILA, HOMOLOG
map locus 11q13 3-

OF ARIX
q13 4

108
M93669_at
Neill J D

109

110

111
rc_AI639444_g_at
Ozanne B W

112
X03369_s_at
191130

TUBULIN, BETA, TUBB
LocusID: 7280, Gene

map locus 6p21 3

113
X59149_at
CH

114
U30938_at
157130

MICROTUBULE-ASSOCIAT-
LocusID: 4133; Gene

PROTEIN 2; ED MAP2
map locus: 2q34-q35

115

116

117
U32314_g_at
266150

PYRUVATE CARBOXYLASE
LocusID: 5091, Gene

DEFICIENCY
map locus 11q13 4-

q13 5

118
M27434_s_at

D

119
K01934mRNA#2_at
HC

120
M10244_at

191290

TYROSINE HYDROXYLASE,
LocusID: 7054, Gene

TH
map locus 11p15 5

121
M15327_atat

122

123
rc_AA799621_at

124
rc_AA799666_at

125
rc_AA892798_at

126
rc_AA893984_at

127
rc_AI638986_s_at

128
rc_AI639294_at

129
rc_H31550_at
JD

64506
CPEB1 cytoplasmic
LocusID: 64506

polyadenytation element binding

protein

[0117]

4

RAT GENES IN RESTENOSIS: 3 DAY UP REGULATED GENES AND HUMAN HOMOLOGUES

A

C

E
F

1
RAT ACCESSION NO.
B
LINK
D
DESCRIPTION
HUMAN LOCUS

2

3

4
M36151cds_i_at

604305

MAJOR HISTOCOMPATIBILITY
LocusID: 3119,

COMPLEX, CLASS II, DQ BETA-1; HLA-
Gene map locus:

DQB1
6p21 3

5
M15562_at

6
rc_AI171966_at

7
X14254cds_g_at

P.
B.

8
X73371_at
Pecht I.

9
M32062_at
DW
146790

Fc FRAGMENT OF IgG, LOW AFFINITY
LocusID: 2122;

IIa, RECEPTOR FOR; FCGR2A
Gene map, locus:

1q21-q23

10
M10072mRNA_s_at
M.

151460
PROTEIN-TYROSINE PHOSPHATASE,
LocusID: 5788;

RECEPTOR-TYPE, C; PTPRC
Gene map locus:

1q31-q32

11

12

13
M98049_s_at
C.
167805
Verrando
PROLIFERATING CELL NUCLEAR
Gene map locus:

P
ANTIGEN; PCNA/PANCREATITIS-
20p12/LocusID:

ASSOCIATED PROTEIN; PAP
5068; Gene map

locus: 2-12

14
J05495_at

T.

15
U82612cds_at
135600
2335

FIBRONECTIN 1; FN1
LocusID: 2335;

Gene map locus:

2q34

16
U82612cds_g_at
135600

FIBRONECTIN 1; FN1
LocusID: 2335;

Gene map locus:

2q34

19
AF100470_g_at
B.
M.

20
J02722cds_at
S.
S.

21
rc_AI179610_at
Koizumi
PA.

S.

22

23

24
M24604_g_at
VJ.
*17674

PROLIFERATING CELL NUCLEAR
LocusID: 5111;

0

ANTIGEN; PCNA
Gene map locus:

20p12

25
U10894_s_at
SA.
605356
CJ.
TYROSINE 3-
LocusID: 7532;

MONOOXYGENASE/TRYPTOPHAN 5-
Gene map locus:

MONOOXYGENASE ACTIVATION
7q11.23

PROTEIN, GAMMA ISOFORM; YWHAG

26
rc_AA998164_s_at
123836
RJ

CYCLIN B1; CCNB1
LocusID: 891; Gene

map locus: 5q12

27
U28938_at
176884
N.
50677
PROTEIN-TYROSINE PHOSPHATASE,
LocusID: 5786;

RECEPTOR-TYPE, ALPHA; PTPRA
Gene map locus:

20p13

28
X17053mRNA_s_at
JR
Charo

I F.

29
AB010119_at
Zhang
42199
MS.
DIc90F: Dynein light chain 90F
LocusID: 42199

M

*Drosophila

30
rc_AI233219_at
601521
Tonnel

ENDOTHELIAL CELL-SPECIFIC
LocusID: 11082

A B.

MOLECULE 1; ESM1

31
rc_AA858520_g_at
Vries
136470

FOLLISTATIN; FST
LocusID: 10468;

C J.

Gene map locus;

5q11.2

32

33

34
U31866_g_at

35
J02720_at

207800

ARGINASE
map locus: 6q23

36

37
rc_AA860039_s_at

38
rc_AA866443_at

39
rc_AA891255_at

40
rc_AA894029_at

[0118]

5

RAT GENES IN RESTENOSIS: 7 DAY UP REGULATED GENES AND HUMAN HOMOLOGUES

A

C

E
F

1
RAT ACCESSION NO.
B
LINKS
D
DESCRIPTION
HUMAN LOCUS

2

3
M36151cds_at

4
X14254cds_at

5
U65217_i_at

6
rc_AI171966 at
HLA-DMB

HLA-DMB: major
LocusID: 3109

histocompatibility complex,

class II, DM beta

7
M61875_s_at

8
J02962_at

9
rc_AA859954_at

10

11

12
M80829_at
TNNT2

TNNT2: troponin T2, cardiac
LocusID: 7139

13
AJ005394_at
1289

COL5A1: collagen, type V,
LocusID: 1289

alpha 1

14
AJ005396_at

15
M12098_s_at

16
L13606_at

17
X51531cds_at

18
X51531cds_g_at

19
rc_AI104561_g_at
ACTC

ACTC: actin, alpha, cardiac
LocusID: 70

muscle

20
rc_AA866452_s_at
ACTC

ACTC: actin, alpha, cardiac
LocusID: 70

muscle

21
X80130cds_f_at
ACTC
*102540

22
X80130cds_i_at

23
rc_AA800206_at

24
rc_AI169327_g_at
TIMP1

TIMP1: tissue inhibitor of
LocusID: 7076

metalloproteinase 1 (erythroid

potentiating activity,

collagenase inhibitor)

25
X83537_at
MMP14

MMP14: matrix
LocusID: 4323

metalloproteinase 14

(membrane-inserted)

26
X98517_at
MMP12

MMP12: matrix
LocusID: 4321

metalloproteinase 12

(macrophage elastase)

27
U57362_at
COL12A1
120320

COL12A1: collagen, type XII,
LocusID: 1303

alpha 1

28
M14656_at
SPP1

SPP1: secreted
LocusID: 6696

phosphoprotein 1

(osteopontin, bone

sialoprotein I, early T-

lymphocyte activation 1)

29
M88469_at
SPON1
T.

SPON1: spondin 1, (f-
LocusID: 10418

spondin) extracellular matrix

protein

30
rc_AA859740_at
HS6ST

HS6ST: heparan sulfate 6-O-
LocusID: 9394

sulfotransferase

31
rc_AA859757_at
COL5A1

COL5A1: collagen, type V,
LocusID: 1289

alpha 1

32
rc_AA859757_g_at

33
rc_AI102814_at
LOX

LOX: lysyl oxidase
LocusID: 4015

34
rc_AA875582_at
LOX
HM

LOX: lysyl oxidase
LocusID: 4015

35
rc_AA875047_at
CCT6A

36
rc_AA875665_at

*603420

CALU: calumenin
LocusID: 813

37
D90258_s_at
5683

PSMA2: proteasome
LocusID: 5683

(prosome, macropain) subunit,

alpha type, 2

38

39

40
S81497_s_at

41
AB005900_at
OLR1

OLR1: oxidised low density
LocusID: 4973

lipoprotein (lectin-like)

receptor 1

42
L07114_at

45
L35271_at
RUNX1

RUNX1: runt-related
LocusID: 861

transcription factor 1 (acute

myeloid leukemia 1, aml1

oncogene)

46
X59864mRNA_at
Verrell

ePl

47
rc_891041_at
JUNB

JUNB: jun B proto-oncogene
LocusID: 3726

48

49

50
AF012891_at
SFRP4

SFRP4: secreted frizzled-
LocusID: 6424

related protein 4

51
rc_AA866465_s_at
NTRK1
#164970
*1913
NIRK1: neurotrophic tyrosine
LocusID: 4914

15
kinase, receptor, type 1

52
D14076_at
1759
Hirokawa
Feron
DNM1: dynamin 1
LocusID: 1759

N
O.

53
J03627_at
S100A10

S100A10: S100 calcium-
LocusID: 6281

binding protein A10 (annexin II

ligand, calpactin I, tight

polypeptide (p11))

54
U23407_at
CRABP2

CRABP2: cellular retinoic acid
LocusID: 1382

binding protein 2

55
rc_AI171796_at
CAPN6

CAPN6: calpain 6
LocusID: 827

56
L38644_at
KPNB1
602738

KPNB1: karyopherin (importin)
LocusID: 3837

beta 1

59
D00753_at
CHD2

CHD2: chromodomain
LocusID: 1106

helicase DNA binding protein

2

60
rc_AA899854_at
TOP2A

TOP2A: topoisomerase (DNA)
LocusID: 7153

II alpha (170kD)

61
M89646_at

62
rc_AI103498_at
RPL5

RPL5: ribosomal protein L5
LocusID: 6125

63
X6O767mRNA_s_at
CDC2

CDC2: cell division cycle 2,
LocusID: 983

G1 to S and G2 to M

64
rc_AI008852_at
EEF1A1

EEF1A1: eukaryotic
LocusID: 1915

translation elongation factor 1

alpha 1

65
rc_AA894059_at
STK18

STK18: serine/threonine
LocusID: 10733

kinase 18

66
AF052695_at
CDC20

CDC20: CDC20 (cell division
LocusID: 991

cycle 20, S. cerevisiae,

homolog)

67
rc_AA859827_at
UMPK

UMPK: uridine

monophosphate kinase

68

69

70
rc_AA945737_at
CXCR4

CXCR4: chemokine (C-X-C
LocusID: 7852

motif), receptor 4 (fusin)

71
X17012mRNA_s_at

72
M15481_g_at
3479
265850

IGF1: insulin-like growth factor
LocusID: 3479

1 (somatomedin C)

73
rc_AA858520_at
Fst

Fst: Foltistatin* rat only
LocusID: 24373

74
M24393_at
MYOG

MYOG: myogenin (myogenic
LocusID: 4656

factor 4)

75
U87983_at
HMMR

HMMR: hyaluronan-mediated
LocusID: 3161

motility receptor (RHAMM)

76
rc_AA894092_at
OSF-2

OSF-2: osteoblast specific
LocusID: 10631

factor 2 (fasciclin I-like)

77
rc_A1233219_at
ESM1

ESM1: endothelial cell-specific
LocusID: 11082

molecule 1

78
U50736_s_at
CARP

CARP: cardiac ankyrin repeat
LocusID: 27063

protein

79
X17053mRNA_s_at
SCYA2
Eb AJ

SCYA2: small inducible
LocusID: 6347

cytokine A2 (monocyte

chemotactic protein 1,

homologous to mouse Sig-je)

80
X89963_at
THBS4

THBS4: thrombospondin 4
LocusID: 7060

81
X02002_at

82
rc_AA874848_s_at
THY1

THY1: Thy-1 cell surface
LocusID: 7070

antigen

83
X63722cds_s_at

84
M84488_at

85
L00191cds#1_s_at

86
U82612cds_at

87
U82612cds_g_at

88
rc_AA893846_at
7143

TNR: tenascin R (restrictin,
LocusID: 7143

janusin)

89
U09361_s_at

90
U09401_s_at

91
U15550_at

92
L20869_at
5068

PAP: pancreatitis-associated
LocusID: 5068

protein

94

95
M23697_at
PLAT

PLAT: plasminogen activator,
LocusID: 5327

tissue

96
M19647_i_at
KLK1

KLK1: kallikrein 1,
LocusID: 3816

renal/pancreas/salivary

97
X74832cds_at
CHRNA1

CHRNA1: cholinergic
LocusID: 1134

receptor, nicotinic, alpha

polypeptide 1 (muscle)

98
X74835cds_at
CHRND

CHRND: cholinergic receptor,
LocusID: 1144

nicotinic, delta polypeptide

99

100

101
U33441mRNA_s_at

102
M31032cds#1_s_at

103
M31O32mRNA#2_at

104
M177O3mRNA_s_at

105
M33976_at

106
rc_AA892775_at
LYZ

LYZ: lysozyme (renal
LocusID: 4069

amyloidosis)

107
X12459_at

108
D14441_at

109
rc_AA859536_at
Basp1
*605940

BASP1: brain abundant,
LocusID: 10409

membrane attached signal

protein 1. *human

110
rc_AI176456_at

4489

MT1A: metallothionein 1A
LocusID: 4489

(functional)

111

112
rc_AA860039_s_at

113
rc_AA860057_at

114
rc_AA866290_at

115
rc_AA866443_at

116
rc_AA893717_at

Identification of genes involved in restenosis and in atherosclerosis

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Provisional Applications (1)