WO2015073709A2 - Détection du risque de maladie cardiovasculaire athéroscléreuse et du risque d'insuffisance cardiaque - Google Patents
Détection du risque de maladie cardiovasculaire athéroscléreuse et du risque d'insuffisance cardiaque Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- This relates to the field of cardiovascular disease, specifically to methods for determining if a subject is at risk for developing atherosclerosis or heart failure.
- Atherosclerotic cardiovascular disease is the leading cause of death in the U.S. and is becoming the leading killer in developing countries.
- the lifetime risk of developing coronary heart disease (CHD) is 1 in 2 for men and 1 in 3 for women.
- CVD ranked highest among all disease categories in hospital discharges; 16 million people in the US have CHD and 7 million have a history of stroke. Each year, an estimated 785,000 Americans will have a first CHD event and 610,000 a first stroke.
- total direct medical costs of CVD are projected to triple, from $273 billion to $818 billion.
- HF heart failure
- Methods are provided for detecting or determining the likelihood that a subject will develop atherosclerotic cardiovascular disease (ASCVD).
- the methods include performing one or more assays that detect a level of ceruloplasmin (CP),
- GDF15 growth/differentiation factor 15
- LPA apolipoprotein(a)
- Tissue inhibitor of metalloproteinases 1 TIMPl
- the detection of an increase in the level of CP, an increase in the level of GDF15, an increase in LPA and an increase in TIPM1 as compared to the respective control indicates that the subject has or will develop ASCVD.
- methods are provided for determining if a pharmaceutical agent is effective for treatment or prevention of ASCVD in a subject.
- the methods include performing one or more assays that detect a level of ceruloplasmin (CP), growth/differentiation factor 15 (GDF15), apolipoprotein(a) (LPA), and Tissue inhibitor of metalloproteinases 1 (TEMPI) in a biological sample from the subject administered the agent; and comparing the level of CP, GDF15, LPA and TEvlPl to a respective control level of CP, GDF15, LPA and TIMPl.
- CP ceruloplasmin
- GDF15 growth/differentiation factor 15
- LPA apolipoprotein(a)
- TEMPI Tissue inhibitor of metalloproteinases 1
- the detection of a decrease in the level of CP, a decrease in the level of GDF15, a decrease in LPA and a decrease in TIPM1, as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- method for detecting or determining the likelihood that a subject will develop heart failure (HF).
- the methods include performing one or more assays that detect a level of natriuretic peptides B (NPPB), growth/differentiation factor 15 (GDF15), myeloperoxidase (MPO) and plasminogen activator inhibitor 1 (SERPINE1) in a biological sample from the subject; and comparing the level of NPPB, GDF15, MPO, and SERPINEl to a respective control level of NPPB, GDF15, MPO, and SERPINE1, respectively.
- the detection of an increase in the level of NPPB, an increase in the level of GDF15, an increase in the level of MPO, and an increase in the level of SERPINEl as compared to the respective control indicates that the subject has or will develop HF.
- methods are provided for determining if a pharmaceutical agent is effective for treatment or prevention of HF in a subject.
- the methods include performing one or more assays that detect a level of natriuretic peptides B (NPPB), growth/differentiation factor 15 (GDF15), myeloperoxidase (MPO) and plasminogen activator inhibitor 1 (SERPINEl) in a biological sample from the subject administered the agent; and comparing the level of NPPB, GDF15, MPO and SERPINEl to a respective control level of NPPB, GDF15, MPO and
- NPPB natriuretic peptides B
- GDF15 growth/differentiation factor 15
- MPO myeloperoxidase
- SERPINEl plasminogen activator inhibitor 1
- SERPINEl The detection of a decrease in the level of NPPB, a decrease in the level of GDF15, a decrease in the level of MPO and a decrease in the level of SERPINEl as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of HF in the subject.
- GENBANK® Accession numbers are provided below. In these entries nucleic and amino acid sequences listed are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand when appropriate. All of the GENBARNK® entries are incorporated herein by reference as available on November 1, 2013. Each GENBANK® Accession number listed herein entry provides an exemplary sequence for the listed proteins. Polypeptides and polynucleotides at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide or nucleic acid sequence shown in these GENBANK® entries, respectively, can be of use in the methods disclosed herein.
- nucleic acid molecule includes single or plural nucleic acid molecules and is considered equivalent to the phrase “comprising at least one nucleic acid molecule.”
- the term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.
- compacts means “includes.”
- comprising A or B means “including A, B, or A and B,” without excluding additional elements.
- Aldosterone antagonist A diuretic drug used for the management of heart failure.
- exemplary aldosterone antagonists include spironolactone, eplerenone, canrenone, prorenone and mexrenone.
- a change in an effective amount of a substance of interest such as a
- the amount of the substance can changed by a difference in the amount of the substance produced, by a difference in the amount of the substance that has a desired function, or by a difference in the activation of the substance.
- the change can be an increase or a decrease.
- the alteration can be in vivo or in vitro.
- altering an amount of a polypeptide or polynucleotide is at least about a 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% increase or decrease in the effective amount (level) of a substance.
- an increase of a polypeptide or polynucleotide is at least about a 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% increase in a polypeptide or polynucleotide as compared to a control, a statistical normal, or a standard value chosen for specific study.
- a decrease of a polypeptide or polynucleotide is at least about a 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% decrease in a polypeptide or polynucleotide as compared to a control, a statistical normal, or a standard value chosen for specific study.
- Angiotensin-converting enzyme (ACE) inhibitor A drug used primarily for the treatment of hypertension and heart failure.
- ACE inhibitors include perindopril, captopril, enalapril, lisinopril and ramipril.
- Angiotensin receptor blockers A group of drugs that act by blocking the effects of the hormone angiotensin ⁇ .
- ARBs are used to treat, for example, hypertension and heart failure.
- Exemplary ARBs include losartan, candesartan, valsartan, irbesartan, telmisartan, eprosartan and olmesartan.
- Antibody A polypeptide including at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen or an antigen- binding fragment thereof.
- Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V H ) region and the variable light (V L ) region. Together, the V H region and the V L region are responsible for binding the antigen recognized by the antibody.
- Antibodies of the present disclosure include those that are specific for the molecules listed.
- antibody includes intact immunoglobulins, as well the variants and portions thereof, such as Fab' fragments, F(ab)' 2 fragments, single chain Fv proteins ("scFv”), and disulfide stabilized Fv proteins ("dsFv").
- scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin.
- immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
- the term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.
- a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
- H heavy chain
- L light chain
- ⁇ lambda
- ⁇ kappa
- IgM immunoglobulin heavy chain classes
- Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”).
- the heavy and the light chain variable regions specifically bind the antigen.
- Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs.”
- VH refers to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.
- VL refers to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
- a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
- Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
- Monoclonal antibodies include humanized monoclonal antibodies.
- a “polyclonal antibody” is an antibody that is derived from different B-cell lines.
- Polyclonal antibodies are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. These antibodies are produced by methods known to those of skill in the art, for instance, by injection of an antigen into a suitable mammal (such as a mouse, rabbit or goat) that induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen, which are then purified from the mammal' s serum.
- a suitable mammal such as a mouse, rabbit or goat
- a “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds an antigen of interest.
- a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin.
- the non-human immunoglobulin providing the CDRs is termed a "donor,” and the human immunoglobulin providing the framework is termed an "acceptor.”
- all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
- Constant regions need not be present, but if they are, they are substantially identical to human immunoglobulin constant regions, e.g. , at least about 85-90%, such as about 95% or more identical.
- Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Patent No. 5,585,089).
- Array An arrangement of molecules, such as biological macromolecules (such as peptides or nucleic acid molecules) or biological samples (such as tissue sections), in addressable locations on or in a substrate.
- a "microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. Arrays are sometimes called chips or biochips.
- the array of molecules makes it possible to carry out a very large number of analyses on a sample at one time.
- one or more molecules (such as an oligonucleotide probe) will occur on the array a plurality of times (such as twice), for instance to provide internal controls.
- the number of addressable locations on the array can vary, for example from at least one, to at least 2, to at least 5, to at least 10, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, least 550, at least 600, at least 800, at least 1000, at least 10,000, or more.
- an array includes nucleic acid molecules, such as oligonucleotide sequences that are at least 15 nucleotides in length, such as about 15-40 nucleotides in length.
- an array includes oligonucleotide probes or primers which can be used to detect ASCVD and HF.
- each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array.
- the feature application location on an array can assume different shapes.
- the array can be regular (such as arranged in uniform rows and columns) or irregular.
- the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position.
- ordered arrays are arranged in a symmetrical grid pattern, but samples could be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters).
- Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity).
- information about the sample at that position such as hybridization or binding data, including for instance signal intensity.
- the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.
- Protein-based arrays include probe molecules that are or include proteins, or where the target molecules are or include proteins, and arrays including antibodies to which proteins are bound, or vice versa.
- an array contains antibodies to ASCVD and HF- associated proteins.
- the array includes positive controls, negative controls, or both, for example molecules specific for detecting ⁇ -actin, 18S RNA, beta-microglobulin, glyceraldehyde- 3-phosphate-dehydrogenase (GAPDH), and other housekeeping genes.
- the array includes 1 to 20 controls, such as 1 to 10 or 1 to 5 controls.
- Atherosclerosis The progressive narrowing and hardening of a blood vessel over time.
- Atherosclerosis is a common form of arteriosclerosis in which deposits of yellowish plaques (atheromas) containing cholesterol, lipoid material and lipophages are formed within the intima and inner media of large and medium-sized arteries.
- Treatment of atherosclerosis includes reversing or slowing the progression of atherosclerosis, for example as measured by the presence of atherosclerotic lesions and/or functional signs of the disease, such as improvement in cardiovascular function as measured by signs (such as peripheral capillary refill), symptoms (such as chest pain and intermittent claudication), or laboratory evidence (such as that obtained by EKG, angiography, or other imaging techniques).
- "Assessing atherosclerosis” indicates determining if a subject of interest has atherosclerosis, determining the prognosis of the subject of interest, and/or determining if a therapeutic regimen administered to the subject is effective in treating the subject.
- Beta blocker A type of drug that targets the beta receptor, which are found on the cells of heart muscles. Beta blockers interfere with binding of epinephrine and other stress hormones to the beta receptor. These drugs are often used for the management of cardiac arrhythmias, prevention of heart attacks and heart failure, and for treating hypertension.
- Bile acid binding resins Agents that lower LDL cholesterol. Bile acids are the breakdown products of cholesterol and are excreted by the liver via the bile. Bile acids are 90% reabsorbed from the intestine and used to re-manufacture cholesterol in the liver. Bile acid binding resins (also referred to as bind acid sequestrants) interfere with this intestinal reabsorption by binding bile acids in the gut and promoting their excretion from the body.
- Blood vessel The vessels through which blood circulates.
- blood vessels are elastic tubular channels that are lined with endothelium.
- Blood vessels include the arteries, veins and capillaries.
- Specific, non-limiting examples of a blood vessel include a vena cava, a thoracic aorta, a saphenous vein, a mammary artery, the brachial artery and a capillary.
- a blood vessel includes the smaller arteries and veins.
- a blood vessel is a capillary of the microvascular circulation.
- Cardiovascular Pertaining to the heart and/or blood vessels.
- Cardiovascular disease Disorders of the heart and blood vessels, such as atherosclerosis (ASCVD), coronary heart disease, cerebrovascular disease, and peripheral vascular disease. Cardiovascular diseases also include, for example, myocardial infarction, stroke, angina pectoris, transient ischemic attacks, and congestive heart failure. Atherosclerosis usually results from the accumulation of fatty material, inflammatory cells, extracellular matrices and plaque. Clinical symptoms and signs indicating the presence of CVD may include one or more of the following: chest pain and other forms of angina, shortness of breath, sweatiness, Q waves or inverted T waves on an EKG, a high calcium score by CT scan, at least one stenotic lesion on coronary angiography, and heart attack. Subclinical ASCVD can be identified by imaging tests (such as CT measures of coronary calcification, or MRI measures of coronary or aortic plaque, and/or ultrasound evidence of carotid plaque or thickening).
- imaging tests such as CT measures of coronary calcification, or
- Cardiovascular risk The likelihood of the development of cardiovascular disease, such as, but not limited to, myocardial ischemia and infarction, intermittent claudication, transient ischemic attacks, ischemic strokes, and other conditions associated with cardiovascular dysfunction.
- the disorder is atherosclerosis.
- atherosclerotic risk is the likelihood of the development of atherosclerosis
- myocardial infarction risk is the likelihood of having a myocardial infarction
- heart failure risk is the likelihood of developing heart failure.
- Cholesterol absorption inhibitor A class of cholesterol lowering drugs that block absorption of cholesterol at the brush border of the intestine without affecting absorption of triglycerides or fat soluble vitamins. These drugs are not systemically absorbed and can lower cholesterol on their own (i.e. without the use of additional drugs).
- An exemplary cholesterol absorption inhibitor is ezetimibe (Ezetrol).
- Cholesterol lowering agent An agent that lowers the level of cholesterol in a subject, such as a pharmaceutical, vitamin, or small molecule.
- Agents include, but are not limited to, niacin, the statins (e.g. , ZOCORTM, LIPITORTM, PRAVACOLTM, LESCORTM, MEVACORTM), bile acid binding resins (e.g. , QUESTRANTM), and fibrates (e.g. LOPIDTM, LIPIDIL MICROTM).
- a housekeeping protein or rRNA such as 18S RNA, beta-microglobulin, GAPDH, and/or ⁇ - actin
- control refers to a sample or standard used for comparison with an experimental sample.
- the control is a sample obtained from a healthy patient or a non-diseased tissue sample obtained from a patient diagnosed with the disorder of interest, such as HF or ASCVD.
- the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with the disorder, or group of samples that represent baseline or normal values, such as the level of specific genes in non-diseased tissue).
- Detecting expression of a gene product Determining the presence of and/or the level of expression of a nucleic acid molecule (such as an mRNA molecule) or a protein encoded by a gene in either a qualitative or quantitative manner.
- exemplary methods include microarray analysis, RT-PCR, Northern blot, Western blot, and mass spectrometry of specimens from a subject, for example measuring levels of a gene product present in blood, serum, or another biological sample as a measure of expression.
- Diagnosis The process of identifying a disease by its signs, symptoms and results of various tests. The conclusion reached through that process is also called "a diagnosis.” Forms of testing commonly performed include blood tests, medical imaging, urinalysis, and biopsy.
- Differential or alteration in expression A difference or change, such as an increase or decrease, in the conversion of the information encoded in a gene into messenger RNA, the conversion of mRNA to a protein, or both.
- the difference is relative to a control or reference value or range of values, such as an amount of gene expression that is expected in a subject who does not have a disorder of interest (for example heart disease, atherosclerosis or myocardial infarction).
- Detecting differential expression can include measuring a change in gene expression or a change in protein levels.
- Diuretic A drug that promotes the production of urine. Diuretics are often used to treat heart failure, hypertension and other diseases.
- RNA such as microRNA, mRNA, rRNA, tRNA, and structural RNA
- gene downregulation or deactivation includes processes that decrease transcription of a gene or translation of mRNA.
- Examples of processes that decrease transcription include those that facilitate degradation of a transcription initiation complex, those that decrease transcription initiation rate, those that decrease transcription elongation rate, those that decrease processivity of transcription and those that increase transcriptional repression.
- Gene downregulation can include reduction of expression above an existing level.
- Examples of processes that decrease translation include those that decrease translational initiation, those that decrease translational elongation and those that decrease mRNA stability.
- Gene downregulation includes any detectable decrease in the production of a gene product.
- production of a gene product decreases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal cell).
- a control is a relative amount of gene expression in a biological sample, such as from a subject that does not have ASCVD or has not had HF.
- Gene expression can be influenced by external signals. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced. In an example, gene expression can be monitored to determine the diagnosis and/or prognosis of a subject with ASCVD or at risk for HF.
- the expression of a nucleic acid molecule in a test sample can be altered relative to a control sample, such as a normal sample from a healthy subject.
- Expression of proteins is the level of protein in a biological sample. Expression includes, but is not limited to, the production of the protein by translation of an mRNA and the half-life of the protein. Expression of proteins is the level of protein in a biological sample. Expression includes, but is not limited to, the production of the protein by translation of an mRNA and the half-life of the protein. Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (e.g., non-disease) situation. Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (e.g., non-disease) situation.
- Alterations in expression include but are not limited to: (1) overexpression; (2) underexpression; or (3) suppression of expression.
- Controls or standards for comparison to a sample, for the determination of differential expression include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have ASCVD) as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.
- Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.
- Fibrates Agents that lower tri-glyceride levels and raise HDL levels. Fibrates, also known as fibric acid derivatives, are particularly useful in diabetic patients whose characteristic lipid abnormality is high tri-glycerides and low HDL. In some patients who have combined lipid abnormalities, fibrates are combined with statins to lower both tri-glycerides and LDL and to raise HDL. Exemplary fibrates include gemfibrozil (LOPIDTM), fenofibrate (Lipidil micro, Lipidil Supra, Lipidil EZ), and bezafibrate (Bezalip).
- LPIDTM gemfibrozil
- fenofibrate Lipidil micro
- Lipidil Supra Lipidil EZ
- Bezalip bezafibrate
- Framingham Risk Score A risk factor score that is used for predicting future risk of coronary artery disease in individuals free of disease, based on the measurement of Framingham risk factors which include age, gender, systolic blood pressure (and use of antihypertensive treatment), cigarette smoking, diabetes, as well as total cholesterol (or low density lipoprotein cholesterol (LDL cholesterol) and high density lipoprotein cholesterol (HDL cholesterol) levels (Wilson et al, Circulation 1998; 97: 1837- 47).
- Gene expression profile (or signature) Differential or altered gene expression can be detected by changes in the detectable amount of gene expression (such as cDNA or mRNA) or by changes in the detectable amount of proteins expressed by those genes.
- a distinct or identifiable pattern of gene expression for instance a pattern of high and low expression of a defined set of genes or gene-indicative nucleic acids such as ESTs.
- a gene expression profile (also referred to as a signature) can be linked to disease progression (such as advanced ASCVD), or to any other distinct or identifiable condition that influences gene expression in a predictable way.
- Gene expression profiles can include relative as well as absolute expression levels of specific genes, and can be viewed in the context of a test sample compared to a baseline or control sample profile (such as a sample from the same tissue type from a subject who does not have ASCVD).
- a gene expression profile in a subject is read on an array (such as a nucleic acid or protein array).
- a gene expression profile can be performed using a commercially available array such as Human Genome GENECHIP® arrays from
- AFFYMETRK® (Santa Clara, CA).
- Heart failure The physiological state in which cardiac output is insufficient in meeting the needs of the body and lungs. This condition is also called “congestive heart failure,” and is most commonly caused when cardiac output is low and the lungs become congested with fluid due to an inability of heart output to properly match venous return. Heart failure can also occur in situations of high output, where the ventricular systolic function is normal but the heart can't process the augmentation of blood volume. This can occur in overload situation (blood or serum infusions), renal diseases, chronic severe anemia, beriberi (vitamin Bi/thiamine deficiency), thyrotoxicosis, Paget' s disease, arteriovenous fistulae, or arteriovenous
- Heart failure includes left sided failure and right sided failure, wherein the left and right ventricles are affected, respectively, and biventricular failure.
- Ischemic heart disease including myocardial infarction
- cigarette smoking smoking
- hypertension obesity
- diabetes diabetes
- valvular heart disease are associated with increased risk of heart failure.
- Viral myocarditis, human immunodeficiency virus infections, connective tissue disease (such as systemic lupus erythematous), drug (***e) abuse, and some chemotherapeutic agents can cause heart failure.
- Hybridization To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule, for example.
- Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the
- Isolated An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells.
- Nucleic acid molecules and proteins that have been "isolated” include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.
- Label An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy.
- a label can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein.
- labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
- a label is conjugated to a binding agent that specifically binds to an ASCVD and HF associated protein, disclosed herein.
- Level of Expression An amount, such as of a protein or an mRNA, that can be measured in a biological sample.
- Lipoprotein A biochemical assembly that contains both proteins and lipids, bound to the proteins, which allow fats to move through the water inside and outside cells.
- HDL contains the highest proportion of protein to cholesterol; its most abundant apolipoproteins are apo A-I and apo ⁇ - ⁇ .
- LDL contains apolipoprotein B, and has a core consisting of linoleate and includes esterified and non-esterified cholesterol molecules.
- LDL particles are approximately 22 nm in diameter and have a mass of about 3 million daltons.
- Lipoprotein a (Lp(a)) is a lipoprotein subclass; lipoprotein a consists of an LDL- like particle and the specific apolipoprotein(a) [apo(a)], which is covalently bound to the apolipoprotein B of the LDL like particle.
- Mammal This term includes both human and non-human mammals. Examples of mammals include, but are not limited to: humans, pigs, cows, goats, cats, dogs, rabbits, rats, and mice.
- a biological marker such as a polypeptide or a polynucleotide, that can be detected in a biological sample from a subject.
- a polypeptide marker can be about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to a reference amino acid sequence, such as a known amino acid sequence provided in a database such as GENBANK® or EMBL®.
- a reference amino acid sequence such as a known amino acid sequence provided in a database such as GENBANK® or EMBL®.
- an antibody that specifically binds a reference sequence of interest will bind the marker in a biological sample.
- a polynucleotide marker can be about 95%, 96%, 97%, 98%, 99% or 100% identical to a reference nucleic acid sequence, such as a known nucleic acid sequence provided in a database such as GENBANK® or EMBL®.
- a probe that specifically hybridizes to a reference sequence under very stringent conditions will bind the polynucleotide marker, or primers designed to amplify the reference sequence will amplify the polynucleotide marker.
- Mass Spectrometry A process used to separate and identify molecules based on their mass. Mass spectrometry ionizes chemical compounds to generate charged molecules or molecule fragments and measures their mass-to-charge ratios.
- time-of-flight mass spectrometry is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass- to-charge ratio.
- Liquid chromatography-mass spectrometry or "LC- MS” is a chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. Liquid chromatography mass spectrometry (LC-MS) separates compounds chromatographically before they are introduced to the ion source and mass spectrometer. It differs from gas chromatography (GC-MS) in that the mobile phase is liquid, usually a mixture of water and organic solvents, instead of gas and the ions fragments. Most commonly, an electrospray ionization source is used in LC-MS.
- GC-MS gas chromatography
- Niacin A B-vitamin that is used as a medication for patients with elevated levels of triglycerides and cholesterol.
- a long-acting preparation of niacin is available as NIASPAN ® .
- Nucleic acid array An arrangement of nucleic acids (such as DNA or RNA) in assigned locations on a matrix, such as that found in cDNA arrays, or oligonucleotide arrays.
- Nucleic acid molecules representing genes Any nucleic acid, for example DNA (intron or exon or both), cDNA, or RNA (such as mRNA), of any length suitable for use as a probe or other indicator molecule, and that is informative about the corresponding gene, such the proteins specified herein.
- PCSK9-targeting drug An agent that targets proprotein convertase subtilisin/kexin type 9 (PCSK9), an enzyme that mediates the post-translational degradation of the LDL receptor, thereby modulating serum levels of LDL cholesterol. Thus, drugs that inhibit PCSK9 also lower cholesterol.
- PCSK9-targeting drugs are in development, including monoclonal antibodies, small molecules and gene silencing agents (e.g., antisense oligonucleotides, locked nucleic acids, and siRNAs).
- Pep tide/Protein/Polypep tide All of these terms refer to a polymer of amino acids and/or amino acid analogs that are joined by peptide bonds or peptide bond mimetics, regardless of length or post-translational modification (such as glycosylation, methylation, ubiquitination, phosphorylation, or the like).
- PCR Polymerase Chain Reaction
- PCR utilizes primers, for example, DNA oligonucleotides 10-100 nucleotides in length, such as about 15, 20, 25, 30 or 50 nucleotides or more in length (such as primers that can be annealed to a complementary target DNA strand by nucleic acid
- Primers can be selected that include at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of a nucleotide sequence of interest. Methods for preparing and using nucleic acid primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990).
- Primers Short nucleic acid molecules, for instance DNA oligonucleotides 10 -100 nucleotides in length, such as about 15, 20, 25, 30 or 50 nucleotides or more in length, such as this number of contiguous nucleotides of a nucleotide sequence encoding a protein of interest or other nucleic acid molecule. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand. Primer pairs can be used for amplification of a nucleic acid sequence, such as by PCR or other nucleic acid amplification methods known in the art.
- PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991,
- a primer includes at least 15 consecutive nucleotides of a nucleotide molecule, such as at least 18 consecutive nucleotides, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of a nucleotide sequence (such as a gene, mRNA or cDNA).
- a nucleotide sequence such as a gene, mRNA or cDNA.
- Such primers can be used to amplify a nucleotide sequence of interest, such as the markers listed in Tables A and/or B, for example using PCR.
- Probe A short sequence of nucleotides, such as at least 8, at least 10, at least 15, at least 20, at least 21, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 or even greater than 100 nucleotides in length, used to detect the presence of a complementary sequence by molecular hybridization.
- oligonucleotide probes include a label that permits detection of oligonucleotide probe:target sequence hybridization complexes.
- Such an oligonucleotide probe can also be used on a nucleic acid array, for example to detect a nucleic acid molecule in a biological sample contacted to the array.
- a probe is used to detect the presence of a nucleic acid molecule for a markers listed in Tables A and/or B.
- Prognosis A prediction of the future course of a disease, such as ASCVD or HF.
- the prediction can include determining the likelihood of a subject to develop complications of
- ASCVD or HF ASCVD or HF, or to survive a particular amount of time (e.g., determine the likelihood that a subject will survive 1, 2, 3 or 5 years), to respond to a particular therapy (e.g., lipid lowering therapy), or combinations thereof.
- a particular therapy e.g., lipid lowering therapy
- purified does not require absolute purity; rather, it is intended as a relative term.
- a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.
- a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation.
- a purified oligonucleotide preparation is one in which the oligonucleotide is more pure than in an environment including a complex mixture of oligonucleotides.
- Sample A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, serum, plasma, urine, fine needle aspirate, tissue biopsy, surgical specimen, and autopsy material.
- Sequence identity/similarity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.
- BLAST Basic Local Alignment Search Tool
- NCBI National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894
- sequence analysis programs blastp, blastn, blastx, tblastn and tblastx Additional information can be found at the NCBI web site.
- BLASTN is used to compare nucleic acid sequences
- BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
- the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
- the percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100
- the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
- the length value will always be an integer.
- the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs may use SEG filtering (Wootton and Federhen, Meth. Enzymol.
- nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
- homologous nucleic acid sequences can, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to a molecule listed in Tables A or B determined by this method.
- cumulative) indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
- Specific binding agent An agent that binds substantially or preferentially only to a defined target such as a protein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinant vector or a small molecule.
- a nucleic acid-specific binding agent binds substantially only to the defined nucleic acid, such as RNA, or to a specific region within the nucleic acid.
- a "specific binding agent” includes an antisense compound (such as an antisense oligonucleotide, siRNA, miRNA, shRNA or ribozyme) that binds substantially to a specified RNA.
- a protein-specific binding agent binds substantially only the defined protein, or to a specific region within the protein.
- a “specific binding agent” includes antibodies and other agents that bind substantially to a specified polypeptide.
- Antibodies can be
- Statin Any of a class of lipid-lowering drugs that reduce serum cholesterol levels by inhibiting a key enzyme involved in the biosynthesis of cholesterol.
- Example statins include atorvastatin (LIPITOR®), fluvastatin (LESCOL®), lovastatin (MEVACOR®, ALTOCOR®, not marketed in the UK), pravastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin (CRESTOR®), simvastatin (ZOCOR®).
- statins There are two groups of statins: (1) Fermentation- derived: lovastatin, simvastatin and pravastatin, and (2) Synthetic statins: fluvastatin, atorvastatin, cerivastatin and rosuvastatin. Generally, statins act by competitively inhibiting 3- hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an enzyme of the HMG-CoA reductase pathway, the body's metabolic pathway for the synthesis of cholesterol.
- HMG CoA 3- hydroxy-3-methylglutaryl coenzyme A
- statin lovastatin
- Subject Living multi-cellular vertebrate organism, a category that includes human and non-human mammals.
- Therapeutically effective amount An amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response.
- a therapeutic agent such as an anticoagulant, or a statin, is administered in therapeutically effective amounts.
- Effective amounts a therapeutic agent can be determined in many different ways, such as assaying for a reduction in atherosclerotic disease or improvement of physiological condition of a subject having vascular disease. Effective amounts also can be determined through various in vitro, in vivo or in situ assays. Therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.
- a pharmaceutical preparation can decrease one or more symptoms of vascular disease, for example decrease a symptom by at least 20%, at least 50%, at least 70%, at least 90%, at least 98%, or even at least 100%, as compared to an amount in the absence of the pharmaceutical preparation.
- Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such a sign, parameter or symptom of vascular disease (for example, ASCVD). Treatment can also induce remission or cure of a condition, such as vascular disease.
- treatment includes preventing a disease, for example by inhibiting the full development of a disease, such as preventing development of vascular disease. Prevention of a disease does not require a total absence of vascular disease. For example, a decrease of at least 50% can be sufficient.
- Upregulated or activation When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in an increase in production of a gene product.
- a gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, gene upregulation or activation includes processes that increase transcription of a gene or translation of mRNA.
- Examples of processes that increase transcription include those that facilitate formation of a transcription initiation complex, those that increase transcription initiation rate, those that increase transcription elongation rate, those that increase processivity of transcription and those that relieve transcriptional repression (for example by blocking the binding of a transcriptional repressor).
- Gene upregulation can include inhibition of repression as well as stimulation of expression above an existing level.
- Examples of processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability.
- Gene upregulation includes any detectable increase in the production of a gene product.
- production of a gene product increases by at least 1.5-fold, such as at least 2-fold, at least 3-fold or at least 4-fold, as compared to a control.
- a control is a relative amount of gene expression in a biological sample, such as from a subject that does not have ASCVD or has not had HF.
- Vasodilator An agent that induces dilation of blood vessels.
- exemplary vasodilators include, for example, hydralazine and minoxidil.
- Methods are provided herein for evaluating cardiovascular risk, for example for determining whether a subject, such as an otherwise healthy subject, or a subject suspected or at risk of having cardiovascular disease, has cardiovascular disease or will likely develop cardiovascular disease, such as ASCVD, in the future,.
- the method can determine if a subject has or will likely develop ASCVD in the future.
- the method can determine if a pharmaceutical agent is effective for treating a subject.
- a biological sample obtained from the subject such as, but not limited to, serum, blood, plasma, saliva, urine, purified cells (for example, blood cells, such as white blood cells, B cells, T cells, or mononuclear cells), a biopsy sample or tissue sample, such as a sample including adipose cells, blood vessels, or heart tissue obtained from the subject are used to predict the subject's risk of vascular disease.
- the subject is apparently healthy, such as a subject who does not exhibit symptoms of vascular disease (for example, does not have clinically evident ASCVD, and/or has not previously had an acute adverse vascular event such as a myocardial infarction or a stroke).
- a healthy subject is one that if examined by a medical professional, would be characterized as healthy and free of symptoms of vascular disease, such as ASCVD.
- the methods disclosed herein can be used to screen subjects for future evaluation or treatment for cardiovascular disease, and to prevent cardiovascular disease.
- the methods determine the likelihood that a subject will develop ASCVD.
- the subject is suspected of having a vascular disease, or is suspected of being at risk of developing a vascular disease, such as ASCVD in the future.
- a vascular disease such as ASCVD in the future.
- such a subject may have elevated cholesterol or tri-glyceride levels, elevated C-reactive protein levels, diabetes, or high blood pressure.
- the methods disclosed herein can be used to confirm a prior clinical suspicion of disease.
- the expression of the markers disclosed herein can be used to assess the efficacy of a therapeutic protocol for the treatment or prevention of ASCVD.
- methods are provided for evaluating the efficacy of a treatment protocol that includes any therapy for atherosclerosis designed to reverse or slow the progression of atherosclerosis, including but not limited to treatment with statins, niacin or other cholesterol-lowering agents, anti-inflammatory agents, aspirin, anti-platelet agents, anticoagulant agents, blood pressure lowering medications, agents for smoking cessation, or any other pharmaceutical compound.
- a sample can be taken from a subject prior to initiation of therapy. After therapy is initiated, an additional sample is taken from the subject. A decrease in the amount of the markers indicates that the therapy is efficacious.
- the subject can be monitored over time to evaluate the continued effectiveness of the therapeutic protocol.
- the effect of different dosages can also be evaluated, by comparing the expression of markers in a sample from the subject receiving a first dose to the expression of the same markers in a sample from the subject receiving a second (different) dose.
- the methods can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times to determine the lowest dose of a pharmaceutical agent that is effective for treating the subject, and/or the shortest duration of administration that is effective for treating the subject.
- the methods can also be used over the course of a therapeutic regimen to monitor the efficacy of a pharmaceutical agent for the treatment of the subject.
- the subject has been determined to be at risk for cardiovascular disease based on risk factors, such as, but not limited to, Framingham risk factors.
- the Framingham Risk Score is a gender- specific algorithm used to estimate the 10-year cardiovascular risk of a subject using specific factors.
- the Framingham Risk Score was first developed based on data obtained from the Framingham Heart Study, to estimate the 10-year risk of developing coronary heart disease (see Third Report of the National Cholesterol
- NCEP Network Education Program
- the method can include evaluation of a subject to determine if the subject is at risk for cardiovascular disease using risk factors, such as, but not limited to, Framingham risk factors and/or guidelines jointly issues by the American Heart Association and American College of Cardiology.
- Framingham risk factors include age, gender, low density lipoprotein (LDL) cholesterol level, whether the subject smokes, blood pressure (and whether the subject is receiving pharmacological treatment for hypertension), total cholesterol level, and high density lipoprotein (HDL) cholesterol level. Programs for this evaluation are available on the internet, such as at the U.S. National Heart, Lung, and Blood Institute (NHLBI) website.
- LDL low density lipoprotein
- HDL high density lipoprotein
- the method does not comprise measuring a lipoprotein, such as LDL or HDL. In further embodiments, the method does not comprise determining a HDL or LDL subpopulation based on size and/or density of the subject's HDL or LDL. In yet other embodiments, the method does not comprise measuring inflammatory markers. In specific, non- limiting examples, the inflammatory marker is C reactive protein.
- the methods disclosed herein can include evaluating the expression of one or more of the following: Table A
- the marker can include a polypeptide or nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide or nucleic acid sequence shown in these GENBANK® entries, respectively, or can be 100% identical to the listed sequence.
- the methods can include assessing expression of 1, 2, 3 or all 4 of the markers listed in
- the methods can further include evaluating the expression of one or more additional markers known to be associated with ASCVD. Proteins and mRNA can be evaluated, such as the level of 1, 2, 3 or all 4 of the markers shown in Table A. In some embodiments, expression of GDF15, CP, TIMP-1 and LPA is assessed. The method can also include evaluating the expression of any markers listed in Table 1 or Table 8.
- Methods are provided for detecting or determining the likelihood that a subject will develop atherosclerotic cardiovascular disease.
- the methods can include performing one or more assays that detects expression of LPA and GDF15 in a biological sample from the subject; and comparing the level of expression of LPA and GDF15 to a respective control level of LPA and GDF15. Detection of an increase in expression of LPA and an increase in expression of GDF15 as compared to the respective control indicates that the subject has or will develop atherosclerotic cardiovascular disease.
- the method further includes performing an assay that detects expression of CP in a biological sample from the subject; and comparing the level of expression of CP to a respective control level of CP. Detection of an increase in expression of CP as compared to the respective control level of LPA indicates that the subject has or will develop ASCVD.
- the method further includes performing an assay that detects expression of TEMPI a biological sample from the subject; and comparing the level of expression of TEMPI to a respective control level of TEMPI. Detection of an increase in expression of TEMP-1 as compared to the respective control level of TEMPI indicates that the subject has or will develop ASCVD.
- expression of all of GDF15, LPA, CP and TEMP-1 is assessed.
- the control can be a standard value of GDF15, LPA, CP and/or TEMP-1, respectively or can be the level of GDF15, LPA, CP and/or TEMP-1, respectively, in one or more subjects known not to have atherosclerotic cardiovascular disease. Proteins and/or mRNA can be evaluated.
- Methods are also provided for determining if a pharmaceutical agent is effective for treatment or prevention of ASCVD in a subject.
- the subject can have ASCVD.
- the subject has intermittent claudication, transient ischemic attacks (TIAs), ischemic strokes, restenosis after angioplasty, transplant
- Atherosclerosis atherosclerosis, unstable angina, or another condition associated with ASCVD.
- the methods can include assessing expression of 1, 2, 3 or all 4 of the markers listed in Table A in any combination.
- methods of determining if a pharmaceutical agent is effective for treatment or prevention of ASCVD in a subject include performing one or more assays that detect expression of LPA and GDF15 in a biological sample from the subject administered the agent; and comparing the level of expression of LPA and
- GDF15 to a respective control level of LPA and GDF15. Detection of a decrease in expression of LPA and a decrease in expression of GDF15 as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects expression of CP in a biological sample from the subject; and comparing the level of expression of CP to a respective control level of CP. Detection of a decrease in expression of CP as compared to the respective control level of CP indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects expression of TEMPI in a biological sample from the subject; and comparing the level of expression of TEMPI to a respective control level of TEMPI . Detection of a decrease in expression of TEMPI as compared to the respective control level of indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the methods include assessing all of the markers listed in Table A. Proteins and or mRNA can be evaluated. Thus, in some embodiments, expression of GDF15, LPA, CP, and TEMPI is assessed.
- control is the level of expression of GDF15, LPA, CP, and TEMPI, respectively in the subject prior to administration of the pharmaceutical agent.
- the sample is a blood, plasma, serum or urine sample.
- the methods include assessing the Framingham risk factors for the subject.
- the one or more assays detect GDF15, LPA, CP, and TEMPI such as by polymerase chain reaction (e.g. , RT-PCR), a microarray analysis or a hybridization reaction.
- polymerase chain reaction e.g. , RT-PCR
- microarray analysis e.g., a microarray analysis or a hybridization reaction.
- the one or more assays detect GDF15 protein, LPA protein, CP protein, and TEMPI protein. These proteins can be detected by any method known to those of skill in the art, such as by mass spectrometry (e.g. , MALDI-TOF mass spectrometry and/or LC- mass spectrometry).
- performing the one or more assays that detect GDF15 protein, LPA protein, CP protein, and TEMPI protein includes contacting the biological sample or a component thereof with an antibody that specifically binds GDF15 protein, an antibody that specifically binds LPA protein, an antibody that specifically binds CP protein, and/or an antibody that specifically binds TEMPI protein.
- the assay is an antibody that specifically binds GDF15 protein, an antibody that specifically binds LPA protein, an antibody that specifically binds CP protein, and/or an antibody that specifically binds TEMPI protein.
- the assay is an antibody that specifically binds GDF15 protein, an antibody that specifically binds L
- the antibody that specifically binds GDF15 protein, the antibody that specifically binds LPA protein, the antibody that specifically binds CP protein, and/or the antibody that specifically binds TEMPI protein is directly labeled, such as with a radioactive marker, a fluorescent marker, an enzyme or a metal.
- the method further includes administering to the subject a therapeutically effective amount of an agent for the treatment or prevention of atherosclerosis if the subject is determined to have an increased likelihood of developing atherosclerosis.
- the agent is a statin, niacin, a fibrate, a bile acid binding resin, a cholesterol absorption inhibitor, a PCSK9-targeting drug, an LDL-targeting drug, or an HDL-targeting drug.
- the method does not comprise measuring lipoprotein other than lipoprotein a.
- the method does not comprise measuring high density lipoproteins, and/or does not comprise measuring inflammatory markers, such as C reactive protein.
- the methods disclosed herein can include evaluating the expression of one or more of the following:
- the marker can include a polypeptide or nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide or nucleic acid sequence shown in these GENBANK® entries, respectively, or can be 100% identical to the listed sequence.
- the methods can include assessing expression of 1, 2, 3, 4, 5 or all 6 of the markers listed in Table B in any combination.
- the methods can further include evaluating the expression of one or more additional markers known to be associated with ASCVD. Proteins and mRNA can be evaluated, such as the level of 1, 2, 3, 4, 5 or all 6 of the markers shown in Table B.
- expression of ANGPTL3, GDF15, LPA, HPX, IGFl, and NPPB is assessed.
- the method can also include evaluating the expression of any markers listed in Table 1 or Table 8.
- Methods are provided for detecting or determining the likelihood that a subject will develop atherosclerotic cardiovascular disease.
- the methods can include performing one or more assays that detects expression of ANGPTL3 and GDF15 in a biological sample from the subject; and comparing the level of expression of ANGPTL3 and GDF15 to a respective control level of ANGPTL3 and GDF15. Detection of a decrease in expression of ANGPTL3 and an increase in expression of GDF15 as compared to the respective control indicates that the subject has or will develop atherosclerotic cardiovascular disease.
- the method further includes performing an assay that detects expression of lipoprotein(a) (LPA) in a biological sample from the subject; and comparing the level of expression of LPA to a respective control level of LPA. Detection of an increase in expression of LPA as compared to the respective control level of LPA indicates that the subject has or will develop ASCVD .
- LPA lipoprotein(a)
- the method further includes performing an assay that detects expression of hemopexin (HPX) a biological sample from the subject; and comparing the level of expression of HPX to a respective control level of HPX. Detection of an increase in expression of HPX as compared to the respective control level of HPX indicates that the subject has or will develop ASCVD .
- HPX hemopexin
- the method further includes performing an assay that detects expression of insulin like growth factor 1 (IGFl) in a biological sample from the subject; and comparing the level of expression of IGFl to a respective control level of IGFl. Detection of a decrease in expression of IGFl as compared to the respective control level of IGFl indicates that the subject has or will develop ASCVD.
- IGFl insulin like growth factor 1
- the method further includes performing an assay that detects expression of natriuretic peptides B (NPPB) in a biological sample from the subject; and comparing the level of expression of NPPB to a respective control level of NPPB. Detection of an increase in expression of NPPB as compared to the respective control level of NPPB indicates that the subject has or will develop ASCVD.
- NPPB natriuretic peptides B
- expression of ANGPTL3, GDF15, LPA, HPX, IGFl, and NPPB is assessed.
- the control can be a standard value of ANGPTL3, GDF15, LPA, HPX, IGFl and/or NPPB, respectively or can be the level of ANGPTL3, GDF15, LPA, HPX, IGFl and/or NPPB, respectively, in one or more subjects known not to have atherosclerotic cardiovascular disease. Proteins and/or mRNA can be evaluated. Methods are also provided for determining if a pharmaceutical agent is effective for treatment or prevention of ASCVD in a subject. In specific non-limiting examples, the subject can have ASCVD. In other embodiments, the subject has intermittent claudication, transient ischemic attacks (TIAs), ischemic strokes, restenosis after angioplasty, transplant
- Atherosclerosis atherosclerosis, unstable angina, or another condition associated with ASCVD.
- the methods can include assessing expression of 1, 2, 3, 4, 5 or all 6 of the markers listed in Table B in any combination.
- methods of determining if a pharmaceutical agent is effective for treatment or prevention of ASCVD in a subject include performing one or more assays that detect expression of ANGPTL3 and GDF15 in a biological sample from the subject administered the agent; and comparing the level of expression of ANGPTL3 and GDF15 to a respective control level of ANGPTL3 and GDF15. Detection of an increase in expression of ANGPTL3 and a decrease in expression of GDF15 as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects expression of LPA in a biological sample from the subject; and comparing the level of expression of LPA to a respective control level of LPA. Detection of a decrease in expression of LPA as compared to the respective control level of LPA indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects expression of HPX in a biological sample from the subject; and comparing the level of expression of HPX to a respective control level of HPX. Detection of a decrease in expression of HPX as compared to the respective control level of indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects expression of IGFl in a biological sample from the subject; and comparing the level of expression of IGFl to a respective control level of IGFl. Detection of an increase in expression of IGFl as compared to the respective control level of IGFl indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects expression of NPPB in a biological sample from the subject; and comparing the level of expression of NPPB to a respective control level of NPPB. Detection of a decrease in expression of NPPB as compared to the respective control level NPPB indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the methods include assessing all of the markers listed in Table B. Proteins and or mRNA can be evaluated. Thus, in some embodiments, expression of ANGPTL3, GDF15, LPA, HPX, IGF1, and NPPB is assessed.
- control is the level of expression of ANGPTL3, GDF15, LPA, HPX, IGF1, and/or NPPB, respectively in the subject prior to administration of the
- the sample is a blood, plasma, serum or urine sample.
- the methods further include assessing the Framingham risk factors for the subject.
- the one or more assays detect ANGPTL3 mRNA, GDF15 mRNA, LPA mRNA, HPX mRNA, IGF1 mRNA, and/or NPPB mRNA, such as by polymerase chain reaction (e.g. , RT-PCR), a microarray analysis or a hybridization reaction.
- polymerase chain reaction e.g. , RT-PCR
- microarray analysis e.g., a microarray analysis or a hybridization reaction.
- the one or more assays detect ANGPTL3 protein, GDF15 protein, LPA protein, HPX protein, IGF1 protein, and/or NPPB protein, such as by mass spectrometry (e.g., MALDI-TOF mass spectrometry and/or LC-mass spectrometry).
- mass spectrometry e.g., MALDI-TOF mass spectrometry and/or LC-mass spectrometry.
- GDF15 protein, LPA protein, HPX protein, IGF1 protein, and/or NPPB protein includes contacting the biological sample or a component thereof with an antibody that specifically binds ANGPTL3 protein, an antibody that specifically binds GDF15 protein, an antibody that specifically binds LPA protein, an antibody that specifically binds HPX protein, an antibody that specifically binds IGF1 protein and/or an antibody that specifically binds NPPB protein.
- the assay is an immunoassay, such as but not limited to a Western blot, an enzyme linked immunosorbent assay, or a radioimmunoassay.
- the antibody that specifically binds ANGPTL3 protein, the antibody that specifically binds GDF15 protein, the antibody that specifically binds LPA protein, the antibody that specifically binds HPX protein, the antibody that specifically binds IGF1 protein, and/or the antibody that specifically binds NPPB protein is directly labeled, such as with a radioactive marker, a fluorescent marker, an enzyme or a metal.
- the method further includes administering to the subject a therapeutically effective amount of an agent for the treatment or prevention of atherosclerosis if the subject is determined to have an increased likelihood of developing atherosclerosis.
- the agent is a statin, niacin, a fibrate, a bile acid binding resin, a cholesterol absorption inhibitor, a PCSK9-targeting drug, an LDL-targeting drug, or an HDL-targeting drug.
- the method does not comprise measuring lipoprotein other than lipoprotein a.
- the method does not comprise measuring high density lipoproteins, and/or does not comprise measuring inflammatory markers, such as C reactive protein.
- a biological sample obtained from the subject such as, but not limited to, serum, blood, plasma, urine, saliva, purified cells (for example, blood cells, such as white blood cells, B cells, T cells, or mononuclear cells), a biopsy or tissue sample, such as a sample including adipose tissue, blood vessels or heart tissue obtained from the subject are used to predict the subject's risk of HF.
- the method disclosed herein can also be used to determine if a pharmaceutical agent is of use to delay or prevent HF in the subject.
- the subject is apparently healthy, such as a subject who does not exhibit symptoms of vascular disease (for example, does not have ASCVD, and/or has not previously had HF).
- a healthy subject is one that if examined by a medical professional, would be characterized as healthy and free of symptoms of vascular disease.
- the subject can be undergoing chemotherapy, be addicted to ***e, or have a medical history of ***e use.
- the subject can be selected based on sex, age, the subject's total cholesterol level, the subject's HDL cholesterol level, blood pressure, smoking history, and/or treatment for hypertension and/or blood pressure.
- the subject does not have HF and does not have clinical ASCVD.
- the subject is at increased risk for HF due to the presence of a
- predisposing condition such as i) hypertension, ii) history of MI, iii) valvular heart disease/left ventricular hypertrophy/structure heart disease, iv) asymptomatic LV systolic dysfunction
- the subject is suspected of having HF, or is suspected of being at risk of developing HF, and of being likely to have HF the future.
- a subject may have diabetes or high blood pressure.
- the subject has been determined to be at risk for
- cardiovascular disease based on risk factors, such as, but not limited to, Framingham risk factors, or guidelines jointly issued by the American Heart Association and American College of risk factors, or guidelines jointly issued by the American Heart Association and American College of risk factors, such as, but not limited to, Framingham risk factors, or guidelines jointly issued by the American Heart Association and American College of risk factors, or guidelines jointly issued by the American Heart Association and American College of risk factors, or guidelines jointly issued by the American Heart Association and American College of
- the method can include evaluating a subject to determine if the subject is at risk for cardiovascular disease using Framingham risk factors.
- risk factors include age, gender, whether the subject smokes, blood pressure, total cholesterol level, and high density lipoprotein cholesterol level (see above).
- the expression of the markers disclosed herein can be used to assess the efficacy of a therapeutic protocol for the prevention of HF.
- methods are provided for evaluating the efficacy of a treatment protocol that includes any therapy for HF designed to reverse or slow the progression of HF, including but not limited to treatment with angiotensin-converting enzyme (ACE) inhibitors, beta blockers, aldosterone antagonists, diuretics, angiotensin receptor blockers (ARBs), or vasodilators.
- ACE angiotensin-converting enzyme
- beta blockers beta blockers
- aldosterone antagonists aldosterone antagonists
- diuretics angiotensin receptor blockers
- ARBs angiotensin receptor blockers
- a sample can be taken from a subject prior to initiation of therapy. After therapy is initiated, an additional sample is taken from the subject. A decrease in the amount of the markers indicates that the therapy is efficacious.
- the subject can be monitored over time to evaluate the continued effectiveness of the therapeutic protocol.
- the effect of different dosages can also be evaluated, by comparing the expression of markers in a sample from the subject receiving a first dose to the expression of the same markers in a sample from the subject receiving a second (different) dose.
- the methods can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times to determine the lowest dose of a pharmaceutical agent that is effective for treating the subject, and/or the shortest duration of administration that is effective for treating the subject.
- the methods can also be used over the course of a therapeutic regimen to monitor the efficacy of a pharmaceutical agent for the treatment of the subject.
- the method can include evaluating the expression of one or more the following:
- the marker can include a polypeptide or nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide or nucleic acid sequence shown in these GENBANK® entries, respectively, or can be
- the methods can include assessing expression of 1, 2, 3 or all 4 of the markers listed in
- the methods can further include evaluating the expression of one or more additional markers known to be associated with HF. Proteins and mRNA can be evaluated, such as the level of 1, 2, 3 or all 4 of the markers shown in Table C.
- the method can also include evaluating the expression of any markers listed in Table 1 or Table 8.
- the methods can include performing one or more assays that detect expression of NPPB and GDF15 in a biological sample from the subject; and comparing the level of expression of NPPB and GDF15 to a respective control level of NPPB and GDF15. Detection of an increase in expression of NPPB and GDF15 as compared to the respective control indicates that the subject has or will develop HF. ®
- the method further includes performing an assay that detects expression of myeloperoxidase (MPO) in a biological sample from the subject; and comparing the level of expression of MPO to a respective control level of MPO. Detection of an increase in expression of MPO as compared to the respective control level of MPO indicates that the subject has or will develop HF.
- MPO myeloperoxidase
- the method further includes performing an assay that detects expression of plasminogen activator inhibitor -1 (SERPINEl) in a biological sample from the subject; and comparing the level of expression of SERPINEl to a respective control level of SERPINEl. Detection of an increase in expression of SERPINEl as compared to the respective control level of SERPINEl indicates that the subject has or will develop HF.
- SERPINEl plasminogen activator inhibitor -1
- the method includes performing one or more assays that detect NPPB, GDF15, MPO and/or SERPINEl.
- the one or more assays detect the expression of NPPB, GDF15, MPO and SERPINEl .
- the control can be a standard value of NPPB, GDF15, MPO and/or SERPINEl, respectively or can be the level of NPPB, GDF15, MPO and/or SERPINEl, respectively, in one or more subjects known not to have HF. Proteins and/or mRNA can be evaluated.
- the methods can include performing one or more assays that detect expression of NPPB and GDF15 in a biological sample from the subject administered the agent; and comparing the level of expression of NPPB and GDF15 to a respective control level of NPPB and GDF15. Detection of a decrease in expression of NPPB and GDF15 as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of HF in the subject.
- the method further includes performing an assay that detects expression of MPO in a biological sample from the subject; and comparing the level of expression of MPO to a respective control level of MPO. Detection of a decrease in expression of MPO as compared to the respective control level of MPO indicates that the pharmaceutical agent is effective for the treatment or prevention of HF in the subject.
- the method further includes performing an assay that detects expression of SERPINEl in a biological sample from the subject; and comparing the level of expression of SERPINEl to a respective control level of SERPINEl. Detection of a decrease in expression of SERPINEl as compared to the respective control level of SERPINEl indicates that the pharmaceutical agent is effective for the treatment or prevention of HF in the subject.
- the methods include assessing all of the markers listed in Table C. Proteins and or mRNA can be evaluated.
- control is the level of expression of NPPB, GDF15, MPO and/or SERPINEI, respectively in the subject prior to administration of the pharmaceutical agent.
- the sample is a blood, plasma, serum or urine sample.
- the methods further include assessing the Framingham risk factors for the subject.
- the one or more assays detect NPPB mRNA, GDF15 mRNA,
- MPO mRNA and/or SERPINEI mRNA such as by polymerase chain reaction (e.g. , RT-PCR), a microarray analysis or a hybridization reaction.
- the one or more assays detect NPPB protein, GDF15 protein, MPO protein and/or SERPINEI protein.
- the assay can be any assay known to one of skill in the art, including mass spectrometry (e.g. , MALDI-TOF mass spectrometry and/or LC-mass spectrometry).
- performing the one or more assays that detect NPPB protein, GDF15 protein, MPO protein and/or SERPINEI protein includes contacting the biological sample or a component thereof with an antibody that specifically binds NPPB protein, an antibody that specifically binds GDF15 protein, an antibody that specifically binds MPO protein, and/or an antibody that specifically binds SERPINEI protein.
- the assay is an immunoassay, such as but not limited to a Western blot, an enzyme linked immunosorbent assay, or a radioimmunoassay.
- the antibody that specifically binds NPPB protein, the antibody that specifically binds GDF15 protein, the antibody that specifically binds MPO protein and/or the antibody that specifically binds SERPINEI protein is directly labeled, such as with a radioactive marker, a fluorescent marker, an enzyme or a metal.
- the method further includes administering to the subject a therapeutically effective amount of an agent for the treatment or prevention of HF if the subject is determined to have an increased likelihood of developing heart failure.
- the agent is an angiotensin-converting enzyme (ACE) inhibitor, a beta blocker, an aldosterone antagonist, a diuretic, an angiotensin receptor blocker (ARB), or a vasodilator.
- the method can include evaluating the expression of one or more of the following:
- the marker can include a polypeptide or nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide or nucleic acid sequence shown in these GENBANK® entries, respectively, or can be 100% identical to the listed sequence.
- the methods can include assessing expression of 1, 2, 3, 4, 5, or all 6 of the markers listed in Table D in any combination.
- the methods can further include evaluating the expression of one or more additional markers known to be associated with HF. Proteins and mRNA can be evaluated, such as the level of 1, 2, 3, 4, 5, or all 6 of the markers shown in Table D.
- the method can also include evaluating the expression of any markers listed in Table 1 or Table 8.
- the methods can include performing one or more assays that detect expression of NPPB and GDF15 in a biological sample from the subject; and comparing the level of expression of NPPB and GDF15 to a respective control level of NPPB and GDF15. Detection of an increase in expression of NPPB and GDF15 as compared to the respective control indicates that the subject has or will develop heart failure.
- the method further includes performing an assay that detects expression of myeloperoxidase (MPO) in a biological sample from the subject; and comparing the level of expression of MPO to a respective control level of MPO. Detection of an increase in expression of MPO as compared to the respective control level of MPO indicates that the subject has or will develop heart failure.
- the method further includes performing an assay that detects expression of tetranectin (CLEC3B) in a biological sample from the subject; and comparing the level of expression of CLEC3B to a respective control level of CLEC3B. Detection of a decrease in expression of CLEC3B as compared to the respective control level of CLEC3B indicates that the subject has or will develop heart failure.
- MPO myeloperoxidase
- the method further includes performing an assay that detects expression of alpha- 1 -acid glycoprotein 1 (ORMl) in a biological sample from the subject; and comparing the level of expression of ORMl to a respective control level of ORMl. Detection of an increase in expression of ORMl as compared to the respective control level of ORMl indicates that the subject has or will develop heart failure.
- ORMl alpha- 1 -acid glycoprotein 1
- the method further includes performing an assay that detects expression of tissue inhibitor of metalloproteinase inhibitor 1 (TIMPl) in a biological sample from the subject; and comparing the level of expression of TIMPl to a respective control level of TIMPl. Detection of a decrease in expression of TIMPl as compared to the respective control level of TIMPl indicates that the subject has or will develop heart failure.
- tissue inhibitor of metalloproteinase inhibitor 1 TIMPl
- the method includes performing one or more assays that detect NPPB, GDF15, MPO, CLEC3B, ORMl, and/or TIMPl.
- the one or more assays detect the expression of NPPB, GDF15, MPO, CLEC3B, ORMl, and TIMPl.
- the control can be a standard value of NPPB, GDF15, MPO, CLEC3B, ORMl, and/or
- TIMPl respectively or can be the level of NPPB, GDF15, MPO, CLEC3B, ORMl, and/or TIMPl, respectively, in one or more subjects known not to have HF. Proteins and/or mRNA can be evaluated.
- the methods can include performing one or more assays that detect expression of NPPB and GDF15 in a biological sample from the subject administered the agent; and comparing the level of expression of NPPB and GDF15 to a respective control level of NPPB and GDF15. Detection of a decrease in expression of NPPB and GDF15 as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- the method further includes performing an assay that detects expression of MPO in a biological sample from the subject; and comparing the level of expression of MPO to a respective control level of MPO. Detection of a decrease in expression of MPO as compared to the respective control level of MPO indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- the method further includes performing an assay that detects expression of CLEC3B in a biological sample from the subject; and comparing the level of expression of CLEC3B to a respective control level of CLEC3B. Detection of an increase in expression of CLEC3B as compared to the respective control level of CLEC3B indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- the method further includes performing an assay that detects expression of alpha- 1 -acid glycoprotein 1 (ORMl) in a biological sample from the subject; and comparing the level of expression of ORMl to a respective control level of ORMl. Detection of a decrease in expression of ORMl as compared to the respective control level of ORMl indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- ORMl alpha- 1 -acid glycoprotein 1
- the method further includes performing an assay that detects expression of TEVIPl in a biological sample from the subject; and comparing the level of expression of TEVIPl to a respective control level of TEVIPl. Detection of an increase in expression of TEVIPl as compared to the respective control level of TEVIPl indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- the methods include assessing all of the markers listed in Table D. Proteins and or mRNA can be evaluated.
- control is the level of expression of NPPB, GDF15, MPO, CLEC3B, ORMl, and/or TIMP1, respectively in the subject prior to administration of the pharmaceutical agent.
- the sample is a blood, plasma, serum or urine sample.
- the methods further include assessing the Framingham risk factors for the subject.
- the one or more assays detect NPPB mRNA, GDF15 mRNA, MPO mRNA, CLEC3B mRNA, ORM1 mRNA, and/or TEMPI mRNA, such as by polymerase chain reaction (e.g. , RT-PCR), a microarray analysis or a hybridization reaction.
- the one or more assays detect NPPB protein, GDF15 protein, MPO protein, CLEC3B protein, ORM1 protein, and/or TEMPI protein, such as by mass spectrometry (e.g. , MALDI-TOF mass spectrometry and/or LC-mass spectrometry).
- mass spectrometry e.g. , MALDI-TOF mass spectrometry and/or LC-mass spectrometry.
- performing the one or more assays that detect NPPB protein, GDF15 protein, MPO protein, CLEC3B protein, ORM1 protein, and/or TIMPl protein includes contacting the biological sample or a component thereof with an antibody that specifically binds NPPB protein, an antibody that specifically binds GDF15 protein, an antibody that specifically binds MPO protein, an antibody that specifically binds CLEC3B protein, an antibody that specifically binds ORM1 protein, and/or an antibody that specifically binds TIMPl protein.
- the assay is an immunoassay, such as but not limited to a Western blot, an enzyme linked immunosorbent assay, or a radioimmunoassay.
- the antibody that specifically binds NPPB protein, the antibody that specifically binds GDF15 protein, the antibody that specifically binds MPO protein, the antibody that specifically binds CLEC3B protein, the antibody that specifically binds ORM1 protein, and/or the antibody that specifically binds TIMPl protein is directly labeled, such as with a radioactive marker, a fluorescent marker, an enzyme or a metal.
- the method further includes administering to the subject a therapeutically effective amount of an agent for the treatment or prevention of heart failure if the subject is determined to have an increased likelihood of developing heart failure.
- the agent is an angiotensin-converting enzyme (ACE) inhibitor, a beta blocker, an aldosterone antagonist, a diuretic, an angiotensin receptor blocker (ARB), or a vasodilator.
- expression of one or more proteins is analyzed by detecting and quantifying the protein in a biological sample.
- one or more proteins corresponding to the markers listed in Tables A, B, C, D or E are analyzed.
- Suitable biological samples include samples containing protein, such as blood, serum, plasma, urine, tissue biopsies, cells, including isolated blood cells, for example peripheral blood mononuclear cells, B cells, T cells, and/or monocytes.
- Detecting an alteration in the amount of one or more of the proteins listed in Tables A, B, C, D or E, using the methods disclosed herein indicates the prognosis or diagnosis of the subject, or indicates if a therapy is effective for treating a subject as described above.
- Expression of proteins is the level of protein in a biological sample. Expression includes, but is not limited to, the production of the protein by translation of an mRNA and the half-life of the protein.
- Any standard immunoassay format (such as ELISA, Western blot, or RIA assay) can be used to measure protein levels. Immunohistochemical techniques can also be utilized. General guidance regarding such techniques can be found in Bancroft and Stevens ⁇ Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and Ausubel et al. ⁇ Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988); these references disclose a number of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
- immunoassays include the use of one or more specific binding agents (such as antibodies) that specifically recognizes and can bind a molecule of interest, such a protein corresponding to a marker listed in Tables A, B, C, D or E.
- binding agents can include a detectable label (such as a radiolabel, fluorophore or enzyme), that permits detection of the binding to the protein and determination of relative or absolute quantities of the molecule of interest in the sample.
- a detectable label such as a radiolabel, fluorophore or enzyme
- the method of detecting the protein in a sample generally includes the steps of contacting the sample with an antibody, which specifically binds to the protein under
- the antibody can be a polyclonal or monoclonal antibody, or fragment thereof.
- the antibody is a humanized antibody.
- the antibody is a chimeric antibody.
- the antibodies can be labeled. Suitable detectable markers are described and known to the skilled artisan.
- various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials can be used.
- suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase.
- suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
- Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
- a non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I, 131 1, 35 S, or 3 H. Additional examples are disclosed above.
- the antibody that binds the protein of interest is unlabeled and a second antibody or other molecule that can bind the antibody that binds the protein of interest is utilized.
- a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody.
- the first antibody is a mouse IgG
- the secondary antibody may be a goat anti- mouse-IgG.
- Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
- Quantitation of proteins can be achieved by immunoassay.
- the amount of proteins can be assessed and optionally in a control sample.
- the amounts of protein in the sample from the subject of interest can be compared to levels of the protein found in samples form control subjects or to another control (such as a standard value or reference value). A significant increase or decrease in the amount can be evaluated using statistical methods known in the art.
- a sandwich ELISA can be used to detect the presence or determine the amount of a protein in a sample.
- a solid surface is first coated with an antibody that specifically binds the protein of interest.
- the test sample containing the protein such as, but not limited to, a blood, plasma, serum, or urine sample
- the antigen is allowed to react with the bound antibody. Any unbound antigen is washed away.
- a known amount of enzyme-labeled protein -specific antibody is then allowed to react with the bound protein. Any excess unbound enzyme-linked antibody is washed away after the reaction.
- the substrate for the enzyme used in the assay is then added and the reaction between the substrate and the enzyme produces a color change.
- the amount of visual color change is a direct measurement of specific enzyme-conjugated bound antibody, and consequently the quantity of the protein present in the sample tested.
- a protein in an alternative example, can be assayed in a biological sample by a competition immunoassay utilizing protein standards labeled with a detectable substance and an unlabeled antibody that specifically binds the protein of interest.
- the biological sample such as, but not limited to, a blood, plasma, serum, or urine sample
- the labeled protein standards and the antibody that specifically binds the protein of interest are combined and the amount of labeled protein standard bound to the unlabeled antibody is determined.
- the amount of protein in the biological sample is inversely proportional to the amount of labeled protein standard bound to the antibody that specifically binds the protein of interest.
- Mass spectrometry is particularly suited to the identification of proteins from biological samples, such those listed in Tables A, B, C, D or E. Mass spectrometry also is particularly useful in the quantitation of peptides in a biological sample, for example using isotopically labeled peptide standards.
- the application of mass spectrometric techniques to identify proteins in biological samples is known in the art and is described, for example, in Akhilesh et al, Nature, 405:837-846, 2000; Dutt et al, Curr. Opin. Biotechnol, 11: 176-179, 2000; Gygi et al, Curr. Opin. Chem.
- Separation of ions according to their m/z ratio can be accomplished with any type of mass analyzer, including quadrupole mass analyzers (Q), time-of-flight (TOF) mass analyzers (for example, linear or reflecting) analyzers, magnetic sector mass analyzers, 3D and linear ion traps ( ⁇ ), Fourier-transform ion cyclotron resonance (FT-ICR) analyzers, Orbitrap analyzers (like LTQ-Orbitrap LC/MS/MS) and combinations thereof (for example, a quadrupole-time-of- flight analyzer, or Q-TOF analyzer).
- Q quadrupole mass analyzers
- TOF time-of-flight
- ⁇ magnetic sector mass analyzers
- ⁇ linear ion traps
- FT-ICR Fourier-transform ion cyclotron resonance
- Orbitrap analyzers like LTQ-Orbitrap LC/MS/MS
- combinations thereof for example, a quadrupole-
- the mass spectrometric technique is tandem mass spectrometry (MS/MS).
- a protein gene product such as those from Tables A, B, C, D or E
- entering the tandem mass spectrometer is selected and subjected to collision induced dissociation (CID).
- CID collision induced dissociation
- the spectrum of the resulting fragment ion is recorded in the second stage of the mass spectrometry, as a so-called CID or ETD spectrum.
- CID or ETD usually causes fragmentation at peptide bonds and different amino acids for the most part yield peaks of different masses
- a CID or ETD spectrum alone often provides enough information to determine the presence of a the protein of Tables A, B, C, D or E.
- Suitable mass spectrometer systems for MS/MS include an ion fragmentor and one, two, or more mass spectrometers, such as those described above.
- suitable ion fragmentors include, but are not limited to, collision cells (in which ions are fragmented by causing them to collide with neutral gas molecules), photo dissociation cells (in which ions are fragmented by irradiating them with a beam of photons), and surface dissociation fragmentor (in which ions are fragmented by colliding them with a solid or a liquid surface).
- Suitable mass spectrometer systems can also include ion reflectors.
- the sample Prior to mass spectrometry, the sample can be subjected to one or more dimensions of chromatographic separation, for example, one or more dimensions of liquid or size exclusion chromatography.
- chromatographic separation include paper chromatography, thin layer chromatography (TLC), liquid chromatography, column
- HPLC high performance liquid chromatography
- FPLC fast protein liquid chromatography
- ion exchange chromatography size exclusion chromatography
- affinity chromatography affinity chromatography
- HPLC high performance liquid chromatography
- nano-RPLC nano-reverse phase liquid chromatography
- PAGE polyacrylamide gel electrophoresis
- CE capillary electrophoresis
- RP-HPLC reverse phase high performance liquid chromatography
- the mass spectrometric technique is directly or indirectly coupled with a one, two or three dimensional liquid chromatography technique, such as column chromatography, high performance liquid chromatography (HPLC or FPLC), reversed phase, ion exchange chromatography, size exclusion chromatography, affinity chromatography (such as protein or peptide affinity chromatography, immunoaffinity chromatography, lectin affinity chromatography, etc.), or one, two or three dimensional polyacrylamide gel electrophoresis (PAGE), or one or two dimensional capillary electrophoresis (CE) to further resolve the biological sample prior to mass spectrometric analysis.
- a one, two or three dimensional liquid chromatography technique such as column chromatography, high performance liquid chromatography (HPLC or FPLC), reversed phase, ion exchange chromatography, size exclusion chromatography, affinity chromatography (such as protein or peptide affinity chromatography, immunoaffinity chromatography, lectin affinity chromatography, etc.), or one, two or three
- SELDI quantitative spectroscopic methods
- SELDI-TOF surface-enhanced laser desorption-ionization time-of-flight
- one version of SELDI uses a chromatographic surface with a chemistry that selectively captures analytes of interest, such as one or more proteins of interest.
- Chromatographic surfaces can be composed of hydrophobic, hydrophilic, ion exchange, immobilized metal, or other chemistries.
- the surface chemistry can include binding functionalities based on oxygen-dependent, carbon-dependent, sulfur-dependent, and/or nitrogen-dependent means of covalent or noncovalent immobilization of analytes.
- the activated surfaces are used to covalently immobilize specific "bait" molecules such as antibodies, receptors, or oligonucleotides often used for biomolecular interaction studies such as protein- protein and protein-DNA interactions.
- analytes bound to the surface can be desorbed and analyzed by any of several means, for example using mass spectrometry.
- mass spectrometry When the analyte is ionized in the process of desorption, such as in laser desorption/ionization mass spectrometry, the detector can be an ion detector.
- Mass spectrometers generally include means for determining the time-of- flight of desorbed ions. This information is converted to mass. However, one need not determine the mass of desorbed ions to resolve and detect them: the fact that ionized analytes strike the detector at different times provides detection and resolution of them.
- the analyte can be detectably labeled (for example with a fluorophore or radioactive isotope). In these cases, the detector can be a fluorescence or radioactivity detector.
- the method may include detection of a protein of interest in a sample using an electrochemical immunoassay method.
- an antibody that specifically binds the protein of interest is conjugated to terminally carboxylated single- wall carbon nanotubes (SWNT), multi-wall carbon nanotubes (MWCNT), or gold nanoparticles (AuNP), which are attached to a conductive surface.
- SWNT terminally carboxylated single- wall carbon nanotubes
- MWCNT multi-wall carbon nanotubes
- AuNP gold nanoparticles
- a sample (such as a blood, plasma or serum sample) is contacted with the SWNTs, MWCNTs, or AuNPs, and protein in the sample binds to the primary antibody.
- a second antibody conjugated directly or indirectly to a redox enzyme such as horseradish peroxidase (HRP), cytochrome c, myoglobin, or glucose oxidase
- HRP horseradish peroxidase
- cytochrome c binds to the primary antibody or to the protein (for example, in a "sandwich” assay).
- the second antibody is conjugated to the enzyme.
- the second antibody and the enzyme are both conjugated to a support (such as a magnetic bead). Signals are generated by adding enzyme substrate (e.g. hydrogen peroxide if the enzyme is HRP) to the solution bathing the sensor and measuring the current produced by the catalytic reduction.
- enzyme substrate e.g. hydrogen peroxide if the enzyme is HRP
- the method includes a first antibody that specifically binds the protein of interest attached to an AuNP sensor surface.
- a sample such as, but not limited to, a blood, plasma, serum, or urine sample
- the AuNP sensor including the first antibody.
- a horseradish peroxidase (HRP)-labeled second antibody that specifically binds the protein of interest (HRP-Ab2) or beads conjugated to both a second antibody that binds the protein of interest and HRP are incubated with the sensor, allowing the second antibody to bind to the protein of interest.
- HRP horseradish peroxidase
- Biocatalytic electrochemical reduction produces a signal via reduction of peroxide activated enzyme following addition of hydrogen peroxide.
- Use of HRP is advantageous for arrays since immobilization of the electroactive enzyme label on the electrode eliminates electrochemical crosstalk between array elements, which can occur when detecting soluble electroactive product.
- iTRAQ® reagents are utilized. Multiple samples can be run simultaneously using different iTRAQ® reagents that label the individual samples with different mass identifiers.
- sample one can be labeled with a mass identifier (or mass tag) that has a molecular weight of 114 amu
- sample two mass identifier or mass tag
- a fragment peptide from sample two will have a predictable mass difference of three amu, compared to the same fragment peptide from sample one. In other words a peptide of identical sequence in sample one and sample two will be three amu heavier. This predictable mass difference can be used both to identify a peptide fragment (and hence the protein from which they were excised) and the relative quantities of each peptide in the samples.
- MRM multiple reaction monitoring
- tryptic peptides are used as markers for the abundance of specific proteins of interest, such as those listed in Tables A, B, C, D or E. This selection is relatively straightforward if the protein has been identified by MS, such that the peptides are observable in a mass spectrometer (for example an LTQ Orbitrap).
- the process of establishing an MRM assay for a protein consists of a number of steps: 1) selection of the appropriate peptide(s) unique to the protein of interest and showing high MS signal response (prototypic peptides) which will help maximize the sensitivity of the assay; 2) selection of predominant peptide fragments specific (MS/MS) for the parent peptide (useful MRM transition); 3) for each peptide-fragment pair, optimization of specific MS parameters (for example, the collision energy) to maximize the signal response/sensitivity; 4) validation of the MRM assay to confirm peptide identity, for example by acquiring a full MS2 spectrum of the peptide in the triple quadrupole MS instrument used for MRM; 5) extraction of the final "coordinates" of the MRM assay, including the selected peptide and peptide fragments, the corresponding mass-to-charge ratios, the fragment intensity ratios, the associated collision energy, and the chromatographic elution time to be optionally used in time-constrained MRM analyses.
- the concentration of the protein of interest such as a protein corresponding to the markers listed in Tables A, B, C, D or E, that is detected can be compared to a control, such as the concentration of the protein in a subject known not to have ASCVD, known not to have had HF, or known not to be at risk for ASCVD and/or HF.
- the control is a standard value, such as a value that represents an average concentration of the protein of interest expected in a subject who does not have ASCVD and/or HF, and/or is not at risk for ASCVD and/or HF.
- Gene expression can be evaluated by detecting mRNA encoding the gene of interest.
- the disclosed methods can include evaluating mRNA encoding one or more of the markers listed in Tables A, B, C, D or E. Any of the methods disclosed above can utilize the detection of mRNA.
- RNA can be isolated from a sample from a subject, such as a biopsy, tissue sample, cardiac tissue, blood vessel, peripheral blood mononuclear cells, or isolated cells, such as white blood cells (B, T or mononuclear cells). RNA can also be isolated from a control, such as the same type of biological tissue from a healthy subject, for example a subject known not to have ASCVD or be at risk for HF, using methods well known to one skilled in the art, including commercially available kits. General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997).
- RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as QIAGEN® (Valencia, CA), according to the manufacturer's instructions.
- QIAGEN® Valencia, CA
- total RNA from cells in culture can be isolated using QIAGEN® RNeasy® mini-columns.
- Other commercially available RNA isolation kits include MASTERPURE® Complete DNA and RNA Purification Kit
- RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test).
- RNA prepared from a biological sample can be isolated, for example, by cesium chloride density gradient
- Methods of gene expression profiling include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, and proteomics-based methods.
- mRNA expression in a sample is quantified using Northern blotting or in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283, 1999); RNAse protection assays (Hod, Biotechniques 13:852-4, 1992); and PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR) (Weis et al. , Trends in Genetics 8:263-4, 1992).
- RT-PCR reverse transcription polymerase chain reaction
- RNA duplexes can be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA- protein duplexes.
- Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
- SAGE Serial Analysis of Gene Expression
- MPSS massively parallel signature sequencing
- RT-PCR can be used to compare mRNA levels in different samples, such as from subject that is undergoing treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and to analyze RNA structure. Methods for quantitating mRNA are well known in the art.
- the method utilizes RT-PCR.
- extracted RNA can be reverse-transcribed using a GENEAMP® RNA PCR kit (Perkin Elmer, Calif., USA), following the manufacturer's instructions.
- TAQMAN® RT-PCR can be performed using commercially available equipment.
- the system can include a thermocycler, laser, charge-coupled device (CCD) camera, and computer.
- the system amplifies samples in a 96-well format on a thermocycler.
- laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD.
- the system includes software for running the instrument and for analyzing the data.
- RT-PCR can be performed using an internal standard.
- the ideal internal standard is expressed at a constant level among different tissues, and is unaffected by an experimental treatment.
- RNAs commonly used to normalize patterns of gene expression are mRNAs for the housekeeping genes GAPDH, ⁇ - actin, and 18S ribosomal RNA.
- RT-PCR is real time quantitative RT-PCR, which measures PCR product accumulation through a dual-labeled fluorogenic probe (e.g., TAQMAN® probe).
- Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR (see Heid et al, Genome Research 6:986-994, 1996).
- Quantitative PCR is also described in U.S. Pat. No. 5,538,848.
- Related probes and quantitative amplification procedures are described in U.S. Pat. No. 5,716,784 and U.S. Pat. No. 5,723,591. Instruments for carrying out quantitative PCR in microtiter plates are available from PE Applied Biosystems (Foster City, CA).
- RNA isolation, purification, primer extension and amplification are given in various published journal articles (see Godfrey et ah, J. Mol. Diag. 2:84 91, 2000; Specht et al., Am. J. Pathol. 158:419-29, 2001). Briefly, a representative protocol for quantitating gene expression using fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification, primer extension and amplification are given in various published journal articles (see Godfrey et ah, J. Mol. Diag. 2:84 91, 2000; Specht et al., Am. J. Pathol. 158:419-29, 2001). Briefly, a representative protocol for quantitating gene expression using fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification, primer extension and amplification are given in various published journal articles (see Godfrey et ah, J. Mol. Diag. 2:84
- RNA repair and/or amplification steps can be included, if necessary, and RNA is reverse transcribed using gene specific promoters followed by RT-PCR.
- the primers used for the amplification are selected so as to amplify a unique segment of the gene of interest (such as mRNA encoding one or more of the markers listed in Tables A, B, C, D or E). In some embodiments, expression of other genes is also detected, such as genes known to be associated with ASCVD and/or HF. Primers that can be used to amplify mRNAs of interest are commercially available or can be designed and synthesized according to well-known methods.
- gene expression is identified or confirmed using the microarray technique.
- the expression profile can be measured in either fresh or paraffin-embedded tissue, using microarray technology.
- nucleic acid sequences of interest are identified or confirmed using the microarray technique.
- RNA RNA isolated from cells or tissues of interest.
- isolated nucleic acids such as cDNA or mRNA
- the source of mRNA typically is total RNA isolated from tissue or cells, and optionally from corresponding tissues or cells from a subject known not to be at risk for ASCVD and/or HF.
- PCR amplified inserts of cDNA clones are applied to a substrate in a dense array.
- the array includes probes specific to markers listed in Tables A, B, C, D or E, or subsets of these markers.
- probes specific for these nucleotide sequences are applied to the substrate, and the array can consist essentially of, or consist of these sequences.
- the microarrayed nucleic acids are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array.
- the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously.
- the miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for genes of interest, such as those in Tables A, B, C, D or E.
- Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as are supplied with Affymetrix
- GENECHIP® technology (Affymetrix, Santa Clara, CA), or Agilent's microarray technology (Agilent Technologies, Santa Clara, CA).
- SAGE Serial analysis of gene expression
- a short sequence tag (about 10-14 base pairs) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously.
- the expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag (see, for example, Velculescu et al., Science 270:484-7, 1995; and Velculescu et al., Cell 88:243-51, 1997).
- ISH In situ hybridization
- ISH is another method for detecting and comparing expression of genes of interest.
- ISH applies and extrapolates the technology of nucleic acid hybridization to the single cell level, and, in combination with the art of cytochemistry, immunocytochemistry and immunohistochemistry, permits the maintenance of morphology and the identification of cellular markers to be maintained and identified, and allows the localization of sequences to specific cells within populations, such as tissues and blood samples.
- ISH is a type of hybridization that uses a complementary nucleic acid to localize one or more specific nucleic acid sequences in a portion or section of tissue (in situ), or, if the tissue is small enough, in the entire tissue (whole mount ISH).
- RNA ISH can be used to assay expression patterns in a tissue, such as one or more of the markers listed in Tables A, B, C, D or E.
- Sample cells or tissues are treated to increase their permeability to allow a probe to enter the cells.
- the probe is added to the treated cells, allowed to hybridize at pertinent temperature, and excess probe is washed away.
- a complementary probe is labeled so that the probe's location and quantity in the tissue can be determined, for example, using autoradiography, fluorescence microscopy or immunoassay.
- the sample may be any sample of interest.
- In situ PCR is the PCR-based amplification of the target nucleic acid sequences prior to ISH.
- an intracellular reverse transcription step is introduced to generate complementary DNA from RNA templates prior to in situ PCR. This enables detection of low copy RNA sequences.
- PCR amplification of target sequences is next performed either in intact cells held in suspension or directly in cytocentrifuge preparations or tissue sections on glass slides.
- fixed cells suspended in the PCR reaction mixture are thermally cycled using conventional thermal cyclers.
- the cells are cytocentrifuged onto glass slides with visualization of intracellular PCR products by ISH or immunohistochemistry.
- In situ PCR on glass slides is performed by overlaying the samples with the PCR mixture under a covers lip which is then sealed to prevent evaporation of the reaction mixture. Thermal cycling is achieved by placing the glass slides either directly on top of the heating block of a conventional or specially designed thermal cycler or by using thermal cycling ovens.
- Detection of intracellular PCR products is generally achieved by one of two different techniques, indirect in situ PCR by ISH with PCR-product specific probes, or direct in situ PCR without ISH through direct detection of labeled nucleotides (such as digoxigenin-11-dUTP, fluorescein-dUTP, 3 H-CTP or biotin-16-dUTP), which have been incorporated into the PCR products during thermal cycling.
- labeled nucleotides such as digoxigenin-11-dUTP, fluorescein-dUTP, 3 H-CTP or biotin-16-dUTP
- RNA (or protein) recovery can also be evaluated. These terms include any constitutively or globally expressed gene (or protein) whose presence enables an assessment of gene (or protein) levels. Such an assessment includes a determination of the overall constitutive level of gene transcription and a control for variations in RNA (or protein) recovery.
- the methods can also evaluate expression of other markers, such as one or more of markers known to be associated with ASCVD and/or HF.
- the concentration of the mRNA of interest is compared to a control, such as the concentration of the mRNA in a subject known not to have ASCVD, known not to have had HF, or known not to be at risk for ASCVD and/or HF.
- a control such as the concentration of the mRNA in a subject known not to have ASCVD, known not to have had HF, or known not to be at risk for ASCVD and/or HF.
- the control is a standard value, such as a value that represents an average concentration of the mRNA of interest expected in a subject who does not have ASCVD and/or HF, and/or is not at risk for ASCVD and/or HF.
- arrays can be used to evaluate gene expression.
- an array that consists essentially of probes or primers specific for the genes listed in Tables A, B, C, D or E
- such an array includes probes or primers specific for these genes, and can further include control probes (for example to confirm the incubation conditions are sufficient).
- the array can consist essentially of probes or primers specific for ANGPTL3 and GDF15, and optionally includes probes or primers specific for LPA, HPX, IGF1, and/or NPPB.
- the array can consist essentially of probes or primers specific for NPPB and GDF15, and optionally includes probes or primers specific for MPO, CLEC3B, ORMl, and/or TIMPl .
- the array can consist essentially of probes or primers specific for NPPB, GDF15, MPO, CLEC3B, ORMl, and TIMPl .
- the array can consist essentially of probes or primers specific for ANGPTL3, GDF15, LPA, HPX, IGF1, and NPPB.
- the array can further include one or more control probes.
- the array may further include additional, such as about 5, 10, 20, 30, 40, 50, 60, or 70 additional nucleic acids, such as other markers with a known associated with ASCVD and/or HF.
- additional nucleic acids such as other markers with a known associated with ASCVD and/or HF.
- Exemplary control probes include GAPDH, ⁇ - actin, and 18S RNA.
- an array is a multi-well plate (e.g. , 96 or 384 well plate).
- the oligonucleotide probes or primers can further include one or more detectable labels, to permit detection of hybridization signals between the probe and target sequence (such as those listed in Tables A, B, C, D or E).
- the solid support of the array can be formed from an organic polymer.
- Suitable materials for the solid support include, but are not limited to: polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene, polyisoprene, polyvinylpyrrolidine,
- suitable characteristics of the material that can be used to form the solid support surface include: being amenable to surface activation such that upon activation, the surface of the support is capable of covalently attaching a biomolecule such as an
- oligonucleotide thereto; amenability to "in situ" synthesis of biomolecules; being chemically inert such that at the areas on the support not occupied by the oligonucleotides or proteins (such as antibodies) are not amenable to non-specific binding, or when non-specific binding occurs, such materials can be readily removed from the surface without removing the oligonucleotides or proteins (such as antibodies).
- a surface activated organic polymer is used as the solid support surface.
- a surface activated organic polymer is a polypropylene material aminated via radio frequency plasma discharge.
- Other reactive groups can also be used, such as carboxylated, hydroxylated, thiolated, or active ester groups.
- a wide variety of array formats can be employed in accordance with the present disclosure.
- One example includes a linear array of oligonucleotide bands, generally referred to in the art as a dipstick.
- Another suitable format includes a two-dimensional pattern of discrete cells (such as 4096 squares in a 64 by 64 array).
- other array formats including, but not limited to slot (rectangular) and circular arrays are equally suitable for use (see U.S. Patent No. 5,981,185).
- the array is a multi-well plate.
- the array is formed on a polymer medium, which is a thread, membrane or film.
- An example of an organic polymer medium is a polypropylene sheet having a thickness on the order of about 1 mil.
- the array can include biaxially oriented polypropylene (BOPP) films, which in addition to their durability, exhibit low background fluorescence.
- BOPP biaxially oriented polypropylene
- a "format” includes any format to which the solid support can be affixed, such as microtiter plates (e.g., multi-well plates), test tubes, inorganic sheets, dipsticks, and the like.
- microtiter plates e.g., multi-well plates
- test tubes e.g., test tubes
- inorganic sheets e.g., inorganic sheets
- dipsticks e.g., inorganic sheets
- the solid support is a polypropylene thread
- one or more polypropylene threads can be affixed to a plastic dipstick-type device
- polypropylene membranes can be affixed to glass slides.
- the particular format is, in and of itself, unimportant.
- the solid support can be affixed thereto without affecting the functional behavior of the solid support or any biopolymer absorbed thereon, and that the format (such as the dipstick or slide) is stable to any materials into which the device is introduced (such as clinical samples and hybridization solutions).
- the arrays of the present disclosure can be prepared by a variety of approaches.
- oligonucleotide or protein sequences are synthesized separately and then attached to a solid support (see U.S. Patent No. 6,013,789).
- sequences are synthesized directly onto the support to provide the desired array (see U.S. Patent No. 5,554,501).
- Suitable methods for covalently coupling oligonucleotides and proteins to a solid support and for directly synthesizing the oligonucleotides or proteins onto the support are known to those working in the field; a summary of suitable methods can be found in Matson et ah, Anal. Biochem. 217:306-10, 1994.
- the oligonucleotides are synthesized onto the support using conventional chemical techniques for preparing oligonucleotides on solid supports (such as PCT applications WO 85/01051 and WO 89/10977, or U.S. Patent No. 5,554,501).
- a suitable array can be produced using automated means to synthesize oligonucleotides in the cells of the array by laying down the precursors for the four bases in a predetermined pattern.
- a multiple-channel automated chemical delivery system is employed to create oligonucleotide probe populations in parallel rows (corresponding in number to the number of channels in the delivery system) across the substrate.
- the substrate can then be rotated by 90° to permit synthesis to proceed within a second set of rows that are now perpendicular to the first set. This process creates a multiple-channel array whose intersection generates a plurality of discrete cells.
- the oligonucleotides can be bound to the polypropylene support by either the 3' end of the oligonucleotide or by the 5' end of the oligonucleotide. In one example, the oligonucleotides are bound to the solid support by the 3' end. However, one of skill in the art can determine whether the use of the 3' end or the 5' end of the oligonucleotide is suitable for bonding to the solid support. In general, the internal complementarity of an oligonucleotide probe in the region of the 3' end and the 5' end determines binding to the support.
- the oligonucleotide probes on the array include one or more labels, that permit detection of oligonucleotide probe:target sequence hybridization complexes. Kits
- Kits are also provided.
- the kit can include probes, primers, or antibodies specific for the genes listed in Tables A, B, C, D or E, and can further include control probes, primers and antibodies (for example to confirm the incubation conditions are sufficient).
- the kit can include a container and a label or package insert on or associated with the container.
- Suitable containers include, for example, bottles, vials, syringes, etc.
- the containers may be formed from a variety of materials such as glass or plastic.
- the container typically holds a composition including one or more of the probes, primers and/or antibodies.
- the container may have a sterile access port.
- a label or package insert indicates that the composition is of use for evaluating if a subject is at risk for HF or ASCVD, or if a therapeutic agent is of use of the treatment of a subject.
- the label or package insert typically will further include instructions for use, such as particular assay conditions.
- the package insert typically includes instructions customarily included in commercial packages of products that contain information about the indications, usage, contraindications and/or warnings concerning the use of such products.
- the instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
- the kits may also include additional components to facilitate the particular application for which the kit is designed.
- the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
- the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
- the methods include performing one or more assays that detect the level of angiopoietin-like 3(ANGPTL3) and growth/differentiation factor 15 (GDF15) in a biological sample from the subject; and comparing the level of
- ANGPTL3 and GDF15 to a respective control level of ANGPTL3 and GDF15. Detection of a decrease in the level of ANGPTL3 and an increase in the level of GDF15 as compared to the respective control indicates that the subject has or will develop ASCVD.
- the method further includes performing an assay that detects the level of lipoprotein(a) (LPA) in a biological sample from the subject; and comparing the level of LPA to a respective control level of LPA. Detection of an increase in the level of LPA as compared to the respective control level of LPA indicates that the subject has or will develop ASCVD.
- LPA lipoprotein(a)
- the method further includes performing an assay that detects the level of hemopexin (HPX) a biological sample from the subject; and comparing the level of HPX to a respective control level of HPX. Detection of an increase in the level of HPX as compared to the respective control level of HPX indicates that the subject has or will develop ASCVD.
- HPX hemopexin
- the method further includes performing an assay that detects the level of insulin like growth factor 1 (IGF1) in a biological sample from the subject; and comparing the level of IGF1 to a respective control level of IGF1. Detection of a decrease in the level of IGF1 as compared to the respective control level of IGF1 indicates that the subject has or will develop ASCVD.
- IGF1 insulin like growth factor 1
- the method further includes performing an assay that detects the level of natriuretic peptides B (NPPB) in a biological sample from the subject; and comparing the level of NPPB to a respective control level of NPPB. Detection of an increase in the level of NPPB as compared to the respective control level of NPPB indicates that the subject has or will develop ASCVD.
- the method includes performing one or more assays that detect the level of all of ANGPTL3, GDF15, LPA, HPX, IGF1, and NPPB.
- the methods include performing one or more assays that detect the level of ANGPTL3 and GDF15 in a biological sample from the subject administered the agent; and comparing the level of ANGPTL3 and GDF15 to a respective control level of ANGPTL3 and GDF15. Detection of an increase in the level of ANGPTL3 and a decrease in the level of GDF15 as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects the level of LPA in a biological sample from the subject; and comparing the level of LPA to a respective control level of LPA. Detection of a decrease in the level of LPA as compared to the respective control level of LPA indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects the level of HPX in a biological sample from the subject; and comparing the level of HPX to a respective control level of HPX. Detection of a decrease in the level of HPX as compared to the respective control level of indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects the level of IGF1 in a biological sample from the subject; and comparing the level of IGF1 to a respective control level of IGF1. Detection of an increase in the level of IGF1 as compared to the respective control level of IGF1 indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method further includes performing an assay that detects the level of NPPB in a biological sample from the subject; and comparing the level of NPPB to a respective control level of NPPB. Detection of a decrease in the level of NPPB as compared to the respective control level of NPPB indicates that the pharmaceutical agent is effective for the treatment or prevention of ASCVD in the subject.
- the method includes performing one or more assays that detect the level of all of ANGPTL3, GDF15, LPA, HPX, IGF1, and NPPB.
- Methods for determining the likelihood that a subject will develop heart failure are also provided.
- the methods include performing one or more assays that detect the level of NPPB and GDF15 in a biological sample from the subject; and comparing the level of NPPB and GDF15 to a respective control level of NPPB and GDF15. Detection of an increase in the level of NPPB and GDF15 as compared to the respective control indicates that the subject has or will develop heart failure.
- the method further includes performing an assay that detects the level of myeloperoxidase (MPO) in a biological sample from the subject; and comparing the level of MPO to a respective control level of MPO. Detection of an increase in the level of MPO as compared to the respective control level of MPO indicates that the subject has or will develop heart failure.
- MPO myeloperoxidase
- the method further includes performing an assay that detects the level of tetranectin (CLEC3B) in a biological sample from the subject; and comparing the level of CLEC3B to a respective control level of CLEC3B. Detection of a decrease in the level of CLEC3B as compared to the respective control level of CLEC3B indicates that the subject has or will develop heart failure.
- CLEC3B tetranectin
- the method further includes performing an assay that detects the level of alpha- 1 -acid glycoprotein 1 (ORM1) in a biological sample from the subject; and comparing the level of ORM1 to a respective control level of ORM1. Detection of an increase in the level of ORM1 as compared to the respective control level of ORM1 indicates that the subject has or will develop heart failure.
- ORM1 alpha- 1 -acid glycoprotein 1
- the method further includes performing an assay that detects the level of metalloproteinase inhibitor 1 (TIMPl) in a biological sample from the subject; and comparing the level of TIMPl to a respective control level of TIMPl. Detection of a decrease in the level of TIMPl as compared to the respective control level of TIMPl indicates that the subject has or will develop heart failure.
- TIMPl metalloproteinase inhibitor 1
- the method includes performing one or more assays that detect all of NPPB, GDF15, MPO, CLEC3B, ORM1, and TIMPl.
- the methods include performing one or more assays that detect the level of NPPB and GDF15 in a biological sample from the subject administered the agent; and comparing the level of NPPB and GDF15 to a respective control level of NPPB and GDF15. Detection of a decrease in the level of NPPB and GDF15 as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- the method further includes performing an assay that detects the level of MPO in a biological sample from the subject; and comparing the level of MPO to a respective control level of MPO. Detection of a decrease in the level of MPO as compared to the respective control level of MPO indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- the method further includes performing an assay that detects the level of CLEC3B in a biological sample from the subject; and comparing the level of CLEC3B to a respective control level of CLEC3B. Detection of an increase in the level of CLEC3B as compared to the respective control level of CLEC3B indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- the method further includes performing an assay that detects the level of alpha- 1 -acid glycoprotein 1 (ORMl) in a biological sample from the subject; and comparing the level of ORMl to a respective control level of ORMl. Detection of a decrease in the level of ORMl as compared to the respective control level of ORMl indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- ORMl alpha- 1 -acid glycoprotein 1
- the method further includes performing an assay that detects the level of TIMPl in a biological sample from the subject; and comparing the level of TIMPl to a respective control level of TIMPl. Detection of an increase in the level of TIMPl as compared to the respective control level of TIMPl indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- a method of detecting or determining the likelihood that a subject will develop atherosclerotic cardiovascular disease comprising: performing one or more assays that detect a level of angiopoietin-like 3 (ANGPTL3) and growth/differentiation factor 15 (GDF15) in a biological sample from the subject; and determining the level of ANGPTL3 and GDF15 to a respective control level of ANGPTL3 and GDF15; wherein detection of a decrease in the level of ANGPTL3 and an increase in the level of GDF15 as compared to the respective control indicates that the subject has or will develop atherosclerotic cardiovascular disease.
- ANGPTL3 angiopoietin-like 3
- GDF15 growth/differentiation factor 15
- Clause 2 The method of clause 1, further comprising: performing an assay that detects a level of lipoprotein(a) (LPA) in a biological sample from the subject; and comparing the level of LPA to a respective control level of LPA; wherein detection of an increase in the level of LPA as compared to the respective control level of LPA indicates that the subject has or will develop atherosclerotic cardiovascular disease.
- LPA lipoprotein(a)
- Clause 3 The method of clause 1 or clause 2, further comprising: performing an assay that detects a level of hemopexin (HPX) a biological sample from the subject; and comparing the level of HPX to a respective control level of HPX; wherein detection of an increase in the level of HPX as compared to the respective control level of HPX indicates that the subject has or will develop atherosclerotic cardiovascular disease.
- HPX hemopexin
- Clause 4 The method of any one of clauses 1-3, further comprising: performing an assay that detects a level of insulin like growth factor 1 (IGFl) in a biological sample from the subject; and comparing the level of IGFl to a respective control level of IGFl; wherein detection of a decrease in the level of IGFl as compared to the respective control level of IGFl indicates that the subject has or will develop atherosclerotic cardiovascular disease.
- IGFl insulin like growth factor 1
- Clause 5 The method of any one of clauses 1-4 further comprising: performing an assay that detects a level of natriuretic peptides B (NPPB) in a biological sample from the subject; andcomparing the level of NPPB to a respective control level of NPPB; wherein detection of an increase in the level of NPPB as compared to the respective control level of NPPB indicates that the subject has or will develop atherosclerotic cardiovascular disease.
- NPPB natriuretic peptides B
- a method of determining if a pharmaceutical agent is effective for treatment or prevention of atherosclerotic cardiovascular disease in a subject comprising: performing one or more assays that a level of angiopoietin-like 3 (ANGPTL3) and growth/differentiation factor
- GDF15 in a biological sample from the subject administered the agent; and comparing the level of ANGPTL3 and GDF15 to a respective control level of ANGPTL3 and GDF15; wherein detection of an increase in the level of ANGPTL3 and a decrease in the level of GDF15 as compared to the respective control indicates that the pharmaceutical agent is effective for the treatment or prevention of atherosclerotic cardiovascular disease in the subject.
- the method of clause 6, further comprising: performing an assay that detects a level of lipoprotein(a) (LPA) in a biological sample from the subject; and comparing the level of LPA to a respective control level of LPA; wherein detection of a decrease in the level of LPA as compared to the respective control level of LPA indicates that the pharmaceutical agent is effective for the treatment or prevention of atherosclerotic cardiovascular disease in the subject.
- LPA lipoprotein(a)
- Clause 8 The method of clause 6 or clause 7, further comprising:
- Clause 9 The method of any one of clauses 6-8, further comprising: performing an assay that detects a level of insulin like growth factor 1 (IGFl) in a biological sample from the subject; and comparing the level of IGFl to a respective control level of IGFl; wherein detection of an increase in the level of IGFl as compared to the respective control level of IGFl indicates that the pharmaceutical agent is effective for the treatment or prevention of atherosclerotic cardiovascular disease in the subject.
- IGFl insulin like growth factor 1
- Clause 10 The method of any one of clauses 6-9, further comprising: performing an assay that detects a level of natriuretic peptides B (NPPB) in a biological sample from the subject; and comparing the level of NPPB to a respective control level of NPPB;wherein detection of a decrease in the level of NPPB as compared to the respective control level NPPB indicates that the pharmaceutical agent is effective for the treatment or prevention of
- NPPB natriuretic peptides B
- Clause 11 The method of any one of clauses 1-5, wherein the control is a standard value of ANGPTL3, GDF15, LPA, HPX, IGFl and/or NPPB, respectively in one or more subjects known not to have atherosclerotic cardiovascular disease.
- ANGPTL3, GDF15, LPA, HPX, IGFl, and/or NPPB respectively in the subject prior to administration of the pharmaceutical agent.
- Clause 13 The method of any one of clauses 1-12, wherein the sample comprises a blood, plasma, serum or urine sample. Clause 14. The method of any one of clauses 1-13, further comprising assessing the Framingham risk factors for the subject.
- Clause 15 The method of any one of clauses 1-14, wherein the one or more assays detect ANGPTL3 mRNA, GDF15 mRNA, LPA mRNA, HPX mRNA, IGF1 mRNA, and/or NPPB mRNA.
- Clause 16 The method of clause 15, wherein the one or more assays comprise a polymerase chain reaction, a microarray analysis or a hybridization reaction.
- Clause 18 The method of any one of clauses 1-14, wherein the one or more assays detect ANGPTL3 protein, GDF15 protein, LPA protein, HPX protein, IGF1 protein, and/or NPPB protein.
- Clause 19 The method of clause 18, wherein the one or more assays comprises mass spectrometry.
- Clause 21 The method of clause 18, wherein performing the one or more assays that detect ANGPTL3 protein, GDF15 protein, LPA protein, HPX protein, IGF1 protein, and/or
- NPPB protein comprises contacting the biological sample or a component thereof with an antibody that specifically binds ANGPTL3 protein, an antibody that specifically binds GDF15 protein, an antibody that specifically binds LPA protein, an antibody that specifically binds HPX protein, an antibody that specifically binds IGF1 protein, and/or an antibody that specifically binds NPPB protein.
- Clause 22 The method of clause 21, wherein the assay is an immunoassay.
- Clause 23 The method of clause 21 or 22, wherein the assay is a Western blot, an enzyme linked immunosorbent assay, or a radioimmunoassay.
- Clause 24 The method of any one of clauses 21-23, wherein the antibody that specifically binds ANGPTL3 protein, the antibody that specifically binds GDF15 protein, the antibody that specifically binds LPA protein, the antibody that specifically binds HPX protein, the antibody that specifically binds IGF1 protein, and/or the antibody that specifically binds
- NPPB protein is directly labeled.
- the label is a radioactive marker, a fluorescent marker, an enzyme or a metal.
- Clause 27 The method of clause 26, wherein the agent is a statin, niacin, a fibrate, a bile acid binding resin, a cholesterol absorption inhibitor, a PCSK9-targeting drug, an LDL-targeting drug, or an HDL- targeting drug.
- the agent is a statin, niacin, a fibrate, a bile acid binding resin, a cholesterol absorption inhibitor, a PCSK9-targeting drug, an LDL-targeting drug, or an HDL- targeting drug.
- Clause 28 The method of any one of clauses 1-27, wherein the method does not comprise measuring lipoprotein other than lipoprotein a.
- Clause 29 The method of clause 28, wherein the method does not comprise measuring high density lipoproteins.
- Clause 30 The method of any one of clauses 1-29, wherein the method does not comprise measuring inflammatory markers.
- a method of detecting or determining the likelihood that a subject will develop heart failure comprising: performing one or more assays that detect a level of natriuretic peptides B (NPPB) and growth/differentiation factor 15 (GDF15) in a biological sample from the subject; and comparing the level of NPPB and GDF15 to a respective control level of NPPB and GDF15; wherein detection of an increase in the level of NPPB and GDF15 as compared to the respective control indicates that the subject has or will develop heart failure.
- NPPB natriuretic peptides B
- GDF15 growth/differentiation factor 15
- Clause 33 The method of clause 32, further comprising: performing an assay that detects a level of myeloperoxidase (MPO) in a biological sample from the subject; and comparing the level of MPO to a respective control level of MPO; wherein detection of an increase in the level of MPO as compared to the respective control level of MPO indicates that the subject has or will develop heart failure.
- MPO myeloperoxidase
- Clause 34 The method of clause 32 or clause 33, further comprising: performing an assay that detects a level of tetranectin (CLEC3B) in a biological sample from the subject; and comparing the level of CLEC3B to a respective control level of CLEC3B; wherein detection of a decrease in the level of CLEC3B as compared to the respective control level of CLEC3B indicates that the subject has or will develop heart failure.
- CLEC3B tetranectin
- Clause 35 The method of any one of clauses 32-34, further comprising: performing an assay that detects a level of alpha- 1 -acid glycoprotein 1 (ORMl) in a biological sample from the subject; and comparing the level of ORMl to a respective control level of ORMl; wherein detection of an increase in the level of ORMl as compared to the respective control level of ORMl indicates that the subject has or will develop heart failure.
- ORMl alpha- 1 -acid glycoprotein 1
- Clause 36 The method of any one of clauses 32-35, further comprising: performing an assay that detects a level of metalloproteinase inhibitor 1 (TIMP1) in a biological sample from the subject; and comparing the level of TIMPl to a respective control level of TIMPl; wherein detection of a decrease in the level of TIMPl as compared to the respective control level of TIMPl indicates that the subject has or will develop heart failure.
- TIMP1 metalloproteinase inhibitor 1
- a method of determining if a pharmaceutical agent is effective for treatment or prevention of heart failure in a subject comprising performing one or more assays that detect a level of Natriuretic peptides B (NPPB) and growth/differentiation factor 15 (GDF15) in a biological sample from the subject administered the agent; and comparing the level of NPPB and GDF15 to a respective control level of NPPB and GDF15; wherein detection of a decrease in the level of NPPB and GDF15 as compared to the respective control indicates that the NPPB and GDF15
- pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- Clause 38 The method of clause 37, further comprising: performing an assay that detects a level of myeloperoxidase (MPO) in a biological sample from the subject; and comparing the level of MPO to a respective control level of MPO; wherein detection of a decrease in the level of MPO as compared to the respective control level of MPO indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- MPO myeloperoxidase
- Clause 39 The method of clause 37 or clause 38, further comprising: performing an assay that detects a level of tetranectin (CLEC3B) in a biological sample from the subject; and comparing the level of CLEC3B to a respective control level of CLEC3B; wherein detection of an increase in the level of CLEC3B as compared to the respective control level of CLEC3B indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- CLEC3B tetranectin
- Clause 40 The method of any one of clauses 37-39, further comprising performing an assay that detects a level of alpha- 1 -acid glycoprotein 1 (ORMl) in a biological sample from the subject; and comparing the level of ORMl to a respective control level of ORMl; wherein detection of a decrease in the level of ORMl as compared to the respective control level of ORMl indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- ORMl alpha- 1 -acid glycoprotein 1
- Clause 41 The method of any one of clauses 37-40, further comprising: performing an assay that detects a level of metalloproteinase inhibitor 1 (TIMPl) in a biological sample from the subject; and comparing the level of TIMPl to a respective control level of TIMPl; wherein detection of an increase in the level of TIMPl as compared to the respective control level of TIMPl indicates that the pharmaceutical agent is effective for the treatment or prevention of heart failure in the subject.
- TIMPl metalloproteinase inhibitor 1
- Clause 42 The method of any one of clauses 32-36, wherein the control is a standard value of NPPB, GDF15, MPO, CLEC3B, ORMl, and/or TIMPl, respectively in one or more subjects known not to have heart failure.
- Clause 43 The method of any one of clauses 37-41, wherein the control is the level of NPPB, GDF15, MPO, CLEC3B, ORMl, and/or TIMPl, respectively in the subject prior to administration of the pharmaceutical agent.
- Clause 44 The method of any one of clauses 32-43, wherein the sample comprises a blood, plasma, serum or urine sample.
- Clause 45 The method of any one of clauses 32-44, further comprising assessing the Framingham risk factors for the subject.
- Clause 46 The method of any one of clauses 32-45, wherein the one or more assays detect NPPB mRNA, GDF15 mRNA, MPO mRNA, CLEC3B mRNA, ORMl mRNA, and/or TIMPl mRNA.
- Clause 47 The method of clause 46, wherein the one or more assays comprise a polymerase chain reaction, a microarray analysis or a hybridization reaction.
- Clause 48 The method of clause 47, wherein the one or more assays comprise reverse transcriptase polymerase chain reaction (RT-PCR).
- RT-PCR reverse transcriptase polymerase chain reaction
- Clause 49 The method of any one of clauses 32-45, wherein the one or more assays detect NPPB protein, GDF15 protein, MPO protein, CLEC3B protein, ORMl protein, and/or TIMP1 protein.
- Clause 50 The method of clause 49, wherein the one or more assays comprises mass spectrometry.
- Clause 51 The method of clause 50, wherein the mass spectrometry is MALDI-TOF mass spectrometry and/or LC-mass spectrometry.
- Clause 52 The method of clause 49, wherein performing the one or more assays that detect NPPB protein, GDF15 protein, MPO protein, CLEC3B protein, ORMl protein, and/or TIMP1 protein comprises contacting the biological sample or a component thereof with an antibody that specifically binds NPPB protein, an antibody that specifically binds GDF15 protein, an antibody that specifically binds MPO protein, CLEC3B protein, an antibody that specifically binds ORMl protein, and/or an antibody that specifically binds TIMP1 protein.
- the assay is an immunoassay.
- Clause 54 The method of clause 52 or 53, wherein the assay is a Western blot, an enzyme linked immunosorbent assay, or a radioimmunoassay.
- Clause 55 The method of any one of clauses 52-54, wherein the antibody that specifically binds NPPB protein, the antibody that specifically binds GDF15 protein, the antibody that specifically binds MPO protein, the antibody that specifically binds CLEC3B protein, the antibody that specifically binds ORMl protein, and/or the antibody that specifically binds TIMP1 protein is directly labeled.
- Clause 56 The method of clause 55, wherein the label is a radioactive marker, a fluorescent marker, an enzyme or a metal.
- Clause 57 The method of any one of clauses 32-36, 42 and 44-56, comprising administering to the subject a therapeutically effective amount of an agent for the treatment or prevention of heart failure if the subject is determined to have an increased likelihood of developing heart failure.
- Clause 58 The method of clause 57, wherein the agent is an angiotensin-converting enzyme (ACE) inhibitor, a beta blocker, an aldosterone antagonist, a diuretic, an angiotensin receptor blocker (ARB), or a vasodilator.
- ACE angiotensin-converting enzyme
- beta blocker an aldosterone antagonist
- ARB angiotensin receptor blocker
- Plasma samples for immunoassay were collected from offspring cohort participants at their 7 th clinic examination and third generation participants at their first examination, and frozen at -80°C until assayed. Participants free of ASCVD (HF) were followed until their next examination approximately 6 years later for the occurrence of new ASVD (HF).
- HF ASCVD
- Immunoassays 47 plasma proteins (see Table 1) were measured by immunoassay in over 7500 FHS offspring and third generation cohort participants to identify biomarker signatures of ASCVD (or HF). Immunoassays were performed using LUMINEX® bead assays. Subjects under the age of 50 were excluded as were subjects with prior ASCVD (for analyses of ASCVD), and those with prior HF (for analyses of HF), leaving a sample size of 2333 for analyses of ASCVD and 2693 for analyses of HF. During follow up, ASCVD events occurred in 209 individuals and HF events occurred in 89. LUMINEX® xMAP®: Antibody pairs were used in a LUMINEX® xMAP® sandwich
- Candidate biomarkers present at low abundances were processed using PROTEOPREP ® 20, an antibody-based resin capable of depleting 95% of the total protein from plasma. Depletion of these high- abundance proteins allows for visualization of proteins co-eluting with, and masked by, the high- abundance proteins and peptides, such as using LC-MS methods.
- the LUMINEX® xMAP® assay is an extension of the enzyme-linked immunosorbent assay (ELISA) performed with multiple analyte- specific capture antibodies bound to a set of fluorescent beads.
- An xMAP® assay can simultaneously quantify up to 100 analytes at abundances as low as pg/ml in multiple samples (Johannisson, 2006). This multiplexed format allows higher throughput and reduced sample use as compared to other ELISA formats.
- the xMAP® technology employed sets of 5.6-micron beads labeled with one of up to 100 unique fluorescent signatures. Prior to analysis, each bead was conjugated to capture antibodies specific to a single analyte through a two-step carbodiimide coupling procedure.
- LUMINEX® bead mixtures were then added and incubated. After aspiration of excess reagent and washing, detection antibodies were added to each sample. An additional incubation and aspiration occurred, followed by resuspension in sample buffer and quantification in a LUMINEX® 200 analyzer.
- the following tables summarize the results of SABRe CVD Initiative analysis to identify protein biomarkers of ASCVD (Table 2 and Table 4) and HF (Table 3 and Table 5).
- the clinical risk factors in the model include age, systolic blood pressure (and use of anti-hypertensive medications), baseline atrial fibrillation, body mass index, diabetes status, gender, HDL cholesterol, smoking status, and total cholesterol level.
- Angiopoietin-like 3 0.8330 0.7164 0.9684 0.0175
- Insulin-like growth factor 1 0.8886 0.7860 1.0047 0.0593
- the Framingham Heart Study is a prospective longitudinal community-based observational cohort study.
- CVD cardiovascular disease
- Candidate protein biomarkers were selected for assay based on the following criteria a) association with atherosclerotic CVD (ASCVD), b) targeting proteins coded for by genes associated with ASCVD in genome-wide association studies, and c) targeting genes associated with ASCVD or its major risk factors in gene expression analyses. A total of 61 proteins were then assayed using a modified enzyme-linked immunosorbent assay sandwich approach, multiplexed on a LUMINEX® xMAP platform (Sigma- Aldrich).
- ASCVD cardiovascular disease
- coronary revascularization percutaneous coronary intervention or bypass surgery
- Biomarkers were log-transformed due to right-skewed distributions. The association of each biomarker and primary outcomes was examined using multivariable Cox proportional hazards regression models. Models for ASCVD were adjusted for age, sex, systolic blood pressure, anti-hypertensive regimen use, diabetes mellitus, body-mass index, smoking status, total and high density lipoprotein cholesterol, and prevalent atrial fibrillation. In addition, HF and were also adjusted for prevalent myocardial infarction. Next, a protein multi-marker approach was taken using a stepwise approach to select proteins associated with the outcome of interest with retention at a p value ⁇ 0.05 after adjusting for risk factors. Iimmunoassays were performed as in Example 1. Subjects under the age of 50 were excluded as were subjects with prior ASCVD (for analyses of ASCVD), and those with prior HF (for analyses of HF). The markers a shown in the table below:
- AIM Alpha-l-microglobulin
- Adipsin pg/mL
- Beta-2-microglobulin pg/mL
- bikunin AMBP-bikunin (BIKUNIN) pg/mL
- Clusterin Clusterin, pg/mL
- cystatin_c Cystatin-C Cystatin-C, pg/mL
- EGF containing fibulin-like extracellular matrix protein 1 EFEMP1
- EFEMP1 EGF containing fibulin-like extracellular matrix protein 1
- gdf_15 Growth/differentiation factor 15
- pg/mL gmp_140 Granule membrane protein 140 (P-selectin)
- pg/mL p5 Glycoprotein V platelet
- igf_l Insulin-like growth factor 1 pg/mL
- igfbpl Insulin-like growth factor-binding protein 1 Insulin-like growth factor-binding protein 2 (IGFBP2) pg/mL igfbp_3 Insulin-like growth factor-binding protein 3, pg/mL klkbl Plasma kallikrein (KLKB1) pg/mL
- leptin_r Leptin receptor pg/mL
- MCAM Melanoma cell adhesion molecule
- resistin Resistin Resistin, pg/mL
- sicam_l Intercellular adhesion molecule 1, pg/mL
- THBS1 thbsl Thrombospondin-1
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Abstract
L'invention concerne des méthodes permettant de déterminer la probabilité qu'un sujet développe une maladie cardiovasculaire athéroscléreuse (ASCVD) ou une insuffisance cardiaque (HF). L'invention concerne également des méthodes permettant de déterminer si un agent pharmaceutique est efficace pour le traitement ou la prévention de l'ASCVD ou de l'HF.
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US11821905B2 (en) | 2015-01-27 | 2023-11-21 | Arterez, Inc. | Biomarkers of vascular disease |
CN112194724A (zh) * | 2020-10-19 | 2021-01-08 | 福州迈新生物技术开发有限公司 | 抗mpo蛋白单克隆抗体、细胞系及其制备方法和应用 |
CN112194724B (zh) * | 2020-10-19 | 2022-01-14 | 福州迈新生物技术开发有限公司 | 抗mpo蛋白单克隆抗体、细胞系及其制备方法和应用 |
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