LPA-ASSOCIATED PROTEIN AND RNA EXPRESSION

BACKGROUND OF THE INVENTION

Lysophospholipids (such as lysophosphatidic acid (LPA)) affect important cellular functions including cellular proliferation, differentiation, survival, migration, adhesion, invasion, and morphogenesis. These functions impact many biological processes including neurogenesis, angiogenesis, wound healing, immunity, and carcinogenesis. LPA acts through specific G protein-coupled receptors (GPCRs) in an autocrine and paracrine fashion. LPA binds to its cognate GPCRs (LPA₁, LPA₂, LPA₃, LPA₄, LPA₅, and LPA₆) and thereby activates intracellular signaling pathways to produce a variety of biological responses. LPA is an important biological effector molecule with a diverse range of physiological actions (e.g., effects on blood pressure, platelet activation, smooth muscle contraction, cell growth, cell rounding, neurite retraction, actin stress fiber formation, cell migration). LPA effects are predominantly receptor mediated and activation of the LPA receptors (LPA₁, LPA₂, LPA₃, LPA₄, LPA₅, and LPA₆) with LPA mediates a range of downstream signaling cascades. Nearly all mammalian cells, tissues, and organs co-express several LPA-receptor subtypes indicating that LPA receptor signaling occurs in a cooperative manner.

There is a need in the art for well-defined biomarkers to determine if a patient has or is at risk for developing an Lysophosphatidic Acid (LPA)-associated disease, to indicate the progressiveness of the disease, and/or to facilitate evaluation of responsiveness to therapy. The invention provided herein addresses these and other needs in the art by providing, inter alia, novel protein biomarkers of LPA-associated disease.

BRIEF SUMMARY OF THE INVENTION

In one aspect, a method of determining an expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 in a subject that has or is at risk for developing an LPA-associated disease is provided. The method includes obtaining a biological sample from the subject and determining an expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 in the biological sample.

In another aspect, a method of determining an expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 in a subject that has or is at risk for developing an LPA-associated disease is provided. The method includes obtaining a biological sample from the subject and determining an expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 in the biological sample.

In another aspect, a method of determining whether a subject has or is at risk of developing an LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker proteins set forth in Table 1 or Table 2 in a subject. (ii) It is determined whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 or a decreased expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 relative to the standard control indicates that the subject has or is at risk of developing an LPA-associated disease. Based at least in part on the expression level in step (ii), it is determined whether the subject has or is at risk for developing an LPA-associated disease.

In another aspect, a method of determining whether a subject has or is at risk of developing an LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 in a subject. (ii) It is determined whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 or a decreased expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to the standard control indicates that the subject has or is at risk of developing an LPA-associated disease. Based at least in part on the expression level in step (ii), it is determined whether the subject has or is at risk for developing an LPA-associated disease.

In another aspect, a method of determining an LPA-associated disease activity in a patient is provided. The method includes (i) determining a first expression level of a protein set forth in Table 1 or Table 2 in the patient at a first time point. (ii) A second expression level of a protein set forth in Table 1 or Table 2 in the patient is determined at a second time point. (iii) The second expression level of a protein set forth in Table 1 or Table 2 is compared to the first expression level of a protein set forth in Table 1 or Table 2, thereby determining the LPA-associated disease activity in the patient.

In another aspect, a method of determining an LPA-associated disease activity in a patient is provided. The method includes (i) determining a first expression level of an RNA set forth in Table 3 or Table 4 in the patient at a first time point. (ii) A second expression level of an RNA set forth in Table 3 or Table 4 is determined in the patient at a second time point. (iii) The second expression level of an RNA set forth in Table 3 or Table 4 is compared to the first expression level of an RNA set forth in Table 3 or Table 4, thereby determining the LPA-associated disease activity in the patient.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes administering to the subject an effective amount of an modulator of an LPA-associated disease marker protein set forth in Table 1 or Table 2, thereby treating an LPA-associated disease in the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes administering to the subject an effective amount of an modulator of an LPA-associated disease marker RNA set forth in Table 3 or Table 4, thereby treating an LPA-associated disease in the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 relative to a standard control. (ii) When an elevated expression level or a decreased expression level of the LPA-associated disease marker protein set forth in Table 1 or Table 2 is found relative to the standard control, an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker protein set forth in Table 1 or Table 2 is administered to the subject, thereby treating the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to a standard control. (ii) When an elevated expression level or a decreased expression level of the LPA-associated disease marker RNA set forth in Table 3 or Table 4 is found relative to the standard control, an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 is administered to the subject, thereby treating the subject.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker proteins set forth in Table 1 or Table 2 in an LPA-associated disease patient. (ii) It is determined whether the expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 relative to the standard control indicates that the LPA-associated disease patient is at risk for progression of the LPA-associated disease. (iii) Based at least in part on the expression level in step (ii), it is determined whether the LPA-associated disease patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 in an LPA-associated disease patient. (ii) It is determined whether the expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to the standard control indicates that the LPA-associated disease patient is at risk for progression of the LPA-associated disease. (iii) Based at least in part on the expression level in step (ii), it is determined whether the LPA-associated disease patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) determining a first expression level of a protein set forth in Table 1 or Table 2 in the patient at a first time point. (ii) A second expression level of a protein set forth in Table 1 or Table 2 in the patient is determined at a second time point. (iii) The second expression level of a protein set forth in Table 1 or Table 2 is compared to the first expression level of a protein set forth in Table 1 or Table 2, wherein when the second expression level of a protein set forth in Table 1 or Table 2 is different from the first level of a protein set forth in Table 1 or Table 2, the patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining an expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 in a subject that has or is at risk for developing an LPA-associated disease is provided. The method includes (i) obtaining a biological sample from the subject; and (ii) determining an expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 in the biological sample.

In another aspect, a method of determining an expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 in a subject that has or is at risk for developing an LPA-associated disease is provided. The method includes (i) obtaining a biological sample from the subject; and (ii) determining an expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 in the biological sample.

In another aspect, a method of determining whether a subject has or is at risk of developing an LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 in a subject. (ii) It is determined whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 or a decreased expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to the standard control indicates that the subject has or is at risk of developing an LPA-associated disease. And (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an LPA-associated disease.

In another aspect, a method of determining whether a subject has or is at risk of developing an LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 in a subject. (ii) It is determined whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 or a decreased expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to the standard control indicates that the subject has or is at risk of developing an LPA-associated disease. And (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an LPA-associated disease.

In another aspect, a method of determining an LPA-associated disease activity in a patient is provided. The method includes (i) determining a first expression level of a protein of SEQ ID NO:1-202 in the patient at a first time point. (ii) A second expression level of a protein of SEQ ID NO:1-202 in the patient is determined at a second time point. (iii) The second expression level of a protein of SEQ ID NO:1-202 is compared to the first expression level of a protein of SEQ ID NO:1-202, thereby determining the LPA-associated disease activity in the patient.

In another aspect, a method of determining an LPA-associated disease activity in a patient is provided. The method includes (i) determining a first expression level of an RNA of SEQ ID NO:203-499 in the patient at a first time point. (ii) A second expression level of an RNA of SEQ ID NO:203-499 is determined in the patient at a second time point. (iii) The second expression level of an RNA of SEQ ID NO:203-499 is compared to the first expression level of an RNA of SEQ ID NO:203-499, thereby determining the LPA-associated disease activity in the patient.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes administering to the subject an effective amount of an modulator of an LPA-associated disease marker protein of SEQ ID NO:1-202, thereby treating an LPA-associated disease in the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes administering to the subject an effective amount of an modulator of an LPA-associated disease marker RNA of SEQ ID NO:203-499, thereby treating an LPA-associated disease in the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to a standard control. And (ii) when an elevated expression level or a decreased expression level of the LPA-associated disease marker protein of SEQ ID NO:1-202 is found relative to the standard control, administering to the subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker protein of SEQ ID NO:1-202, thereby treating the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to a standard control. And (ii) when an elevated expression level or a decreased expression level of the LPA-associated disease marker RNA of SEQ ID NO:203-499 is found relative to the standard control, administering to the subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker RNA of SEQ ID NO:203-499, thereby treating the subject.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker proteins set forth of SEQ ID NO:1-202 in an LPA-associated disease patient. (ii) It is determined whether the expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to the standard control indicates that the LPA-associated disease patient is at risk for progression of the LPA-associated disease; and (iii) based at least in part on the expression level in step (ii), determining whether the LPA-associated disease patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 in an LPA-associated disease patient. (ii) It is determined whether said expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to the standard control indicates that the LPA-associated disease patient is at risk for progression of said LPA-associated disease; and (iii) based at least in part on the expression level in step (ii), determining whether the LPA-associated disease patient is at risk for progression of the LPA-associated disease.

In another aspect, a complex in vitro is provided. The complex includes a marker protein binding agent bound to a LPA-associated disease marker protein of SEQ ID NO:1-202 or fragment thereof, wherein the LPA-associated disease marker protein is extracted from a human subject having or at risk of developing an LPA-associated disease.

In another aspect, a complex in vitro is provided. The complex includes a marker RNA binding agent bound to a LPA-associated disease marker RNA of SEQ ID NO:203-499 or fragment thereof, wherein the LPA-associated disease marker RNA is extracted from a human subject having or at risk of developing an LPA-associated disease.

In another aspect, a kit is provided. The kit includes (a) a marker protein binding agent capable of binding to a substance within a biological sample from a human subject having or at risk of developing an LPA-associated disease; wherein the substance is an LPA-associated disease marker protein of SEQ ID NO:1-202 or fragment thereof. And (b) a detecting reagent or a detecting apparatus capable of indicating binding of the marker protein binding agent to the substance.

In another aspect, a kit is provided. The kit includes (a) a marker RNA binding agent capable of binding to a substance within a biological sample from a human subject having or at risk of developing an LPA-associated disease; wherein the substance is an LPA-associated disease marker RNA of SEQ ID NO:203-499 or fragment thereof. And (b) a detecting reagent or a detecting apparatus capable of indicating binding of the marker RNA binding agent to the substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: LPA receptor expression in cells. Expression of LPA receptors 1-6 was assayed by qRT-PCR in primary human fibroblasts, endothelial cells, epithelial cells, and pericytes.

FIG. 2: Experimental Design for Gene Expression and Secreted Protein Analysis.

FIG. 3: LPA-Induced mRNAs in Primary Pulmonary Fibroblasts.

FIG. 4: Comparison of 1 μM LPA induction in Fibroblasts and Epithelial Cells.

FIG. 5: Selected Differentially Secreted Proteins from Fibroblasts.

FIG. 6: LPA-Induced Proteins Independently Associated With IPF.

DETAILED DESCRIPTION OF THE INVENTION
I. Definitions

The term “disease” refers to any deviation from the normal health of a mammal and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g., infection, gene mutation, genetic defect, etc.) has occurred, but symptoms are not yet manifested. According to the present invention, the methods disclosed herein are suitable for use in a patient that is a member of the Vertebrate class, Mammalia, including, without limitation, primates, livestock and domestic pets (e.g., a companion animal). Typically, a patient will be a human patient.

The terms “pulmonary disease,” “pulmonary disorder,” “lung disease,” etc. are used interchangeably herein. The term is used to broadly refer to lung disorders characterized by difficulty breathing, coughing, airway discomfort and inflammation, increased mucus, and/or pulmonary fibrosis.

The term “LPA-associated disease” is used to broadly refer to disorders or symptoms of disease associated with LPA function. In embodiments, the disease is caused by, or a symptom of the disease is caused by aberrant LPA function. LPA function as described herein refers to any cellular function affected by LPA and includes without limitation cellular proliferation, differentiation, survival, migration, adhesion, invasion, morphogenesis, neurogenesis, angiogenesis, wound healing, immunity, and carcinogenesis.

The term “associated” or “associated with” in the context of a substance (e.g., LPA) or substance activity (e.g., LPA activity) or substance function (e.g., LPA function) associated with a disease means that the disease is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function (i.e., LPA, LPA activity, LPA function). For example, a disease associated with LPA or a symptom of an LPA-associated disease or condition associated with an increase or decrease in LPA activity may be a disease or symptom that results (entirely or partially) from an increase or decrease in LPA activity (e.g. increase or decrease in LPA activity or increase or decrease in activity of an LPA signal transduction or signaling pathway). The term “signaling pathway” as used herein refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components.

Non-limiting examples of LPA-associated diseases are idiopathic pulmonary fibrosis, pulmonary fibrosis, bronchiolitis obliterans, chronic lung transplant rejection, scleroderma, primary focal segmental glomerulosclerosis (FSGC) or membranoproliferative glomerulonephritis (MPGN), idiopathic interstitial pneumonia, interstitial lung disease in systemic sclerosis, a fibrosis condition of the lung, autoimmune lung diseases, benign prostate hypertrophy, coronary or myocardial infarction, atrial fibrillation, cerebral infarction, myocardial fibrosis, musculoskeletal fibrosis, post-surgical adhesions, liver cirrhosis, renal fibrotic disease, fibrotic vascular disease, scleroderma, Hermansky-Pudlak syndrome, neurofibromatosis, Alzheimer's disease, diabetic retinopathy, or skin lesions, lymph node fibrosis associated with HIV, chronic obstructive pulmonary disease (COPD), inflammatory pulmonary fibrosis, rheumatoid arthritis; rheumatoid spondylitis; osteoarthritis; gout, other arthritic conditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome; myofacial pain syndrome (MPS); Shigellosis; asthma; adult respiratory distress syndrome; inflammatory bowel disease; Crohn's disease; psoriasis; eczema; ulcerative colitis; glomerular nephritis; scleroderma; chronic thyroiditis; Grave's disease; Ormond's disease; autoimmune gastritis; myasthenia gravis; autoimmune hemolytic anemia; autoimmune neutropenia; thrombocytopenia; pancreatic fibrosis; chronic active hepatitis including hepatic fibrosis; acute or chronic renal disease; renal fibrosis; diabetic nephropathy; irritable bowel syndrome; pyresis; restenosis; cerebral malaria; stroke or ischemic injury; neural trauma; Alzheimer's disease; Huntington's disease; Parkinson's disease; acute or chronic pain; allergies, including allergic rhinitis or allergic conjunctivitis; cardiac hypertrophy, chronic heart failure; acute coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter's syndrome; acute synoviitis; muscle degeneration, bursitis; tendonitis; tenosynoviitis; herniated, ruptured, or prolapsed intervertebral disk syndrome; osteopetrosis; thrombosis; silicosis; pulmonary sarcosis; bone resorption diseases, such as osteoporosis or multiple myeloma-related bone disorders; cancer, including but not limited to metastatic breast carcinoma, colorectal carcinoma, malignant melanoma, gastric cancer, or non-small cell lung cancer; graft-versus-host reaction; or auto-immune diseases, such as multiple sclerosis, lupus or fibromyalgia; AIDS or other viral diseases such as Herpes Zoster, Herpes Simplex I or II, influenza virus, Severe Acute Respiratory Syndrome (SARS) or cytomegalovirus; or diabetes mellitus, proliferative disorders (including both benign or malignant hyperplasia), acute myelogenous leukemia, chronic myelogenous leukemia, Kaposi's sarcoma, metastatic melanoma, multiple myeloma, breast cancer, including metastatic breast carcinoma; colorectal. carcinoma; malignant melanoma; gastric cancer; non-small cell lung cancer (NSCLC); bone metastases; pain disorders including neuromuscular pain, headache, cancer pain, dental pain, or arthritis pain; angiogenic disorders including solid tumor angiogenesis, ocular neovascularization, or infantile hemangioma; conditions associated with the cyclooxygenase or lipoxygenase signaling pathways, including conditions associated with prostaglandin endoperoxide synthase-2 (including edema, fever, analgesia, or pain); organ hypoxia; thrombin-induced platelet aggregation; or protozoal diseases. For example, IPF and scleroderma (or systemic sclerosis) associated interstitial lung disease (SSc-ILD) share overlapping pathologic pathways, most notably the activation and proliferation of fibroblasts, expression of fibrogenic cytokines and growth factors, and progressive interstitial fibrosis.

The terms “subject,” “patient,” “individual,” and the like as used herein are not intended to be limiting and can be generally interchanged. That is, an individual described as a “patient” does not necessarily have a given disease, but may be merely seeking medical advice.

The term “subject” as used herein includes all members of the animal kingdom prone to suffering from the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human.

A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a patient suspected of having an LPA-associated disease and compared to samples from a known LPA-associated disease patient, or a known normal (non-disease) individual. A control can also represent an average value gathered from a population of similar individuals, e.g., LPA-associated disease patients or healthy individuals with a similar medical background, same age, weight, etc. A control value can also be obtained from the same individual, e.g., from an earlier-obtained sample, prior to disease, or prior to treatment. One of skill will recognize that controls can be designed for assessment of any number of parameters.

One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

As used herein, the terms “pharmaceutically” acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. For the present invention, the dose will generally refer to the amount of LPA-associated disease treatment, agonist or antagonist. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment can refer to any delay in onset, reduction in the frequency or severity of symptoms, amelioration of symptoms, improvement in patient comfort and/or respiratory function, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving a given treatment, or to the same patient prior to, or after cessation of, treatment.

“Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, “treatment” as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease's spread; relieve the disease's symptoms, fully or partially remove the disease's underlying cause, shorten a disease's duration, or do a combination of these things.

“Treating” and “treatment” as used herein include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.

The term “prevent” refers to a decrease in the occurrence of LPA-associated disease symptoms in a patient. As indicated above, the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.

The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

The term “diagnosis” refers to a relative probability that an LPA-associated disease is present in the subject. Similarly, the term “prognosis” refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present invention, prognosis can refer to the likelihood that an individual will develop an LPA-associated disease, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, survival, etc.). The terms are not intended to be absolute, as will be appreciated by any one of skill in the field of medical diagnostics.

The terms “correlating” and “associated,” in reference to determination of a LPA-associated disease risk factor, refers to comparing the presence or amount of the risk factor (e.g., decreased or increased expression of an LPA-associated biomarker protein) in an individual to its presence or amount in persons known to suffer from, or known to be at risk of, the LPA-associated disease, or in persons known to be free of LPA-associated disease, and assigning an increased or decreased probability of having/developing the LPA-associated disease to an individual based on the assay result(s).

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids, including ribonucleic acids (RNA) and deoxyribonucleic acids (DNA), and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids (e.g., genomic sequences or subsequences or coding sequences) or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length.

An example of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. As will be appreciated by one of skill in the art, the software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information (ncbi.nlm.nih.gov).

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, often silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. Typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

A “labeled protein or polypeptide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the labeled protein or polypeptide may be detected by detecting the presence of the label bound to the labeled protein or polypeptide. Alternatively, methods using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.

“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen, e.g., a specific bacterial antigen. Typically, the “variable region” contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding. See Paul, Fundamental Immunology (2003).

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.

Antibodies can exist as intact immunoglobulins or as any of a number of well-characterized fragments that include specific antigen-binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′₂, a dimer of Fab which itself is a light chain joined to V_H-C_H1 by a disulfide bond. The F(ab)′₂may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

The term “aptamer” as provided herein refers to short oligonucleotides (e.g. deoxyribonucleotides), which fold into diverse and intricate molecular structures that bind with high affinity and specificity to proteins, peptides, and small molecules in a non-Watson Crick manner. An aptamer can thus be used to detect or otherwise target nearly any molecule of interest, including an LPA-associated disease marker protein. Methods of constructing and determining the binding characteristics of aptamers are well known in the art. For example, such techniques are described in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459. Aptamers are typically at least 5 nucleotides, 10, 20, 30 or 40 nucleotides in length, and can be composed of modified nucleic acids to improve stability. Flanking sequences can be added for structural stability, e.g., to form 3-dimensional structures in the aptamer. Aptamers can be selected in vitro from very large libraries of randomized sequences by the process of systemic evolution of ligands by exponential enrichment (SELEX as described in Ellington A D, Szostak J W (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818-822; Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505-510) or by developing SOMAmers (slow off-rate modified aptamers) (Gold L et al. (2010) Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5(12):e15004). Applying the SELEX and the SOMAmer technology includes for instance adding functional groups that mimic amino acid side chains to expand the aptamers' chemical diversity. As a result high affinity aptamers for almost any protein target are enriched and identified.

A “biomarker,” “marker protein, “biomarker protein,” “marker RNA, or “biomarker RNA” as provided herein refers to any assayable characteristics or compositions that are used to identify, predict, or monitor a condition (e.g., an LPA-associated disease or lack thereof) or a therapy for said condition in a subject or sample. A biomarker is, for example, a protein or combination of proteins, an RNA or a combination of RNAs whose presence, absence, or relative amount is used to identify a condition (e.g. an LPA-associated disease) or status of a condition (e.g. an LPA-associated disease) in a subject or sample. Biomarkers identified herein are measured to determine levels, expression, activity, or to detect fragments, variants or homologs of said biomarkers. Variants include amino acid or nucleic acid variants or post translationally modified variants. In embodiments, the marker protein is a protein or fragment thereof as set forth in Table 1, Table 2 or Table 5. In embodiments, the marker protein is a homolog of the protein listed in Table 1, Table 2 or Table 5. In embodiments, the marker protein is a protein of SEQ ID NO:1-202 or fragment thereof. In embodiments, the marker protein is a homolog of the protein of SEQ ID NO:1-202. The marker proteins provided herein are identified by accession numbers referring to the corresponding amino acid and/or nucleic acid sequence of the individual marker proteins. Therefore, a person of ordinary skill in the art will immediately recognize the sequences of the marker proteins provided herein.

In embodiments, the marker RNA is an RNA of SEQ ID NO:203-499 or fragment thereof. In embodiments, the marker RNA is a homolog of the RNA of SEQ ID NO:203-499. In embodiments, the marker RNA complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 20 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 30 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 40 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 50 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof In embodiments, the marker RNA includes a sequence of at least 60 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 70 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 80 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 90 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes a sequence of at least 100 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA includes about 50 nucleotides complementary to SEQ ID NO:203-499 or fragments thereof. In embodiments, the marker RNA is sequence at least 50%, 55%, 60%, 65,%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:203-499. In embodiments, the marker RNA has a sequence at least 50%, 55%, 60%, 65,%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the complement of SEQ ID NO:203-499.

In embodiments, the marker RNA is an RNA or fragment thereof as set forth in Table 3 or Table 4. In embodiments, the marker RNA is a homolog of the RNA listed in Table 3 or Table 4. The marker RNAs provided herein are identified by accession numbers referring to the corresponding nucleic acid sequence of the individual marker RNAs. Therefore, a person of ordinary skill in the art will immediately recognize the sequences of the marker RNAs provided herein.

In some examples of the disclosed methods, when the expression level of a biomarker(s) is assessed, the level is compared with control expression level of the biomarker(s). By control level is meant the expression level of a particular biomarker(s) from a sample or subject lacking a disease (e.g. an LPA-associated disease), at a selected stage of a disease or disease state, or in the absence of a particular variable such as a therapeutic agent. Alternatively, the control level comprises a known amount of biomarker. Such a known amount correlates with an average level of subjects lacking a disease, at a selected stage of a disease or disease state, or in the absence of a particular variable such as a therapeutic agent. A control level also includes the expression level of one or more biomarkers from one or more selected samples or subjects as described herein. For example, a control level includes an assessment of the expression level of one or more biomarkers in a sample from a subject that does not have a disease (e.g. an LPA-associated disease), is at a selected stage of progression of a disease (e.g. an LPA-associated disease), or has not received treatment for a disease. Another exemplary control level includes an assessment of the expression level of one or more biomarkers in samples taken from multiple subjects that do not have a disease, are at a selected stage of progression of a disease, or have not received treatment for a disease.

When the control level includes the expression level of one or more biomarkers in a sample or subject in the absence of a therapeutic agent, the control sample or subject is optionally the same sample or subject to be tested before or after treatment with a therapeutic agent or is a selected sample or subject in the absence of the therapeutic agent. Alternatively, a control level is an average expression level calculated from a number of subjects without a particular disease. A control level also includes a known control level or value known in the art.

In one particular example, a biomarker is a protein or combination of proteins whose expression level in a subject or sample is indicative of an LPA-associated disease or an LPA-associated disease activity. The expression level of a biomarker or a combination of a plurality of biomarkers may be increased or decreased compared to a control level. For example, the expression level of a biomarker or a combination of a plurality of biomarkers as provided herein may be increased or decreased in a subject compared to the expression level of the same subject at an earlier time point. Therefore, the expression level of a biomarker as provided herein may be indicative of a specific disease stage. Alternatively, the biomarker may be indicative of the efficacy of treatment. In other words, the expression level of a biomarker may be indicative of whether a patient is responsive to a treatment. The biomarker may further be indicative of the activity of a disease, wherein the activity of a disease refers to the change of one or more biomarker expression levels over the course of the disease.

The terms “agonist,” “activator,” “upregulator,” etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein. The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein. The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of the antagonist.

II. Methods

Provided herein, inter alia, are biomarkers (e.g. protein biomarkers, RNA biomarkers) for diagnosing LPA-associated diseases as well as evaluation of progression, activity and treatment of LPA-associated diseases. Further provided herein are therapeutic targets for ameliorating LPA-associated diseases and compositions including an LPA-associated biomarker bound to a marker protein binding agent (e.g., an antibody).

In embodiments, the determining includes (a) contacting the LPA-associated disease marker protein with a marker protein binding agent in the biological sample, thereby forming a disease marker protein-binding agent complex; and (b) detecting the disease marker protein-binding agent complex. In embodiments, the marker protein binding agent includes a detectable moiety. In embodiments, the marker protein binding agent includes a capturing moiety. In embodiments, the capturing moiety is a cleavable capturing moiety. In embodiments, the detecting includes contacting the disease marker protein-binding agent complex with a capturing agent, thereby forming a captured disease marker protein-binding agent complex. In embodiments, the detecting further includes (1) contacting the captured disease marker protein-binding agent complex with a tagging moiety; thereby forming a tagged disease marker protein-binding agent complex; and (2) separating the tagged disease marker protein-binding agent complex from the biological sample. In embodiments, the detecting further includes after the separating of step (2) separating the capturing binding agent from the tagged disease marker protein-binding agent complex, thereby forming a cleaved disease marker protein-binding agent complex. In embodiments, the detecting further includes (3) separating the marker protein binding agent from the cleaved disease marker protein-binding agent complex; thereby forming a released marker protein binding agent; and (4) determining an amount of the released marker protein binding agent.

In embodiments, the method includes selecting a subject that has or is at risk for developing an LPA-associated disease. The selected subject may be treated for an LPA-associated disease. In some embodiments, the subject is not treated for an LPA-associated disease. The subject may be part of a plurality of subjects participating in a clinical trial. Wherein the subject is part of a clinical trial, the selecting is at least part based on the determining of an expression level as provided herein.

An LPA-associated disease marker protein is a biomarker protein useful to identify, predict, or monitor an LPA-associated disease or lack thereof or a therapy for an LPA-associated disease in a subject or sample. A person of ordinary skill in the art will immediately recognize that determining an expression level of an LPA-associated disease marker protein or an LPA-associated marker RNA described herein includes determining the level of one or more LPA-associated disease marker proteins or LPA-associated disease marker RNAs in a sample (e.g. patient biological sample such as a blood-derived biological sample). Thus is some embodiments, the expression level of a plurality of LPA-associated disease marker proteins or LPA-associated disease marker RNAs is determined. Wherein the expression level of a plurality of LPA-associated disease marker proteins or LPA-associated disease marker RNAs is determined, the level of at least two (e.g. 3, 4, 5, 6, 7, 8, 9, 10 etc.) LPA-associated disease marker proteins or LPA-associated disease marker RNAs is determined and the at least two LPA-associated disease marker proteins or LPA-associated disease marker RNAs are independently different.

In some embodiments, the LPA-associated disease is a fibrotic pulmonary disease. In embodiments, the fibrotic pulmonary disease is idiopathic pulmonary fibrosis or familial interstitial pneumonia. In embodiments, the fibrotic pulmonary disease is a progressive form of idiopathic pulmonary fibrosis. In other embodiments, the LPA-associated disease marker protein is a progressive fibrotic pulmonary disease marker protein or a progressive fibrotic pulmonary disease marker RNA. A progressive fibrotic pulmonary disease maker protein or RNA is a biomarker protein or RNA indicative of a fibrotic pulmonary disease patient having or being at risk of developing progressive fibrotic pulmonary disease (e.g., a progressive form of a fibrotic pulmonary disease). Thus, in some embodiments, the subject has or is at risk for developing a progressive fibrotic pulmonary disease. In other embodiments, the progressive fibrotic pulmonary disease is idiopathic pulmonary fibrosis. A progressive fibrotic pulmonary disease is a disease wherein certain clinical or physiological parameters decline over the course of the disease. Commonly used parameters to determine fibrotic pulmonary disease progression include for example, breathing metrics, such as forced expiratory volume (FEV1), vital capacity (VC), forced vital capacity (FVC), FEV1/FVC and diffusing capacity of carbon monoxide (DL_CO). For example, a fibrotic pulmonary disease patient showing a 5% decline in FVC as compared to a control level may be considered a progressive fibrotic pulmonary disease patient. A control level may be the FVC of the same patient measured at an earlier stage of the fibrotic pulmonary disease or the FVC calculated from a number of subjects lacking the fibrotic pulmonary disease. Thus, in some embodiments, the FVC of a progressive fibrotic pulmonary disease patient is at least 5% less than a control level.

In embodiments, the biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of the subject. In embodiments, the blood-derived biological sample is whole blood, serum or plasma.

The methods provided herein including embodiments thereof further include treating a subject for LPA-associated diseases. As described above the expression level of one or more LPA-associated disease marker proteins or LPA-associated disease marker RNAs is determined. In embodiments, the expression level of one or more LPA-associated disease marker RNAs is determined. In embodiments, an expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 is determined. In embodiments, an expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 is determined.

The expression levels of the one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 may be increased or decreased in an LPA-associated disease patient. The expression levels of the one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 may be increased or decreased in an LPA-associated disease patient. In some embodiments, the one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 is increased. In some embodiments, the one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 is decreased. In some embodiments, the one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 is increased. In some embodiments, the one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 is decreased. In embodiments, the expression level of the LPA-associated disease marker protein of SEQ ID NO:1-202 is elevated relative to a standard control. In embodiments, the expression level of the LPA-associated disease marker protein of SEQ ID NO:1-202 is deceased relative to a standard control. In embodiments, the expression level of the LPA-associated disease marker RNA of SEQ ID NO:203-499 is elevated relative to a standard control. In embodiments, the expression level of the LPA-associated disease marker RNA of SEQ ID NO:203-499 is deceased relative to a standard control.

A treatment regimen for an LPA-associated disease patient with modulated expression levels of one or more LPA-associated disease marker proteins or LPA-associated disease marker RNAs as disclosed herein (e.g. in Table 1, 2, 3, 4 or 5) may be administering to the patient an effective amount of a modulator affecting the one or more increased or decreased biomarker protein or biomarker RNA expression levels. Therefore, in some embodiments, the method includes administering to the subject an effective amount of a modulator of a modulator of the LPA-associated disease marker protein set forth in Table 1 or Table 2. In embodiments, the method includes administering to the subject an effective amount of a modulator of a modulator of the LPA-associated disease marker protein of SEQ ID NO:1-202. In embodiments, the modulator is an antagonist. In embodiments, the antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the expression level of the LPA-associated disease marker protein set forth in Table 1 or Table 2 is elevated relative to a standard control. In embodiments, the expression level of the LPA-associated disease marker protein of SEQ ID NO:1-202 is elevated relative to a standard control. In embodiments, the modulator is an agonist. In embodiments, the agonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the expression level of the LPA-associated disease marker protein of SEQ ID NO:1-202 is decreased relative to a standard control. In embodiments, the expression level of the LPA-associated disease marker protein set forth in Table 1 or Table 2 is decreased relative to a standard control. In embodiments, the method includes administering to the subject an effective amount of a further therapeutic agent.

The methods provided herein may include combinatorial treatment of a LPA-associated disease patient with a modulator of one or more LPA-associated disease marker proteins and a fibrotic pulmonary disease treatment. Thus, the method includes administering to the subject an effective amount of a further therapeutic agent. Exemplary therapeutic agents include, but are not limited to, the agents selected from the group consisting of steroids (including but not limited to prednisolone), cytotoxic agents (including but not limited to azathioprine and cyclophosphamide), bardoxolone, LPA agonists (including but not limited to AM152); Torisel (temsirolimus); PI3K inhibitors; pentraxin or serum amyloid P (including but not limited to Pentraxin-2 (PTX-2 or PRM-151)); MEK inhibitors (including but not limited to ARRY-162 and ARRY-300); p38 inhibitors; PAI-1 inhibitors (including but not limited to Tiplaxtinin); agents that reduce the activity of transforming growth factor-beta (TGF-β) (including but not limited to GC-1008 (Genzyme/Medlmmune); lerdelimumab (CAT-152; Trabio, Cambridge Antibody); metelimumab (CAT-192, Cambridge Antibody); LY-2157299 (Eli Lilly); ACU-HTR-028 (Opko Health)) including antibodies that target one or more TGF-β isoforms, inhibitors of TGF-β receptor kinases TGFBR1 (ALK5) and TGFBR2, and modulators of post-receptor signaling pathways; chemokine receptor signaling; endothelin receptor antagonists including inhibitors that target both endothelin receptor A and B and those that selectively target endothelin receptor A (including but not limited to ambrisentan; avosentan; bosentan; clazosentan; darusentan; BQ-153; FR-139317, L-744453; macitentan; PD-145065; PD-156252; PD163610; PS-433540; S-0139; sitaxentan sodium; TBC-3711; zibotentan); agents that reduce the activity of connective tissue growth factor (CTGF) (including but not limited to FG-3019, FibroGen), and including other CTGF-neutralizing antibodies; matrix metalloproteinase (MMP) inhibitors (including but not limited to MMPI-12, PUP-1 and tigapotide triflutate); agents that reduce the activity of epidermal growth factor receptor (EGFR) including but not limed to erlotinib, gefitinib, BMS-690514, cetuximab, antibodies targeting EGF receptor, inhibitors of EGF receptor kinase, and modulators of post-receptor signaling pathways; agents that reduce the activity of platelet derived growth factor (PDGF) (including but not limited to Imatinib mesylate (Novartis)) and also including PDGF neutralizing antibodies, antibodies targeting PDGF receptor (PDGFR), inhibitors of PDGFR kinase activity, and post-receptor signaling pathways; agents that reduce the activity of vascular endothelial growth factor (VEGF) (including but not limited to axitinib, bevacizumab, BIBF-1120, CDP-791, CT-322, IMC-18F1, PTC-299, and ramucirumab) and also including VEGF-neutralizing antibodies, antibodies targeting the VEGF receptor 1 (VEGFR1, Flt-1) and VEGF receptor 2 (VEGFR2, KDR), the soluble form of VEGFR1 (sFlt) and derivatives thereof which neutralize VEGF, and inhibitors of VEGF receptor kinase activity; inhibitors of multiple receptor kinases such as BIBF-1120 which inhibits receptor kinases for vascular endothelial growth factor, fibroblast growth factor, and platelet derived growth factor; agents that interfere with integrin function (including but not limited to STX-100 and IMGN-388) and also including integrin targeted antibodies; agents that interfere with the pro-fibrotic activities of IL-4 (including but not limited to AER-001, AMG-317, APG-201, and sIL-4Rα) and IL-13 (including but not limited to AER-001, AMG-317, anrukinzumab, CAT-354, cintredekin besudotox, MK-6105, QAX-576, SB-313, SL-102, and TNX-650) and also including neutralizing anti-bodies to either cytokine, antibodies that target IL-4 receptor or IL-13 receptor, the soluble form of IL-4 receptor or derivatives thereof that is reported to bind and neutralize both IL-4 and IL-13, chimeric proteins including all or part of IL-13 and a toxin particularly pseudomonas endotoxin, signaling though the JAK-STAT kinase pathway; agents that interfere with epithelial mesenchymal transition including inhibitors of mTor (including but not limited to AP-23573 or rapamycin); agents that reduce levels of copper such as tetrathiomolybdate; agents that reduce oxidative stress including N-acetyl cysteine and tetrathiomolybdate; and interferon gamma, inhibitors of phosphodiesterase 4 (PDE4) (including but not limited to Roflumilast); inhibitors of phosphodiesterase 5 (PDES) (including but not limited to mirodenafil, PF-4480682, sildenafil citrate, SLx-2101, tadalafil, udenafil, UK-369003, vardenafil, and zaprinast); or modifiers of the arachidonic acid pathway including cyclooxygenase and 5-lipoxegenase inhibitors (including but not limited to Zileuton), compounds that reduce tissue remodeling or fibrosis including prolyl hydrolase inhibitors (including but not limited to 1016548, CG-0089, FG-2216, FG-4497, FG-5615, FG-6513, fibrostatin A (Takeda), lufironil, P-1894B, and safironil) and peroxisome proliferator-activated receptor (PPAR)-gamma agonists (including but not limited to pioglitazone and rosiglitazone), and combinations thereof. In some embodiments, the therapeutic agent is an anti-fibrotic drug. In some embodiments, the therapeutic agent is an idiopathic pulmonary fibrosis drug. In other embodiment, the idiopathic pulmonary fibrosis drug is a mucolytic drug.

In embodiments, the LPA-associated disease marker protein is SEQ ID NO:9, SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:47, SEQ ID NO:58, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:102, SEQ ID NO:118, SEQ ID NO:131, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO:160, SEQ ID NO:164, SEQ ID NO:183 or SEQ ID NO:185.

For the methods of determining an expression level of an LPA-associated diseases marker RNA, the determining may include (a) contacting the LPA-associated disease marker RNA with a marker RNA binding agent in the biological sample, thereby forming a disease marker RNA-binding agent complex; and (b) detecting the disease marker RNA-binding agent complex. In embodiments, the marker RNA binding agent includes a detectable moiety. In embodiments, the marker RNA binding agent includes a capturing moiety. In embodiments, the capturing moiety is a cleavable capturing moiety. In embodiments, the detecting includes contacting the disease marker RNA-binding agent complex with a capturing agent, thereby forming a captured disease marker RNA-binding agent complex. In embodiments, the detecting includes (1) contacting the captured disease marker RNA-binding agent complex with a tagging moiety; thereby forming a tagged disease marker RNA-binding agent complex; and (2) separating the tagged disease marker RNA-binding agent complex from the biological sample. In embodiments, the detecting further includes after the separating of step (2) separating the capturing binding agent from the tagged disease marker RNA-binding agent complex, thereby forming a cleaved disease marker RNA-binding agent complex. In embodiments, the detecting includes (3) separating the marker RNA binding agent from the cleaved disease marker RNA-binding agent complex; thereby forming a released marker RNA binding agent; and (4) determining an amount of the released marker RNA binding agent.

In embodiments, the method includes selecting a subject that has or is at risk for developing an LPA-associated disease. In embodiments, the biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of the subject. In embodiments, the blood-derived biological sample is whole blood, serum or plasma.

In embodiments, the method includes administering to the subject an effective amount of a modulator of the LPA-associated disease marker RNA set forth in Table 3 or Table 4. In embodiments, the method includes administering to the subject an effective amount of a modulator of the LPA-associated disease marker RNA of SEQ ID NO:203-499. In embodiments, the modulator is an antagonist. In embodiments, the antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the expression level of the LPA-associated disease marker RNA set forth in Table 3 or Table 4 is elevated relative to a standard control. In embodiments, the expression level of the LPA-associated disease marker RNA of SEQ ID NO:203-499 is elevated relative to a standard control. In embodiments, the modulator is an agonist. In embodiments, the agonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the expression level of the LPA-associated disease marker RNA set forth in Table 3 or Table 4 is decreased relative to a standard control. In embodiments, the expression level of the LPA-associated disease marker RNA of SEQ ID NO:203-499 is decreased relative to a standard control. In embodiments, the method includes administering to the subject an effective amount of a further therapeutic agent.

In embodiments, the LPA-associated disease marker RNA is SEQ ID NO:209, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:222, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:303, SEQ ID NO:309, SEQ ID NO:353, SEQ ID NO:364, SEQ ID NO:378, SEQ ID NO:396, SEQ ID NO:417, SEQ ID NO:432 or SEQ ID NO:448.

In another aspect, a method of determining whether a subject has or is at risk of developing an LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 in a subject. (ii) It is determined whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 or a decreased expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to the standard control indicates that the subject has or is at risk of developing an LPA-associated disease. And (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an LPA-associated disease.

In embodiments, the method includes selecting a subject that has or is at risk for developing an LPA-associated disease. In embodiments, the expression level of one or more LPA-associated disease marker proteins set forth in Table 1 or Table 2 is detected from a biological sample of the subject. In embodiments, the expression level of one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 is detected from a biological sample of the subject. In embodiments, the biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of the subject. In embodiments, the blood-derived biological sample is whole blood, serum or plasma. In embodiments, the method includes administering to the subject an effective amount of a modulator of the LPA-associated disease marker protein set forth in Table 1 or Table 2. In embodiments, the method includes administering to the subject an effective amount of a modulator of the LPA-associated disease marker protein of SEQ ID NO:1-202. In embodiments, the modulator is an antagonist. In embodiments, the antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the modulator is an agonist. In embodiments, the agonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the method includes administering to the subject an effective amount of a further therapeutic agent.

In another aspect, a method of determining whether a subject has or is at risk of developing an LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 in a subject. (ii) It is determined whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 or a decreased expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to the standard control indicates that the subject has or is at risk of developing an LPA-associated disease. (iii) Based at least in part on the expression level in step (ii), it is determined whether the subject has or is at risk for developing an LPA-associated disease.

In another aspect, a method of determining whether a subject has or is at risk of developing an LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 in a subject. (ii) It is determined whether the expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 or a decreased expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to the standard control indicates that the subject has or is at risk of developing an LPA-associated disease. And (iii) based at least in part on the expression level in step (ii), determining whether the subject has or is at risk for developing an LPA-associated disease.

In embodiments, the method includes selecting a subject that has or is at risk for developing an LPA-associated disease. In embodiments, the expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 is detected from a biological sample of the subject. In embodiments, the expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 is detected from a biological sample of the subject. In embodiments, the biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of the subject. In embodiments, the blood-derived biological sample is whole blood, serum or plasma. In embodiments, the method includes administering to the subject an effective amount of a modulator of the LPA-associated disease marker RNA set forth in Table 3 or Table 4. In embodiments, the method includes administering to the subject an effective amount of a modulator of the LPA-associated disease marker RNA of SEQ ID NO:203-499. In embodiments, the modulator is an antagonist. In embodiments, the antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the modulator is an agonist. In embodiments, the agonist is a peptide, small molecule, nucleic acid, antibody or aptamer. In embodiments, the method includes administering to the subject an effective amount of a further therapeutic agent.

In another aspect, a method of determining an LPA-associated disease activity in a patient is provided. The method includes (i) determining a first expression level of a protein of SEQ ID NO:1-202 in the patient at a first time point. (ii) A second expression level of a protein of SEQ ID NO:1-202 in the patient is determined at a second time point. (iii) The second expression level of a protein of SEQ ID NO:1-202 is compared to the first expression level of a protein of SEQ ID NO:1-202, thereby determining the LPA-associated disease activity in the patient.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker proteins set forth of SEQ ID NO:1-202 in an LPA-associated disease patient. (ii) It is determined whether the expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to the standard control indicates that the LPA-associated disease patient is at risk for progression of the LPA-associated disease; and (iii) based at least in part on the expression level in step (ii), determining whether the LPA-associated disease patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 in an LPA-associated disease patient. (ii) It is determined whether the expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to the standard control indicates that the LPA-associated disease patient is at risk for progression of the LPA-associated disease. (iii) Based at least in part on the expression level in step (ii), it is determined whether the LPA-associated disease patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) detecting an expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 in an LPA-associated disease patient. (ii) It is determined whether said expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to the standard control indicates that the LPA-associated disease patient is at risk for progression of said LPA-associated disease; and (iii) based at least in part on the expression level in step (ii), determining whether the LPA-associated disease patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) determining a first expression level of a protein set forth in Table 1 or Table 2 in the patient at a first time point. (ii) A second expression level of a protein set forth in Table 1 or Table 2 in the patient is determined at a second time point. (iii) The second expression level of a protein set forth in Table 1 or Table 2 is compared to the first expression level of a protein set forth in Table 1 or Table 2, wherein when the second expression level of a protein set forth in Table 1 or Table 2 is different from the first level of a protein set forth in Table 1 or Table 2, the patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) determining a first expression level of a protein of SEQ ID NO:1-202 in the patient at a first time point. (ii) A second expression level of a protein of SEQ ID NO:1-202 in the patient is determined at a second time point. (iii) The second expression level of a protein of SEQ ID NO:1-202 is compared to the first expression level of a protein of SEQ ID NO:1-202, wherein when the second expression level of a protein of SEQ ID NO:1-202 is different from the first level of a protein of SEQ ID NO:1-202, the patient is at risk for progression of the LPA-associated disease. T

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) determining a first expression level of an RNA set forth in Table 3 or Table 4 in the patient at a first time point. (ii) A second expression level of an RNA set forth in Table 3 or Table 4 in the patient is determined at a second time point. (iii) The second expression level of an RNA set forth in Table 3 or Table 4 is compared to the first expression level of an RNA set forth in Table 3 or Table 4, wherein when the second expression level of an RNA set forth in Table 3 or Table 4 is different from the first level of an RNA set forth in Table 3 or Table 4, the patient is at risk for progression of the LPA-associated disease.

In another aspect, a method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease is provided. The method includes (i) determining a first expression level of an RNA of SEQ ID NO:203-499 in the patient at a first time point. (ii) A second expression level of an RNA of SEQ ID NO:203-499 in the patient is determined at a second time point. (iii) The second expression level of an RNA of SEQ ID NO:203-499 is compared to the first expression level of an RNA of SEQ ID NO:203-499, wherein when the second expression level of an RNA of SEQ ID NO:203-499 is different from the first level of an RNA of SEQ ID NO:203-499, the patient is at risk for progression of the LPA-associated disease.

The patient at risk for progression of the LPA-associated disease may have received treatment for the LPA-associated disease prior to the determining in step (i). Where the patient at risk for progression of the LPA-associated disease has received treatment for the LPA-associated disease prior to the determining in step (i) the treatment may be altered after the determining in step (i) and before the determining in step (ii). Alternatively, the patient at risk for progression of the LPA-associated disease may not have received treatment for the LPA-associated disease prior to the determining in step (i). Where the patient at risk for progression of the LPA-associated disease has not received treatment for the LPA-associated disease prior to the determining in step (i) the patient may receive treatment after the determining in step (i). Thus, in some embodiments, the method includes administering an LPA-associated disease treatment after the determining in step (i). In some embodiments, the method further includes determining a rate of progression of the LPA-associated disease in the patient based on the comparing.

In embodiments, the determining the first expression level of a protein of SEQ ID NO:1-202 or an RNA of SEQ ID NO:203-499 and the second expression level of a protein of SEQ ID NO:1-202 or an RNA of SEQ ID NO:203-499 includes normalizing the first expression level of a protein of SEQ ID NO:1-202 or an RNA of SEQ ID NO:203-499 and the second expression level of a protein of SEQ ID NO:1-202 or an RNA of SEQ ID NO:203-499 to a protein or RNA expressed from a standard gene in the patient. In some further embodiments, the standard gene is a so-called housekeeping gene, as is commonly known in the art, such as GAPDH or beta-actin. In embodiments, the standard gene is non-differentially expressed. Where the standard gene is non-differentially expressed, the expression level of the standard gene remains unchanged over the time course of the disease. In some embodiment, the first expression level is detected from a first biological sample of the subject and the second expression level is detected from a second biological sample of the subject. In some embodiment, the first biological sample is a first bodily fluid sample and the second biological sample is a second bodily fluid sample.

III. Methods of Treatment

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes administering to the subject an effective amount of an modulator of an LPA-associated disease marker protein of SEQ ID NO:1-202, thereby treating an LPA-associated disease in the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes administering to the subject an effective amount of an modulator of an LPA-associated disease marker RNA set forth in Table 3 or Table 4, thereby treating an LPA-associated disease in the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes administering to the subject an effective amount of an modulator of an LPA-associated disease marker RNA of SEQ ID NO:203-499, thereby treating an LPA-associated disease in the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to a standard control. And (ii) when an elevated expression level or a decreased expression level of the LPA-associated disease marker protein of SEQ ID NO:1-202 is found relative to the standard control, administering to the subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker protein of SEQ ID NO:1-202, thereby treating the subject.

In some embodiments, the determining whether the expression level is modulated relative to a standard control includes determining whether the expression level is elevated or suppressed relative to other LPA-associated disease patients. Therefore a standard control as referred to herein may include or may be an average value gathered from a population of LPA-associated disease patients. In other embodiments, a standard control is an average value gathered from a population of normal patients.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to a standard control. (ii) When an elevated expression level or a decreased expression level of the LPA-associated disease marker RNA set forth in Table 3 or Table 4 is found relative to the standard control, an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 is administered to the subject, thereby treating the subject.

In another aspect, a method of treating an LPA-associated disease in a subject in need thereof is provided. The method includes (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to a standard control. And (ii) when an elevated expression level or a decreased expression level of the LPA-associated disease marker RNA of SEQ ID NO:203-499 is found relative to the standard control, administering to the subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker RNA of SEQ ID NO:203-499, thereby treating the subject.

IV. Kits and Compositions

The invention provides kits for detection of LPA-associated disease marker proteins or LPA-associated disease marker RNAs or fragments thereof in a subject. The kit can be for personal use or provided to medical professionals. The kit can be a kit for diagnosing or prognosing and LPA-associated disease, or for monitoring the progression of disease or the efficacy of treatment.

In one aspect, a kit is provided. The kit includes (a) a marker protein binding agent (e.g., an aptamer, optionally labeled) capable of binding to a substance within a biological sample (e.g., whole blood, serum or plasma) from a human subject having or at risk of developing an LPA-associated disease; wherein the substance is an LPA-associated disease marker protein of SEQ ID NO:1-202 or fragment thereof. And (b) a detecting reagent or a detecting apparatus capable of indicating binding of the marker protein binding agent to the substance.

The kits provided herein can further include assay containers (tubes), buffers, or enzymes necessary for carrying out the detection assay. In embodiments, the kit further includes a sample collection device for collecting a sample from a subject. In embodiments, the human subject has an LPA-associated disease.

In embodiments, the kit includes components to examine more than one LPA-associated disease marker protein or RNA or fragment thereof. For example, the kit can include more than one marker protein binding agent or marker RNA binding agent capable of binding to one or more LPA-associated disease marker proteins or fragments thereof of SEQ ID NO:1-202 or one or more LPA-associated disease marker RNAs or fragments thereof of SEQ ID NO:203-499. In embodiments, the kit includes a plurality of marker protein binding agents or a plurality of marker RNA binding agents. Where the kit includes a plurality of marker protein binding agents or marker RNA binding agents a plurality of LPA-associated disease marker proteins or RNAs or fragments thereof (e.g., of SEQ ID NO:1-202 or SEQ ID NO:203-499) are detected.

The kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the testing agent, can be suitably reacted or aliquoted. Kits can also include components for comparing results such as a suitable control sample, for example a positive and/or negative control. The kit can also include a collection device for collecting and/or holding the sample from the subject. The collection device can include a sterile swab or needle (for collecting blood), and/or a sterile tube (e.g., for holding the swab or a bodily fluid sample).

V. Methods of Detection

The methods provided herein include the step of determining (detecting) an expression level of an LPA-associated disease marker protein or fragment thereof or an LPA-associated disease marker RNA or fragment thereof. Methods for detecting and identifying proteins or RNAs and their interactions with other proteins or nucleic acid molecules involve conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed., 1986).

“Determining an expression level of a protein”, “determining a level of expression of a protein”, “determining an expression level of an RNA” or “determining a level of expression of an RNA” as provided herein includes methods and technologies well known in the art. For example, capture arrays for expression profiling may be used to determine an expression level of a protein or an RNA. Capture arrays employ high affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides, nucleic acid aptamers or complementary nucleic acids (e.g., RNA or DNA), to bind and detect specific target ligands in high throughput manner. Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially (BD Biosciences, San Jose, Calif.; Clontech, Mountain View, Calif.; BioRad; Sigma, St. Louis, Mo.). Antibodies for capture arrays are made either by conventional immunization (polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; Biolnvent, Lund, Sweden; Affitech, Walnut Creek, Calif.; Biosite, San Diego, Calif.). In addition to the conventional antibodies, Fab and scFv fragments, single V-domains from camelids or engineered human equivalents (Domantis, Waltham, Mass.) are optionally useful in arrays.

The term scaffold refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity. The variants are produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display. Such ligand-binding scaffolds or frameworks include Affibodies based on S. aureus protein A (Affibody, Bromma, Sweden), Trinectins based on fibronectins (Phylos, Lexington, Mass.) and Anticalins based on the lipocalin structure (Pieris Proteolab, Freising-Weihenstephan, Germany). These are used on capture arrays in a similar fashion to antibodies and have advantages of robustness and ease of production.

Nonprotein capture molecules, notably the nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, Colo.). Aptamers are selected from libraries of oligonucleotides by the Selex™ procedure (SomaLogic, Boulder, Colo.) and their interaction with protein is enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand reduces the cross reactivity of aptamers due to the specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains are used to detect binding. Other nonprotein capture molecules include complementary nucleic acid molecules (e.g., RNA, DNA) capable of hybridizing to the marker RNA. In embodiments, the complementary nucleic acid molecules are attached to a detectable moiety.

Protein analytes binding to antibody arrays are detected directly or indirectly, for example, via a secondary antibody. Direct labeling is used for comparison of different samples with different colors. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label-free detection methods, including mass spectrometry, surface plasmon resonance and atomic force microscopy, avoid alteration of ligand. What is required from any method is optimal sensitivity and specificity, with low background to give high signal to noise. Since analyte concentrations cover a wide range, sensitivity has to be tailored appropriately. Serial dilution of the sample or use of antibodies of different affinities are solutions to this problem. Proteins of interest are frequently those in low concentration in body fluids and extracts, requiring detection in the pg range or lower, such as cytokines or the low expression products in cells.

An alternative to an array of capture molecules is one made through molecular imprinting technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, Calif.).

Another methodology which is useful diagnostically and in expression profiling is the ProteinChip® array (Ciphergen, Fremont, Calif.), in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins.

Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and are used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrates, etc. Generally they require an expression library, cloned into E. coli, yeast or similar from which the expressed proteins are then purified, e.g., via a His tag and immobilized. Cell free protein transcription/translation is a viable alternative for synthesis of proteins which do not express well in bacterial or other in vivo systems.

In embodiments, determining (detecting) an expression level of an LPA-associated disease marker protein or fragment thereof as provided herein includes contacting an LPA-associated disease marker protein with a marker protein binding agent. A “marker protein binding agent” as provided herein refers to a substance capable of binding an LPA-associated disease marker protein. The marker protein binding agent may be a nucleic acid or a protein. In embodiments, the marker protein binding agent is an aptamer. In embodiments, the marker protein binding agent is a peptide. In embodiments, the marker protein binding agent is a small molecule. In embodiments, the marker protein binding agent is an antibody. In embodiments, the LPA-associated disease marker protein or fragment thereof is contacted with a marker protein binding agent in a biological sample (e.g., whole blood, serum or plasma). In embodiments, the marker protein binding agent includes a detectable moiety. In embodiments, the detectable moiety is a fluorescent moiety. In embodiments, the marker protein binding agent includes a capturing moiety. A “capturing moiety” refers to a protein or nucleic acid, which is covalently, through a linker or a chemical bond, or noncovalently attached to the marker protein binding agent and is capable of interacting with a capturing agent. An example of a capturing moiety useful for the methods provided herein is biotin. In embodiments, the capturing moiety is biotin. In embodiments, the capturing moiety is a cleavable capturing moiety. In embodiments, the capturing moiety is photocleavable biotin.

In embodiments, determining (detecting) an expression level of an LPA-associated disease marker RNA or fragment thereof as provided herein includes contacting LPA-associated disease marker RNA with a marker RNA binding agent. A “marker RNA binding agent” as provided herein refers to a substance capable of binding an LPA-associated disease marker RNA. The marker RNA binding agent may be a nucleic acid or a protein. In embodiments, the marker RNA binding agent is an aptamer. In embodiments, the marker RNA binding agent is a peptide. In embodiments, the marker RNA binding agent is a small molecule. In embodiments, the marker RNA binding agent is an antibody. In embodiments, the LPA-associated disease marker RNA or fragment thereof is contacted with a marker RNA binding agent in a biological sample (e.g., whole blood, serum or plasma). In embodiments, the marker RNA binding agent includes a detectable moiety. In embodiments, the detectable moiety is a fluorescent moiety. In embodiments, the marker RNA binding agent includes a capturing moiety. A “capturing moiety” refers to a protein or nucleic acid, which is covalently, through a linker or a chemical bond, or noncovalently attached to the marker RNA binding agent and is capable of interacting with a capturing agent. An example of a capturing moiety useful for the methods provided herein is biotin. In embodiments, the capturing moiety is biotin. In embodiments, the capturing moiety is a cleavable capturing moiety. In embodiments, the capturing moiety is photocleavable biotin.

A “capturing agent” as provided herein refers to an agent capable of binding a capturing moiety. The interaction between the capturing moiety and the capturing agent may be a high affinity interaction, wherein the capturing moiety and the capturing agent bind to each other (e.g., biotin, streptavidin). An example of a capturing agent useful for the methods provided herein are streptavidin coated beads. In embodiments, the capturing agent is a streptavidin coated bead. Without limitation any suitable affinity binding pairs known in the art may be used as capturing moiety and capturing agent in the methods provided herein. For example, the capturing moiety may be an antibody and the capturing agent may be an antigen-coated bead. In embodiments, the capturing moiety is biotin and the capturing agent is a streptavidin coated bead.

The marker protein binding agent may bind non-covalently to the LPA-associated disease marker protein through ionic, van der Waals, electrostatic or hydrogen bonds. Upon binding of the marker protein binding agent to the LPA-associated disease marker protein a disease marker-protein binding agent complex is formed. The methods provided herein including embodiments thereof include detecting the disease marker-protein binding agent complex, thereby determining the expression level of a LPA-associated disease marker protein or fragment thereof in a biological sample. Thus, in embodiments, the determining includes (a) contacting an LPA-associated disease marker protein with a marker protein binding agent in the biological sample, thereby forming a disease marker protein-binding agent complex; and (b) detecting the disease marker protein-binding agent complex. The disease marker protein-binding agent complex may be separated from the sample and unbound components contained therein by contacting the disease marker protein-binding agent complex with a capturing agent as described above (e.g., streptavidin-coated beads). Thus, in embodiments, the detecting includes contacting the disease marker protein-binding agent complex with a capturing agent, thereby forming a captured disease marker protein-binding agent complex. The captured disease marker protein-binding agent complex may be washed to remove any unbound components.

The marker RNA binding agent may bind non-covalently to the LPA-associated disease marker RNA through ionic, van der Waals, electrostatic or hydrogen bonds. Upon binding of the marker RNA binding agent to the LPA-associated disease marker RNA a disease marker-RNA binding agent complex is formed. The methods provided herein including embodiments thereof include detecting the disease marker-RNA binding agent complex, thereby determining the expression level of an LPA-associated disease marker RNA or fragment thereof in a biological sample. Thus, in embodiments, the determining includes (a) contacting an LPA-associated disease marker RNA with a marker RNA binding agent in the biological sample, thereby forming a disease marker RNA-binding agent complex; and (b) detecting the disease marker RNA-binding agent complex. The disease marker RNA-binding agent complex may be separated from the sample and unbound components contained therein by contacting the disease marker RNA-binding agent complex with a capturing agent as described above (e.g., streptavidin-coated beads). Thus, in embodiments, the detecting includes contacting the disease marker RNA-binding agent complex with a capturing agent, thereby forming a captured disease marker RNA-binding agent complex. The captured disease marker RNA-binding agent complex may be washed to remove any unbound components.

For the methods provided herein, the LPA-associated disease marker protein or fragment thereof or an LPA-associated disease marker RNA may be contacted with a tagging moiety. A “tagging moiety” as provided herein is a composition capable of non-covalently binding to the LPA-associated disease marker protein or RNA or fragment thereof. In embodiments, the tagging moiety is biotin. Upon binding to the LPA-associated disease marker protein or RNA or fragment thereof, the tagging moiety may bind through high affinity interaction with a tagging agent (e.g., streptavidin). In embodiments, the LPA-associated disease marker protein or RNA or fragment thereof is contacted with a tagging moiety after the formation of a captured disease marker protein-binding agent complex. In embodiments, the LPA-associated disease marker protein or fragment thereof is contacted with a tagging moiety before the formation of a captured disease marker protein-binding agent complex. In embodiments, the LPA-associated disease marker protein or fragment thereof is contacted with a tagging moiety at the same time as the formation of a captured disease marker protein-binding agent complex. In embodiments, the detecting further includes (1) contacting the captured disease marker protein-binding agent complex with a tagging moiety; thereby forming a tagged disease marker protein-binding agent complex; and (2) separating the tagged disease marker protein-binding agent complex from the biological sample.

Once the tagged disease marker protein-binding agent complex is separated from the biological sample, the interaction between capturing moiety (e.g., photocleavable biotin) and capturing agent (e.g., streptavidin-coated beads) is reversed (e.g. through cleavage of the photocleavable biotin) and a cleaved disease marker protein-binding agent complex is formed. The cleaved disease marker protein-binding agent complex includes a LPA-associated disease marker protein or fragment thereof bound to a marker protein binding agent and a tagging moiety. Thus, in embodiments, the detecting further includes after the separating of step (2) separating the capturing binding agent from the tagged disease marker protein-binding agent complex, thereby forming a cleaved disease marker protein-binding agent complex.

The cleaved disease marker protein-binding agent complex may be contacted with a tagging agent (e.g., streptavidin-coated beads) and the tagging moiety (e.g., biotin) bound to the LPA-associated disease marker protein or fragment thereof may form a high affinity interaction with the tagging agent. The cleaved disease marker protein-binding agent complex may be captured by a tagging agent (e.g., streptavidin-coated beads) and the marker protein binding agent may be subsequently separated, (e.g., eluted by affinity chromatography) from the cleaved disease marker protein-binding agent complex. Once the marker protein binding agent (e.g., aptamer) is released (separated) from the cleaved disease marker protein-binding agent complex it may be quantified using standard techniques know in the art to quantify labeled nucleic acids molecules (e.g., hybridization to a custom DNA microarray). Thus, in embodiments, the detecting further includes (3) separating the marker protein binding agent from the cleaved disease marker protein-binding agent complex; thereby forming a released marker protein binding agent; and (4) determining an amount of released marker protein binding agent.

VI. Marker Proteins and RNAs

The LPA-associated disease marker proteins and LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 1, 2, 3, 4 or 5) are applicable to all methods, kits and compositions described herein. For the methods, kits and compositions described herein the expression level of one or more LPA-associated disease marker proteins or LPA-associated disease marker RNAs may be determined (detected). In embodiments, the expression level of at least one LPA-associated disease marker protein or LPA-associated disease marker RNA is determined (detected). In embodiments, the expression level of a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of LPA-associated disease marker proteins or LPA-associated disease marker RNAs is determined (detected). Where the expression level of an LPA-associated disease marker protein or an LPA-associated disease marker RNA is determined (detected), the expression level of a combination of any one of the LPA-associated disease marker proteins or LPA-associated disease marker RNAs provided herein is determined (detected).

For the methods, kits and compositions described herein the expression level of one or more LPA-associated disease marker proteins and one or more LPA-associated disease marker RNAs may be determined (detected). In embodiments, the expression level of at least one LPA-associated disease marker protein and at least one LPA-associated disease marker RNA is determined (detected). In embodiments, the expression level of a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of LPA-associated disease marker proteins and a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of LPA-associated disease marker RNAs is determined (detected). For example, the expression level of a combination of LPA-associated disease marker proteins set forth in Table 1 or 2 or of SEQ ID NO:1-202 may be determined (detected) in combination with one or more LPA-associated disease marker RNAs set forth in Table 3 or 4 or of SEQ ID NO:203-499.

For example, the expression level of a combination of LPA-associated disease marker proteins set forth in Table 1 or 2 or of SEQ ID NO:1-202 may be determined (detected) in the methods, kits or compositions provided herein. In embodiments, the LPA-associated disease marker protein is CXCL3/CXCL2, PAI-1, ANGPT1, P41, GREM1, NID2, NET4, NXPH1, THBS1, TIMP-3, BASI, CAPG, CTAP-III, DDR2, IGFBP-2, MMP-1, MMP-9, NAP-2, CCLS or SERPINE2. In embodiments, the LPA-associated disease marker protein is CXCL3/CXCL2. In embodiments, the LPA-associated disease marker protein is PAI-1. In embodiments, the LPA-associated disease marker protein is ANGPT1. In embodiments, the LPA-associated disease marker protein is P41. In embodiments, the LPA-associated disease marker protein is GREM1. In embodiments, the LPA-associated disease marker protein is NID2. In embodiments, the LPA-associated disease marker protein is NET4. In embodiments, NXPH1. In embodiments, the LPA-associated disease marker protein is THBS1. In embodiments, the LPA-associated disease marker protein is TIMP-3. In embodiments, the LPA-associated disease marker protein is BASI. In embodiments, the LPA-associated disease marker protein is CAPG. In embodiments, the LPA-associated disease marker protein is CTAP-III. In embodiments, the LPA-associated disease marker protein is DDR2. In embodiments, the LPA-associated disease marker protein is IGFBP-2. In embodiments, the LPA-associated disease marker protein is MMP-1. In embodiments, the LPA-associated disease marker protein is MMP-9. In embodiments, the LPA-associated disease marker protein is NAP-2. In embodiments, the LPA-associated disease marker protein is CCL5. In embodiments, the LPA-associated disease marker protein is SERPINE2.

In embodiments, the expression level of CXCL3/CXCL2 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of PAI-1 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of ANGPT1 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of P41 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of GREM1 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of NID2 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of NET4 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of NXPH1 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected).

In embodiments, the expression level of THBS1 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of TIMP-3 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of BASI and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of CAPG and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of CTAP-III and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of DDR2 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected).

In embodiments, the expression level of IGFBP-2 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of MMP-1 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of MMP-9 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of NAP-2 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of CCL5 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected). In embodiments, the expression level of SERPINE2 and one or more LPA-associated disease marker proteins as provided herein (e.g., set forth in Table 1 or 2 or of SEQ ID NO:1-202) is determined (detected).

For example, the expression level of a combination of LPA-associated disease marker RNAs set forth in Table 3 or 4 or of SEQ ID NO:203-499 may be determined (detected) in the methods, kits or compositions provided herein. In embodiments, the LPA-associated disease marker RNA is CYR61, KRTAP1-5, DKK1, MCP-1/CCL-2, HBEGF, CTGF, KPRP, IL6TNFAIP3, KRTAP4-12, BIRC3, MIR218-1, VGLL3, IER3, GPR37, DUSP1, EFNB2, HAS2, PDCD1LG2, SERPINB2, UGCG, CHIC2, PTX3, CNST or TXNIP. In embodiments, the LPA-associated disease marker RNA is CYR61. In embodiments, the LPA-associated disease marker RNA is KRTAP1-5. In embodiments, the LPA-associated disease marker RNA is DKK1. In embodiments, the LPA-associated disease marker RNA is MCP-1/CCL-2. In embodiments, the LPA-associated disease marker RNA is HBEGF. In embodiments, the LPA-associated disease marker RNA is CTGF. In embodiments, the LPA-associated disease marker RNA is KPRP. In embodiments, the LPA-associated disease marker RNA is IL6TNFAIP3. In embodiments, the LPA-associated disease marker RNA is KRTAP4-12. In embodiments, the LPA-associated disease marker RNA is BIRC3. In embodiments, the LPA-associated disease marker RNA is MIR218-1.

In embodiments, the LPA-associated disease marker RNA is VGLL3. In embodiments, the LPA-associated disease marker RNA is IER3. In embodiments, the LPA-associated disease marker RNA is GPR37. In embodiments, the LPA-associated disease marker RNA is DUSP1. In embodiments, the LPA-associated disease marker RNA is EFNB2. In embodiments, the LPA-associated disease marker RNA is HAS2. In embodiments, the LPA-associated disease marker RNA is PDCD1LG2. In embodiments, the LPA-associated disease marker RNA is SERPINB2. In embodiments, the LPA-associated disease marker RNA is UGCG. In embodiments, the LPA-associated disease marker RNA is CHIC2. In embodiments, the LPA-associated disease marker RNA is PTX3. In embodiments, the LPA-associated disease marker RNA is CNST. In embodiments, the LPA-associated disease marker RNA is TXNIP.

In embodiments, the expression level of CYR61 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of KRTAP1-5 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of DKK1 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of MCP-1/CCL-2 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected).

In embodiments, the expression level of HBEGF and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of CTGF and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of KPRP and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of IL6TNFAIP3 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of KRTAP4-12 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of BIRC3 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of MIR218-1 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected).

In embodiments, the expression level of VGLL3 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of IER3 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of GPR37 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of DUSP1 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of EFNB2 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of HAS2 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of PDCD1LG2 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of SERPINB2 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected).

In embodiments, the expression level of UGCG and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of CHIC2 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of PTX3 and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of CNST F and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected). In embodiments, the expression level of TXNIP and one or more LPA-associated disease marker RNAs as provided herein (e.g., set forth in Table 3 or 4 or of SEQ ID NO:203-499) is determined (detected).

VII. Specific Embodiments

LPA Receptor Expression in Cells

Expression of LPA receptors 1-6 was assayed by qRT-PCR in primary human fibroblasts, endothelial cells, epithelial cells, and pericytes. LPA1 is the most abundantly expressed isoform in fibroblasts, epithelial cells, and pericytes. LPA6 is the dominant isoform in endothelial cells and is present in all cell types tested and LPA2, LPA6 are predominant in circulating cells (FIG. 1).

Experimental Design for Gene Expression and Secreted Protein Analysis

Changes in gene expression after 3 hours of LPA induction were profiled on Affymetrix Human Gene ST 2.0 microarrays; differentially expressed genes identified by ANOVA with FDR <0.05 and fold-change >1.2. Process networks associated with differentially expressed genes were defined using MetaCore. Changes in secreted protein expression after 24 hours of LPA induction were profiled on an aptamer-based proteomic platform (1129 proteins); differentially expressed proteins identified by t-test.

Gene Expression after LPA Induction

FIG. 4 shows a comparison of 1 μM LPA induction in fibroblasts and epithelial cells. LPA induced significant changes in gene expression in fibroblasts and epithelial cells as shown in Tables 3 and 4. Changes were broadly inhibited by addition of a selective LPA1 receptor antagonist. LPA-induced both common and cell-specific gene expression changes in fibroblasts and epithelial cells as shown in Tables 3 and 4. LPA-induced pathways include without limitation, cytoskeletal remodeling and immune response in fibroblasts and epithelial cells, cell adhesion in fibroblasts, and epithelial-mesenchymal transition in epithelial cells. LPA induced changes in secreted protein expression in fibroblasts and epithelial cells are shown in Tables 1, 2, and 5. Changes could be inhibited by an LPA1 receptor antagonist. Several LPA-induced proteins were observed as differentially expressed in plasma of IPF patients compared to controls (Table 5).

LPA1 is significantly expressed in cells relevant to fibrosis, including fibroblasts, epithelial cells, and pericytes. LPA6 is predominant in endothelial cells, but is present in all cell types tested. Unbiased characterization of gene and secreted protein expression identified common, as well as cell-specific, effects. LPA-induced genes and proteins were associated with pathways implicated in fibrosis. Several have also been independently associated with IPF. Treatment with a selective LPA1 receptor antagonist inhibited LPA-dependent changes.

Treatment of Cells with LPA, DNA Isolation and mRNA Expression Analysis of Marker RNAs Shown in Table 3 and 4:

Total RNA was isolated from normal human lung fibroblasts, human bronchial epithelial cells, and human pulmonary alveolar epithelial cells treated for 4 hours with LPA and/or LPAR1 inhibitor. Each treatment condition was assayed in triplicate. Samples were profiled on Affymetrix Human Gene ST Arrays 1.0 and/or 2.0 after preparation using the Ambion WT Expression Kit according to manufacturers' instructions.

mRNA Expression Data Analysis of Marker RNAs Shown in Table 3 and 4

Data normalization and all subsequent analyses were performed in Partek Genomics Suite (Partek, St. Louis, Mo.). Data were normalized using the Robust Multi-array Average (RMA) algorithm. Differentially expressed genes were identified by ANOVA with correction for multiple comparisons with p-value with FDR<0.05. A fold-change cut-off was not strictly applied, although the majority of the differentially expressed genes identified have fold-changes greater than 1.2. Genes were included if they were statistically significant for (1) LPA-induction and (2) response to treatment with an LPAR1 inhibitor.

Protein Analysis

Treatment of cells with LPA, isolation supernatant, analysis of secreted protein expression using soma panel was performed as follows: Supernatant was collected from in vitro cultures of normal human lung fibroblasts, human bronchial epithelial cells, and human pulmonary alveolar epithelial cells treated for 24 hours with LPA and/or LPAR1 inhibitor. Each treatment condition was assayed in triplicate. 1129 proteins were profiled using a broad aptamer-based proteomic platform (SomaLogic, Colo., US). Briefly, the technology uses DNA aptamers containing chemically modified nucleotides as highly specific protein binding reagents in a multiplexed assay that transforms the quantity of each targeted protein into a corresponding quantify of the aptamer. Protein quantities are then detected on a microarray platform and recorded as Relative Fluorescence Units (RFU).

In Tables 1-5 The differentially expressed secreted proteins set forth in Tables 1, 2, and 5 and the differentially expressed mRNAs set forth in Table 3 and 4 were identified by visual inspection of individual dot-plots.

Cell Culture Conditions

For the gene and protein expression analysis primary fibroblasts (NHLF product #CC-2512 from Lonza group LTD) and primary epithelial cells (NHBE Product #CC-2540 from Lonza; HPAEpiC Product #3200 from Sciencell (USA, California)) were cultured as recommended by the manufacturer, except they were cultured on collagen coated plates. As part of the induction protocol cells were starved overnight in low (0.2 or 0.1% serum) or no serum (0%), prior to stimulation with LPA.

VIII. References

1. Shea B S and Tager A M. Pro Am Thorac Soc 2012. 9(3):102-110.

2. Swaney J S et al. BJP 2010. 160:1699-1713.

3. Montesi S B et al. BMC Pulm Med 2014. 14:5.

4. Tokumura A et al. Int J Med Sci 2009. 6(4):168-176.

5. Elsheikh E et al. Hepatology 2013. 58(S1):92A-207A.

6. Antoniou K M et al. Chest 2006. 130(4):982-988.

7. Takizawa H et al. Clin Exp Immunol 1997. 107(1):175-181.

8. Needleman B W et al. Arthr Rheumat 1992. 35:67-72.

9. Collard H R et al. Am J Physiol Lung Cell Mol Physiol 2010. 299(1):L3-7.

10. Suga M et al. Eur Resp J 1999. 14(2):376-382.

11. Caporali R et al. Sarcoidosis Vasc Diffuse Lung Dis 2004. 21(2):111-118.

12. Yanaba K et al. Ann Rheum Dis 2006. 65(1):124-126.

13. Choi E S et al. Am J Respir Crit Care Med 2004. 170(5):508-515.

14. Yamashita C M et al. Am J Pathol 2011. 179(4):1733-45.

IX. Tables

TABLE 1

Secreted Proteins

SEQ

effect of

ID

effect
LPAR1

NO:
Target
UniProt
EntrezGeneID
EntrezGeneSymbol
of LPA
inhibitor

1
41
P11171
2035
EPB41
up
down

2
Activin A
P08476
3624
INHBA
up
down

3
ADAM 9
Q13443
8754
ADAM9
up
down

4
ADAM12
O43184
8038
ADAM12

down

5
Aflatoxin B1
O43488
8574
AKR7A2

up

aldehyde

reductase

6
ALCAM
Q13740
214
ALCAM
up
down

7
Aminoacylase-1
Q03154
95
ACY1
up
up

8
Angiogenin
P03950
283
ANG
up
down

9
Angiopoietin-1
Q15389
284
ANGPT1
up
down

10
Angiopoietin-2
O15123
285
ANGPT2
down
up

11
Angiostatin
P00747
5340
PLG
down

12
ANGL4
Q9BY76
51129
ANGPTL4
up

13
annexin I
P04083
301
ANXA1
up

14
Apo D
P05090
347
APOD

up

15
Apo E2
P02649
348
APOE
down

16
Apo E3
P02649
348
APOE
down

17
AREG
P15514
374
AREG
up

18
ART
O00253
181
AGRP

up

19
ARTS1
Q9NZ08
51752
ERAP1
up
down

20
ASM3A
Q92484
10924
SMPDL3A
up

21
ATS1
Q9UHI8
9510
ADAMTS1
down
up

22
B7-H1
Q9NZQ7
29126
CD274
up
down

23
BASI
P35613
682
BSG
up

24
BFL1
Q16548
597
BCL2A1
up
down

25
BGH3
Q15582
7045
TGFBI
up
down

26
BGN
P21810
633
BGN
up
down

27
Bone
P07585
1634
DCN
up
down

proteoglycan II

28
BSSP4
Q9GZN4
64063
PRSS22
up
down

29
C1r
P00736
715
C1R

up

30
Cadherin-2
P19022
1000
CDH2

up

31
calgranulin B
P06702
6280
S100A9

up

32
CAPG
P40121
822
CAPG
up
up

33
Carbonic
P23280
765
CA6

up

anhydrase 6

34
Caspase-3
P42574
836
CASP3
up
up

35
Cathepsin A
P10619
5476
CTSA
up
down

36
Cathepsin H
P09668
1512
CTSH
up
down

37
Cathepsin S
P25774
1520
CTSS
up
down

38
Cathepsin V
O60911
1515
CTSL2
up
down

39
CATZ
Q9UBR2
1522
CTSZ
up
down

40
CK2-A1:B
P68400
1457
CSNK2A1
up

P67870
1460
CSNK2B

41
Coactosin-like
Q14019
23406
COTL1

down

protein

42
Coagulation
P00742
2159
F10
down
up

Factor Xa

43
Cofilin-1
P23528
1072
CFL1
up

44
COMMD7
Q86VX2
149951
COMMD7
up

45
CRDL1
Q9BU40
91851
CHRDL1
up
down

46
CRP
P02741
1401
CRP

down

47
CTAP-III
P02775
5473
PPBP
up
down

48
CXCL16 soluble
Q9H2A7
58191
CXCL16
up

49
Cyclophilin A
P62937
5478
PPIA
up
up

50
Cystatin M
Q15828
1474
CST6
up
down

51
DAF
P08174
1604
CD55
up
down

52
DBNL
Q9UJU6
28988
DBNL
up
down

53
DcR3
O95407
8771
TNFRSF6B
up
down

54
DC-SIGN
Q9NNX6
30835
CD209

down

55
DERM
Q07507
1805
DPT
down
up

56
Desmoglein-1
Q02413
1828
DSG1

up

57
discoidin domain
Q08345
780
DDR1
up
down

receptor 1

58
Discoidin domain
Q16832
4921
DDR2
up
down

receptor 2

59
DKK1
O94907
22943
DKK1
up
down

60
DKK3
Q9UBP4
27122
DKK3
up
down

61
Dkk-4
Q9UBT3
27121
DKK4
up
down

62
DLRB1
Q9NP97
83658
DYNLRB1

up

63
DPP2
Q9UHL4
29952
DPP7
up
down

64
eIF-5A-1
P63241
1984
EIF5A
down
up

65
Elafin
P19957
5266
PI3
up

66
EMR2
Q9UHX3
30817
EMR2
up
down

67
ENA-78
P42830
6374
CXCL5
up
down

68
Endocan
Q9NQ30
11082
ESM1
up
down

69
Endothelin-
P42892
1889
ECE1
up
down

converting

enzyme 1

70
Eotaxin
P51671
6356
CCL11
up
down

71
EPHA3
P29320
2042
EPHA3
up
down

72
EphB4
P54760
2050
EPHB4
up

73
Ephrin-A5
P52803
1946
EFNA5
up
down

74
Ephrin-B3
Q15768
1949
EFNB3
up
down

75
EPI
O14944
2069
EREG
up
down

76
Epithelial cell
P29317
1969
EPHA2
up
down

kinase

77
FCAR
P24071
2204
FCAR
down

78
FGF-18
O76093
8817
FGF18
down
up

79
FGF-20
Q9NP95
26281
FGF20
up
down

80
FGF7
P21781
2252
FGF7
up
down

81
Flt-3
P36888
2322
FLT3
up
up

82
FST
P19883
10468
FST
up
down

83
FSTL3
O95633
10272
FSTL3
up
down

84
Galectin-3
P17931
3958
LGALS3
up
up

85
GAPDH liver
P04406
2597
GAPDH
up
up

86
GCP-2
P80162
6372
CXCL6
up
down

87
G-CSF
P09919
1440
CSF3
up
down

88
GDF-9
O60383
2661
GDF9
down
up

89
gp130 soluble
P40189
3572
IL6ST
up
down

90
GREM1
O60565
26585
GREM1
up
down

91
GRN
P28799
2896
GRN
up
down

92
Gro-a
P09341
2919
CXCL1
up
down

93
Gro-b/g
P19876
None
CXCL3 CXCL2
up
down

P19875

94
GV
P39877
5322
PLA2G5
up
down

95
HAI-1
O43278
6692
SPINT1

up

96
HGF
P14210
3082
HGF
up
down

97
Histone H2A.z
P0C0S5
3015
H2AFZ

down

98
hnRNP A/B
Q99729
3182
HNRNPAB

up

99
HNRPQ
O60506
10492
SYNCRIP

up

100
ICOS
Q9Y6W8
29851
ICOS

down

101
IDE
P14735
3416
IDE
up

102
IGFBP-2
P18065
3485
IGFBP2
down
up

103
IGFBP-3
P17936
3486
IGFBP3
up
down

104
IGFBP-7
Q16270
3490
IGFBP7
up
down

105
IGF-I sR
P08069
3480
IGF1R
up
down

106
IGF-II receptor
P11717
3482
IGF2R
up
down

107
IL-1 R4
Q01638
9173
IL1RL1
up
down

108
IL-11
P20809
3589
IL11
up
down

109
IL-12 RB2
Q99665
3595
IL12RB2
up
down

110
IL-16
Q14005
3603
IL16

up

111
IL-17D
Q8TAD2
53342
IL17D
up
down

112
IL-6
P05231
3569
IL6
up
down

113
IL-6 sRa
P08887
3570
IL6R
up
down

114
IL-8
P10145
3576
IL8
up
down

115
IP-10
P02778
3627
CXCL10
up
down

116
JAG1
P78504
182
JAG1
up
down

117
JAG2
Q9Y219
3714
JAG2
up
down

118
KPCI
P41743
5584
PRKCI
up

119
Layilin
Q6UX15
143903
LAYN
up
down

120
LG3BP
Q08380
3959
LGALS3BP
up
down

121
LGMN
Q99538
5641
LGMN
up
down

122
LRIG3
Q6UXM1
121227
LRIG3
up
down

123
MATN2
O00339
4147
MATN2

down

124
MCP-1
P13500
6347
CCL2
up
down

125
MCP-3
P80098
6354
CCL7
up
down

126
Met
P08581
4233
MET
up
down

127
MICA
Q29983
4276
MICA
up
down

128
MICB
Q29980
4277
MICB
up
down

129
Midkine
P21741
4192
MDK
up

130
MIP-3a
P78556
6364
CCL20
up
down

131
MMP-1
P03956
4312
MMP1
up
down

132
MMP-10
P09238
4319
MMP10
up
down

133
MMP-12
P39900
4321
MMP12
up
down

134
MMP-13
P45452
4322
MMP13
up
down

135
MMP-3
P08254
4314
MMP3
up
down

136
MMP-9
P14780
4318
MMP9
up
down

137
MRC2
Q9UBG0
9902
MRC2
up
down

138
NAGK
Q9UJ70
55577
NAGK
up

139
NAP-2
P02775
5473
PPBP
up
down

140
NCAM-L1
P32004
3897
L1CAM
up
down

141
NET4
Q9HB63
59277
NTN4
up
down

142
Neurotrophin-3
P20783
4908
NTF3
up
down

143
NID2
Q14112
22795
NID2
up
down

144
NKp46
O76036
9437
NCR1
up
down

145
Nogo Receptor
Q9BZR6
65078
RTN4R
up
down

146
NovH
P48745
4856
NOV
up
down

147
NRP1
O14786
8829
NRP1
up
down

148
NRX3B
Q9HDB5
9369
NRXN3
up
down

149
NXPH1
P58417
30010
NXPH1
up
down

150
OMD
Q99983
4958
OMD
up
down

151
ON
P09486
6678
SPARC
up
down

152
PAI-1
P05121
5054
SERPINE1
up
down

153
PARK7
Q99497
11315
PARK7
up
down

154
PCSK7
Q16549
9159
PCSK7
up
down

155
PDE11
Q9HCR9
50940
PDE11A

down

156
PDGF-AA
P04085
5154
PDGFA
up
down

157
PDGF-BB
P01127
5155
PDGFB
up

158
PGP9.5
P09936
7345
UCHL1

down

159
PHI
P06744
2821
GPI

up

160
Protease nexin I
P07093
5270
SERPINE2
up
down

161
PSD7
P51665
5713
PSMD7

down

162
PTHrP
P12272
5744
PTHLH
up
down

163
RAC1
P63000
5879
RAC1
up
down

164
RANTES
P13501
6352
CCL5
up
down

165
RAP
P30533
4043
LRPAP1
up

166
RGMB
Q6NW40
285704
RGMB
up
down

167
ROBO2
Q9HCK4
6092
ROBO2
up
down

168
ROR1
Q01973
4919
ROR1

down

169
SARP-2
Q8N474
6422
SFRP1
up
down

170
SCGF-alpha
Q9Y240
6320
CLEC11A
up
down

171
SDF-1
P48061
6387
CXCL12
up
down

172
Semaphorin 3A
Q14563
10371
SEMA3A
up
down

173
sFRP-3
Q92765
2487
FRZB
down
up

174
SLIK5
O94991
26050
SLITRK5
up
down

175
Sphingosine
Q9NYA1
8877
SPHK1
up
up

kinase 1

176
Spondin-1
Q9HCB6
10418
SPON1
down
up

177
SSRP1
Q08945
6749
SSRP1

up

178
ST4S6
Q7LFX5
51363
CHST15
up
down

179
suPAR
Q03405
5329
PLAUR
up
down

180
TAJ
Q9NS68
55504
TNFRSF19
up
down

181
TFPI
P10646
7035
TFPI
up
down

182
TGF-b R III
Q03167
7049
TGFBR3
up
down

183
Thrombospondin-
P07996
7057
THBS1
up
down

1

184
TIG2
Q99969
5919
RARRES2
up
down

185
TIMP-3
P35625
7078
TIMP3
up
down

186
TNF sR-I
P19438
7132
TNFRSF1A
up
down

187
TNF sR-II
P20333
7133
TNFRSF1B
up
down

188
TNFSF15
O95150
9966
TNFSF15
up
down

189
TrATPase
P13686
54
ACP5
down
up

190
TRY3
P35030
5646
PRSS3

up

191
TSG-6
P98066
7130
TNFAIP6
up
down

192
TSLP
Q969D9
85480
TSLP
up
down

193
TSP2
P35442
7058
THBS2
up
down

194
TWEAK
O43508
8742
TNFSF12
down
up

195
TXD12
O95881
51060
TXNDC12
up
down

196
UNC5H3
O95185
8633
UNC5C
up
down

197
uPA
P00749
5328
PLAU
up
down

198
URB
Q76M96
151887
CCDC80
up
down

199
VEGF
P15692
7422
VEGFA
up
down

200
VEGF121
P15692
7422
VEGFA
up
down

201
VEGF-C
P49767
7424
VEGFC
up
down

202
WKFN1
Q96D09
114928
GPRASP2
down
up

TABLE 2

Secreted Proteins

Secreted protein overlap with plasma proteins

differentially expressed in IPF patients

IPF vs.

control

SEQ

(fold-

ID NO:
Protein

change)

93
CXCL3
Gro-b/g
1.62

CXCL2

152
PAI-1
Serpine peptidase inhibitor, clade E
1.53

9
ANGPT1
Angiopoietin-1
1.36

118
P41
P41
1.53

90
GREM1
Gremlin 1
1.57

143
NID2
Nidogen 2 (osteonidogen)
1.45

141
NET4
Transmembrane protein 53
1.28

149
NXPH1
Neuroexophilin 1
1.41

183
THBS1
Thrombospondin-1
1.47

185
TIMP-3
TIMP metallopeptidase inhibitor 3
1.43

23
BASI
Extracellular matrix metalloproteinase
1.76

32
CAPG
Macrophage-capping protein
1.90

47
CTAP-III
Pro-platelet basic protein
1.43

58
DDR2
Discoidin domain receptor tyrosine
1.25

kinase 2

102
IGFBP-2
Insulin-like growth factor-binding
−1.27

protein 2

131
MMP-1
Matrix metallopeptidase 1
1.51

136
MMP-9
Matrix metallopeptidase 9
1.49

139
NAP-2
Napsin B aspartic peptidase
1.53

164
CCL5
RANTES
1.55

160
SERPINE2
protease nexin I
1.99

TABLE 3

Gene expression from fibroblasts and/or epithelial cells

effect of

SEQ

effect of
LPAR1

ID NO:
Gene Symbol
RefSeq
LPA
inhibitor

203
ABL2
NM_007314
up
down

204
AKAP12
NM_005100
up
down

205
AMOTL2
NM_016201
up
down

206
ARRDC3
NM_020801
down
up

207
ARRDC4
NM_183376
down
up

208
BAZ1A
NM_013448
up
down

209
BIRC3
NM_001165
up
down

210
C3orf52
NM_024616
up
down

211
C7orf49
NR_024185
down
up

212
CDK6
NM_001259
up
down

213
CDR2
NM_001802
up
down

214
CHIC2
NM_012110
up
down

215
CRIM1
NM_016441
up
down

216
CTGF
NM_001901
up
down

217
CYR61
NM_001554
up
down

218
DKK1
NM_012242
up
down

219
DUSP1
NM_004417
up
down

220
DUSP14
NM_007026
up
down

221
DUSP5
NM_004419
up
down

222
EFNB2
NM_004093
up
down

223
ELL2
NM_012081
up
down

224
ENC1
NM_003633
up
down

225
EPHA2
NM_004431
up
down

226
ERRFI1
NM_018948
up
down

227
ETS1
NM_001143820
up
down

228
FAM86B1
NR_003494
down
up

229
FERMT2
NM_006832
up
down

230
FGF5
NM_004464
up
down

231
FOSB
NM_006732
up
down

232
FOSL1
NM_005438
up
down

233
FST
NM_006350
up
down

234
GADD45B
NM_015675
up
down

235
GPCPD1
NM_019593
down
up

236
HAS2
NM_005328
up
down

237
HBEGF
NM_001945
up
down

238
HIVEP2
NM_006734
up
down

239
ICAM1
NM_000201
up
down

240
INHBA
NM_002192
up
down

241
JMY
NM_152405
down
up

242
KPRP
NM_001025231
up
down

243
KRTAP1-5
NM_031957
up
down

244
LIF
NM_002309
up
down

245
LMO7
NM_005358
up
down

246
MAP2K3
NM_145109
up
down

247
MPV17L2
NM_032683
down
up

248
MYADM
NM_001020818
up
down

249
NAV3
NM_014903
up
down

250
NFKB2
NM_002502
up
down

251
NFKBIZ
NM_031419
up
down

252
PLAUR
NM_002659
up
down

253
PRDM1
NM_001198
up
down

254
RAPH1
NM_213589
up
down

255
RASA2
NM_006506
up
down

256
SEMA7A
NM_003612
up
down

257
SERPINB9
NM_004155
up
down

258
SERPINE1
NM_000602
up
down

259
SGK1
NM_001143676
up
down

260
SH3RF1
NM_020870
up
down

261
SLC38A2
NM_018976
up
down

262
SMURF2
NM_022739
up
down

263
SPOCD1
NM_144569
up
down

264
TAGLN3
NM_013259
up
down

265
THBS1
NM_003246
up
down

266
TMEM2
NM_013390
up
down

267
TNFAIP3
NM_006290
up
down

268
TSC22D2
NM_014779
up
down

269
TXNIP
NM_006472
down
up

270
UGCG
NM_003358
up
down

271
VGLL3
NM_016206
up
down

272
ACPP
NM_001099
up
down

273
ADAMTS1
NM_006988
up
down

274
ADAMTS6
NM_197941
up
down

275
AHCTF1
NM_015446
up
down

276
AMACR
NM_014324
down
up

277
ANKRD13A
NM_033121
up
down

278
APBB3
NM_133174
down
up

279
ARHGAP31
NM_020754
up
down

280
ARHGEF9
NM_015185
down
up

281
ARID3B
NM_006465
up
down

282
ARL4D
NM_001661
down
up

283
ARL5B
NM_178815
up
down

284
ARSJ
NM_024590
up
down

285
ASAP1
NM_018482
up
down

286
ASB13
NM_024701
down
up

287
ATP8B1
NM_005603
up
down

288
ATXN7L1
NM_020725
down
up

289
B3GNT2
NM_006577
up
down

290
BCAR3
NM_003567
up
down

291
BCL3
NM_005178
up
down

292
BIRC2
NM_001166
up
down

293
BMP2
NM_001200
up
down

294
BRIX1
NM_018321
up
down

295
C10orf78
NM_145247
down
up

296
C16orf52
NM_001164579
up
down

297
C2CD2
NM_015500
up
down

298
C5orf33
NM_001085411
down
up

299
C6orf130
AJ420538
down
up

300
C6orf70
NM_018341
down
up

301
CALD1
NM_033138
up
down

302
CCDC113
NM_014157
down
up

303
CCL2
NM_002982
up
down

304
CD274
NM_014143
up
down

305
CDK7
NM_001799
up
down

306
CHST2
NM_004267
up
down

307
CIRBP
NM_001280
down
up

308
CITED2
NM_006079
up
down

309
CNST
NM_152609
up
down

310
COL4A5
NM_000495
down
up

311
CRY1
NM_004075
up
down

312
CSF2
NM_000758
up
down

313
CSGALNACT2
NM_018590
up
down

314
CSRP2BP
NM_020536
down
up

315
DAB2
NM_001343
up
down

316
DCUN1D3
NM_173475
up
down

317
DDAH1
NM_012137
up
down

318
DDX21
NM_004728
up
down

319
DNAJB4
NM_007034
up
down

320
DOK5
NM_018431
up
down

321
DOT1L
NM_032482
up
down

322
DSN1
NM_001145316
down
up

323
DTX4
NM_015177
up
down

324
DZIP1
NM_198968
down
up

325
EDN1
NM_001955
up
down

326
EHD1
NM_006795
up
down

327
EIF2C2
NM_012154
up
down

328
ELF1
NM_172373
up
down

329
ELK3
NM_005230
up
down

330
EMP1
NM_001423
up
down

331
ENOSF1
NM_017512
down
up

332
ENTPD7
NM_020354
up
down

333
EPM2AIP1
NM_014805
down
up

334
EREG
NM_001432
up
down

335
ESM1
NM_007036
up
down

336
EYA1
NM_000503
down
up

337
FAM120C
NM_017848
down
up

338
FAM126B
NM_173822
up
down

339
FAM180A
NM_205855
up
down

340
FAM46C
NM_017709
up
down

341
FBXL4
NM_012160
down
up

342
FGF2
NM_002006
up
down

343
FGF7
NM_002009
up
down

344
FHL2
NM_201555
up
down

345
FLRT2
NM_013231
down
up

346
FMN1
NM_001103184
up
down

347
FOXN2
NM_002158
up
down

348
FSTL1
NM_007085
up
down

349
GALT
NM_000155
down
up

350
GJC1
NM_005497
up
down

351
GLIPR1
NM_006851
up
down

352
GPR176
NM_007223
up
down

353
GPR37
NM_005302
up
down

354
GUSBP3
NR_027386
down
up

355
GZF1
NM_022482
down
up

356
H1F0
NM_005318
down
up

357
HDDC3
NM_198527
down
up

358
HIST2H2BF
NM_001024599
down
up

359
HMGCR
NM_000859
down
up

360
HMGCS1
NM_001098272
down
up

361
HMMR
NM_001142556
down
up

362
HS6ST1
NM_004807
down
up

363
HSD17B7P2
NR_003086
down
up

364
IER3
NM_003897
up
down

365
IGF2BP2
NM_006548
up
down

366
IL6
NM_000600
up
down

367
IL7R
NM_002185
up
down

368
JMJD6
NM_001081461
up
down

369
KCNRG
NM_173605
down
up

370
KCTD10
NM_031954
up
down

371
KCTD11
NM_001002914
up
down

372
KCTD18
NM_152387
down
up

373
KIAA0586
NM_014749
down
up

374
KIAA0776
NM_015323
down
up

375
KIAA0922
NM_001131007
up
down

376
KIAA1377
NM_020802
down
up

377
KLF6
NM_001300
up
down

378
KRTAP4-12
NM_031854
up
down

379
LACTB
NM_032857
up
down

380
LATS2
NM_014572
up
down

381
LIAS
NM_006859
down
up

382
LIMD1
NM_014240
up
down

383
LOC100130691
NR_026966
down
up

384
LOC100130876
AK130278
up
down

385
LOC100131860
AK097109
down
up

386
LOC100133299
AY358688
up
down

387
LOC100133315
NR_029192
down
up

388
LOC400464
AK127420
down
up

389
LRCH1
NM_001164211
up
down

390
LRRCC1
NM_033402
down
up

391
MAP3K8
NM_005204
up
down

392
MAP4K3
NM_003618
up
down

393
MAST4
NM_001164664
up
down

394
MBLAC2
NM_203406
down
up

395
MBNL1
NM_021038
up
down

396
MIR218-1
NR_029631
up
down

397
MIR221
NR_029635
up
down

398
MRM1
NM_024864
down
up

399
MSH5
NM_002441
down
up

400
MYO1E
NM_004998
up
down

401
NAB2
NM_005967
up
down

402
NAPEPLD
NM_001122838
down
up

403
NCEH1
NM_001146276
up
down

404
NDEL1
NM_001025579
up
down

405
NEU3
NM_006656
down
up

406
NFAT5
NM_138714
up
down

407
NFKB1
NM_003998
up
down

408
NFKBIE
NM_004556
up
down

409
NFX1
NM_002504
down
up

410
NPC1
NM_000271
up
down

411
NRIP1
NM_003489
up
down

412
NUDT12
NM_031438
down
up

413
NUFIP2
NM_020772
up
down

414
ORC5
NM_002553
down
up

415
PALB2
NM_024675
down
up

416
PATZ1
NM_014323
down
up

417
PDCD1LG2
NM_025239
up
down

418
PDP1
NM_001161778
up
down

419
PEA15
NM_003768
up
down

420
PEX1
NM_000466
down
up

421
PEX11B
NM_003846
down
up

422
PIK3R3
NM_003629
down
up

423
PMS1
NM_000534
down
up

424
PPM1K
NM_152542
down
up

425
PPP1R3B
NM_024607
up
down

426
PPP1R3C
NM_005398
down
up

427
PRDM6
NM_001136239
down
up

428
PRKCD
NM_006254
up
down

429
PRSS22
NM_022119
up
down

430
PTCD2
NM_024754
down
up

431
PTPN12
NM_002835
up
down

432
PTX3
NM_002852
up
down

433
RAB8B
NM_016530
up
down

434
RAI14
NM_001145525
up
down

435
RBMS1
NM_016836
up
down

436
RCOR3
NM_001136223
down
up

437
REV3L
NM_002912
down
up

438
RND3
NM_005168
up
down

439
RNF19A
NM_183419
up
down

440
RP2
NM_006915
up
down

441
RWDD2B
NM_016940
down
up

442
SAMD4A
NM_015589
up
down

443
SCN8A
NM_014191
up
down

444
SDC4
NM_002999
up
down

445
SEC24A
NM_021982
up
down

446
SEC24B
NM_006323
up
down

447
SENP8
NM_145204
down
up

448
SERPINB2
NM_001143818
up
down

449
SESN1
NM_014454
down
up

450
SGK223
NM_001080826
up
down

451
SGMS2
NM_001136258
up
down

452
SIRT5
NM_031244
down
up

453
SKIL
NM_005414
up
down

454
SLC16A14
NM_152527
down
up

455
SLC24A1
NM_004727
down
up

456
SLITRK6
NM_032229
down
up

457
SPOPL
NM_001001664
up
down

458
SRBD1
NM_018079
down
up

459
SRF
NM_003131
up
down

460
SRGAP1
NM_020762
up
down

461
ST3GAL3
NM_174963
down
up

462
STK17B
NM_004226
up
down

463
STK36
NM_015690
down
up

464
STX11
NM_003764
up
down

465
STXBP5
NM_001127715
up
down

466
SYNE1
NM_182961
up
down

467
TACC2
NM_206862
down
up

468
TBX5
NM_181486
down
up

469
TK2
NM_004614
down
up

470
TMEM192
NM_001100389
down
up

471
TNFRSF12A
NM_016639
up
down

472
TP53BP2
NM_005426
up
down

473
TP53INP1
NM_033285
down
up

474
TRIB1
NM_025195
up
down

475
TRIO
NM_007118
up
down

476
TSC22D3
NM_198057
down
up

477
TSEN2
NM_025265
down
up

478
UAP1
NM_003115
up
down

479
USP13
NM_003940
down
up

480
USP40
NM_018218
down
up

481
USP53
NM_019050
up
down

482
VCL
NM_014000
up
down

483
VEGFC
NM_005429
up
down

484
ZBTB1
NM_001123329
up
down

485
ZNF143
NM_003442
up
down

486
ZNF18
NM_144680
down
up

487
ZNF25
NM_145011
down
up

488
ZNF331
NM_018555
down
up

489
ZNF362
NM_152493
down
up

490
ZNF43
NM_003423
down
up

491
ZNF469
NM_001127464
up
down

492
ZNF717
NM_001128223
down
up

493
ZNF76
NM_003427
down
up

494
ZNF800
NM_176814
up
down

495
ZNF804A
NM_194250
up
down

496
ZNRD1-AS
AF032109
down
up

497
ZRANB1
NM_017580
up
down

498
ZSWIM6
NM_020928
up
down

499
ZYX
NM_003461
up
down

TABLE 4A

Differentially expressed mRNAs (List of selected LPA-induced

genes in fibroblasts (average of 3 experiments)).

Effect of 1 μM

Effect of 1
BMS-986020

μM LPA
relative to

(fold-
induced cells

Gene
change)
(fold-change)

CYR61
cysteine-rich, angiogenic inducer, 61
6.52
−7.53

KRTAP1-5
keratin associated protein 1-5
6.49
−6.89

DKK1
dickkopf homolog 1
6.36
−6.16

MCP-1/
chemokine (C-C motif) ligand 2
5.58
−5.99

CCL2

LIF
leukemia inhibitory factor
5.15
−5.07

HBEGF
heparin-binding EGF-like growth factor
5.11
−5.41

CTGF
connective tissue growth factor
4.71
−4.62

KPRP
keratinocyte proline-rich protein
4.31
−3.81

IL6
interleukin 6
4.19
−4.08

TNFAIP3
tumor necrosis factor, α-induced
3.44
−3.68

protein 3

KRTAP4-12
keratin associated protein 4-12
3.23
−3.90

BIRC3
baculoviral IAP repeat-containing 3
3.17
−3.33

MIR218-1
microRNA 218-1
3.07
−2.82

VGLL3
vestigial like 3
2.96
−2.97

IER3
immediate early response 3
2.94
−3.28

GPR37
G protein-coupled receptor 37
2.80
−3.13

DUSP1
dual specificity phosphatase 1
2.76
−2.56

EFNB2
ephrin-B2
2.61
−2.63

HAS2
hyaluronan synthase 2
2.61
−2.89

PDCD1LG2
programmed cell death 1 ligand 2
2.52
−2.51

SERPINB2
serpin peptidase inhibitor, clade B
2.44
−2.64

UGCG
UDP-glucose ceramide
2.41
−2.52

glucosyltransferase

CHIC2
cysteine-rich hydrophobic domain 2
2.36
−2.40

PTX3
pentraxin 3
2.34
−2.49

PAI-1
serpin peptidase inhibitor, clade E
2.28
−2.43

CNST
consortin, connexin sorting protein
2.26
−2.32

TXNIP
thioredoxin interacting protein
−3.79
4.41

TABLE 4B

Differentially expressed mRNAs as listed in Table 4A including

sequence identifiers (SEQ ID NO:) (List of selected LPA-induced

genes in fibroblasts (average of 3 experiments)).

SEQ ID NO:
Gene Symbol

211
CYR61

243
KRTAP1-5

218
DKK1

303
MCP-1/CCL-2

237
HBEGF

216
CTGF

242
KPRP

267
IL6TNFAIP3

378
KRTAP4-12

209
BIRC3

396
MIR218-1

271
VGLL3

364
IER3

353
GPR37

219
DUSP1

222
EFNB2

236
HAS2

417
PDCD1LG2

448
SERPINB2

270
UGCG

214
CHIC2

432
PTX3

309
CNST

269
TXNIP

TABLE 5

LPA-induced proteins independently associated with IPF.

IPF vs.

control

(fold-

Protein
change)

CXCL2/3
Gro-b/g
1.62

ANGPT1
Angiopoietin-1
1.36

p41
P41
1.53

GREM1
Gremlin 1
1.57

NID2
Nidogen 2 (osteonidogen)
1.45

NET4
Transmembrane protein 53
1.28

NXPH1
Neuroexophilin 1
1.41

PAI-1
Serpine peptidase inhibitor,
1.53

clade E

TSP-1
Thrombospondin-1
1.47

TIMP-3
TIMP metallopeptidase
1.43

inhibitor 3

CTAP-III
Pro-platelet basic protein
1.43

DDR2
Discoidin domain receptor
1.25

tyrosine kinase 2

MMP-1
Matrix metallopeptidase 1
1.51

MMP-9
Matrix metallopeptidase 9
1.49

NAP-2
Napsin B aspartic peptidase
1.53

X. Embodiments
Embodiments I
Embodiment 1

A method of determining an expression level of an LPA-associated disease marker protein as set forth in Table 1 or Table 2 in a subject that has or is at risk for developing an LPA-associated disease, said method comprising: (i) obtaining a biological sample from said subject; and (ii) determining an expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 in said biological sample.

Embodiment 2

The method of embodiment 1, wherein said determining comprises: (a) contacting a LPA-associated disease marker protein with a marker protein binding agent in said biological sample, thereby forming a disease marker protein-binding agent complex; and (b) detecting said disease marker protein-binding agent complex.

Embodiment 3

The method of embodiment 1, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 4

The method of embodiment 1, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject.

Embodiment 5

The method of embodiment 4, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 6

The method of embodiment 1, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker protein set forth in Table 1 or Table 2.

Embodiment 7

The method of embodiment 6, wherein said modulator is an antagonist.

Embodiment 8

The method of embodiment 7, wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 9

The method of embodiment 7, wherein said expression level of said LPA-associated disease marker protein set forth in Table 1 or Table 2 is elevated relative to a standard control.

Embodiment 10

The method of embodiment 6, wherein said modulator is an agonist.

Embodiment 11

The method of embodiment 10, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 12

The method of embodiment 10, wherein said expression level of said LPA-associated disease marker protein set forth in Table 1 or Table 2 is decreased relative to a standard control.

Embodiment 13

The method of embodiment 6, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 14

A method of determining an expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 in a subject that has or is at risk for developing an LPA-associated disease, said method comprising: (i) obtaining a biological sample from said subject; and (ii) determining an expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 in said biological sample.

Embodiment 15

The method of embodiment 14, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 16

The method of embodiment 14, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject.

Embodiment 17

The method of embodiment 16, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 18

The method of embodiment 14, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker RNA set forth in Table 3 or Table 4.

Embodiment 19

The method of embodiment 18, wherein said modulator is an antagonist.

Embodiment 20

The method of embodiment 19, wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 21

The method of embodiment 19, wherein said expression level of said LPA-associated disease marker RNA set forth in Table 3 or Table 4 is elevated relative to a standard control.

Embodiment 22

The method of embodiment 18, wherein said modulator is an agonist.

Embodiment 23

The method of embodiment 22, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 24

The method of embodiment 22, wherein said expression level of said LPA-associated disease marker RNA set forth in Table 3 or Table 4 is decreased relative to a standard control.

Embodiment 25

The method of embodiment 18, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 26

A method of determining whether a subject has or is at risk of developing an LPA-associated disease, said method comprising: (i) detecting an expression level of one or more LPA-associated disease marker proteins set forth in Table 1 or Table 2 in a subject; (ii) determining whether said expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 or a decreased expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 relative to said standard control indicates that said subject has or is at risk of developing an LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said subject has or is at risk for developing an LPA-associated disease.

Embodiment 27

The method of embodiment 26, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 28

The method of embodiment 26, wherein said expression level of one or more LPA-associated disease marker proteins set forth in Table 1 or Table 2 is detected from a biological sample of said subject.

Embodiment 29

The method of embodiment 28, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject.

Embodiment 30

The method of embodiment 29, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 31

The method of embodiment 26, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker protein set forth in Table 1 or Table 2.

Embodiment 32

The method of embodiment 31, wherein said modulator is an antagonist.

Embodiment 33

The method of embodiment 32, wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 34

The method of embodiment 31, wherein said modulator is an agonist.

Embodiment 35

The method of embodiment 34, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 36

The method of embodiment 31, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 37

A method of determining whether a subject has or is at risk of developing an LPA-associated disease, said method comprising: (i) detecting an expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 in a subject; (ii) determining whether said expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 or a decreased expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to said standard control indicates that said subject has or is at risk of developing an LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said subject has or is at risk for developing an LPA-associated disease.

Embodiment 38

The method of embodiment 37, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 39

The method of embodiment 37, wherein said expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 is detected from a biological sample of said subject.

Embodiment 40

The method of embodiment 39, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject.

Embodiment 41

The method of embodiment 40, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 42

The method of embodiment 37, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker RNA set forth in Table 3 or Table 4.

Embodiment 43

The method of embodiment 42, wherein said modulator is an antagonist.

Embodiment 44

The method of embodiment 43, wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 45

The method of embodiment 42, wherein said modulator is an agonist.

Embodiment 46

The method of embodiment 45, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 47

The method of embodiment 42, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 48

A method of determining an LPA-associated disease activity in a patient, said method comprising: (i) determining a first expression level of a protein set forth in Table 1 or Table 2 in said patient at a first time point; (ii) determining a second expression level of a protein set forth in Table 1 or Table 2 in said patient at a second time point; (iii) comparing said second expression level of a protein set forth in Table 1 or Table 2 to said first expression level of a protein set forth in Table 1 or Table 2, thereby determining said LPA-associated disease activity in said patient.

Embodiment 49

A method of determining an LPA-associated disease activity in a patient, said method comprising: (i) determining a first expression level of an RNA set forth in Table 3 or Table 4 in said patient at a first time point; (ii) determining a second expression level of an RNA set forth in Table 3 or Table 4 in said patient at a second time point; (iii) comparing said second expression level of an RNA set forth in Table 3 or Table 4 to said first expression level of an RNA set forth in Table 3 or Table 4, thereby determining said LPA-associated disease activity in said patient.

Embodiment 50

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising administering to said subject an effective amount of an modulator of an LPA-associated disease marker protein set forth in Table 1 or Table 2, thereby treating an LPA-associated disease in said subject.

Embodiment 51

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising administering to said subject an effective amount of an modulator of an LPA-associated disease marker RNA set forth in Table 3 or Table 4, thereby treating an LPA-associated disease in said subject.

Embodiment 52

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising: (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 relative to a standard control; and (ii) when an elevated expression level or a decreased expression level of said LPA-associated disease marker protein set forth in Table 1 or Table 2 is found relative to said standard control, administering to said subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker protein set forth in Table 1 or Table 2, thereby treating said subject.

Embodiment 53

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising: (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to a standard control; and (ii) when an elevated expression level or a decreased expression level of said LPA-associated disease marker RNA set forth in Table 3 or Table 4 is found relative to said standard control, administering to said subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker RNA set forth in Table 3 or Table 4, thereby treating said subject.

Embodiment 54

A method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease, the method comprising: (i) detecting an expression level of one or more LPA-associated disease marker proteins set forth in Table 1 or Table 2 in an LPA-associated disease patient; (ii) determining whether said expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker protein set forth in Table 1 or Table 2 relative to said standard control indicates that said LPA-associated disease patient is at risk for progression of said LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said LPA-associated disease patient is at risk for progression of said LPA-associated disease.

Embodiment 55

A method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease, the method comprising: (i) detecting an expression level of one or more LPA-associated disease marker RNAs set forth in Table 3 or Table 4 in an LPA-associated disease patient; (ii) determining whether said expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker RNA set forth in Table 3 or Table 4 relative to said standard control indicates that said LPA-associated disease patient is at risk for progression of said LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said LPA-associated disease patient is at risk for progression of said LPA-associated disease.

Embodiments II
Embodiment 1

A method of determining an expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 in a subject that has or is at risk for developing an LPA-associated disease, said method comprising: (i) obtaining a biological sample from said subject; and (ii) determining an expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 in said biological sample.

Embodiment 2

The method of embodiment 1, wherein said determining comprises: (a) contacting said LPA-associated disease marker protein with a marker protein binding agent in said biological sample, thereby forming a disease marker protein-binding agent complex; and (b) detecting said disease marker protein-binding agent complex.

Embodiment 3

The method of embodiment 2, wherein said marker protein binding agent comprises a detectable moiety.

Embodiment 4

The method of embodiment 2, wherein said marker protein binding agent comprises a capturing moiety.

Embodiment 5

The method of embodiment 4, wherein said capturing moiety is a cleavable capturing moiety.

Embodiment 6

The method of one of embodiments 2-5, wherein said detecting comprises contacting said disease marker protein-binding agent complex with a capturing agent, thereby forming a captured disease marker protein-binding agent complex.

Embodiment 7

The method of embodiment 6, wherein said detecting further comprises: (1) contacting said captured disease marker protein-binding agent complex with a tagging moiety; thereby forming a tagged disease marker protein-binding agent complex; and (2) separating said tagged disease marker protein-binding agent complex from said biological sample.

Embodiment 8

The method of embodiment 7, wherein said detecting further comprises after said separating of step (2) separating said capturing binding agent from said tagged disease marker protein-binding agent complex, thereby forming a cleaved disease marker protein-binding agent complex.

Embodiment 9

The method of embodiment 8, wherein said detecting further comprises: (3) separating said marker protein binding agent from said cleaved disease marker protein-binding agent complex; thereby forming a released marker protein binding agent; and (4) determining an amount of said released marker protein binding agent.

Embodiment 10

The method of one of embodiments 1-9, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 11

The method of one of embodiments 1-10, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject.

Embodiment 12

The method of embodiment 11, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 13

The method of one of embodiments 1-12, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker protein of SEQ ID NO:1-202.

Embodiment 14

The method of embodiment 13, wherein said modulator is an antagonist.

Embodiment 15

The method of embodiment 14 wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer

Embodiment 16

The method of embodiment 14, wherein said expression level of said LPA-associated disease marker protein of SEQ ID NO:1-202 is elevated relative to a standard control.

Embodiment 17

The method of embodiment 13, wherein said modulator is an agonist.

Embodiment 18

The method of embodiment 17, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 19

The method of embodiment 17, wherein said expression level of said LPA-associated disease marker protein of SEQ ID NO:1-202 is decreased relative to a standard control.

Embodiment 20

The method of one of embodiments 13-19, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 21

The method of one of embodiments 1-20, wherein said LPA-associated disease marker protein is SEQ ID NO:9, SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:47, SEQ ID NO:58, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:102, SEQ ID NO:118, SEQ ID NO:131, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO:160, SEQ ID NO:164, SEQ ID NO:183 or SEQ ID NO:185.

Embodiment 22

A method of determining an expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 in a subject that has or is at risk for developing an LPA-associated disease, said method comprising: (i) obtaining a biological sample from said subject; and (ii) determining an expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 in said biological sample.

Embodiment 23

The method of embodiment 22, wherein said determining comprises: (a) contacting said LPA-associated disease marker RNA with a marker RNA binding agent in said biological sample, thereby forming a disease marker RNA-binding agent complex; and (b) detecting said disease marker RNA-binding agent complex.

Embodiment 24

The method of embodiment 23, wherein said marker RNA binding agent comprises a detectable moiety.

Embodiment 25

The method of embodiment 23, wherein said marker RNA binding agent comprises a capturing moiety.

Embodiment 26

The method of embodiment 25, wherein said capturing moiety is a cleavable capturing moiety.

Embodiment 27

The method of one of embodiments 23-26, wherein said detecting comprises contacting said disease marker RNA-binding agent complex with a capturing agent, thereby forming a captured disease marker RNA-binding agent complex.

Embodiment 28

The method of embodiment 27, wherein said detecting further comprises: (1) contacting said captured disease marker RNA-binding agent complex with a tagging moiety; thereby forming a tagged disease marker RNA-binding agent complex; and (2) separating said tagged disease marker RNA-binding agent complex from said biological sample.

Embodiment 29

The method of embodiment 28, wherein said detecting further comprises after said separating of step (2) separating said capturing binding agent from said tagged disease marker RNA-binding agent complex, thereby forming a cleaved disease marker RNA-binding agent complex.

Embodiment 30

The method of embodiment 29, wherein said detecting further comprises: (3) separating said marker RNA binding agent from said cleaved disease marker RNA-binding agent complex; thereby forming a released marker RNA binding agent; and (4) determining an amount of said released marker RNA binding agent.

Embodiment 31

The method of one of embodiments 22-30, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 32

The method of one of embodiments 22-31, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject

Embodiment 33

The method of embodiment 32, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 34

The method of one of embodiments 22-33, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker RNA of SEQ ID NO:203-499.

Embodiment 35

The method of embodiment 34, wherein said modulator is an antagonist.

Embodiment 36

The method of embodiment 35, wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 37

The method of embodiment 35, wherein said expression level of said LPA-associated disease marker RNA of SEQ ID NO:203-499 is elevated relative to a standard control.

Embodiment 38

The method of embodiment 34, wherein said modulator is an agonist.

Embodiment 39

The method of embodiment 38, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 40

The method of embodiment 38, wherein said expression level of said LPA-associated disease marker RNA of SEQ ID NO:203-499 is decreased relative to a standard control.

Embodiment 41

The method of one of embodiments 34-40, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 42

The method of one of embodiments 22-41, wherein said LPA-associated disease marker RNA is SEQ ID NO:209, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:222, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:303, SEQ ID NO:309, SEQ ID NO:353, SEQ ID NO:364, SEQ ID NO:378, SEQ ID NO:396, SEQ ID NO:417, SEQ ID NO:432 or SEQ ID NO:448.

Embodiment 43

A method of determining whether a subject has or is at risk of developing an LPA-associated disease, said method comprising: (i) detecting an expression level of one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 in a subject; (ii) determining whether said expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 or a decreased expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to said standard control indicates that said subject has or is at risk of developing an LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said subject has or is at risk for developing an LPA-associated disease.

Embodiment 44

The method of embodiment 43, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 45

The method of embodiment 43, wherein said expression level of one or more LPA-associated disease marker proteins of SEQ ID NO:1-202 is detected from a biological sample of said subject.

Embodiment 46

The method of embodiment 45, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject.

Embodiment 47

The method of embodiment 46, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 48

The method of embodiment 43, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker protein of SEQ ID NO:1-202.

Embodiment 49

The method of embodiment 48, wherein said modulator is an antagonist.

Embodiment 50

The method of embodiment 49, wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 51

The method of embodiment 48, wherein said modulator is an agonist.

Embodiment 52

The method of embodiment 51, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 53

The method of embodiment 48, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 54

A method of determining whether a subject has or is at risk of developing an LPA-associated disease, said method comprising: (i) detecting an expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 in a subject; (ii) determining whether said expression level is increased or decreased relative to a standard control, wherein an elevated expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 or a decreased expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to said standard control indicates that said subject has or is at risk of developing an LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said subject has or is at risk for developing an LPA-associated disease.

Embodiment 55

The method of embodiment 54, further comprising selecting a subject that has or is at risk for developing an LPA-associated disease.

Embodiment 56

The method of embodiment 54, wherein said expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 is detected from a biological sample of said subject.

Embodiment 57

The method of embodiment 56, wherein said biological sample is a blood-derived biological sample, a urine-derived biological sample or a saliva-derived biological sample of said subject.

Embodiment 58

The method of embodiment 57, wherein said blood-derived biological sample is whole blood, serum or plasma.

Embodiment 59

The method of embodiment 54, further comprising administering to said subject an effective amount of a modulator of said LPA-associated disease marker RNA of SEQ ID NO:203-499.

Embodiment 60

The method of embodiment 59, wherein said modulator is an antagonist.

Embodiment 61

The method of embodiment 60, wherein said antagonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 62

The method of embodiment 59, wherein said modulator is an agonist.

Embodiment 63

The method of embodiment 62, wherein said agonist is a peptide, small molecule, nucleic acid, antibody or aptamer.

Embodiment 64

The method of embodiment 59, further comprising administering to said subject an effective amount of a further therapeutic agent.

Embodiment 65

A method of determining an LPA-associated disease activity in a patient, said method comprising: (i) determining a first expression level of a protein of SEQ ID NO:1-202 in said patient at a first time point; (ii) determining a second expression level of a protein of SEQ ID NO:1-202 in said patient at a second time point; (iii) comparing said second expression level of a protein of SEQ ID NO:1-202 to said first expression level of a protein of SEQ ID NO:1-202, thereby determining said LPA-associated disease activity in said patient.

Embodiment 66

A method of determining an LPA-associated disease activity in a patient, said method comprising: (i) determining a first expression level of an RNA of SEQ ID NO:203-499 in said patient at a first time point; (ii) determining a second expression level of an RNA of SEQ ID NO:203-499 in said patient at a second time point; (iii) comparing said second expression level of an RNA of SEQ ID NO:203-499 to said first expression level of an RNA of SEQ ID NO:203-499, thereby determining said LPA-associated disease activity in said patient.

Embodiment 67

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising administering to said subject an effective amount of an modulator of an LPA-associated disease marker protein of SEQ ID NO:1-202, thereby treating an LPA-associated disease in said subject.

Embodiment 68

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising administering to said subject an effective amount of an modulator of an LPA-associated disease marker RNA of SEQ ID NO:203-499, thereby treating an LPA-associated disease in said subject.

Embodiment 69

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising: (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to a standard control; and (ii) when an elevated expression level or a decreased expression level of said LPA-associated disease marker protein of SEQ ID NO:1-202 is found relative to said standard control, administering to said subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker protein of SEQ ID NO:1-202, thereby treating said subject.

Embodiment 70

A method of treating an LPA-associated disease in a subject in need thereof, said method comprising: (i) determining whether a subject expresses an elevated level or a decreased level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to a standard control; and (ii) when an elevated expression level or a decreased expression level of said LPA-associated disease marker RNA of SEQ ID NO:203-499 is found relative to said standard control, administering to said subject an LPA-associated disease treatment, an antagonist or an agonist of an LPA-associated disease marker RNA of SEQ ID NO:203-499, thereby treating said subject.

Embodiment 71

A method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease, the method comprising: (i) detecting an expression level of one or more LPA-associated disease marker proteins set forth of SEQ ID NO:1-202 in an LPA-associated disease patient; (ii) determining whether said expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker protein of SEQ ID NO:1-202 relative to said standard control indicates that said LPA-associated disease patient is at risk for progression of said LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said LPA-associated disease patient is at risk for progression of said LPA-associated disease.

Embodiment 72

A method of determining whether an LPA-associated disease patient is at risk for progression of the LPA-associated disease, the method comprising: (i) detecting an expression level of one or more LPA-associated disease marker RNAs of SEQ ID NO:203-499 in an LPA-associated disease patient; (ii) determining whether said expression level is modulated relative to a standard control, wherein a modulated expression level of an LPA-associated disease marker RNA of SEQ ID NO:203-499 relative to said standard control indicates that said LPA-associated disease patient is at risk for progression of said LPA-associated disease; and (iii) based at least in part on said expression level in step (ii), determining whether said LPA-associated disease patient is at risk for progression of said LPA-associated disease.

Embodiment 73

A complex in vitro comprising a marker protein binding agent bound to a LPA-associated disease marker protein of SEQ ID NO:1-202 or fragment thereof, wherein said LPA-associated disease marker protein is extracted from a human subject having or at risk of developing an LPA-associated disease.

Embodiment 74

The complex of embodiment 73, wherein said subject has an LPA-associated disease.

Embodiment 75

A complex in vitro comprising a marker RNA binding agent bound to a LPA-associated disease marker RNA of SEQ ID NO:203-499 or fragment thereof, wherein said LPA-associated disease marker RNA is extracted from a human subject having or at risk of developing an LPA-associated disease.

Embodiment 76

The complex of embodiment 75, wherein said subject has an LPA-associated disease.

Embodiment 77

A kit comprising: (a) a marker protein binding agent capable of binding to a substance within a biological sample from a human subject having or at risk of developing an LPA-associated disease; wherein said substance is an LPA-associated disease marker protein of SEQ ID NO:1-202 or fragment thereof; (b) a detecting reagent or a detecting apparatus capable of indicating binding of said marker protein binding agent to said substance.

Embodiment 78

A kit comprising: (a) a marker RNA binding agent capable of binding to a substance within a biological sample from a human subject having or at risk of developing an LPA-associated disease; wherein said substance is an LPA-associated disease marker RNA of SEQ ID NO:203-499 or fragment thereof; (b) a detecting reagent or a detecting apparatus capable of indicating binding of said marker RNA binding agent to said substance.

	Number	Date	Country
Parent	PCT/US2015/031427	May 2015	US
Child	15351719		US

LPA-ASSOCIATED PROTEIN AND RNA EXPRESSION

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