Patent Publication Number: US-2006019890-A1

Title: Method for treating cardiac remodeling following myocardial injury

Description:
This application claims priority to U.S. provisional application Ser. No. 60/537,221. The 60/537,221 provisional application is herein incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION  
      The present invention concerns methods of treatment using one or more natriuretic peptides or derivatives thereof. More specifically, the invention concerns methods of treating or preventing cardiac dysfunction in a subject after said subject has undergone myocardial injury.  
     BACKGROUND  
      Myocardial infarction is a major cause of significant disability and death in the United States and in many other countries around the world, and accounts for approximately ⅔ of all heart failure. Hunt et al, AMERICAN COLLEGE OF CARDIOLOGY/AMERICAN Heart Association. ACC/AHA guidelines for the evaluation and management of chronic heart failure in the adult: executive summary. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to revise the 1995 Guidelines for the Evaluation and Management of Heart Failure). Journal of the American College of Cardiology 2001; 38: 2101-2113. Several disease-initiating events (e.g. myocardial infarction, untreated hypertension, congenital mutations of contractile proteins) can result in a common heart disease phenotype that consists of dilation of the cardiac chambers, resulting in reduction in contractile function (i.e., a decrease in the fraction of total blood ejected from each chamber during systole) that leads to the clinical syndrome of heart failure. This phenotype generally involves a compensatory aspect that results from myocardial infarction when the normal compensatory hypertrophy of surviving, non-infarcted myocardium is insufficient. Often this compensatory mechanism is a result of the profibrotic response associated with cardiac injury.  
      Available therapies for heart dysfunction are insufficient, and new methods of treatment are needed. The heart responds to infarction by hypertrophy of surviving cardiac muscle in an attempt to maintain normal contraction. However, when the hypertrophy is insufficient to compensate, cardiac remodeling and reduced cardiac function result, leading to heart failure and death. Despite important advances in medical therapies for preventing cardiac dysfunction and heart failure after myocardial infarction, these problems remain a significant unsolved public health problem.  
      No pharmacological therapy for post MI cardiac remodeling is curative or satisfactory, and many patients die or, in selected cases, undergo heart transplantation. Presently available pharmacological therapies for reducing cardiac dysfunction and reducing mortality in patients with heart failure fall into three main categories: angiotensin-converting enzyme (ACE) inhibitors, beta adrenergic receptor (OAR) antagonists, and aldosterone antagonists. Despite reducing mortality, patients treated with these medicines remain at significantly increased rislc for death compared to age-matched control patients without heart failure. ACE inhibitors, βAR antagonists and (at least one type of) aldosterone receptor antagonist can significantly reduce the incidence and extent of cardiac dysfunction and heart failure after myocardial infarction.  
      ACE inhibitors are associated with cough in 10% of patients and can result in renal failure in the setting of bilateral renal artery stenosis or other severe kidney disease. βAR antagonists are associated with impotence and depression, and are contraindicated in patients with asthma; furthermore, patients may develop worsened heart failure, hypotension, bradycardia, heart block, and fatigue with initiation of βAR antagonists. Aldosterone receptor antagonism causes significant hyperkalemia and painful gynecomastia in 10% of male patients. Agents without a demonstrated mortality benefit are also associated with problems; most notable is the consistent finding that many cardiac stimulants improve symptoms, but actually increase mortality, likely by triggering lethal cardiac arrhythmias. In summary, presently available pharmacological therapies are ineffective and are limited by significant unwanted side effects, and so development of new therapies with improved efficacy and less severe side effects is an important public health goal.  
     SUMMARY OF THE INVENTION  
      The present invention is directed to the use of natriuretic peptides for the prevention and/or treatment of cardiac remodeling in a subject that has undergone myocardial injury. In a preferred embodiment, the natriuretic peptide(s) comprise brain natriuretic peptide (BNP), also known as nesiritide. In another embodiment, the invention is directed to the treatment of cardiac dysfunction, said treatment comprising the administration of a therapeutically effective amount of natriuretic peptide to a subject that has undergone myocardial injury.  
      In another related embodiment, the invention is directed to a method of alleviating or reversing the effect of TGFβ mediated cell activation in cardiac tissue on the expression of one or more genes associated with fibrosis, comprising contacting one or more cells or tissues in which the expression of said genes is altered as a result of TGFβ mediated activation, with BNP. In another related embodiment, the targeted gene(s) associated with fibrosis are selected from the group consisting essentially of Collagen1, Collagent 3, Fibronectin, CTGF, PAI-1, and TIMP3.  
      In another embodiment, the invention is directed to a method of inhibiting the production of Collagen 1, Collagen 3 or Fibronectin proteins by the administration of a therapeutically effective amount of BNP to a subject in need thereof.  
      In another related embodiment, the invention is directed to a method of inhibiting TGFβ mediated myofibroblast conversion by administration of a therapeutically effective amount of BNP to a mammalian subject in need thereof.  
      In another related embodiment, the invention is directed to a method of alleviating or reversing the effect of TGFβ mediated cell activation in cardiac tissue on the expression of one or more genes associated with cell proliferation, comprising contacting one or more cells or tissues in which the expression of said genes is altered as a result of TGFβ mediated activation, with BNP. In another related embodiment, the targeted gene(s) associated with cell proliferation are selected from the group consisting essentially of PDGFA, IGF1, FGF18, and IGFBP10.  
      In another related embodiment, the invention is directed to a method of alleviating or reversing the effect of TGFβ mediated cell activation in cardiac tissue on the expression of one or more genes associated with inflammation, comprising contacting one or more cells or tissues in which the expression of said genes is altered as a result of TGFβ mediated activation, with BNP. In another related embodiment, the targeted gene(s) associated with inflammation are selected from the group comprise COX1, IL6, TNFα-inducted protein 6, TNF superfamily, member 4. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1 . Gene expression changes induced by TGFβ and BNP in human cardiac fibroblasts at 24 and 48 h. Histograms show the number of gene expression changes that were up-regulated and down-regulated by TGFβ and BNP treatment. Hybridizations using fluorescently-labeled cDNA probes compare untreated (control) to TGFβ-treated cells and control to BNP-treated cells. See Experimental for details related to the gene expression values. Histogram bars: 24 h (white) and 48 h (black).  
       FIG. 2 . Effects of BNP on TGFβ-induced gene expression in human cardiac fibroblasts. Hybridizations using fluorescently-labeled cDNA probes compare TGFβ-treated to TGFβ BNP-treated cells at 24 and 48 h. Strong and weak effects represent 1.8- and 1.5-fold gene expression levels, respectively. See Experimental for details related to statistical significance. Histogram bars: no effect (white), weak effect (grey), and strong effect (black).  
       FIG. 3 . Gene expression patterns in TGFβ-treated human cardiac fibroblasts. Data was generated using the hierarchical clustering algorithm contained in Spotfire™ software. Each row represents one of 524 genes, and each column represents the results from duplicate hybridizations: (A) control vs. TGFβ, 24 h; (B) control vs. TGFβ, 48 h; (C) TGFβ vs. TGFβ+BNP 24 h; (D) TGFβ vs. TGFβ+BNP 48 h; (E) control vs. BNP 24 h; and (F) control vs. BNP 48 h. Normalized data values depicted in shades of red and green represent elevated and repressed expression, respectively. See Table 2 in Experimental section for gene identities and expression values.  
       FIG. 4 . Gene expression clusters in human cardiac fibroblasts: (A) fibrosis and ECM, (B) cell proliferation, and (C) inflammation. See  FIG. 4  legend for descriptions of the hybridizations and gene expression color codes.  
       FIG. 5 . Effects of BNP on TGFβ-induced Collagen 1 (A and B) and Fibronectin (C and D) mRNA and protein levels in cultured human cardiac fibroblasts. Histograms show control cells (white), cells treated with BNP (gray), cells treated with TGFβblack), and cells co-treated with BNP and TGFβ(hatched). (A and C) Real-time RT-PCR expression levels were normalized to 18S rRNA and plotted relative to the level in the 6 h control cells. Error bars reflect duplicate biological replicates; real-time RT-PCR reactions were performed in triplicate. (B and D) Western blot analyses are presented as mean±SD from three separate experiments; *p&lt;0.01 vs. control; **p&lt;0.01 vs. TGFβ.  
       FIG. 6 . Effects of BNP on TGFβ-induced fibrotic and inflammatory genes. Real-time RT-PCR expression levels were normalized to 18S rRNA and plotted relative to the level in the 6 h control cells. See  FIG. 5  for key to histogram bar labels and error bars.  
       FIG. 7 . Effect of PKG and MEK inhibitors on BNP-dependent inhibition of TGFβ signaling in human cardiac fibroblasts. (A) Western analysis of ERK phosphorylation. Cells were treated with BNP (0.5 μmol/L) in the presence or absence of KT5823 (1 μmol/L) or U0126 (10 μmol/L) for 15 min. (B) Western blot and (C) real-time RT-PCR analysis to detect Collagen 1 expression. Cells were treated with 5 ng/ml TGFβ and/or BNP (100 nmol/L, three times daily) in the presence or absence of KT5823 (1 μmol/L), U0126 (0.1-10 μmol/L) or PD98059 (10 μmol/L) for 48 h. Control (C); KT5823 (KT); U0126 (U); TGFβ (TGF).  
       FIG. 8 . Summary of BNP effects on gene expression in TGFβ-stimulated human cardiac fibroblasts.  
       FIG. 9 . Effects of BNP on TGFβ-stimulated fibroblast proliferation. Histograms show fold induction of BrdU labeled cells treated with TGFβ alone, BNP alone or co-treated with BNP and TGFβ. Cells were co-treated with BNP and TGFβ for 24 h, then labeled with BrdU and cultured for an additional 24 h. Pooled data represent the mean±SD from three individual experiments: *p&lt;0.01 vs. the control; **p&lt;0.05 vs. TGFβ.  
       FIG. 10 . Changes in plasma aldosterone level. The increased plasma aldosterone level by L-NAME/AngII was reduced by BNP (p&lt;0.05, n=7)  
       FIG. 11 . Changes in heart/body weight ratio. BNP abolished L-NAME/AngII-induced increase in heart/body weight ratio (p&lt;0.01, n=12)  
       FIG. 12 . Real time RT-PCR results. Expression of mRNA of collagen I (A), collagen III (B) and fibronectin (C) in the heart. BNP abolished the fibrotic genes that enhanced by L-NAME plus Angiotensin II (p&lt;0.01 in all cases).  
       FIG. 13 . Cardiac function parameters including heart rate (A), stroke volume (B), ejection fraction (C), cardiac output (D), stroke work (E), maximum dP/dt (F), minimum dP/fy (G), and arterial elastance (H). L-NAME/AngII induced deterioration of cardiac function. Administration of BNP significantly improved cardiac function as judged by increases in stroke volume, ejection fraction, cardiac output, stroke work and decrease in arterial elastance (p&lt;0.001, n=8). BNP also increased maximum dP/dt (p&lt;0.05) and minimum dP/dt. BNP had no effect on heart rate.  
    
    
     DETAILED DESCRIPTION  
      A. Definitions  
      As used herein, any reference to “reversing the effect of TGF-β-mediated cell activation on the expression of a gene associated with fibrosis” means partial or complete reversal the effect of TGF-β-mediated cell activation of that gene, relative to a normal sample of the same cell or tissue type. It is emphasized that total reversal (i.e. total return to the normal expression level) is not required, although is advantageous, under this definition.  
      The term “cardiac remodeling” generally refers to the compensatory or pathological response following myocardial injury. Cardiac remodeling is viewed as a key determinant of the clinical outcome in heart disorders. It is characterized by a structural rearrangement of the cardiac chamber wall that involves cardiomyocyte hypertrophy, fibroblast proliferation, and increased deposition of extracellular matrix (ECM) proteins. Cardiac fibrosis is a major aspect of the pathology typically seen in the failing heart. The proliferation of interstitial fibroblasts and increased deposition of extracellular matrix components results in myocardial stiffness and diastolic dysfunction, which ultimately leads to heart failure. A number of neurohumoral or growth factors have been implicated in the development of cardiac fibrosis. These include angiotensin II (AII), endothelin-1 (ET-1), cardiotrophin-1 (CT-1), norepinephrine (NE), aldosterone, FGF2, PDGF, and transforming growth factored (TGFβ). TGFβ expression is also stimulated by AII and ET-1 in cardiac myocytes and fibroblasts, further supporting its involvement in cardiac fibrosis.  
      The term “cardiac dysfunction” refers to the pathological decline in cardiac performance following myocardial injury. Cardiac dysfunction may be manifested through one or more parameters or indicia including changes to stroke volume, ejection fraction, end diastolic fraction, stroke work, arterial elastance, or an increase in heart weight to body weight ratio.  
      The terms “differentially expressed gene,” “differential gene expression” and their synonyms, which are used interchangeably, refer to a gene whose expression is activated to a higher or lower level in a test sample relative to its expression in a normal or control sample. For the purpose of this invention, “differential gene expression” is considered to be present when there is at least an about 2.5-fold, preferably at least about 4-fold, more preferably at least about 6-fold, most preferably at least about 10-fold difference between the expression of a given gene in normal and test samples.  
      “Myocardial injury” means injury to the heart. It may arise from myocardial infarction, cardiac ischemia, cardiotoxic compounds and the like. Myocardial injury may be either an acute or nonacute injury in terms of clinical pathology. In any case it involves damage to cardiac tissue and typically results in a structural or compensatory response.  
      As used herein, “natriuretic peptides” means a composition that includes one or more of an Atrial natriuretic peptide (ANP), a Brain natriuretic peptide (BNP), or a C-type natriuretic peptide (CNP). It is contemplated that analogues and variants of these peptides be included in the definition. Examples of such include anaritide (ANP analogue of different length) or combinations of natriuretic peptide including but not limited to ANP/BNP, ANP/CNP, an BNP/CNP variants. Preferably, natriuretic peptide means BNP (nesiritide).  
      The terms “treating” or “alleviating” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In the treatment of a fibroproliferative disease, a therapeutic agent may directly decrease the pathology of the disease, or render the disease more susceptible to treatment by other therapeutic agents.  
      The term “subject” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the subject is human.  
      Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.  
      A “therapeutically effective amount”, in reference to the treatment of cardiac or renal fibrosis, e.g. when inhibitors of the present invention are used, refers to an amount capable of invoking one or more of the following effects: (1) inhibition (i.e., reduction, slowing down or complete stopping) of the development or progression of fibrosis and/or sclerosis; (2) inhibition (i.e., reduction, slowing down or complete stopping) of consequences of or complications resulting from such fibrosis and/or sclerosis; and (3) relief, to some extent, of one or more symptoms associated with the fibrosis and/or sclerosis, or symptoms of consequences of or complications resulting from such fibrosis and/or sclerosis.  
      B. Modes of Carrying out the Invention  
      Natriuretic peptides comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are predominantly produced in the heart and exert vasorelaxant, natriuretic, and anti-growth activities. Binding of ANP and BNP to type-A natriuretic peptide receptor (NPRA) leads to the generation of cyclic guanosine monophosphate (cGMP), which mediates most biological effects of the peptides. Mice lacking NPRA exhibit cardiac hypertrophy, fibrosis, hypertension and increased expression of fibrotic genes including TGFβ1, TGFβ3 and Collagen 1. Furthermore, targeted disruption of the BNP gene in mice results in cardiac fibrosis and enhanced fibrotic response to ventricular pressure overload, suggesting that BNP is involved in cardiac remodeling.  
      TGFβ mediates fibrosis by modulating fibroblast proliferation and ECM production, particularly of collagen and fibronectin. TGFβ also promotes the phenotypic transformation of fibroblasts into myofibroblasts characterized by expression of α-smooth muscle actin. Studies have demonstrated that increased myocardial TGFβ expression is associated with cardiac hypertrophy and fibrosis. Moreover, functional blockade of TGFβ prevents myocardial fibrosis and diastolic dysfunction in pressure overloaded rats, indicating that TGFβ has a crucial role in the process of myocardial remodeling, particularly in cardiac fibrosis. However, the implication of natriuretic peptide(s) in this process has not been previously explored.  
      The present invention is directed to the treatment or prevention of cardiac remodeling following myocardial injury. In a preferred embodiment, the myocardial injury comprises an acute myocardial infarction. Preferably the administration of natriuretic peptide occurs as soon as possible after the injury event.  
      In another embodiment, the invention involves the treatment of cardiac dysfunction in a subject in need thereof comprising the administration of a natriuretic peptide to a subject in need thereof wherein said administration occurs after said subject has undergone myocardial injury.  
      The manner of administration and formulation of the natriuretic peptide(s) useful in the invention will depend on the nature of the condition, the severity of the condition, the particular subject to be treated, and the judgment of the practitioner; formulation will depend on mode of administration. The peptides of the invention are conveniently administered by oral administration by compounding them with suitable pharmaceutical excipients so as to provide tablets, capsules, syrups, and the like. Suitable formulations for oral administration may also include minor components such as buffers, flavoring agents and the like. Typically, the amount of active ingredient in the formulations will be in the range of about 5%-95% of the total formulation, but wide variation is permitted depending on the carrier. Suitable carriers include sucrose, pectin, magnesium stearate, lactose, peanut oil, olive oil, water, and the like.  
      The peptides useful in the invention may also be administered through suppositories or other transmucosal vehicles. Typically, such formulations will include excipients that facilitate the passage of the compound through the mucosa such as pharmaceutically acceptable detergents.  
      The peptides may also be administered by injection, including intravenous, intramuscular, subcutaneous, intrarticular or intraperitoneal injection. Preferably the natriuretic peptide(s) are administered intravenously. Typical formulations for such use are liquid formulations in isotonic vehicles such as Hank&#39;s solution or Ringer&#39;s solution.  
      Alternative formulations include aerosol inhalants, nasal sprays, liposomal formulations, slow-release formulations, and the like, as are known in the art.  
      Any suitable formulation may be used. A compendium of art-known formulations is found in  Remington&#39;s Pharmaceutical Sciences , latest edition, Mack Publishing Company, Easton, Pa. Reference to this manual is routine in the art.  
      The dosages of the peptide(s) of the invention will depend on a number of factors which will vary from patient to patient. The dose regimen will vary, depending on the conditions being treated and the judgment of the practitioner. Further information regarding related formulations and dosages for brain natriuretic peptide can be found in the package insert or the latest version of Physicians Desk Reference (PDR) for nesiritide or the Natrecor® product.  
      It should be noted that the peptides useful for the invention can be administered as individual active ingredients, or as mixtures of several different compounds. In addition, the peptide(s) can be used as single therapeutic agents or in combination with other therapeutic agents. Drugs that could be usefully combined with these compounds include natural or synthetic corticosteroids, particularly prednisone and its derivatives, monoclonal antibodies targeting cells of the immune system or genes associated with the development or progression of fibrotic diseases, and small molecule inhibitors of cell division, protein synthesis, or mRNA transcription or translation, or inhibitors of immune cell differentiation or activation.  
      As implicated above, although the peptide(s) of the invention may be used in humans, they are also available for veterinary use in treating non-human mammalian subjects.  
      Further details of the invention will be apparent from the Experimental section as provided below.  
     EXPERIMENTAL  
      In vitro  
      Cell Culture  
      Two lots of primary human cardiac fibroblasts, derived from an 18-year old Caucasian male (lot 1) and a 56-year old Caucasian male (lot 2), were provided by Cambrex Bio Science (Walkersville, Md.). Cells stained positive for α-smooth muscle actin and vimenfin antibodies corroborating their identity as cardiac fibroblasts and myofibroblasts. Both lots were used for the real-time RT-PCR studies; lot 1 was used for the microarray analysis. Cells at passage 3-5 were cultured in FGM containing 15% FBS. At confluence, cells were split and cultured in 6-well plates for 24 h. Cells were changed to serum-free medium and treated with human BNP (American Peptide Company, Sunnyvale, Calif.) in the presence or absence of 5 ng/ml of TGFβ (R&amp;D systems, Minneapolis, Minn.) for 6, 24 and 48 h. BNP and/or TGFβ-treated cells were also incubated in the presence of cGMP-dependent protein kinase (PKG) inhibitor KT5823 (1 μmol/L, Calbiochem, San Diego, Calif.), MAP kinase kinase (MEK) inhibitor U0126 (0.1-10 μmol/L, Sigma, St. Louis, Mo.) or PD98059 (10 μmol/L, Sigma) for 48 h. BNP (100 nmol/L) was added into the medium three times a day, such that the total calculated concentrations of exogenous BNP were 200 nmol/L, 600 nmol/L, and 900 nmol/L at 6, 24, and 48 h, respectively. This dosing protocol was necessary to maintain the levels of BNP in culture, since two distinct clearance pathways are responsible for the rapid degradation of natriuretic peptides. Without this treatment regime, it was found that BNP was significantly degraded in the cardiac fibroblasts; 50% of added BNP was metabolized within 24 h as measured by immunoreactive assays and cGMP stimulation cell bioassays.  
      Intracellular cGMP Assay  
      Cells were cultured in 6-well plates for 24 h, then changed to serum-free medium, and pre-incubated with 0.1 mmol/L of 3-isobutyl-1-methylxanthine (IBMX) for 1 h before treating with 10 −9 -10 −6  mol/L of BNP for 10 min. The medium was aspirated and 0.5 ml of cold PBS was added into each well. Cells were scraped and mixed with 2 volumes of cold ethanol by vortex. After a 5 min room temperature incubation, the precipitate was removed by centrifugation at 1500×g for 10 min. The supernatant was dried by vacuum centrifugation, and levels of cGMP were measured using the cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, Mich.).  
      BrdU incorporation  
      Cells were placed in 96-well plates and cultured for 24 h before changing to serum-free medium. Cells were treated with BNP (100 nmol/L, three times a day) in the presence or absence of 5 ng/ml of TGF-β for 24 h. Subsequently, 10 μmol/L of 5-bromo-2′-deoxyuridine (BrdU) was added to the cells, and they were cultured for an additional 24 h. BrdU incorporation was detected using the Cell Proliferation ELISA kit (Roche, Indianapolis, Ind.). Data was analyzed by ANOVA using the Newman-Keuls test to assess significance.  
      cDNA Microarray  
      Gene expression profiles were determined from cDNA microarrays containing 8,600 elements derived from clones isolated from normalized cDNA libraries or purchased from ResGen (Invitrogen Life Technologies, Carlsbad, Calif.). DNA for spotting was generated by PCR amplification using 5′amino-modified primers (BD Biosciences Clontech, Palo Alto, Calif.) derived from flanking vector sequences. Amplified DNA was purified in a 96-well format using Qiagen&#39;s Qiaquick columns (Valencia, Calif.) according to the manufacturer&#39;s recommendations. Samples were eluted in Milli-Q purified water, dried to completion and resuspended in 7 μl of 3×SSC. A fluorescent assay using PicoGreen (Molecular Probes, Eugene, Oreg.) was randomly performed on 12% of the PCR products to determine the average yield after purification; yields were ˜1.5 μg of DNA which corresponds to a concentration of 214 μg/ml. Purified DNA was arrayed from 384-well microtiter plates onto lysine-coated glass slides using an OmniGrid II microarrayer (GeneMachines, San Carlos, Calif.). After printing, DNA was cross-linked to the glass with 65 mjoules UV irradiation and reactive amines were blocked by treatment with succinic anhydride.  
      mRNA Isolation, Labeling, and Hybridizations  
      Total RNA was extracted from cells using Qiagen&#39;s RNeasy kit; two wells from a 6 well plate were pooled to yield a total of 4×10 5  cells per treatment. RNA was amplified using a modified Eberwine protocol 5  that incorporated a polyA tail into the amplified RNA. Fluorescently-labeled cDNA probes were generated by reverse transcription of 4 μg of RNA with SuperScript II (Invitrogen Life Technologies, Carlsbad, Calif.) using anchored dT primers in the presence of Cy3 or Cy5 dUTP (Amersham, Piscataway, N.J.). Labeled cDNA probe pairs were precipitated with ethanol and purified using Qiaquick columns. Twenty μg each of poly(A) DNA, yeast tRNA, and human Cot1 DNA (Applied Genetics, Melbourne, Fla.) was added to the eluant. The samples were dried to completion and resuspended in 12.5 μl 3×SSC, 0.1% SDS. Probes were heated to 95° C. for 5 minutes, applied to the arrays under a 22 mm 2  cover slip and allowed to hybridize for at least 16 h at 65° C. The arrays were washed at 55° C. for 10 minutes in 2×SSC, 0.1% SDS, followed by two washes at room temperature in 1×SSC (10 min) and 0.2×SSC (15 min). Hybridization of each fluorophore was quantified using an Axon GenePix 4000A scanner.  
      Microarray Data Analysis  
      Differential expression values were expressed as the ratio of the median of background-subtracted fluorescent intensity of the experimental RNA to the median of background-subtracted fluorescent intensity of the control RNA. For ratios greater than or equal to 1.0, the ratio was expressed as a positive value. For ratios less than 1.0, the ratio was expressed as the negative reciprocal (i.e., a ratio of 0.5=−2.0). Median ratios were normalized to 1.0 using two pools of 3000 randomly chosen cDNAs in each pool. Six replicates of each of the two pools were printed in 4 evenly distributed blocks of the array. Expression data was rejected if neither channel produced a signal of at least 2.0-fold over background. Differential expression ratios were determined as the mean of the two values from dye-swapped duplicates.  
      A statistically significant differential expression threshold value was empirically determined according to the method of Yang et al. 53  Seven independent self-self-hybridizations were performed in which the same RNA sample was labeled with Cy3 dUTP and Cy5 dUTP and hybridized to arrays containing 8,448 elements. Only elements that gave a signal greater than 2.0-fold over background in at least one of the dyes were considered in the analysis. Expression ratios were converted to log (2)  and normalized to a mean=0. Combining data from all hybridizations, the 3 standard deviation limit was equivalent to a 1.48 fold change (+/−0.563 log (2) ). Of the 45,633 elements analyzed, 0.85% fell outside this threshold. Therefore, at this standard deviation limit, genes with fold changes greater than 1.48 can be considered differentially expressed at a 99% confidence level for any given hybridization. The percentage of elements that reproducibly fell outside the 3 standard deviation limit between any two duplicates of the seven self-self hybridizations was determined by comparing all 21 pair-wise combinations. An average of 18.9 elements +/−15.6 per hybridization duplicated at a fold change of 1.5, corresponding to a false positive rate of 0.29%. At a fold change of 1.8, an average of 0.71 elements +/−0.97 duplicated, corresponding to a false positive rate of 0.01%. A 1.8-fold threshold value was used to identify differentially expressed genes, except in  FIG. 3 , a 1.5-fold threshold value was used to designate “weak effects”.  
      Real-Time RT-PCR  
      Real-time RT-PCR 18  was performed in a two-step manner. cDNA synthesis and real-time detection were carried out in a PTC-100™ Thermal Cycler (MJ Research Inc, Waltham, Mass.) and an ABI Prism™ 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), respectively. Random hexamers (Qiagen, Valencia, Calif.) were used to generate cDNA from 200 ng RNA as described in Applied Biosystems User Bulletin #2. TaqMan™ PCR Core Reagent Kit or TaqMan™ Universal PCR Master Mix (Applied Biosystems) were used in subsequent PCR reactions according to the manufacturer&#39;s protocols. Relative quantitation of gene expression was performed using the relative standard curve method. All real-time RT-PCR reactions were performed in triplicate.  
      Sequence specific primers and probes were designed using Primer Express Version 2 software (Applied Biosystems). Sequences of primers and probes can be found in Table 1 below. Expression levels were normalized to 18S rRNA. The selection of 18S rRNA as an endogenous control was based on an evaluation of the ΔC T  levels (Applied Biosystems document # 4308134C) of 6 “housekeeping” genes: Cyclophilin A, 18S, GAPDH, β-actin, β-Glucuronidase, and Hypoxanthine Guanine Phosphoribosyl Transferase. The ΔC T  levels of 18S did not differ significantly between treatment conditions; thus, they were expressed at constant levels between samples.  
                   TABLE 1                          Real-time PCR primers and probes.           Western blot analysis                             Gene   Forward   Probe   Reverse                                         18S   5′-GCCGCTAGACGTGAAATTCTTG-   5′-6FAM-AGCGGCGCAAGACGGACCAG-TAMRA-3′   5′-CATTCTTGGCAAATGCTTTCG-               3′       3′               Collagen1   5′-GGAATTGGGCTTCGACGTT-3′   5′-6FAM-TCTGCTTGCTGTAAACTCCCTGCATCCC-   5′-TTCAGTTTGGGTTGCTTGTCTGT               TAMRA-3′   -3′               Fibronectin   5′-AGATCTACCTGTACACCTTGAAT   5′-6FAM-TGTCGTCATGGACGCCTCCA-TAMRA-3′   5′-CATGATACCAGCAAGGAATTGG-           GACA-3′       3′               TIMP3   5′-TGTGTCATGTGAGGCTGTAATAT   5′-6FAM-CACATCCCGCCATTTTGCTGAATCAA-   5′-GGCTAGAAGTATTTTGCTCTCCA           GTG-3′   TAMRA-3′   TTC-3′               PAI-1   5′-GGCTGACTTCACGAGTCTTTCA-   5′-6FAM-ACCAGAGGCTCTCGACGTCCCGG-   5′-GTTCACCTCGATCTTCACTTTCT           3′   TAMRA-3′   G-3′               CTGF   5′-TGTGTGAGGAGCGCAAGGA-3′   5′-6FAM-CTGCCCTCGCGGCTTACCGA-TAMRA-3′   5′-TAGTTGGGTCTGGGCCAAAC-3′               IL11   5′-AGAACAGCGAATTAAATGTGTCA   5′-6FAM-AGACAAATGGCCCTCAAGTGGA-   5′-CCCAGTTACGCAAGCATCCA-3′           TACA-3′   TAMRA-3′               COX2   5′-GCTCAAACATGATGTTTGCATTC   5′-6FAM-TTGCCCAGCACTTCAGGCATCAG-   5′-GCCCTCGCTTATGATCTGTCTT-           -3′   TAMRA-3′   3′               IL6   5′-ATGTAGCATGGCCACCTCAGAT-   5′-6FAM-TGGTCAGAAACCTGTCCACTGGGCA-   5′-TAACGCTCATACTTTTAGTTCTG           3′   TAMRA-3′   CATAGA-3′               a-smooth   5′-CCCCAGAGACCCTGTTGCA3′   5′6FAM-GCCAGCAGACTCCATGCCGA-TAMRA-3′   5′-TGATGCTGTTGTAGGTGGTTTCA       muscle actin           -3′                  
 
 Cells were cultured in 6-well plates and treated with BNP (100 nM, three times daily) in the presence or absence of 5 ng/ml TGFβ for 48 h. Lysis was induced with 0.2 ml of buffer containing 20 mM Tris-HCL, pH 7.9, 137 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM NaF, 1 mM β-glycerophosphate, and protease inhibitor cocktail. The protein concentration of each lysate was measured using coomassie protein reagent from PIERCE. Twenty μg of protein from each sample was loaded and electrophoresed on 4-12% gradient polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (Invitrogen, San Diego, Calif.). The membranes were incubated with rabbit anti-human Collagen 1 antibody (Cortex Biochem, San Leandro, Calif.), HRP-conjugated anti-human Fibronectin antibody, or goat anti-Actin antibody (Santa Cruz Biotehnology, Santa Cruz, Calif.) in TBST buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20, and 5% nonfat dried milk at 4° C. for ˜16 h. For ERK phosphorylation, cells were treated with 0.5 μmol/L BNP in the presence of 1 μmol/L KT5823 or 10 μmol/L U0126 for 15 min; the membranes were incubated with rabbit anti-human phospho-ERK ½ antibody or rabbit anti-human ERK ½ antibody (Cell Signaling, Beverly, Mass.). For secondary antibody detection, membranes were incubated with HRP-conjugated anti-rabbit antibody or anti-goat antibody at room temperature for 1 h and washed 3 times with TBST buffer. The blots were soaked in ECL Plus reagent for 5 min and exposed to KODAK x-ray film. Signals were identified and quantified using a Typhoon Scanner and Densitometer from Amersham Biosciences (Piscataway, N.J.). Data was analyzed by ANOVA using the Newman-Keuls test to assess significance. 
 
 Results 
 
 cGMP Production in Cardiac Fibroblasts 
 
      To determine if NPRA was expressed in the cultured fibroblast cells, cGMP accumulation assays were utilized. BNP dose-dependently induced intracellular cyclic GMP production in cardiac fibroblasts with an EC50 of 50 nmol/L. These results are consistent with the report of Cao and Gardner showing NPRA expression in cardiac fibroblasts.  
      Effects of BNP on TGFβ-Induced Fibroblast Proliferation  
      To examine the effects of TGFβ and BNP on cell proliferation, BrdU incorporation was measured in cardiac fibroblasts treated with TGFβ in the presence or absence of BNP. TGFβ modestly increased (˜50%) cardiac fibroblast proliferation, and BNP inhibited TGFβ-induced proliferation by ˜65% ( FIG. 9 ).  
      Effects of BNP on TGFβ-Induced Gene Expression  
      In order to determine the effects of BNP on gene expression profiles induced by TGFT in cardiac fibroblasts, a microarray analysis was performed. Fluorescently-labeled cDNA probes were prepared from pooled mRNAs generated from duplicate wells of cells from four groups: unstimulated (control), TGFβ-treated, BNP-treated, and co-treated with TGFβ and BNP for 24 and 48 h (as described above). Arrays were probed in duplicate for a total of 12 hybridizations (6 at each time point): control compared to TGFβ-treated, TGFβ-treated compared to TGFβ+BNP-treated, and control compared to BNP-treated.  
      It was observed that BNP had no significant effects on gene expression in unstimulated human cardiac fibroblasts ( FIG. 1 ). In contrast, TGFβ induced 394 and 501 gene expression changes at 24 and 48 h, respectively. These differentially expressed genes represent ˜7-8% of the target genes on the array. Interestingly, BNP had dramatic effects on the gene expression changes induced by TGFβ ( FIG. 2 ). Approximately, 88% and 85% of TGFβ-regulated gene expression events were opposed by BNP at 24 and 48 h, respectively. These results demonstrate that BNP has strikingly different effects on gene expression in TGFβ stimulated fibroblasts compared to unstimulated cells.  
      Gene Expression Clustering  
      To identify different gene expression patterns following TGFβ stimulation, we performed a hierarchical cluster analysis. A visualization of this analysis is shown in  FIG. 3 . A complete listing of differentially expressed genes is provided in Table 2. The clustered expression patterns showed temporal effects of TGFβ responsive genes (compare A to B). In addition, the dramatic effects of BNP in opposing TGFβ induced up- and down-regulated gene changes were revealed in the clusters (compare A and B to C and D). The insignificant effects of BNP on gene expression in unstimulated cardiac fibroblast cells were evident in groups E and F.  
      Genes were grouped according to functional categories by using a combination of gene expression clustering and functional annotations. A cluster of genes involved in fibrosis and ECM production was up-regulated in cells stimulated with TGFβ; these genes were down-regulated when treated with BNP ( FIG. 4   a ). This cluster includes extracellular matrix components: Collagen 1a2 (COL1A2), Collagen 15A (COL15A), Collagen 7A1 (COL7A1), Microfibril-associated glycoprotein-2 (MAGP2), Matrilin 3 (MATN3), Fibrillin 1 (FBN1), and Cartilage oligomeric matrix protein (COMP). Also included in the cluster are known markers of fibrosis such as TIMP3, CTGF, IL11, and SERPINE1 (PAI-1). Furthermore, the cluster revealed that BNP opposed TGFβ-induction of myofibroblast markers including α-smooth muscle actin 2 (ACTA2) and non-muscle myosin heavy chain (MYH9).  
      Many genes involved in cell proliferation were also regulated by TGFβ and were opposed by BNP ( FIG. 4B ). For example, TGFβ induced the expression of positive regulators of cell proliferation, including PDGFA, IGFBP10, IGF1, and Parathyroid hormone-like hormone (PTHLH). It was also found that TGFβ down-regulated both positive and negative regulators of proliferation, such as, CDC25B and Cullin 5 (CUL5), respectively. All of these TGFβ-regulated gene events were opposed by BNP.  
      BNP affected TGFβ-induced genes involved in inflammation ( FIG. 4C ). For example, BNP reversed TGFβ-induction of PTGS2 (COX2), TNF α-induced protein 6 (TNFAIP6), and TNF superfamily, member 4 (TNFSF4) ( FIG. 4C  and data not shown). TNFAIP6 and TNFSF4 were not included in  FIG. 4C , since some of the data points at 48 h did not meet acceptable criteria (see Experimental); at 24 h both genes were elevated ˜3-fold by TGFβ and opposed by BNP. TGFβ also down-regulated many pro-inflammatory genes including IL1B, CCR2 (MCP1-R), CXCL1 (GRO1), CXCL3 (GRO3), and CCL13 (MCP4), which were reversed by BNP. The significance of these inflammatory changes is discussed below.  
               TABLE 2                          Expression data for differentially expressed genes in TGFβ-treated human cardiac fibroblasts. Median       differential expression values are shown for each hybridization: control vs. TGFβ 24 h (column 2); control vs.       TGFβ 48 h (column 3); TGFβ vs. TGFβ + BNP 24 h (column 4); TGFβ vs.       TGFβ + BNP 48 h (column 5); control vs. BNP 24 h (column 6); and control vs. BNP 48 h (column 7).                                                             TGF           TGF                           TGF   BNP   BNP   TGF   BNP   BNP       Clone ID   24 h   24 h   24 h   48 h   48 h   48 h   Symbol   Name   Accession                                                             P00777_A03   2.5   −2.8   1.1   1.5   −1.6   1.1       EST           P00777_A04   8.9   −5.7   1.2   3.3   −2.4   1       EST       P00777_A12   2.1   −2.4   −1   1.8   −1.9   −1.1       EST       P01061_E01   2.7   −3   1   2.6   −2.8   −1       EST       P01061_B10   −2.7   2.3   1.1   −4   2.4   −1.2       EST       P01077_A08   −1.8   3.1   1.3   −2.2   1.9   1.2       No Sequence       P01111_A08   −1.3   1.4   1.3   −1.8   1.7   1.1       EST       P01113_E11   −1.7   1.8   1.1   −1.8   1.6   −1       EST       P01111_F07   −4.5   5.5   1.3   −5.3   4.2   1.1       EST       P01111_A07   2   −2.7   1.3   1.4   −1.5   −1.1       EST       P01110_G03   −1.2   1.5   1.3   −3.9   2.1   1.1       No Sequence       P01108_G07   4.2   −4.4   −1.1   3.9   −4.5   −1       EST       P01099_G03   −1.9   1.9   1.1   −2.2   1.9   1.2       EST       P01113_B03   6.4   −5.1   1   4.3   −3.7   −1       EST       P01080_A11   4   −3   1   4.2   −4.1   −1       EST       P01076_E01   −1.7   1.8   1.1   −1.8   1.8   −1.1       EST       P01075_H09   −3.1   3.6   1.4   −2.9   3.2   1.4       No Sequence       P01139_D10   3   −2.6   1.1   2.1   −2.1   1       EST       P01132_B01   −2.1   2   1   −1.4   1.3   1       EST       P01123_H03   2.2   −2.2   1.2   1.9   −1.9   1.1       EST       P01117_D08   −1.7   1.5   1.1   −4.9   2.4   −1       EST       P01115_F08   −2.2   1.6   −1   −2.3   1.7   −1       EST       P01081_F02   2.4   −1.8   1.2   2.4   −2.1   1.1       No Sequence       P01087_A12   2.4   −2   1   2.6   −2.6   −1       EST       P01077_A02   2.2   −2   1   1.4   −1.3   −1       No Sequence       P01136_G11   −2   2.5   1.3   −3   2.5   1       EST       P01130_B03   −3.3   3.5   1.1   −4.2   5.3   1.1       EST       P01124_A05   −1.2   −1   1.1   −1.8   1.5   1       EST       P01124_A10   2.1   −2   −1   2.7   −2.5   −1.1       EST       P01124_B04   −1.9   2   1.3   −1.6   1.7   1.1       EST       P01120_G06   −2.3   2.2   −1.1   −2.4   2.2   −1.1       EST       P01117_B11   1.8   −2.4   1   2.4   −2   1       EST       P01116_A02   −3.1   2.7   1.1   −3.7   2.2   −1.4       EST       P01088_C10   2.1   −2   −1   1.6   −2.1   −1.1       EST       P01093_C04   2.6   −2.3   1   1.8   −1.9   −1       EST       P01095_H01   −1.8   1.8   1   −1.4   1.2   1       EST       P01099_D03   1.9   −1.8   1.1   1.1   −1.2   1.1       EST       P01100_A07   1   −1   1.1   −3   1.7   −1.1       EST       P01100_D09   −1.6   1.6   −1   −2.1   1.8   −1.1       EST       P01101_C11   −2.4   1.7   −1   −1.4   1.6   1       No Sequence       P01101_E11   −1.4   1.5   1.1   −2   1.8   −1       EST       P01103_H04   −3.2   2.9   1.1   −5.6   4.3   −1       EST       P01104_A09   −1.9   1.6   1.1   −1.8   1.5   1       No Sequence       P01104_E03   −2.5   2.3   −1   −2.8   2   −1.1       EST       P01104_G04   2.5   −2   −1   1.1   −1.3   −1.1       EST       P01104_G12   −3.7   2.7   −1.1   −4.9   3.2   −1       EST       P01105_A05   2.3   −2.3   1.3   1.3   −1.3   1       EST       P01105_D09   1.8   −1.1   1.1   1.8   −2.1   −1       EST       P01109_A01   −1.4   1.4   1.2   −2.2   1.7   1.1   A2M   alpha-2-macroglobulin   NM_000014       P01109_G11   1.4   −1   1.1   2   −1.6   1   ABCG1   ATP-binding cassette, sub-   NM_004915                                       family G (WHITE), member 1       P01092_E08   2.3   −2   1.2   1.5   −1.3   1.1   ACLY   ATP citrate lyase   NM_001096       P01088_C02   −1.9   1.8   1.2   −2.1   2   1   ACO1   aconitase 1, soluble   NM_002197       P00777_G09   2.6   −2.2   −1.5   1   1   −1.3   ACTA1   actin, alpha 1, skeletal muscle   NM_001100       P01094_F04   2.6   −2.5   −1.4   −1   −1   −1.4   ACTA2   actin, alpha 2, smooth muscle,   NM_001613                                       aorta       P01091_G04   1.9   −1.6   1.1   1.2   −1.3   −1   ACTR3   ARP3 actin-related protein 3   NM_005721                                       homolog (yeast)       P01096_D02   −1.3   1.5   1.1   −2.3   2.2   1   ADAMTS1   a disintegrin-like and   NM_006988                                       metalloprotease (reprolysin                                       type) with thrombospondin                                       type 1 motif, 1       P01097_D04   1.7   −1.9   −1   2.1   −1.8   −1.1   ADAMTS6   a disintegrin-like and   NM_014273                                       metalloprotease (reprolysin                                       type) with thrombospondin                                       type 1 motif, 6       P01092_D03   −6.5   6   −1.1   −6.3   6.5   −1   ADFP   adipose differentiation-related   NM_001122                                       protein       P01070_D09   −5   4.1   1.3   −9.7   3.8   1.3   ADH1B   alcohol dehydrogenase IB   NM_000668                                       (class I), beta polypeptide       P01134_D11   −1.7   2   1.3   −3.6   1.6   1.2   ADH1C   alcohol dehydrogenase 1C   NM_000669                                       (class I), gamma polypeptide       P01070_D05   −1.3   −1.4   1.2   −2.2   1.7   1.1   ADH5   alcohol dehydrogenase 5   NM_000671                                       (class III), chi polypeptide       P01094_D10   −2.3   2.5   1.1   −2.2   1.8   −1   ADORA2B   adenosine A2b receptor   NM_000676       P01124_F09   −1.5   1.6   1.1   −1.8   1.9   1   AHR   aryl hydrocarbon receptor   NM_001621       P01101_B03   −2.4   1   1   −3   2.8   1.1   AKAP2   A kinase (PRKA) anchor   NM_007203                                       protein 2       P01120_C03   −1.9   2   1.2   −1.2   1.5   1.2   AKR1B1   aldo-keto reductase family 1,   NM_001628                                       member B1 (aldose reductase)       P01134_B08   −2.7   2.6   1.1   −1.4   1.9   1.2   AKR1B10   aldo-keto reductase family 1,   NM_020299                                       member B10 (aldose                                       reductase)       P01069_C01   −2.8   3.1   1.2   −2.2   2.6   1.1   AKR1C1   aldo-keto reductase family 1,   NM_001353                                       member C1 (dihydrodiol                                       dehydrogenase 1; 20-alpha (3-                                       alpha)-hydroxysteroid                                       dehydrogenase)       P01081_A11   −2.3   3.3   1.6   −2.2   1.9   1.3   AKR1C2   aldo-keto reductase family 1,   NM_001354                                       member C2 (dihydrodiol                                       dehydrogenase 2; bile acid                                       binding protein; 3-alpha                                       hydroxysteroid                                       dehydrogenase, type III)       P01143_D10   −2.8   3.2   1.3   −2.1   2.7   1.1   AKR1C2   aldo-keto reductase family 1,   NM_001354                                       member C2 (dihydrodiol                                       dehydrogenase 2; bile acid                                       binding protein; 3-alpha                                       hydroxysteroid                                       dehydrogenase, type III)       P01106_C11   −2.3   2.8   1.2   −2   2.5   1.1   AKR1C3   aldo-keto reductase family 1,   NM_003739                                       member C3 (3-alpha                                       hydroxysteroid                                       dehydrogenase, type II)       P01094_D12   −2.8   3.6   1.2   −2.5   1.7   1.2   ALDH1A3   aldehyde dehydrogenase 1   NM_000693                                       family, member A3       P01094_E01   −1.4   1.8   1.1   −2.1   1.6   1.1   ALDH3A2   aldehyde dehydrogenase 3   NM_000382                                       family, member A2       P01140_G11   −1.9   2.7   1.4   −2.5   1.8   1.1   ALDH3A2   aldehyde dehydrogenase 3   NM_000382                                       family, member A2       P01118_A12   −1.9   1.6   1.1   −2.6   2.2   1   ALEX1   ALEX1 protein   NM_016608       P01096_E12   −2.4   2   1   −2.1   2.2   1   ANG   angiogenin, ribonuclease,   NM_001145                                       RNase A family, 5       P01145_E08   −2   2.3   1.2   −2.9   2.6   −1   ANGPT1   angiopoietin 1   NM_001146       P01091_G02   −1.2   1.5   1.2   −2.7   2   1.1   ANGPT2   angiopoietin 2   NM_001147       P01094_D06   −2.1   1.9   −1   −1.9   1.3   −1.1   ANK3   ankyrin 3, node of Ranvier   NM_001149                                       (ankyrin G)       P01128_A07   −1.5   1.8   1.2   −2.2   2.3   1.2   AOX1   aldehyde oxidase 1   NM_001159       P01116_H05   −1.1   1.4   1.2   −2   1.8   1   APELIN   apelin; peptide ligand for APJ   NM_017413                                       receptor       P01103_F06   2.4   −2.4   −1.1   1.4   −1.5   −1.1   APG3   autophagy Apg3p/Aut1p-like   NM_022488       P01123_A07   3.2   −3   −1   1.5   −1.8   −1   APOA1   apolipoprotein A-I   NM_000039       P01105_G06   −2.2   1.8   −1.1   −4.5   5.7   1.1   APOC1   apolipoprotein C-I   NM_001645       P01124_G03   −1.3   1.4   1   −2.4   2   1.1   APOE   apolipoprotein E   NM_000041       P01105_B02   −1.6   1.8   −1   −2.9   1.9   1.2   ARHGAP6   Rho GTPase activating protein 6   NM_001174       P01064_G03   −1.1   1.3   1.1   −2   1.6   1.2   ARHGEF16   Rho guanine exchange factor   NM_014448                                       (GEF) 16       P01110_E10   −2   2.1   1.2   −2.3   1.9   1   ARHGEF3   Rho guanine nucleotide   NM_019555                                       exchange factor (GEF) 3       P01142_C03   −1.6   1.8   1.5   −1.9   1.7   1.2   ARHI   ras homolog gene family,   NM_004675                                       member I       P01138_A09   1.9   −2.2   −1.1   1.8   −1.9   −1.1   ARL4   ADP-ribosylation factor-like 4   NM_005738       P01064_G12   −1.7   1.8   1.1   −1.8   1.6   −1   ARNT2   aryl-hydrocarbon receptor   NM_014862                                       nuclear translocator 2       P01088_H09   −1.5   1.7   1.2   −1.8   1.6   1.1   ASAH1   N-acylsphingosine   NM_004315                                       amidohydrolase (acid                                       ceramidase) 1       P01105_F06   2.9   −2.8   1.1   2.1   −2.4   −1.2   ASNS   asparagine synthetase   NM_001673       P01070_E06   1.8   −1.5   −1.3   1.6   −1.4   1   ATF3   activating transcription factor 3   NM_001674       P01122_G07   −1.2   1.7   1.2   −1.8   1.5   1.3   AXIN2   axin 2 (conductin, axil)   NM_004655       P01115_D06   −1.4   1.6   1   −2   1.5   −1.1   B3GALT2   UDP-Gal:betaGlcNAc beta   NM_003783                                       1,3-galactosyltransferase,                                       polypeptide 2       P01128_A08   −1.6   1.7   1   −2.4   1.7   −1   B3GALT3   UDP-Gal:betaGlcNAc beta   NM_003781                                       1,3-galactosyltransferase,                                       polypeptide 3       P01095_F06   2.4   −2.2   1.1   1.3   −1.5   −1   BAI3   brain-specific angiogenesis   NM_001704                                       inhibitor 3       P01094_C02   −1.8   2   1.2   −2.4   2.7   −1   BF   B-factor, properdin   NM_001710       P01134_E02   −1.7   1.8   1   −2.2   1.6   −1   BFSP1   beaded filament structural   NM_001195                                       protein 1, filensin       P01081_D08   −1.2   1.7   1.2   −3.5   1.8   1.2   BIRC1   baculoviral IAP repeat-   NM_004536                                       containing 1       P01094_B06   −2.6   2.9   1.1   −4   2.5   −1   BMP4   bone morphogenetic protein 4   NM_001202       P01145_A02   −3.2   2.3   1   −3.6   3.2   −1.1   BNIP2   BCL2/adenovirus E1B 19 kDa   NM_004330                                       interacting protein 2       P01075_F05   −1.5   1.5   1.2   −1.8   2   1.2   BRE   brain and reproductive organ-   NM_004899                                       expressed (TNFRSF1A                                       modulator)       P01124_B10   −1.3   1.5   1.3   −2.2   1.6   1.2   BST1   bone marrow stromal cell   NM_004334                                       antigen 1       P01094_B08   −1.8   1.6   −1.1   −1.2   1.3   −1.1   BTD   biotinidase   NM_000060       P01093_E08   −2   1.5   −1.1   −1.9   2.7   1.1   C1R   complement component 1, r   NM_001733                                       subcomponent       P01077_E12   −1.4   1.6   1.1   −1.8   1.9   −1.1   C1S   complement component 1, s   NM_001734                                       subcomponent       P01097_G03   1.9   −1.7   −1   1   −1.5   −1.1   C20orf14   chromosome 20 open reading   NM_012469                                       frame 14       P01140_A07   2.3   −3.2   −1   3   −2.6   −1   C20orf97   chromosome 20 open reading   NM_021158                                       frame 97       P01069_E02   −1.7   1.6   1.1   −3.3   3.2   1.1   C6   complement component 6   NM_000065       P01077_E10   −3.1   2.9   1.1   −8.2   4.7   −1   C7   complement component 7   NM_000587       P01099_C10   −1.8   2.1   1.2   −2.7   3.5   1.1   CA12   carbonic anhydrase XII   NM_001218       P01117_G05   −3   2.4   −1.1   −2.2   2.3   1.1   CAMK2B   calcium/calmodulin-dependent   NM_001220                                       protein kinase (CaM kinase) II                                       beta       P01114_A05   −2.7   3.9   1.2   −3.5   2.7   1   CAMK2D   calcium/calmodulin-dependent   NM_001221                                       protein kinase (CaM kinase) II                                       delta       P01080_B05   −2.3   3   1.1   −2.3   2.1   1.1   CAMK2D   calcium/calmodulin-dependent   NM_001221                                       protein kinase (CaM kinase) II                                       delta       P01063_E07   −1.6   2   1.2   −1.8   1.6   1.1   CASP1   caspase 1, apoptosis-related   NM_001223                                       cysteine protease (interleukin                                       1, beta, convertase)       P01093_G08   −2.4   2.3   −1.2   −2.1   2.4   1   CAV1   caveolin 1, caveolae protein   NM_001753                                       22 kDa       P01093_E04   1.8   −1.7   −1.1   1.6   −1.9   −1.1   CBS   cystathionine-beta-synthase   NM_000071       P01064_D02   −1.5   1.6   −1.3   −2.2   2.8   −1.1   CCL13   chemokine (C—C motif) ligand   NM_005408                                       13       P01072_E08   −1.3   1.4   −1.2   −2.2   3.2   −1.1   CCL7   chemokine (C—C motif) ligand 7   NM_006273       P01127_H03   1.1   1.2   −1.3   −2   2.9   −1   CCL8   chemokine (C—C motif) ligand 8   NM_005623       P01070_A04   −1.4   1.9   1.2   −3.2   2.4   1.1   CCR2   chemokine (C—C motif)   NM_000647                                       receptor 2       P01138_B02   −1.2   1.3   1.3   −3.6   1.5   1   CCRL1   chemokine (C—C motif)   NM_016557                                       receptor-like 1       P01069_H09   −1.9   1.9   1.3   −3.6   1.8   1.2   CD36   CD36 antigen (collagen type I   NM_000072                                       receptor, thrombospondin                                       receptor)       P01072_E03   −2.8   2.7   1.2   −2.9   2.8   1.2   CDC25B   cell division cycle 25B   NM_004358       P01093_H07   2   −4.3   1.2   2.1   −2   −1   CDH2   cadherin 2, type 1, N-cadherin   NM_001792                                       (neuronal)       P01129_E07   1.7   −1.4   1.1   2   −1.9   −1.1   CDH4   cadherin 4, type 1, R-cadherin   NM_001794                                       (retinal)       P01130_H07   2.1   −2.4   −1.1   1.9   −1.8   −1   CDH5   cadherin 5, type 2, VE-   NM_001795                                       cadherin (vascular epithelium)       P01116_H02   −3.3   2.1   1.1   −2   2.4   1.1   CDK5RAP2   CDK5 regulatory subunit   NM_018249                                       associated protein 2       P01102_B02   −2.1   2.5   1   −3.4   3.2   −1.1   CDSN   comeodesmosin   NM_001264       P01140_G02   −1.4   1.3   1.1   −2.9   2.4   1   CEACAM5   carcinoembryonic antigen-   NM_004363                                       related cell adhesion molecule 5       P01094_A06   −1.6   1.3   1.3   −4.2   2.9   1   CEACAM5   carcinoembryonic antigen-   NM_004363                                       related cell adhesion molecule 5       P01062_G02   −1.3   1.5   1.3   −2.9   2.1   1.1   CEACAM6   carcinoembryonic antigen-   NM_002483                                       related cell adhesion molecule                                       6 (non-specific cross reacting                                       antigen)       P01099_B05   −1.8   1.8   1.1   −2.9   3   −1.1   CEACAM7   carcinoembryonic antigen-   NM_006890                                       related cell adhesion molecule 7       P01090_E04   −1.3   1.6   1.3   −1.9   1.8   −1   CEBPD   CCAAT/enhancer binding   NM_005195                                       protein (C/EBP), delta       P01070_A01   −2.6   3.1   −1   −9.2   9.2   1.1   CHI3L1   chitinase 3-like 1 (cartilage   NM_001276                                       glycoprotein-39)       P01125_G02   −2.9   2   1   −5   6.2   1   CHI3L2   chitinase 3-like 2   NM_004000       P01134_F10   8   −6.3   1.2   19.5   −8   1.1   CILP   cartilage intermediate layer   NM_003613                                       protein, nucleotide                                       pyrophosphohydrolase       P01089_A12   −1.9   2.1   1   −2.1   2.1   −1   CITED2   Cbp/p300-interacting   NM_006079                                       transactivator, with Glu/Asp-                                       rich carboxy-terminal domain, 2       P01076_A07   2.1   −1.8   −1   1.4   −1.2   1.1   CKAP4   cytoskeleton-associated   NM_006825                                       protein 4       P01104_C09   2.2   −2.4   1.1   4.3   −2.8   1   CKLF   chemokine-like factor   NM_016326       P01103_G05   −1.4   1.6   1.3   −2.5   1.5   1.3   CLDN1   claudin 1   NM_021101       P01105_D03   −3   2.7   1.3   −2.6   2   −1   CLECSF2   C-type (calcium dependent,   NM_005127                                       carbohydrate-recognition                                       domain) lectin, superfamily                                       member 2 (activation-induced)       P01064_F09   2.2   −1.5   1.2   1.2   −1.2   1.1   CNN1   calponin 1, basic, smooth   NM_001299                                       muscle       P01090_A03   −1.1   1.3   1.2   −2.2   1.6   1.1   CNTNAP1   contactin associated protein 1   NM_003632       P01069_F02   1.2   1.2   1.2   3.2   −3   1   COL15A1   collagen, type XV, alpha 1   NM_001855       P01077_E08   1.8   −1.5   1   1.9   −1.9   −1   COL1A2   collagen, type I, alpha 2   NM_000089       P01093_F03   1.7   −2.3   1   1.9   −2.1   −1   COL4A2   collagen, type IV, alpha 2   NM_001846       P01105_C12   1.8   −1.5   1.4   3.1   −2.3   1.1   COL7A1   collagen, type VII, alpha 1   NM_000094                                       (epidermolysis bullosa,                                       dystrophic, dominant and                                       recessive)       P01120_G04   2.7   −2.1   1.2   3.8   −3.6   1.1   COL8A2   collagen, type VIII, alpha 2   M60832       P01084_A12   −4.9   4.7   1.1   −9.9   6   1   COLEC12   collectin sub-family member   NM_030781                                       12       P01082_H06   1.3   −1.3   1.2   3.3   −2.4   1.2   COMP   cartilage oligomeric matrix   NM_000095                                       protein                                       (pseudoachondroplasia,                                       epiphyseal dysplasia 1,                                       multiple)       P01129_C12   1.4   −1.5   1.3   2.6   −1.6   1.3   COMP   cartilage oligomeric matrix   NM_000095                                       protein                                       (pseudoachondroplasia,                                       epiphyseal dysplasia 1,                                       multiple)       P01076_C09   −2.2   2.7   1.2   −2.1   1.6   1.1   COPB   coatomer protein complex,   NM_016451                                       subunit beta       P01085_D11   −4   4.2   1.1   −7.7   4.1   1.1   CPA4   carboxypeptidase A4   NM_016352       P01104_A07   −1.9   2   1.2   −2.5   2.2   1   CPD   carboxypeptidase D   NM_001304       P01077_G01   1.9   −1.8   1.1   1.7   −1.9   1.1   CRABP2   cellular retinoic acid binding   NM_001878                                       protein 2       P01095_E03   −1.8   1.8   1.2   −2.1   2   −1.1   CREG   cellular repressor of E1A-   NM_003851                                       stimulated genes       P01124_E01   −2.2   2.1   1   −2.5   2.2   −1   CREM   cAMP responsive element   NM_001881                                       modulator       P01120_B01   1.8   −1.6   1.2   3.9   −3.4   1.1   CRLF1   cytokine receptor-like factor 1   NM_004750       P01120_D10   −1.5   1.9   1.3   −3.5   2.4   1.1   CROT   camitine O-   NM_021151                                       octanoyltransferase       P01124_F10   −1.2   1.3   1.2   −1.8   1.7   1.1   CRYAA   crystallin, alpha A   NM_000394       P00777_A08   −2   1.6   1.1   −2.6   2.5   −1.1   CRYAB   crystallin, alpha B   NM_001885       P01077_E04   −2.1   1.8   1.1   −2.5   2.6   −1.1   CRYAB   crystallin, alpha B   NM_001885       P01125_B11   −1.8   1.2   1.1   −1.8   1.8   1   CSF1   colony stimulating factor 1   NM_000757                                       (macrophage)       P01108_G05   3.8   −3   1.1   2.3   −2.4   1   CSPG2   chondroitin sulfate   NM_004385                                       proteoglycan 2 (versican)       P01075_F12   −1.5   1.6   1.1   −2   2   1.1   CSRP2   cysteine and glycine-rich   NM_001321                                       protein 2       P01145_A03   −2.1   2.4   1   −3.7   3.4   −1.1   CST4   cystatin S   NM_001899       P00777_D03   2.5   −2   1.1   1.1   −1.4   −1.2   CTGF   connective tissue growth factor   NM_001901       P01077_D08   2.6   −3.5   −1.2   1.8   −2.7   −1.2   CTGF   connective tissue growth factor   NM_001901       P01069_D11   2   −2.1   1.2   1.9   −1.4   1.2   CTH   cystathionase (cystathionine   NM_001902                                       gamma-lyase)       P01099_B01   −1.7   2   1.1   −2   1.6   −1   CTNNAL1   catenin (cadherin-associated   NM_003798                                       protein), alpha-like 1       P01093_G10   −1.4   1.4   1.1   −1.8   2   1   CTSC   cathepsin C   NM_001814       P01077_G03   −1   1.3   1.2   −1.8   1.7   1.2   CTSH   cathepsin H   NM_004390       P01069_H12   −1.5   1.5   1.1   −2.3   2.6   −1   CTSK   cathepsin K (pycnodysostosis)   NM_000396       P01093_G09   −2.5   2.1   1.1   −2   2.3   −1   CTSL   cathepsin L   NM_001912       P01112_D02   −1.6   1.8   1.2   −2.9   2.1   −1   CUGBP2   CUG triplet repeat, RNA   NM_006561                                       binding protein 2       P01131_G04   −1.3   1.6   1.3   −2.2   1.7   1.3   CUGBP2   CUG triplet repeat, RNA   NM_006561                                       binding protein 2       P01090_H01   −2   1.8   1.3   −1.5   1.9   1.3   CUL5   cullin 5   NM_003478       P01085_C05   −3.8   3.4   1.1   −5.5   5   −1   CXCL1   chemokine (C—X—C motif)   NM_001511                                       ligand 1 (melanoma growth                                       stimulating activity, alpha)       P01093_A02   −3.7   3.1   1   −5.8   5.4   −1   CXCL1   chemokine (C—X—C motif)   NM_001511                                       ligand 1 (melanoma growth                                       stimulating activity, alpha)       P01125_H11   −2.4   2   1.1   −2.3   2.1   1   CXCL3   chemokine (C—X—C motif)   NM_002090                                       ligand 3       P01136_B01   −4.5   4.4   1.1   −8.4   10   1.1   CXCL6   chemokine (C—X—C motif)   NM_002993                                       ligand 6 (granulocyte                                       chemotactic protein 2)       P01069_D07   −2.4   2.3   1.3   −2   1.7   1   CYB5   cytochrome b-5   NM_001914       P00777_A11   2   −2.5   −1   1.8   −2   −1   CYR61   cysteine-rich, angiogenic   NM_001554                                       inducer, 61       P00777_C11   1.8   −2.5   −1   1.8   −1.9   −1.1   CYR61   cysteine-rich, angiogenic   NM_001554                                       inducer, 61       P00777_C12   2   −2.6   −1.1   1.9   −1.9   −1.1   CYR61   cysteine-rich, angiogenic   NM_001554                                       inducer, 61       P01108_B04   2.3   −2.4   1.1   1.9   −1.9   −1.1   CYR61   cysteine-rich, angiogenic   NM_001554                                       inducer, 61       P01130_H03   2   −2.4   −1.1   1.9   −1.8   −1.1   CYR61   cysteine-rich, angiogenic   NM_001554                                       inducer, 61       P01100_C06   2.2   −2.4   −1.1   1.8   −1.9   −1   DACT1   dapper homolog 1, antagonist   NM_016651                                       of beta-catenin ( xenopus )       P01069_C07   1.7   −1.4   1.1   2.3   −2.1   1.1   DAF   decay accelerating factor for   NM_000574                                       complement (CD55, Cromer                                       blood group system)       P01129_B04   −2.8   2.5   1.2   −4.3   3.5   1   DAPK1   death-associated protein   NM_004938                                       kinase 1       P01092_G02   −2.7   2.6   1.1   −3.8   2.8   1.2   DAPK1   death-associated protein   NM_004938                                       kinase 1       P01065_A02   −1.8   1.9   −1   −1.8   1.7   −1.1   DDX38   DEAD/H (Asp-Glu-Ala-   NM_014003                                       Asp/His) box polypeptide 38       P01105_A10   −3.7   2.9   −1   −7   5.4   −1   DKK1   dickkopf homolog 1 ( Xenopus     NM_012242                                         laevis )       P01113_E05   2.8   −2.4   −1.1   1.8   −2.1   −1.1   DLC1   deleted in liver cancer 1   NM_006094       P01093_C11   −1.8   1.9   −1   −4.5   3.3   −1   DPP4   dipeptidylpeptidase 4 (CD26,   NM_001935                                       adenosine deaminase                                       complexing protein 2)       P01073_G11   −1.8   1.7   1   −1.6   1.5   1   DPYSL2   dihydropyrimidinase-like 2   NM_001386       P01090_F08   1.4   −1.5   −1.1   2   −1.9   −1.1   DSCR1   Down syndrome critical region   NM_004414                                       gene 1       P01122_D11   1.7   −1.2   1.3   1.9   −2.1   1.3   EBAF   endometrial bleeding   NM_003240                                       associated factor (left-right                                       determination, factor A;                                       transforming growth factor                                       beta superfamily)       P01123_B11   −1.8   1.7   1   −2.3   1.9   1   ECM2   extracellular matrix protein 2,   NM_001393                                       female organ and adipocyte                                       specific       P01124_E11   −1.6   1.9   1.2   −2.1   1.9   1.2   EDG1   endothelial differentiation,   NM_001400                                       sphingolipid G-protein-coupled                                       receptor, 1       P01103_G08   −1.8   1.8   1.1   −2.4   2.4   1   EDG2   endothelial differentiation,   NM_001401                                       lysophosphatidic acid G-                                       protein-coupled receptor, 2       P01093_C01   −2.1   1.5   −1.1   −2.9   1.9   −1.3   EDN1   endothelin 1   NM_001955       P01105_H10   −1.9   1.9   1   −2.2   2.3   −1   EFEMP1   EGF-containing fibulin-like   NM_004105                                       extracellular matrix protein 1       P01064_A03   −1.4   1.9   1.2   −2   2   1.1   EFNB3   ephrin-B3   NM_001406       P01093_B07   −1.8   1.7   1.3   −1.5   1.3   1.1   EGR2   early growth response 2 (Krox-   NM_000399                                       20 homolog,  Drosophila )       P01121_C03   −2   2   1.2   −1.2   1.5   1.2   EHD3   EH-domain containing 3   NM_014600       P01065_E02   1.9   −1.6   1.2   3.4   −3.3   1.1   ELN   elastin (supravalvular aortic   NM_000501                                       stenosis, Williams-Beuren                                       syndrome)       P01096_H11   −3.4   3.7   1.1   −3.5   3.2   −1   EPAS1   endothelial PAS domain   NM_001430                                       protein 1       P01102_E11   −2   2.1   1.2   −2.5   2.1   −1   EPB41L2   erythrocyte membrane protein   NM_001431                                       band 4.1-like 2       P01104_A05   −2.3   3.3   1.1   −2.2   2.3   1.1   EPI64   EBP50-PDZ interactor of 64 kD   NM_031937       P01130_H01   −2   2   1.1   −2.5   3.9   −1   EPOR   erythropoietin receptor   NM_000121       P01077_A07   −1.6   2.7   1.4   −2.3   2.1   1.2   ETV5   ets variant gene 5 (ets-related   NM_004454                                       molecule)       P01097_C06   −5.9   4.9   −1   −15.8   14   −1.1   EVI2B   ecotropic viral integration site   NM_006495                                       2B       P01077_A01   1.8   −1.8   1.1   1.3   −1.4   1   EXT1   exostoses (multiple) 1   NM_000127       P01069_F04   −1.7   1.6   1.2   −2.1   1.7   1.1   F2R   coagulation factor II (thrombin)   NM_001992                                       receptor       P01128_B02   1.8   −1.9   1.1   −1   −1.1   −1   F3   coagulation factor III   NM_001993                                       (thromboplastin, tissue factor)       P01132_G03   1.9   −1.7   1.2   1.8   −1.6   1.1   FACL3   fatty-acid-Coenzyme A ligase,   NM_004457                                       long-chain 3       P01096_A03   1.8   −2   1   1.8   −2   1   FACL3   fatty-acid-Coenzyme A ligase,   NM_004457                                       long-chain 3       P01083_D07   2.2   −1.6   1.2   1.4   −1.3   1   FADS1   fatty acid desaturase 1   NM_013402       P01093_B02   −2   1.6   1.1   −3.4   3.4   1   FBLN1   fibulin 1   NM_001996       P01123_A08   3.4   −3   1.2   1.6   −1.9   −1   FBLN5   fibulin 5   NM_006329       P01068_H09   1.4   −1.4   1   2.2   −2   −1   FBN1   fibrillin 1 (Marfan syndrome)   NM_000138       P01084_E10   1.9   −1.7   1.3   −1.1   −1.1   1.1   FGF18   fibroblast growth factor 18   NM_003862       P01093_B03   −4.2   4.9   1.2   −5.9   5.6   1   FGF7   fibroblast growth factor 7   NM_002009                                       (keratinocyte growth factor)       P01092_C04   −3.2   2.9   1.1   −3.1   2.3   −1   FGL2   fibrinogen-like 2   NM_006682       P01126_F06   −5   5.2   −1.1   −6.5   4.9   −1.1   FMO2   flavin containing   NM_001460                                       monooxygenase 2       P01078_G11   −1.9   2.1   1.2   −3.1   2.2   1.1   FMO3   flavin containing   NM_006894                                       monooxygenase 3       P01088_F09   2   −1.8   1.2   1.4   −1.5   1.1   FOXD1   forkhead box D1   NM_004472       P01120_B03   −1.8   1.9   1.3   −1.2   1.5   1.2   FRA   Fos-related antigen   NM_024816       P01138_B06   −1.8   1.5   1   −1.4   1.8   −1   FTHL17   ferritin, heavy polypeptide-like   NM_031894                                       17       P01068_G11   2.7   −2.1   1.3   2.4   −2.5   1.1   FUT4   fucosyltransferase 4 (alpha   NM_002033                                       (1,3) fucosyltransferase,                                       myeloid-specific)       P01077_A05   1.8   −1.5   1   1.5   −1.2   1   FYN   FYN oncogene related to   NM_002037                                       SRC, FGR, YES       P01124_G01   −1.9   1.9   1.1   −1.4   1.2   1   FZD7   frizzled homolog 7   NM_003507                                       ( Drosophila )       P01083_B09   3.2   −4   −1   4.2   −3.5   −1   GABARAPL2   GABA(A) receptor-associated   NM_007285                                       protein-like 2       P01106_B05   −1.8   1.5   1   −1.1   2.4   1.2   GALT   galactose-1-phosphate   NM_000155                                       uridylyltransferase       P01092_G07   2.5   −2.3   −1.2   1.8   −2.2   −1.1   GARS   glycyl-tRNA synthetase   NM_002047       P01085_D09   −2.9   4.1   1.4   −5.3   2.6   1.3   GAS1   growth arrest-specific 1   NM_002048       P01063_E09   −2   1.7   1.1   −2   1.8   1.2   GBP2   guanylate binding protein 2,   NM_004120                                       interferon-inducible       P01123_D12   −1.9   1.4   −1.2   −2.7   2.7   −1.1   GBP2   guanylate binding protein 2,   NM_004120                                       interferon-inducible       P01135_C03   −1.8   1.9   1.2   −2.7   2.4   1.1   GCNT1   glucosaminyl (N-acetyl)   NM_001490                                       transferase 1, core 2 (beta-                                       1,6-N-                                       acetylglucosaminyltransferase)       P01127_B01   −2.8   2.2   −1.1   −2.9   3.7   −1   GDF5   growth differentiation factors 5   NM_000557                                       (cartilage-derived                                       morphogenetic protein-1)       P01065_A06   −1.7   1.7   1.1   −1.8   1.6   1   GGA3   golgi associated, gamma   NM_014001                                       adaptin ear containing, ARF                                       binding protein 3       P01076_H05   −2   2.4   1.2   −2.7   1.9   1.1   GM2A   GM2 ganglioside activator   NM_000405                                       protein       P01062_E04   −2.3   1.9   −1   −2.1   1.9   −1   GNPI   glucosamine-6-phosphate   NM_005471                                       isomerase       P01138_C10   −2.1   2.2   −1.1   −2   1.9   −1.1   GNPI   glucosamine-6-phosphate   NM_005471                                       isomerase       P01074_D06   3.5   −4   −1   5.7   −3.6   1.1   GOLGA4   golgi autoantigen, golgin   NM_002078                                       subfamily a, 4       P01083_C04   −1.1   1.2   1.2   −1.8   1.5   1.1   GOLPH2   golgi phosphoprotein 2   NM_016548       P01125_G10   1.8   −1.9   −1   1.6   −1.9   −1.1   GOLPH4   golgi phosphoprotein 4   NM_014498       P01131_F08   1.7   −2.3   −1.2   1.8   −1.6   −1.2   GOT1   glutamic-oxaloacetic   NM_002079                                       transaminase 1, soluble                                       (aspartate aminotransferase 1)       P01080_A01   −1.2   1.9   1.3   −3.6   1.8   1.2   GPM6B   glycoprotein M6B   NM_005278       P01082_E09   −2.2   2.3   1.2   −2.9   2.3   1   GPNMB   glycoprotein (transmembrane)   NM_002510                                       nmb       P01087_G08   −3   2.3   1   −4.9   4   −1   GPNMB   glycoprotein (transmembrane)   NM_002510                                       nmb       P01140_E04   −1.8   1.6   1.3   −2.5   1.8   1.1   GPRK5   G protein-coupled receptor   NM_005308                                       kinase 5       P01068_E08   −3.2   1.8   −1.1   −1.9   2.9   1.1   GSTM1   glutathione S-transferase M1   NM_000561       P01068_E09   −1.8   1.5   1.1   −1.7   2.3   1.1   GSTM3   glutathione S-transferase M3   NM_000849                                       (brain)       P01086_A10   −2.4   1.5   1.1   −1.9   2.7   1.1   GSTM5   glutathione S-transferase M5   NM_000851       P01080_C03   1.7   −1.8   1.1   2.1   −1.9   −1   GTPBP2   GTP binding protein 2   NM_019096       P01108_A05   −1.2   1.5   1.2   −1.9   1.7   1.1   GYPC   glycophorin C (Gerbich blood   NM_002101                                       group)       P01121_B02   −1.6   1.2   −1   −1.9   1.9   1.1   HAGE   DEAD-box protein   NM_018665       P01133_H11   −1.2   1.8   1.4   −2.1   1.7   1.2   HAS2   hyaluronan synthase 2   NM_005328       P01101_C10   −1.8   1.6   −1   −1.4   1.5   −1   HEBP1   heme binding protein 1   NM_015987       P01137_B02   1.8   −1.5   1   1.6   −1.3   1   HERPUD1   homocysteine-inducible,   NM_014685                                       endoplasmic reticulum stress-                                       inducible, ubiquitin-like domain                                       member 1       P01136_A05   2   −2.1   1.1   2   −1.8   −1   HERPUD1   homocysteine-inducible,   NM_014685                                       endoplasmic reticulum stress-                                       inducible, ubiquitin-like domain                                       member 1       P01083_G12   1.6   −1.1   1.1   2.2   −1.5   1.2   HEYL   hairy/enhancer-of-split related   NM_014571                                       with YRPW motif-like       P01126_B01   −1.3   1.4   1.1   −1.8   1.8   −1   HFL1   H factor (complement)-like 1   NM_002113       P01075_H10   −3.6   6.2   1.3   −5.3   3.8   1.3   HGF   hepatocyte growth factor   NM_000601                                       (hepapoietin A; scatter factor)       P01110_C10   1.9   −1.6   1.3   1.3   −1.2   1.1   HMGCR   3-hydroxy-3-methylglutaryl-   NM_000859                                       Coenzyme A reductase       P01112_G07   2   −1.7   1.3   −1   −1.1   −1   HMGCS1   3-hydroxy-3-methylglutaryl-   NM_002130                                       Coenzyme A synthase 1                                       (soluble)       P01064_F02   −2   2.6   1.1   −3.1   3   1   HNMT   histamine N-methyltransferase   NM_006895       P01078_F05   −2.1   2.4   1.2   −2.1   2.6   1.1   HPN   hepsin (transmembrane   NM_002151                                       protease, serine 1)       P01107_H06   1.8   −1.9   −1.2   1.4   −1.7   −1.3   IARS   isoleucine-tRNA synthetase   NM_002161       P01100_C10   −1.2   1.5   1.3   −1.8   1.7   1.1   ICOS   inducible T-cell co-stimulator   NM_012092       P01124_A06   −1.7   2   1.3   −1.8   1.7   −1   ID2   inhibitor of DNA binding 2,   NM_002166                                       dominant negative helix-loop-                                       helix protein       P01072_H03   1.8   −1.6   1.1   1.6   −1.6   1.2   ID4   inhibitor of DNA binding 4,   NM_001546                                       dominant negative helix-loop-                                       helix protein       P01088_C01   −2.4   2.2   1   −2.5   2.2   −1   IDH2   isocitrate dehydrogenase 2   NM_002168                                       (NADP+), mitochondrial       P01130_F01   4.5   −2.9   1.3   1.8   −1.7   1   IGF1   insulin-like growth factor 1   NM_000618                                       (somatomedin C)       P01063_D10   2.1   1   1.2   3.8   −1.9   1.3   IGF1   insulin-like growth factor 1   NM_000618                                       (somatomedin C)       P00777_D09   −2.6   2.2   1.1   −2.9   3.2   1.2   IGFBP4   insulin-like growth factor   NM_001552                                       binding protein 4       P01130_B02   12.3   −11.1   1.2   6.1   −5.4   1.1   IL11   interleukin 11   NM_000641       P01088_D05   −2   2   1.2   −1.8   1.4   1.1   IL1B   interleukin 1, beta   NM_000576       P01063_E06   −3   3.3   1.1   −6.7   6.1   1.1   IL1R1   interleukin 1 receptor, type I   NM_000877       P01110_E12   −1.4   2.3   1.3   −2.5   1.5   1.1   IL1R1   interleukin 1 receptor, type I   NM_000877       P01145_A04   −3   2.4   −1   −4.2   2.7   −1   IL6ST   interleukin 6 signal transducer   NM_002184                                       (gp130, oncostatin M receptor)       P01091_B03   −1.9   1.9   −1   −1.3   1.3   1   IMPA2   inositol(myo)-1(or 4)-   NM_014214                                       monophosphatase 2       P01063_E03   1.7   −1.7   −1.2   2.4   −1.7   1.1   INDO   indoleamine-pyrrole 2,3   NM_002164                                       dioxygenase       P01082_F07   2.1   −2.6   −1.1   2.2   −1.5   1.2   INHBA   inhibin, beta A (activin A,   NM_002192                                       activin AB alpha polypeptide)       P01130_D09   2.1   −1.7   −1   1.7   −1.7   −1.1   INPP4B   inositol polyphosphate-4-   NM_003866                                       phosphatase, type II, 105 kDa       P01067_B04   2   −1.7   1.2   1.6   −1.3   1.1   INSIG1   insulin induced gene 1   NM_005542       P01074_G10   −1.7   1.7   −1   −4.7   3.1   −1.1   IQGAP2   IQ motif containing GTPase   NM_006633                                       activating protein 2       P01061_E02   2.6   −2.6   1   2.4   −2.5   −1   ISGF3G   interferon-stimulated   NM_006084                                       transcription factor 3, gamma                                       48 kDa       P01140_B08   1.8   −1.7   1.2   3   −1.8   1   ITGA11   integrin, alpha 11   NM_012211       P01088_C11   −1.5   1.8   1.2   −1.8   1.9   1.1   ITGAM   integrin, alpha M (complement   NM_000632                                       component receptor 3, alpha;                                       also known as CD11b (p170),                                       macrophage antigen alpha                                       polypeptide)       P01081_E02   2.3   −1.8   1.2   2.2   −2.2   1   JUNB   jun B proto-oncogene   NM_002229       P01072_G01   1.6   −1.5   1.2   1.9   −1.6   1.1   JUP   junction plakoglobin   NM_002230       P01079_A01   −1.9   2.1   −1.1   −1.5   1.5   −1   JWA   vitamin A responsive;   NM_006407                                       cytoskeleton related       P01122_A09   1.1   1.3   1.2   −1.9   1.6   1.1   KCNE3   potassium voltage-gated   NM_005472                                       channel, lsk-related family,                                       member 3       P01113_F02   −1.8   1.9   1.2   −2.4   2.3   1.1   KHDRBS3   KH domain containing, RNA   NM_006558                                       binding, signal transduction                                       associated 3       P01074_B01   −1.6   1.2   1.1   −1.9   1.7   1   KIAA0102   KIAA0102 gene product   NM_014752       P01104_A04   −3.2   3.8   −1   −3   3.4   −1   KIAA1049   KIAA1049 protein   NM_014972       P01120_B02   −1.6   1.5   1.1   −1.8   1.7   1   KIF1B   kinesin family member 1B   NM_015074       P01088_C06   −1.6   1.6   1.2   −1.9   1.8   1   KRT4   keratin 4   NM_002272       P01085_D06   −1.8   1.7   1.2   −3.8   4.1   1   LAMA4   laminin, alpha 4   NM_002290       P01131_H02   −1.4   1.4   1.1   −2   1.9   −1.1   LAMC1   laminin, gamma 1 (formerly   NM_002293                                       LAMB2)       P01131_H10   −2.4   1.8   −1.1   −2.1   1.5   1   LCN2   lipocalin 2 (oncogene 24p3)   NM_005564       P01100_H05   −2.8   2.7   1.2   −5   2.7   1   LEPR   leptin receptor   NM_002303       P01088_B02   −2.3   2.4   1.1   −2.6   2.1   −1   LGALS3   lectin, galactoside-binding,   NM_002306                                       soluble, 3 (galectin 3)       P01081_B11   −3.5   1.3   1.1   −4.6   4.4   1   LHFP   lipoma HMGIC fusion partner   NM_005780       P01107_D06   2.2   −2   −1   1.7   −1.8   −1.1   LIMK2   LIM domain kinase 2   NM_005569       P01085_G06   1.2   −1.4   −1.1   1.9   −2.1   −1   LMO7   LIM domain only 7   NM_005358       P01085_D05   −2.1   2.2   1.2   −3.9   3.7   1.1   LOC56270   hypothetical protein 628   NM_019613       P01082_E01   2.1   −1.5   1.2   1.8   −1.6   1.2   LOX   lysyl oxidase   NM_002317       P01083_H02   −1.4   1.5   1.1   −2   2   1   LPHN2   latrophilin 2   NM_012302       P01131_D06   −1.6   1.7   1.2   −2.4   1.8   1.2   LRP4   low density lipoprotein   AB011540                                       receptor-related protein 4       P01072_F03   1.8   −1.2   −1   2.2   −1.6   −1   LTBP2   latent transforming growth   NM_000428                                       factor beta binding protein 2       P01088_C04   −2.3   2.3   1.1   −4.4   4.7   1.1   LTF   lactotransferrin   NM_002343       P01063_A11   −2.3   2.4   −1   −4.8   3.9   −1   LUM   lumican   NM_002345       P01135_G05   −2.4   2.4   1.2   −1.7   1.6   −1   LY96   lymphocyte antigen 96   NM_015364       P01085_C04   −2   1.8   1.2   −2   1.5   1   MADH3   MAD, mothers against   NM_005902                                       decapentaplegic homolog 3                                       ( Drosophila )       P01091_G10   1.8   −1.4   1.2   2.2   −2.1   1.2   MADH7   MAD, mothers against   NM_005904                                       decapentaplegic homolog 7                                       ( Drosophila )       P01089_C01   1.2   −1.2   −1   1.8   −1.6   −1.2   MAGP2   Microfibril-associated   NM_003480                                       glycoprotein-2       P01084_A09   1.8   −1.6   1.2   1.4   −1.6   −1   MAP3K2   mitogen-activated protein   NM_006609                                       kinase kinase kinase 2       P01073_E08   −2   2.4   −1   −2.3   1.8   −1   MAP3K5   mitogen-activated protein   NM_005923                                       kinase kinase kinase 5       P01066_F10   2   −2   1.1   1.9   −1.7   1.1   MAPK7   mitogen-activated protein   NM_002749                                       kinase 7       P01076_B12   1.9   −2.1   −1.1   1.7   −1.7   −1.1   MAPRE2   microtubule-associated   NM_014268                                       protein, RP/EB family, member 2       P01134_C04   3.1   −2.1   1.1   2.8   −3.3   −1   MATN3   matrilin 3   NM_002381       P01145_A05   −1.7   1.9   −1   −2.6   2.1   −1   ME1   malic enzyme 1, NADP(+)-   NM_002395                                       dependent, cytosolic       P01072_D11   −3.3   3.7   −1   −3.5   3.1   1   MEST   mesoderm specific transcript   NM_002402                                       homolog (mouse)       P01121_F04   −1.9   2.1   1.3   −2.1   1.8   1   MGC1203   hypothetical protein MGC1203   NM_024296       P01068_F12   −2.9   2.8   1.1   −2.6   2.4   −1   MGST1   microsomal glutathione S-   NM_020300                                       transferase 1       P01091_B06   −1.8   1.6   −1   −1.5   1.6   −1.1   MGST2   microsomal glutathione S-   NM_002413                                       transferase 2       P01099_H09   −2.4   2.3   1.1   −2.4   2   1.2   MID1   midline 1 (Opitz/BBB   NM_000381                                       syndrome)       P01062_H05   −1.4   2.2   1.3   −2.4   2.4   1.3   MME   membrane metallo-   NM_000902                                       endopeptidase (neutral                                       endopeptidase,                                       enkephalinase, CALLA, CD10)       P01125_H08   1   1   1.1   2.6   −2.1   −1   MMP11   matrix metalloproteinase 11   NM_005940                                       (stromelysin 3)       P01072_D02   2.8   −2.6   −1.3   1.7   −2   −1.2   MTHFD2   methylene tetrahydrofolate   NM_006636                                       dehydrogenase (NAD+                                       dependent),                                       methenyltetrahydrofolate                                       cyclohydrolase       P01125_A10   −1.6   1.6   1.2   −1.8   1.5   1.1   MTMR4   myotubularin related protein 4   NM_004687       P01130_C09   1.9   −1.7   1.3   1.1   −1.1   1.1   MUCDHL   mucin and cadherin-like   NM_017717       P01102_A12   1.4   −1.3   1.2   2.5   −1.6   1.1   MVK   mevalonate kinase (mevalonic   NM_000431                                       aciduria)       P01133_F05   1.9   −1.8   1   1.4   −1.4   −1.1   MYH9   myosin, heavy polypeptide 9,   NM_002473                                       non-muscle       P01100_B07   −2   2.4   1.1   −5.5   2.6   −1.1   MYOZ2   myozenin 2   NM_016599       P01072_C06   −1.6   1.6   1   −2.6   2.6   1.1   NCK1   NCK adaptor protein 1   NM_006153       P01086_B12   −1.2   1.4   −1   −1.8   1.6   −1.1   NCOA3   nuclear receptor coactivator 3   NM_006534       P01135_C12   3.3   −3   1.3   3.1   −2.1   1.2   NEDD9   neural precursor cell   NM_006403                                       expressed, developmentally                                       down-regulated 9       P01112_A08   2.5   −2.1   1.2   1.7   −1.8   1.1   NET-6   transmembrane 4 superfamily   NM_014399                                       member tetraspan NET-6       P01103_E02   −1.7   2.1   1.2   −2.5   2.1   −1   NFIA   nuclear factor I/A   AL096888       P01073_E06   −1.9   1.9   1   −2.1   1.8   −1.1   NFIB   nuclear factor I/B   NM_005596       P01064_C02   −1.9   2   1.2   −3.3   2.5   1   NID2   nidogen 2 (osteonidogen)   NM_007361       P01131_E08   2.3   −1.6   1.3   5.1   −3   1.3   NINJ2   ninjurin 2   NM_016533       P01072_D01   2.2   −2.2   1.1   2.2   −2.1   −1   NK4   natural killer cell transcript 4   NM_004221       P01121_G06   −2.2   2.1   −1.1   −2.5   2.2   −1.1   NOL3   nucleolar protein 3 (apoptosis   NM_003946                                       repressor with CARD domain)       P01104_C08   6.9   −6.1   1.1   5.8   −5.8   1.1   NOX4   NADPH oxidase 4   NM_016931       P01107_D11   −1.7   1.6   −1   −1.8   1.8   1   NPC2   Niemann-Pick disease, type   NM_006432                                       C2       P01132_G06   2.4   −2   1.3   1.5   −1.6   1.1   NPR3   natriuretic peptide receptor   NM_000908                                       C/guanylate cyclase C                                       (atrionatriuretic peptide                                       receptor C)       P01096_F08   −1.5   1.6   1.2   −2.1   2   1.1   NPTX2   neuronal pentraxin II   NM_002523       P01126_E07   −1.5   2   1.2   −2   1.7   1.1   NR2F2   nuclear receptor subfamily 2,   NM_021005                                       group F, member 2       P01064_G11   −1.5   1.6   1   −2.1   1.5   −1   NRCAM   neuronal cell adhesion   NM_005010                                       molecule       P01097_E11   1.9   −1.8   1.1   1.6   −1.8   −1   NS1-BP   NS1-binding protein   NM_006469       P01103_C04   2.4   −2.2   −1   1.3   −1.5   −1   NUDT3   nudix (nucleoside diphosphate   NM_006703                                       linked moiety X)-type motif 3       P01072_B11   2.6   −2.6   −1.1   2.3   −2.3   −1.2   ODC1   omithine decarboxylase 1   NM_002539       P01082_E10   −1.4   2   1.3   −5.7   1.9   1.2   OGN   osteoglycin (osteoinductive   NM_014057                                       factor, mimecan)       P01119_G07   −2.1   2.2   1.1   −2.2   1.6   −1   OSBPL1A   oxysterol binding protein-like   NM_018030                                       1A       P01075_F01   2.3   −1.6   −1   3.9   −3.7   −1   OSF-2   osteoblast specific factor 2   NM_006475                                       (fasciclin I-like)       P01129_A10   2.2   −1.6   −1   4.1   −3.6   −1   OSF-2   osteoblast specific factor 2   NM_006475                                       (fasciclin I-like)       P01126_B11   −2   1.5   −1.2   1.1   1.7   1.2   OXA1L   oxidase (cytochrome c)   NM_005015                                       assembly 1-like       P01071_D09   −1.9   1.6   −1.1   1.1   1.7   1.3   OXA1L   oxidase (cytochrome c)   NM_005015                                       assembly 1-like       P01085_C08   −1.3   1.3   1.1   −2.2   1.6   −1   OXTR   oxytocin receptor   NM_000916       P01125_D04   2.1   −1.7   1.2   4.2   −2.3   1.3   PACE4   paired basic amino acid   NM_002570                                       cleaving system 4       P01090_D03   −1.4   1.2   −1   −2.4   1.8   −1.2   PARG1   PTPL1-associated RhoGAP 1   NM_004815       P01122_G06   2.6   −2.4   1.1   1.9   −2   −1   PAWR   PRKC, apoptosis, WT1,   NM_002583                                       regulator       P01120_F04   −1.8   2.3   1.3   −2   1.7   1.1   PBF   papillomavirus regulatory   NM_018660                                       factor PRF-1       P01071_G08   −2   1.5   −1   −1.4   1.7   1   PBP   prostatic binding protein   NM_002567       P01064_A09   1.1   −1.2   1.2   1.9   −1.5   1.2   PCDH1   protocadherin 1 (cadherin-like   NM_002587                                       1)       P01066_G05   −1.4   1.6   1.3   −3.2   2.4   1.2   PDE1A   phosphodiesterase 1A,   NM_005019                                       calmodulin-dependent       P01128_B03   1.8   −1.7   1.1   −1.1   −1   −1   PDE5A   phosphodiesterase 5A, cGMP-   NM_001083                                       specific       P01087_E02   3.4   −2.4   1.1   3   −3.7   −1   PDGFA   platelet-derived growth factor   NM_002607                                       alpha polypeptide       P01081_F07   −2.3   2.1   1.1   −2.2   2.1   1.1   PDGFRA   platelet-derived growth factor   NM_006206                                       receptor, alpha polypeptide       P01142_D01   −1.1   −1.8   1.2   −2.2   1.9   1.2   PDGFRL   platelet-derived growth factor   NM_006207                                       receptor-like       P01064_G02   1.3   −1.1   1.3   2.3   −2   1.2   PDGFRL   platelet-derived growth factor   NM_006207                                       receptor-like       P01137_F04   −1.8   2   1.1   −2   1.4   1.1   PDP   pyruvate dehydrogenase   NM_018444                                       phosphatase       P01071_H07   1.8   −1.9   1   1.3   −1   1.1   PFKP   phosphofructokinase, platelet   NM_002627       P01064_H07   −1.8   1.7   1   −1.6   1.5   1   PHF3   PHD finger protein 3   NM_015153       P01131_G12   1.2   −1   1.2   1.8   −1.7   1.2   PIGB   phosphatidylinositol glycan,   NM_004855                                       class B       P01074_H07   −1.8   1.9   1.2   −1.9   1.6   1   PIK3R1   phosphoinositide-3-kinase,   AF279367                                       regulatory subunit, polypeptide                                       1 (p85 alpha)       P01068_A02   −2.4   1.7   −1.1   −1.4   2.1   1   PIR   Pirin   NM_003662       P01112_H01   1.8   −1.6   1.4   1.3   −1.4   −1   PIST   PDZ/coiled-coil domain   NM_020399                                       binding partner for the rho-                                       family GTPase TC10       P01118_H09   −2.4   2.1   −1   −1.8   1.9   1   PITPNM   phosphatidylinositol transfer   NM_004910                                       protein, membrane-associated       P01110_G02   −1.3   1.6   1.3   −4   2   1   PKIB   protein kinase (cAMP-   NM_032471                                       dependent, catalytic) inhibitor                                       beta       P01146_A11   1.4   −1.5   −1   1.8   −1.9   −1   PLA2G4C   phospholipase A2, group IVC   NM_003706                                       (cytosolic, calcium-                                       independent)       P01124_G10   3   −2.5   1   2.5   −3.2   −1.2   PLA2R1   phospholipase A2 receptor 1,   NM_007366                                       180 kDa       P01070_G08   1.8   −1.7   −1   1.9   −2.3   −1.1   PLAU   plasminogen activator,   NM_002658                                       urokinase       P01064_F01   −1.8   2.3   1.2   −1.7   1.9   1.2   PLCL1   phospholipase C-like 1   NM_006226       P01118_E04   2.4   −1.8   1.3   2.3   −1.9   1.1   PLEK2   pleckstrin 2   NM_016445       P01072_A03   5.2   −5.1   1.3   2.1   −1.6   1.2   PLN   phospholamban   NM_002667       P01084_A08   2.8   −2.2   1.1   1.9   −1.8   1.1   PLOD2   procollagen-lysine, 2-   NM_000935                                       oxoglutarate 5-dioxygenase                                       (lysine hydroxylase) 2       P01063_E04   1.6   −1.7   −1.2   2.4   −1.6   1.1   PLP2   proteolipid protein 2 (colonic   NM_002668                                       epithelium-enriched)       P01130_B04   −3.6   4.3   1   −5.6   5.1   1.1   PMP2   peripheral myelin protein 2   NM_002677       P01131_C08   −2.2   1.6   1.1   −3.4   2.3   1.1   PNUTL2   peanut-like 2 ( Drosophila )   NM_004574       P01106_F02   1.5   −1.3   1.2   1.8   −1.7   1.1   PODXL   podocalyxin-like   NM_005397       P01074_B08   2.8   −1.8   1.2   2.2   −2.8   1.1   POLD3   polymerase (DNA directed),   BC020587                                       delta 3       P01080_A04   −1.3   1.4   1   −1.8   1.5   1.1   PP   pyrophosphatase (inorganic)   NM_021129       P01123_E01   −2.9   3.2   1.3   −3.1   2.8   1.3   PPAP2B   phosphatidic acid phosphatase   NM_003713                                       type 2B       P01064_B12   −1.5   1.7   1.2   −2.2   1.5   −1   PPARG   peroxisome proliferative   NM_005037                                       activated receptor, gamma       P01136_D03   −5.4   3.3   1.1   −5.3   4.2   −1   PPL   periplakin   NM_002705       P01131_H04   1.2   −1.4   −1.2   2   −1.6   −1.2   PPP2R4   protein phosphatase 2A,   NM_021131                                       regulatory subunit B′ (PR 53)       P01087_D04   −1.2   1.3   −1.1   −1.9   1.5   −1.1   PRKCM   protein kinase C, mu   NM_002742       P01128_H07   2.3   −2.2   1.3   1.4   −1.4   1.1   PRPS1   phosphoribosyl pyrophosphate   NM_002764                                       synthetase 1       P01062_F06   −1.6   1.5   1.1   −4.4   3.6   1   PSG1   pregnancy specific beta-1-   NM_006905                                       glycoprotein 1       P01133_G04   −2   1.9   1.2   −5.5   4.8   −1   PSG1   pregnancy specific beta-1-   NM_006905                                       glycoprotein 1       P01131_G08   −1.4   1.4   1.2   −2.6   2.6   1.1   PSG11   pregnancy specific beta-1-   NM_002785                                       glycoprotein 11       P01141_B07   −1.4   1.8   1.3   −4.1   4   1.1   PSG4   pregnancy specific beta-1-   NM_002780                                       glycoprotein 4       P01079_F07   −1.5   1.5   1.1   −2   1.5   −1   PTGER4   prostaglandin E receptor 4   NM_000958                                       (subtype EP4)       P01131_C07   −2.8   1.7   −1.1   −2.2   1.8   −1.1   PTGIS   prostaglandin I2 (prostacyclin)   NM_000961                                       synthase       P01102_D10   2.3   −2.8   −1.1   1.1   −1.2   −1.1   PTGS1   prostaglandin-endoperoxide   NM_000962                                       synthase 1 (prostaglandin G/H                                       synthase and cyclooxygenase)       P01087_D05   3   −2.6   1.1   1.3   −1.2   −1.1   PTGS2   prostaglandin-endoperoxide   NM_000963                                       synthase 2 (prostaglandin G/H                                       synthase and cyclooxygenase)       P01106_G06   1.8   −1.5   1.2   3.1   −1.5   1.1   PTHLH   parathyroid hormone-like   NM_002820                                       hormone       P01071_G12   −1.9   1.4   −1   −3.7   3.6   −1.1   PTN   pleiotrophin (heparin binding   NM_002825                                       growth factor 8, neurite                                       growth-promoting factor 1)       P01128_H08   −2.3   2.4   1.1   −1.5   1.3   −1   PTTG1   pituitary tumor-transforming 1   NM_004219       P01095_A03   −2.4   2.4   1.2   −1.4   1.2   1   PTTG1   pituitary tumor-transforming 1   NM_004219       P01097_G06   −1.7   1.7   −1   −2.2   1.6   −1   PUS1   pseudouridylate synthase 1   NM_025215       P01076_C04   2.5   −1.7   1.2   3.1   −2.6   1.2   QPCT   glutaminyl-peptide   NM_012413                                       cyclotransferase (glutaminyl                                       cyclase)       P01129_C05   −2.1   1.8   −1   −1.5   2.1   1.2   RAB13   RAB13, member RAS   NM_002870                                       oncogene family       P01115_G01   −1.8   1.5   1.1   −1.6   2.2   1.2   RAB13   RAB13, member RAS   NM_002870                                       oncogene family       P01110_E09   1.4   −1.2   1.2   1.8   −1.6   1   RAI   RelA-associated inhibitor   NM_006663       P01100_E02   −1.5   1.5   1.1   −3.3   2.7   1   RAI3   retinoic acid induced 3   NM_003979       P01082_A01   −2.4   1.8   1.1   −2.6   2.3   1.1   RARRES3   retinoic acid receptor   NM_004585                                       responder (tazarotene                                       induced) 3       P01117_H10   −1.8   1.6   1.1   −2.5   2   1   RASSF5   Ras association (RalGDS/AF-   NM_031437                                       6) domain family 5       P01108_C07   1.4   −1.4   1.1   2.5   −1.9   1.2   RBP1   retinol binding protein 1,   NM_002899                                       cellular       P01136_C04   2.6   −1.8   1.1   2.3   −1.6   1.2   RGS2   regulator of G-protein   NM_002923                                       signalling 2, 24 kDa       P01145_A10   −1.2   1.2   −1.1   −2   1.6   −1.1   RGS4   regulator of G-protein   NM_005613                                       signalling 4       P01090_D02   −1.3   1.2   −1.1   −3   1.9   −1.3   RGS4   regulator of G-protein   NM_005613                                       signalling 4       P01081_H10   −2.2   1.6   1   −6.7   4.7   −1   RGS5   regulator of G-protein   NM_003617                                       signalling 5       P01071_E04   −1.9   1.4   −1.1   −3.8   3.2   −1.1   RNASE1   ribonuclease, RNase A family,   NM_002933                                       1 (pancreatic)       P01088_G09   −1   1   1   −1.8   1.8   −1.1   RPL5   ribosomal protein L5   NM_000969       P01127_E10   1.8   −1.6   1.1   1.7   −1.5   −1   RRAS   related RAS viral (r-ras)   NM_006270                                       oncogene homolog       P01122_B03   −2   2   1.1   −2.4   3.1   1.2   RRP4   homolog of Yeast RRP4   NM_014285                                       (ribosomal RNA processing 4),                                       3′-5′-exoribonuclease       P01104_D09   2.1   −1.7   1.1   2   −1.8   1   RTP801   HIF-1 responsive RTP801   NM_019058       P01121_G04   2.1   −1.8   1.1   4.1   −3.2   1.1   RUVBL2   RuvB-like 2 ( E. coli )   NM_006666       P01087_B06   −1.4   1   −1.2   −1.9   2.4   −1.2   S100A10   S100 calcium binding protein   NM_002966                                       A10 (annexin II ligand,                                       calpactin I, light polypeptide                                       (p11))       P01064_F10   1.5   −1.5   −1.3   1.8   −1.5   −1.2   S100A11   S100 calcium binding protein   NM_005620                                       A11 (calgizzarin)       P00777_A05   −1.9   1.7   1.1   −2.3   2.4   1.1   S100A4   S100 calcium binding protein   NM_002961                                       A4 (calcium protein,                                       calvasculin, metastasin,                                       murine placental homolog)       P00777_A06   −1.9   1.8   1.1   −2.6   2.7   1.1   S100A4   S100 calcium binding protein   NM_002961                                       A4 (calcium protein,                                       calvasculin, metastasin,                                       murine placental homolog)       P01143_A11   −1.7   1.7   1.1   −2.4   2.4   1.1   S100A4   S100 calcium binding protein   NM_002961                                       A4 (calcium protein,                                       calvasculin, metastasin,                                       murine placental homolog)       P01141_F03   1.5   −1.2   1.3   3.9   −1.7   1.3   SAA2   serum amyloid A2   NM_030754       P01061_F04   −3.1   4   1.3   −2.2   2.8   1.3   SAT   spermidine/spermine N1-   NM_002970                                       acetyltransferase       P01124_B03   −2.9   3.7   1.4   −2.1   2.5   1.4   SAT   spermidine/spermine N1-   NM_002970                                       acetyltransferase       P01140_G05   2   −2.1   1.1   1.3   −1.3   −1   SC5DL   sterol-C5-desaturase (ERG3   NM_006918                                       delta-5-desaturase homolog,                                       fungal)-like       P01066_H04   4.1   −2.7   1.2   3   −2   1.2   SCD   stearoyl-CoA desaturase   NM_005063                                       (delta-9-desaturase)       P01140_D11   4.7   −3.8   1.2   3.5   −2.4   1   SCD   stearoyl-CoA desaturase   NM_005063                                       (delta-9-desaturase)       P01119_B12   −1.6   1.9   1.2   −3.9   2.3   1.1   SCDGF-B   spinal cord-derived growth   NM_025208                                       factor-B       P01087_A04   −1.1   1.3   1.2   −4   2   1.2   SCG2   secretogranin II (chromogranin   NM_003469                                       C)       P01096_B12   2.6   −1.9   1.2   2.8   −2.5   −1   SCRG1   scrapie responsive protein 1   NM_007281       P01071_B04   −1.7   1.7   −1.1   −2.6   2.3   1   SDC4   syndecan 4 (amphiglycan,   NM_002999                                       ryudocan)       P01063_H09   −1.8   1.7   1   −1.8   1.6   −1   SDCBP   syndecan binding protein   NM_005625                                       (syntenin)       P01076_C05   1.8   −1.5   1.2   1   −1.2   −1   SEC23A   Sec23 homolog A ( S. cerevisiae )   NM_006364       P01096_G04   −3.6   2.5   −1   −3   6   1.3   SELENBP1   selenium binding protein 1   NM_003944       P01119_G09   −3.2   2.4   1.1   −2.5   5.8   1.4   SELENBP1   selenium binding protein 1   NM_003944       P01076_B03   −1.6   1.4   −1   −2   1.5   −1.1   SEPP1   selenoprotein P, plasma, 1   NM_005410       P01062_D11   3   −2.9   −1   4.3   −3.3   −1   SERPINE1   serine (or cysteine) proteinase   NM_000602                                       inhibitor, clade E (nexin,                                       plasminogen activator inhibitor                                       type 1), member 1       P01090_H11   −1.3   1.2   1.2   −1.9   2.1   1.3   SFRP1   secreted frizzled-related   NM_003012                                       protein 1       P01078_F01   −1.8   2.4   −1.1   −1.6   1.6   1.1   SFRP4   secreted frizzled-related   NM_003014                                       protein 4       P01087_A06   −2.9   1.9   −1.2   −3   2.2   −1.3   SGNE1   secretory granule,   NM_003020                                       neuroendocrine protein 1 (7B2                                       protein)       P01106_G05   1.8   −1.7   1.3   2.9   −2.2   1.2   SKIL   SKI-like   NM_005414       P01102_A06   −1.8   2   1.3   −3.2   2.8   1.3   SLC11A3   solute carrier family 11   NM_014585                                       (proton-coupled divalent metalion                                       transporters), member 3       P01105_A03   1.9   −1.7   1.1   1.5   −1.4   −1   SLC1A4   solute carrier family 1   NM_003038                                       (glutamate/neutral amino acid                                       transporter), member 4       P01143_D11   −2.7   2.5   1.3   −2.1   2.8   1.1   SLC25A11   solute carrier family 25   NM_003562                                       (mitochondrial carrier;                                       oxoglutarate carrier), member                                       11       P01111_H03   1.8   −1.7   1   1.9   −2   −1   SLC7A11   solute carrier family 7,   NM_014331                                       (cationic amino acid                                       transporter, y+ system)                                       member 11       P01138_A08   3   −2.9   −1   2.3   −2.4   −1.1   SLC7A5   solute carrier family 7 (cationic   NM_003486                                       amino acid transporter, y+                                       system), member 5       P01088_E10   3.1   −2.7   1.1   2.6   −2.3   1   SLC7A5   solute carrier family 7 (cationic   NM_003486                                       amino acid transporter, y+                                       system), member 5       P01112_E05   −1.6   1.3   1   −3   2.2   −1   SLIT3   slit homolog 3 ( Drosophila )   NM_003062       P01136_F07   −1.1   1.4   1.2   −2.1   1.9   1.1   SLIT3   slit homolog 3 ( Drosophila )   NM_003062       P01079_G03   −3.1   3.4   −1   −3.9   3.5   −1   SNAI2   snail homolog 2 ( Drosophila )   NM_003068       P01140_F07   2.9   −2.6   1.1   2.2   −2.5   1.1   SNF1LK   SNF1-like kinase       P01083_A04   −3.2   3.2   1   −9.3   7   −1.1   SNK   serum-inducible kinase   NM_006622       P01085_F06   −1.2   1.2   1.1   −2.6   1.7   1.1   SOD3   superoxide dismutase 3,   NM_003102                                       extracellular       P01074_H12   1   1.1   1.1   −2.6   1.5   1.1   SPINT2   serine protease inhibitor,   NM_021102                                       Kunitz type, 2       P01108_B02   −2.5   2.6   1.2   −4.2   2.2   −1   SPRY1   sprouty homolog 1, antagonist   AF041037                                       of FGF signaling ( Drosophila )       P01095_F04   −2.6   2   −1.1   −1.8   1.8   −1.1   SQRDL   sulfide quinone reductase-like   NM_021199                                       (yeast)       P01128_E07   1.9   −2   1   2.6   −2.7   −1   SRPUL   sushi-repeat protein   NM_014467       P01073_B02   −1.7   1.7   1.2   −2.5   1.9   −1   SRPX   sushi-repeat-containing   NM_006307                                       protein, X chromosome       P01104_F12   −2.1   2.5   1.2   −2.2   2.3   −1.1   SSBP2   single-stranded DNA binding   NM_012446                                       protein 2       P01069_C06   1.9   −1.3   −1   2.7   −2.6   −1   SSR1   signal sequence receptor,   NM_003144                                       alpha (translocon-associated                                       protein alpha)       P01130_F10   −1.3   1.6   1.1   −2.3   2.3   −1.1   STC1   stanniocalcin 1   NM_003155       P01130_B11   2.1   −2   −1   1.7   −1.8   −1.1   STCH   stress 70 protein chaperone,   NM_006948                                       microsome-associated, 60 kDa       P01074_E03   1.7   −1.3   −1   −1.9   1.5   −1.1   STE   sulfotransferase, estrogen-   NM_005420                                       preferring       P01127_G01   −1.4   1.5   1.2   −1.9   1.5   1.2   STK17B   serine/threonine kinase 17b   NM_004226                                       (apoptosis-inducing)       P01125_C11   −2   1.6   −1   −2.2   2.1   −1   STK25   serine/threonine kinase 25   NM_006374                                       (STE20 homolog, yeast)       P01076_D03   −2.7   2.9   1.1   −2.1   1.8   1   STK38   serine/threonine kinase 38   NM_007271       P01105_E03   −2.8   2.7   −1.1   −2.7   2.5   −1.1   STMN1   stathmin 1/oncoprotein 18   NM_005563       P01069_A08   −1.5   1.5   1.1   −1.8   1.5   1   STOM   stomatin   NM_004099       P01102_E10   −1.5   1.6   1.1   −2.3   1.5   1   SVIL   supervillin   NM_003174       P01062_H06   −1.5   2.1   1.2   −2.9   2.7   1.2   TACSTD2   tumor-associated calcium   NM_002353                                       signal transducer 2       P01098_E05   1.9   −1.8   1.2   1.2   −1.3   1.1   TAF13   TAF13 RNA polymerase II,   NM_005645                                       TATA box binding protein                                       (TBP)-associated factor,                                       18 kDa       P01101_B02   −1.9   1.4   1.1   −2   1.8   1   TCF7L1   transcription factor 7-like 1 (T-   NM_031283                                       cell specific, HMG-box)       P01061_C01   −1.7   1.6   1.3   −2   2.4   1.1   TF   transferrin   NM_001063       P01144_C03   −3.4   3.6   1.2   −4   2.9   1.1   TFPI   tissue factor pathway inhibitor   NM_006287                                       (lipoprotein-associated                                       coagulation inhibitor)       P01071_A04   −1.4   1.6   1.4   −2.3   2.3   1.1   TFPI2   tissue factor pathway inhibitor 2   NM_006528       P01085_B12   −1.4   1.4   1.2   −2.1   1.7   1.1   TGFB2   transforming growth factor,   NM_003238                                       beta 2       P01061_C08   −3.5   3.8   1.2   −4.7   4   1.2   TGFBR3   transforming growth factor,   NM_003243                                       beta receptor III (betaglycan,                                       300 kDa)       P01078_B04   1.8   −1.7   −1   1.8   −1.9   −1.2   THBS2   thrombospondin 2   NM_003247       P01124_G04   2.8   −2.5   −1   2.4   −3.1   −1.3   TIMP3   tissue inhibitor of   NM_000362                                       metalloproteinase 3 (Sorsby                                       fundus dystrophy,                                       pseudoinflammatory)       P01086_F06   2.1   −2.4   −1   2.6   −3.1   −1.2   TIMP3   tissue inhibitor of   NM_000362                                       metalloproteinase 3 (Sorsby                                       fundus dystrophy,                                       pseudoinflammatory)       P01071_A06   −3   3   −1   −2.5   2.6   −1   TM4SF1   transmembrane 4 superfamily   NM_014220                                       member 1       P01099_E08   −1.6   1.8   1   −1.8   1.5   −1.1   TncRNA   trophoblast-derived noncoding                                       RNA       P01126_E09   −1.7   2   1   −3.7   4.2   1.1   TNFAIP2   tumor necrosis factor, alpha-   NM_006291                                       induced protein 2       P01085_A06   −1.5   1.6   −1   −2.4   1.9   1.1   TNFAIP3   tumor necrosis factor, alpha-   NM_006290                                       induced protein 3       P01138_G10   1.8   −1.7   1.2   2.1   −2   1   TNFRSF12A   tumor necrosis factor receptor   NM_016639                                       superfamily, member 12A       P01078_E05   −2.1   3   1.4   −2.6   2.5   1.2   TNFSF10   tumor necrosis factor (ligand)   NM_003810                                       superfamily, member 10       P01144_C11   −2   2   1   −1.9   1.4   1.2   TOP2A   topoisomerase (DNA) II alpha   NM_001067                                       170 kDa       P01140_D03   2.1   −1.7   −1   1.2   −1.6   −1   TTID   titin immunoglobulin domain   NM_006790                                       protein (myotilin)       P01070_H07   −2.9   2.3   1.1   −3   2.6   −1.1   TXNRD1   thioredoxin reductase 1   NM_003330       P01089_D01   1.7   −3   1.1   2.5   −2.3   −1   UCHL1   ubiquitin carboxyl-terminal   NM_004181                                       esterase L1 (ubiquitin                                       thiolesterase)       P01123_D07   −1.9   2.4   1.1   −2   1.8   1.1   UGCG   UDP-glucose ceramide   NM_003358                                       glucosyltransferase       P01089_F07   1.5   −2.6   1.2   2.4   −1.7   1.2   UMPK   undine monophosphate kinase   NM_012474       P01070_F11   2.1   −3.1   1.1   2.4   −1.9   1   UMPS   uridine monophosphate   NM_000373                                       synthetase (orotate                                       phosphoribosyl transferase                                       and orotidine-5′-                                       decarboxylase)       P01061_B02   −2.7   2.4   1   −4.2   2.3   −1.3   VCAM1   vascular cell adhesion   NM_001078                                       molecule 1       P01141_C06   2.7   −1.8   1.3   1.4   −1.2   1.2   WISP1   WNT1 inducible signaling   NM_003882                                       pathway protein 1       P00777_C09   −1.6   1.8   1.1   −5   4   −1   WISP2   WNT1 inducible signaling   NM_003881                                       pathway protein 2       P00777_C10   −2.2   2.1   1.1   −5.6   4.4   −1   WISP2   WNT1 inducible signaling   NM_003881                                       pathway protein 2       P01126_H07   −1.8   1.6   1.1   −3   3.9   −1   WISP2   WNT1 inducible signaling   NM_003881                                       pathway protein 2       P01142_D08   3.7   −3   1.3   3.6   −2.8   1.1   XRCC4   X-ray repair complementing   NM_003401                                       defective repair in Chinese                                       hamster cells 4       P01104_H07   −1.7   1.7   1.2   −1.9   1.5   1.1   ZFPM2   zinc finger protein, multitype 2   NM_012082       P01064_H12   −1.4   1.5   1.1   −1.8   1.5   −1   ZNF142   zinc finger protein 142 (clone   NM_005081                                       pHZ-49)       P01075_E02   1.5   −1.2   1.1   1.9   −1.9   1   ZNF193   zinc finger protein 193   NM_006299                  
 
 Validation of Microarray by Real-Time RT-PCR and Western Blot Analyses 
 
      Representative microarray data was validated using real-time RT-PCR and Western analyses. TGFβ induced Collagen 1 mRNA levels in human cardiac fibroblasts at 6, 24, and 48 h; this induction was blocked by BNP at all 3 time points ( FIG. 5A ). Collagen 1 protein synthesis was also induced (˜3-fold) at 48 h, and BNP inhibited this stimulation by ˜75% ( FIG. 5B ). BNP also inhibited TGFβ-induced Fibronectin mRNA and protein expression at 48 h ( FIG. 5C ,D). These data corroborate the microarray results, with the exception of Fibronectin, which did not exceed the array differential expression threshold value, most likely due to the lower sensitivity of the microarray compared to real-time RT-PCR. The effects of BNP on TGFβ stimulation of pro-fibrotic genes CTGF, PAI-1, TIMP3, IL11, and ACTA2 were also confirmed by real-time RT-PCR ( FIG. 6 ). Additional verification was obtained for the pro-inflammatory genes COX2 and IL6 at 6, 24, and 48 h ( FIG. 6 ). Again, most likely due to sensitivity issues, IL6 was not included in  FIG. 4C , since it did not exceed the array differential expression threshold value.  
      In addition, real-time RT-PCR assays were performed for 9 genes on primary cultures of human cardiac fibroblasts from a second independent donor lot of fibroblasts (see Table 3). The effects of BNP on TGFβ-induced gene expression in both donors were similar, although donor lot 2 was slightly less responsive to TGFβ. Taken together, these results confirm the microarray data using independent assay methods, as well as, multiple human cardiac fibroblast donors.  
               TABLE 3                          Real-time RT-PCR validation of microarray data using human cardiac fibroblasts from       two separate donors (lot 1 and lot 2). Expression levels are normalized to 18s RNA and       are shown relative to the control samples. Standard deviations reflect duplicate       biological replicates; real-time RT-PCR reactions were performed in triplicate.                                         Gene   Control   BNP   TGFβ   TGFβ + BNP   Time (h)   Lot                                                 Collagen 1   1.0 ± 0.05   1.0 ± 0.05   1.9 ± 0.04   1.2 ± 0.01   6   1           1.0 ± 0.06   1.1 ± 0.13   3.3 ± 0.05   1.3 ± 0.26   24   1           1.0 ± 0.11   1.0 ± 0.26   1.5 ± 0.09   1.2 ± 0.01   24   2           1.0 ± 0.13   1.2 ± 0.03   3.8 ± 0.38   1.3 ± 0.03   48   1           1.0 ± 0.20   1.0 ± 0.01   2.5 ± 0.32   1.3 ± 0.18   48   2       Fibronectin   1.0 ± 0.04   0.9 ± 0.19   1.1 ± 0.17   1.0 ± 0.29   6   1           1.0 ± 0.21   1.0 ± 0.10   1.0 ± 0.05   1.0 ± 0.18   24   1           1.0 ± 0.19   0.9 ± 0.24   1.0 ± 0.02   1.0 ± 0.12   24   2           1.0 ± 0.04   1.1 ± 0.04   2.2 ± 0.38   1.3 ± 0.35   48   1           1.0 ± 0.01   1.0 ± 0.11   2.0 ± 0.39   1.5 ± 0.02   48   2       SERPINE1/PAI-1   1.0 ± 0.07   0.7 ± 0.08   7.3 ± 0.44   1.7 ± 0.37   6   1           1.0 ± 0.01   0.7 ± 0.01   8.5 ± 0.08   0.7 ± 0.10   24   1           1.0 ± 0.10   0.7 ± 0.11   2.4 ± 0.06   1.1 ± 0.10   24   2           1.0 ± 0.22   0.9 ± 0.00   8.4 ± 1.33   0.9 ± 0.13   48   1           1.0 ± 0.17   0.8 ± 0.03   2.6 ± 0.03   0.9 ± 0.06   48   2       CTGF   1.0 ± 0.15   0.9 ± 0.24   3.5 ± 0.08   0.9 ± 0.03   6   1           1.0 ± 0.28   1.0 ± 0.29   3.3 ± 0.25   0.7 ± 0.25   24   1           1.0 ± 0.09   1.5 ± 0.44   2.2 ± 0.16   1.5 ± 0.04   24   2           1.0 ± 0.45   1.4 ± 0.13   3.1 ± 0.01   1.1 ± 0.01   48   1           1.0 ± 0.32   1.3 ± 0.12   2.1 ± 0.14   1.0 ± 0.24   48   2       IL11   1.0 ± 0.20   1.1 ± 0.04   13.3 ± 0.89    2.1 ± 0.06   6   1           1.0 ± 0.13   1.2 ± 0.07   32.3 ± 0.82    1.1 ± 0.14   24   1           1.0 ± 0.06   1.0 ± 0.05   7.7 ± 0.81   2.1 ± 0.18   24   2           1.0 ± 0.23   0.7 ± 0.10   17.6 ± 0.22    1.0 ± 0.08   48   1           1.0 ± 0.09   0.8 ± 0.09   5.9 ± 0.18   1.2 ± 0.10   48   2       TIMP3   1.0 ± 0.01   0.9 ± 0.11   1.4 ± 0.03   1.0 ± 0.12   6   1           1.0 ± 0.31   1.0 ± 0.12   2.6 ± 0.26   1.0 ± 0.23   24   1           1.0 ± 0.13   0.7 ± 0.09   1.5 ± 0.12   1.3 ± 0.14   24   2           1.0 ± 0.26   0.9 ± 0.00   3.0 ± 0.34   1.0 ± 0.09   48   1           1.0 ± 0.08   0.6 ± 0.00   1.7 ± 0.13   0.8 ± 0.01   48   2       IL6   1.0 ± 0.06   0.9 ± 0.02   3.6 ± 0.27   1.3 ± 0.14   6   1           1.0 ± 0.13   0.9 ± 0.21   1.7 ± 0.14   0.8 ± 0.03   24   1           1.0 ± 0.09   0.9 ± 0.07   1.4 ± 0.05   1.0 ± 0.11   24   2           1.0 ± 0.13   0.9 ± 0.03   1.6 ± 0.12   0.9 ± 0.05   48   1           1.0 ± 0.17   0.9 ± 0.06   1.4 ± 0.17   0.9 ± 0.17   48   2       PTGS2/COX-2   1.0 ± 0.01   1.2 ± 0.22   9.0 ± 1.49   1.8 ± 0.05   6   1           1.0 ± 0.08   1.2 ± 0.38   3.5 ± 0.67   1.2 ± 0.19   24   1           1.0 ± 0.07   1.1 ± 0.05   4.9 ± 0.36   1.4 ± 0.18   24   2           1.0 ± 0.10   1.0 ± 0.12   2.2 ± 0.12   1.3 ± 0.03   48   1           1.0 ± 0.19   1.0 ± 0.06   5.4 ± 0.92   1.2 ± 0.01   48   2       ACTA2   1.0 ± 0.03   0.8 ± 0.12   1.1 ± 0.11   0.9 ± 0.20   6   1           1.0 ± 0.14   0.9 ± 0.11   2.2 ± 0.00   0.9 ± 0.07   24   1           1.0 ± 0.04   0.9 ± 0.25   2.3 ± 0.12   1.6 ± 0.41   24   2           1.0 ± 0.17   1.0 ± 0.03   1.0 ± 0.19   1.0 ± 0.21   48   1           1.0 ± 0.05   0.7 ± 0.11   2.5 ± 0.13   1.0 ± 0.12   48   2                  
 
      In a related study, a gene microassay profile of rat heart tissue was conducted. The results of this study are shown in  FIG. 12 . Fibrotic and extracellular matrix associated genes were stimulated in vivo by L-NAME plus angiotensin II. MRNA expression for collagen I, collagen III, and fibronectin was markedly reduced by the administration of BNP.  
      MEK/ERK Pathway Involved in BNP&#39;s Anti-Fibrotic Role  
      Natriuretic peptides were previously shown to stimulate ERK activity in cardiac myocytes and vascular endothelial cells. The MEK/ERK pathway has been linked to the repression of TGFβ/Smad signaling. To determine whether PKG or ERK signaling is involved in BNP-dependent attenuation of TGFβ signaling, cultured cells were treated with BNP and/or TGFβ in the presence of a PKG inhibitor (KT5823) or two different MEK inhibitors (U0126, PD98059). BNP induced ERK phosphorylation was completely blocked by KT5823 and U0126, indicating that BNP activates ERK via PKG and MEK signaling cascades ( FIG. 7   a ). Both MEK inhibitors (U0126, PD98059) reversed BNP inhibition of TGFβ-induced Collagen-1 expression analyzed by Western blot ( FIG. 7   b ) and real-time RT-PCR ( FIG. 7   c ). A similar result was demonstrated for PAI-1 using real-time RT-PCR. These findings suggest that the ERK pathway plays an important role in BNP-dependent inhibition of the fibrotic response induced by TGFβ in human cardiac fibroblasts.  
      Fibrosis and ECM  
      One of the key features of cardiac fibrosis is the increased deposition of the ECM. The dynamic turnover of ECM proteins is controlled by several regulatory mechanisms: de novo biosynthesis of ECM components, proteolytic degradation of ECMs by matrix metalloproteinases (MMPs), and inhibition of MMP activities by endogenous inhibitors, TIMPs. All of these processes have been shown to be profoundly affected by TGFβ. The results provided herein suggest that TGFβ-induced ECM deposition in human cardiac fibroblasts occurs largely by increasing ECM gene expression, including Fibronectin, COL1A2, COL15A, COL7A1, MAGP2, MATN3, FBN1, and COMP. Fibronectin and collagen expression in cardiac fibroblasts has been well-established in the fibrotic response, however, this is the first report of TGFβ induction of other ECM genes including MAGP2, MATN3, FBN1 and COMP, further corroborating TGFβ&#39;s role in ECM induction. Interestingly, COMP, which is a member of the thrombospondin family, has been shown to have a direct interaction with Fibronectin, 25  supporting its role in fibrotic processes. We also found Thombospondin 2, which is involved in the activation of latent TGFβ 26  regulated by TGFβ in our studies and opposed by BNP (Table 2). Also sharing close identity with the latent TGFβ family of binding proteins is FBN1, a component of extracellular microfibrils. The opposing effects of BNP on these gene regulatory events, suggests that BNP modulates cardiac fibrosis.  
      In addition to the suppression of TGFβ-induced ECM biosynthesis, BNP may also modulate the degradation of ECM proteins by opposing elevated TIMP3 levels in TGFβ-stimulated cells. The TIMP family of proteins is believed to play significant roles in controlling extracellular matrix remodeling. Elevation of TIMP3 expression has been observed in animal models of myocardial infarction, suggesting that it may be a contributor to matrix remodeling in the failing heart.  
      Another hallmark of the fibrotic process is the transformation of cardiac fibroblasts to myofibroblasts and the induction of pro-fibrotic mediators. Myofibroblasts acquire contractile properties similar to smooth muscle cells. The results provided above demonstrate that BNP inhibited TGFβ-induction of several myofibroblast markers including ACTA2 and MYH9. BNP also inhibited TGFβ pro-fibrotic mediators, such as, CTGF, PAI-1, and IL11. CTGF and PAI-1 are well-established downstream signaling genes of the TGFβ pathway, and IL11 has been associated with tissue remodeling and fibrosis. IL11 expression in cardiac fibroblasts also seems to contribute to TGFβ-mediated fibrosis. The use of BNP to suppress this response should result in a protective effect.  
      Collectively, these effects of BNP on gene expression in TGFβ-stimulated cells demonstrate a role for BNP in anti-fibrotic processes in cardiac fibroblasts. In striking contrast to TGFβ-treated cells, BNP had no significant effects in unstimulated fibroblasts. This is consistent with the physiological actions of BNP, working only in opposition to other hormonal systems such as the renin-angiotensin-aldosterone system.  
      Changes in Cell Proliferation  
      The effects of TGFβ on cell growth is cell-type dependent. As provided above, TGFβ stimulated cardiac fibroblast proliferation. Whether TGFβ has a direct effect on cell cycle or an indirect effect through other mechanisms is unclear. However, cDNA microarray analysis revealed that BNP markedly inhibits the expression of a number of TGFβ-induced growth factors or growth factor-like genes including PDGFA, IGF1, FGF18, and IGFBP10 (CYR61). The up-regulation of these genes by TGFβ could partially explain the induction of cell proliferation, suggesting that it may be mediated indirectly through the stimulation of growth factor productions. TGFβ also induced the expression of PTHLH (PTHrP), which has known chronotropic and vasodilatory effects. In osteoblast-like cells PTHrP can induce cell proliferation. Interestingly, in the myocardium, PTHrP levels are increased in congestive heart failure (CHF).  
      The growth inhibitory effects of natriuretic peptides have previously been reported. Cao and Gardner first demonstrated that natriuretic peptides inhibit PDGF, FGF2, and mechanical stretch-induced DNA synthesis in neonatal rat cardiac fibroblasts. Consistent with these findings, natriuretic peptides and cyclic GMP have been reported to inhibit cell proliferation induced by angiontensin II, endothelin-1, and norepinephrine in many cell types including cardiac fibroblasts, vascular smooth muscle cells, endothelial cells, and mesangial cells. The results provided herein suggest an important role for BNP in regulating fibroblast growth during cardiac remodeling.  
      Changes in Inflammatory Genes  
      Cardiac expression of cytokines is thought to contribute to a decrease in left ventricle contractile performance and deleterious remodeling. Although similar effects have been observed with ANP, reported herein for the first time is that brain natriuretic peptide blocks TGFβ stimulation of several pro-inflammatory genes including COX2, IL6, TNFAIP6, and TNFSF4.  
      TGFβ has a dual effect in the regulation of inflammatory processes. For example, it increases COX2 expression and prostaglandin E2 release in pulmonary artery smooth muscle cells, airway smooth muscle cells, and intestinal epithelial cells. On the other hand, TGFβ down-regulates the production of MCP-1 and complement components (C3 and C4) in human proximal tubular epithelial cells and macrophages. The results provided herein corroborates the dual effect of TGFβ in the modulation of inflammatory gene expression in cardiac fibroblasts. From these results, it was found that while TGFβ induced some inflammatory genes, it down-regulated others, such as, IL1b, MCP1-R, GRO1, GRO3, and MCP4. Both effects are reversed by BNP. However, in the absence of TGFβ stimulation, BNP had no significant effect on the expression of inflammatory genes. It is likely that a balance of pro- and anti-inflammatory stimuli is important in the process of cardiac remodeling.  
      Signaling Mechanism Underlying BNP&#39;s Anti-Fibrotic Role  
      Studies aimed at elucidating the mechanism of BNP&#39;s inhibition of a fibrotic response indicate that the ERK signaling pathway plays an important role. The results provided herein demonstrate that BNP phosphorylates ERK via PKG-dependent signaling in primary human cardiac fibroblasts. Moreover, this activation attenuates the TGFβ-induced fibrotic response as measured by Collagen 1 expression. This is consistent with previous studies showing that ERK activation is required for both the anti-hypertrophic effect of ANP in cardiac myocytes, and the inhibition of TGFβ signaling in mammary and lung epithelial cells.  
      In Vivo Studies  
      In a related study, an in vivo model for acute myocardial injury was used to explore the effects of BNP. Male Sprague Dawley rats ranging in weight from 225 to 250 gm were utilized. Acute myocardial injury was induced by administration of Nω-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg/day)salt (1% NaCl) plus angiotensin II (AngII, 0.5 mg/kg/day) in the rats. The L-NAME was administered in drinking water from day 1 to day 14. Angiotensin II was continuously infused subcutaneously with an osmotic pump from day 11 to day 14. Rat BNP (400 mg/kg/min) was intravenously infused through an external infusion pump from day 10 to day 14.  
      Systolic blood pressure, plasma level of aldosterone, cardiac function heart/body weight ration and gene expression in the heart were analyzed. Systolic blood pressure was monitored via tail cuff technique with an IITC blood pressure recording system. Cardiac function was monitored via a Millar ARIA Pressure Volume Conductance System with an 1.4 F catheter. Gene expression as referenced above with results provided in  FIG. 12  were monitored by RT-PCR with an ABI Prism™ 7700 sequence detection system.  
      It was observed that BNP had no effect on systolic blood pressure raised by L-NAME+AngII but significantly attenuated aldosterone1.25.2±0.2 vs. 6.6±0.16 ng/ml, p&lt;0.05). See  FIG. 10 . As shown in  FIG. 13 , BNP improved cardiac function by significantly increase in stroke volume (2.68±0.23 vs. 4.74±0.73 ul, p&lt;0.05), ejection fraction (13.6±1.1 vs. 20.4±2.4% p&lt;0.05), and diastolic volume (19.0±0.9 vs 22.4±1.1 ul, p&lt;0.05) and stroke work (223.0±29.4 vs 531.5±99.1 mmH*ul, p&lt;0.05), and decrease in arterial elastance (6.50±5.7 vs 42.6±5.1 mmHg/ul, p&lt;0.01). As shown in  FIG. 11 , BNP significantly reduced the heart/body weigh ratio (0.0039±0.002 vs. 0.0029±0.001, p&lt;0.05) and as referenced above, abolished the profibrotic phenotype indicated by decreasing expression of collagen I (p&lt;0.01), collagen III (p&lt;0.05) and fibronectin (p&lt;0.05).  
     SUMMARY  
      Along with the endothelin pathway, the renin-angiotensin and aldosterone system, the fibrosis-promoting TGFβ pathway is important in the pathophysiology of heart failure. BNP appears to oppose TGFβ-regulated gene expression related to fibrosis and myofibroblast conversion. Furthermore, BNP&#39;s opposition to the TGFβ-stimulated fibrotic response is dependent on the PKG and the MEK/ERK pathways. This finding is consistent with the observation that BNP deficient mice show increased fibrosis and Collagen 1 expression. In addition to BNP&#39;s global effects on fibrosis, it may also have effects on other processes, such as inflammation and proliferation ( FIG. 8 ). These findings support a beneficial role for BNP in the prevention of cardiac fibrosis and the treatment of cardiac diseases. They also provide the first demonstration that BNP has a direct effect on cardiac fibroblasts to oppose a TGFβ-induced fibrotic response, suggesting that BNP functions as an anti-fibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.  
      Independent from the antifibrotic effect, the in vivo studies as provided herein indicate that BNP may be used to reduce cardiac remodeling and prevent subsequent heart failure. BNP may also be useful as a cardioprotective agent to improve cardiac function post acute myocardial injury such as myocardial infarction.  
      All references cited throughout the specification are expressly incorporated herein by reference. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, and the like. All such modifications are within the scope of the claims appended hereto.