(12) Patent Application Publication (10) Pub. No.: US 2006/0019890 A1 Kapoun Et Al

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US 2006OO19890A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0019890 A1 KapOun et al. (43) Pub. Date: Jan. 26, 2006

(54) METHOD FOR TREATING CARDIAC Related U.S. Application Data REMODELING FOLLOWING MYOCARDIAL INJURY (60) Provisional application No. 60/537,221, filed on Jan. 15, 2004. (76) Inventors: Ann M. Kapoun, Mountain View, CA Publication Classification (US); George F. Schreiner, Los Altos, CA (US); Faquan Liang, San (51) Int. Cl. Francisco, CA (US); Zhihe Li, Foster A6IK 38/17 (2006.01) City, CA (US) (52) U.S. Cl...... 514/12 (57) ABSTRACT Correspondence Address: Samuel M. Kais The invention concerns methods for treating cardiac remod Scios Inc. eling in a Subject who has undergone myocardial injury, Said 6500 Paseo Padre Parkway method comprising the administration of natriuretic peptide Fremont, CA 94555 (US) to said subject. Preferably the natriuretic peptide is brain natriuretic peptide. The invention also concerns methods for (21) Appl. No.: 11/038,826 treating Structural heart disorders arising from myocardial injury, Said method comprising the administration of a (22) Filed: Jan. 18, 2005 natriuretic peptide to a patient in need thereof. Patent Application Publication Jan. 26, 2006 Sheet 1 of 13 US 2006/0019890 A1 Figure 1

TGFB treated BNP treated Patent Application Publication Jan. 26, 2006 Sheet 2 of 13 US 2006/0019890 A1 Figure 2

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Fibrosis Myofibroblast Transformation CTGF Cullagen 1, PA-1, IL 11, Fibronectin, Fibrillin, or-Smooth Muscle Actin, TIMP3 non-muscle myosin heavy chain

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Control BNP TGFB TGFB+ BNP Patent Application Publication Jan. 26, 2006 Sheet 10 of 13 US 2006/0019890 A1 Figure 10

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METHOD FOR TREATING CARDIAC inhibitors, BAR antagonists and (at least one type of) aldos REMODELING FOLLOWING MYOCARDIAL terone receptor antagonist can Significantly reduce the inci INJURY dence and extent of cardiac dysfunction and heart failure 0001. This application claims priority to U.S. provisional after myocardial infarction. application Ser. No. 60/537,221. The 60/537,221 provisional 0006 ACE inhibitors are associated with cough in 10% application is herein incorporated by reference in its entirety. of patients and can result in renal failure in the Setting of bilateral renal artery Stenosis or other Severe kidney disease. FIELD OF THE INVENTION BAR antagonists are associated with impotence and depres 0002 The present invention concerns methods of treat Sion, and are contraindicated in patients with asthma, fur ment using one or more natriuretic peptides or derivatives thermore, patients may develop worsened heart failure, thereof. More Specifically, the invention concerns methods hypotension, bradycardia, heart block, and fatigue with of treating or preventing cardiac dysfunction in a Subject initiation of BAR antagonists. Aldosterone receptor antago after Said Subject has undergone myocardial injury. nism causes significant hyperkalemia and painful gyneco mastia in 10% of male patients. Agents without a demon Strated mortality benefit are also associated with problems, BACKGROUND most notable is the consistent finding that many cardiac 0003) Myocardial infarction is a major cause of signifi Stimulants improve Symptoms, but actually increase mortal cant disability and death in the United States and in many ity, likely by triggering lethal cardiac arrhythmias. In Sum other countries around the World, and accounts for approxi mary, presently available pharmacological therapies are mately 2/3 of all heart failure. Hunt et al., AMERICAN ineffective and are limited by Significant unwanted Side COLLEGE OF CARDIOLOGY/AMERICAN Heart ASSO effects, and So development of new therapies with improved ciation. ACC/AHA guidelines for the evaluation and man efficacy and leSS Severe side effects is an important public agement of chronic heart failure in the adult: executive health goal. Summary. A report of the American College of Cardiology/ American Heart Association Task Force on Practice Guide SUMMARY OF THE INVENTION lines (Committee to revise the 1995 Guidelines for the Evaluation and Management of Heart Failure). Journal of 0007. The present invention is directed to the use of the American College of Cardiology 2001; 38: 2101-2113. natriuretic peptides for the prevention and/or treatment of Several disease-initiating events (e.g. myocardial infarction, cardiac remodeling in a Subject that has undergone myocar untreated hypertension, congenital mutations of contractile dial injury. In a preferred embodiment, the natriuretic pep proteins) can result in a common heart disease phenotype tide(s) comprise brain natriuretic peptide (BNP), also known that consists of dilation of the cardiac chambers, resulting in as neSiritide. In another embodiment, the invention is reduction in contractile function (i.e., a decrease in the directed to the treatment of cardiac dysfunction, Said treat fraction of total blood ejected from each chamber during ment comprising the administration of a therapeutically Systole) that leads to the clinical Syndrome of heart failure. effective amount of natriuretic peptide to a Subject that has This phenotype generally involves a compensatory aspect undergone myocardial injury. that results from myocardial infarction when the normal 0008. In another related embodiment, the invention is compensatory hypertrophy of Surviving, non-infarcted myo directed to a method of alleviating or reversing the effect of cardium is insufficient. Often this compensatory mechanism TGFB mediated cell activation in cardiac tissue on the is a result of the profibrotic response associated with cardiac expression of one or more genes associated with fibrosis, injury. comprising contacting one or more cells or tissues in which 0004 Available therapies for heart dysfunction are insuf the expression of Said genes is altered as a result of TGFB ficient, and new methods of treatment are needed. The heart mediated activation, with BNP. In another related embodi responds to infarction by hypertrophy of Surviving cardiac ment, the targeted gene(s) associated with fibrosis are muscle in an attempt to maintain normal contraction. How Selected from the group consisting essentially of Collagen1, ever, when the hypertrophy is insufficient to compensate, Collagent 3, Fibronectin, CTGF, PAI-1, and TIMP3. cardiac remodeling and reduced cardiac function result, 0009. In another embodiment, the invention is directed to leading to heart failure and death. Despite important a method of inhibiting the production of Collagen 1, Col advances in medical therapies for preventing cardiac dyS lagen 3 or Fibronectin proteins by the administration of a function and heart failure after myocardial infarction, these therapeutically effective amount of BNP to a subject in need problems remain a significant unsolved public health prob thereof. lem. 0005 No pharmacological therapy for post MI cardiac 0010. In another related embodiment, the invention is remodeling is curative or Satisfactory, and many patients die directed to a method of inhibiting TGFB mediated myofi or, in Selected cases, undergo heart transplantation. Presently broblast conversion by administration of a therapeutically available pharmacological therapies for reducing cardiac effective amount of BNP to a mammalian subject in need dysfunction and reducing mortality in patients with heart thereof. failure fall into three main categories: angiotensin-convert 0011. In another related embodiment, the invention is ing enzyme (ACE) inhibitors, beta adrenergic receptor directed to a method of alleviating or reversing the effect of (OAR) antagonists, and aldosterone antagonists. Despite TGFB mediated cell activation in cardiac tissue on the reducing mortality, patients treated with these medicines expression of one or more genes associated with cell pro remain at Significantly increased rislc for death compared to liferation, comprising contacting one or more cells or tissues age-matched control patients without heart failure. ACE in which the expression of Said genes is altered as a result of US 2006/OO19890 A1 Jan. 26, 2006

TGFB mediated activation, with BNP. In another related 0.018 FIG. 6. Effects of BNP on TGFB-induced fibrotic embodiment, the targeted gene(s) associated with cell pro and inflammatory genes. Real-time RT-PCR expression lev liferation are Selected from the group consisting essentially els were normalized to 18S rRNA and plotted relative to the of PDGFA, IGF1, FGF18, and IGFBP10. level in the 6 h control cells. See FIG. 5 for key to histogram bar labels and error bars. 0012. In another related embodiment, the invention is directed to a method of alleviating or reversing the effect of 0.019 FIG. 7. Effect of PKG and MEK inhibitors on TGFB mediated cell activation in cardiac tissue on the BNP-dependent inhibition of TGFB signaling in human expression of one or more genes associated with inflamma cardiac fibroblasts. (A) Western analysis of ERK phospho tion, comprising contacting one or more cells or tissues in rylation. Cells were treated with BNP (0.5umol/L) in the which the expression of Said genes is altered as a result of presence or absence of KT5823 (1 umol/L) or UO126 (10 TGFB mediated activation, with BNP. In another related umol/L) for 15 min. (B) Western blot and (C) real-time embodiment, the targeted gene(s) associated with inflam RT-PCR analysis to detect Collagen 1 expression. Cells were mation are Selected from the group comprise COX1, IL6, treated with 5 ng/ml TGFB and/or BNP (100 nmol/L, three TNFC.-inducted protein 6, TNF Superfamily, member 4. times daily) in the presence or absence of KT5823 (1 umol/L), UO126 (0.1-10 umol/L) or PD98059 (10 umol/L) BRIEF DESCRIPTION OF THE DRAWINGS for 48 h. Control (C); KT5823 (KT); UO126 (U); TGFB 0013 FIG. 1. Gene expression changes induced by (TGF). TGFB and BNP in human cardiac fibroblasts at 24 and 48 h. 0020 FIG. 8. Summary of BNP effects on gene expres Histograms show the number of gene expression changes sion in TGFB-stimulated human cardiac fibroblasts. that were up-regulated and down-regulated by TGFB and 0021 FIG. 9. Effects of BNP on TGFB-stimulated fibro BNP treatment. Hybridizations using fluorescently-labeled blast proliferation. Histograms show fold induction of BrdU cDNA probes compare untreated (control) to TGFB-treated labeled cells treated with TGFB alone, BNP alone or co cells and control to BNP-treated cells. See Experimental for treated with BNP and TGFB. Cells were co-treated with BNP details related to the gene expression values. Histogram and TGFB for 24 h, then labeled with BrdU and cultured for bars: 24 h (white) and 48 h (black). an additional 24 h. Pooled data represent the meant-SD from 0014 FIG. 2. Effects of BNP on TGFB-induced gene three individual experiments: p<0.01 vs. the control; expression in human cardiac fibroblasts. Hybridizations **p-0.05 vs. TGFB. using fluorescently-labeled cDNA probes compare TGFB treated to TGFB BNP-treated cells at 24 and 48 h. Strong and 0022 FIG. 10. Changes in plasma aldosterone level. The weak effects represent 1.8- and 1.5-fold gene expression increased plasma aldosterone level by L-NAME/Ang|I was levels, respectively. See Experimental for details related to reduced by BNP (p<0.05, n=7) Statistical significance. Histogram bars: no effect (white), 0023 FIG. 11. Changes in heart/body weight ratio. BNP weak effect (grey), and strong effect (black). abolished L-NAME/Ang|I-induced increase in heart/body 0015 FIG. 3. Gene expression patterns in TGFB-treated weight ratio (p<0.01, n=12) human cardiac fibroblasts. Data was generated using the 0024 FIG. 12. Real time RT-PCR results. Expression of hierarchical clustering algorithm contained in Spotfire TM mRNA of collagen I (A), collagen III (B) and fibronectin (C) Software. Each row represents one of 524 genes, and each in the heart. BNP abolished the fibrotic genes that enhanced column represents the results from duplicate hybridizations: by L-NAME plus Angiotensin II (p<0.01 in all cases). (A) control vs. TGFB, 24 h; (B) control vs. TGFB, 48 h; (C) TGFB vs. TGFB+BNP 24 h; (D) TGFB vs. TGFB+BNP 48 0025 FIG. 13. Cardiac function parameters including h; (E) control vs. BNP 24 h; and (F) control vs. BNP 48 h. heart rate (A), stroke volume (B), ejection fraction (C), Normalized data values depicted in Shades of red and green cardiac output (D), stroke work (E), maximum dP/dt (F), represent elevated and repressed expression, respectively. minimum dP/fy (G), and arterial elastance (H). L-NAME/ See Table 2 in Experimental Section for gene identities and Ang induced deterioration of cardiac function. Adminis expression values. tration of BNP significantly improved cardiac function as judged by increases in Stroke Volume, ejection fraction, 0016 FIG. 4. Gene expression clusters in human cardiac cardiac output, Stroke work and decrease in arterial elastance fibroblasts: (A) fibrosis and ECM, (B) cell proliferation, and (p<0.001, n=8). BNP also increased maximum dP/dt (C) inflammation. See FIG. 4 legend for descriptions of the (p<0.05) and minimum dP/dt. BNP had no effect on heart hybridizations and gene expression color codes. rate. 0017 FIG.5. Effects of BNP on TGFB-induced Collagen 1 (A and B) and Fibronectin (C and D) mRNA and protein DETAILED DESCRIPTION levels in cultured human cardiac fibroblasts. Histograms A. Definitions show control cells (white), cells treated with BNP (gray), cells treated with TGFBblack), and cells co-treated with 0026. As used herein, any reference to “reversing the BNP and TGFB(hatched). (A and C) Real-time RT-PCR effect of TGF-B-mediated cell activation on the expression expression levels were normalized to 18S rRNA and plotted of a gene associated with fibrosis” means partial or complete relative to the level in the 6 h control cells. Error bars reflect reversal the effect of TGF-B-mediated cell activation of that duplicate biological replicates; real-time RT-PCR reactions gene, relative to a normal Sample of the same cell or tissue were performed in triplicate. (B and D) Western blot analy type. It is emphasized that total reversal (i.e. total return to SeS are presented as meantSD from three Separate experi the normal expression level) is not required, although is ments; *p-0.01 vs. control; **p-0.01 vs. TGFB. advantageous, under this definition. US 2006/OO19890 A1 Jan. 26, 2006

0027. The term “cardiac remodeling' generally refers to liferative disease, a therapeutic agent may directly decrease the compensatory or pathological response following myo the pathology of the disease, or render the disease more cardial injury. Cardiac remodeling is viewed as a key Susceptible to treatment by other therapeutic agents. determinant of the clinical outcome in heart disorders. It is characterized by a structural rearrangement of the cardiac 0033. The term “subject” for purposes of treatment refers chamber wall that involves cardiomyocyte hypertrophy, to any animal classified as a mammal, including humans, fibroblast proliferation, and increased deposition of extra domestic and farm animals, and Zoo, Sports, or pet animals, cellular matrix (ECM) proteins. Cardiac fibrosis is a major Such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, aspect of the pathology typically Seen in the failing heart. etc. Preferably, the Subject is human. The proliferation of interstitial fibroblasts and increased 0034 Administration “in combination with one or more deposition of extracellular matrix components results in further therapeutic agents includes simultaneous (concur myocardial StiffneSS and diastolic dysfunction, which ulti rent) and consecutive administration in any order. mately leads to heart failure. A number of neurohumoral or 0035 A “therapeutically effective amount”, in reference growth factors have been implicated in the development of to the treatment of cardiac or renal fibrosis, e.g. when cardiac fibrosis. These include angiotensin II (AII), endot inhibitors of the present invention are used, refers to an helin-1 (ET-1), cardiotrophin-1 (CT-1), norepinephrine amount capable of invoking one or more of the following (NE), aldosterone, FGF2, PDGF, and transforming growth effects: (1) inhibition (i.e., reduction, slowing down or factored (TGFB). TGFB expression is also stimulated by AII complete stopping) of the development or progression of and ET-1 in cardiac myocytes and fibroblasts, further Sup fibrosis and/or Sclerosis; (2) inhibition (i.e., reduction, slow porting its involvement in cardiac fibrosis. ing down or complete stopping) of consequences of or 0028. The term “cardiac dysfunction” refers to the patho complications resulting from Such fibrosis and/or Sclerosis, logical decline in cardiac performance following myocardial and (3) relief, to Some extent, of one or more symptoms injury. Cardiac dysfunction may be manifested through one asSociated with the fibrosis and/or Sclerosis, or Symptoms of or more parameters or indicia including changes to Stroke consequences of or complications resulting from Such fibro Volume, ejection fraction, end diastolic fraction, Stroke sis and/or Sclerosis. work, arterial elastance, or an increase in heart weight to body weight ratio. B. Modes of Carrying out the Invention 0036 Natriuretic peptides comprise a family of vasoac 0029. The terms “differentially expressed gene,”“differ tive hormones that play important roles in the regulation of ential gene expression' and their synonyms, which are used cardiovascular and renal homeostasis. Atrial natriuretic pep interchangeably, refer to a gene whose expression is acti tide (ANP) and brain natriuretic peptide (BNP) are predomi Vated to a higher or lower level in a test Sample relative to nantly produced in the heart and exert vasorelaxant, natri its expression in a normal or control Sample. For the purpose uretic, and anti-growth activities. Binding of ANP and BNP of this invention, “differential gene expression' is consid to type-A natriuretic peptide receptor (NPRA) leads to the ered to be present when there is at least an about 2.5-fold, generation of cyclic guanosine monophosphate (cGMP), preferably at least about 4-fold, more preferably at least which mediates most biological effects of the peptides. Mice about 6-fold, most preferably at least about 10-fold differ lacking NPRA exhibit cardiac hypertrophy, fibrosis, hyper ence between the expression of a given gene in normal and tension and increased expression of fibrotic genes including test Samples. TGFB1, TGFB3 and Collagen 1. Furthermore, targeted dis 0030) “Myocardial injury' means injury to the heart. It ruption of the BNP gene in mice results in cardiac fibrosis may arise from myocardial infarction, cardiac ischemia, and enhanced fibrotic response to Ventricular pressure over cardiotoxic compounds and the like. Myocardial injury may load, Suggesting that BNP is involved in cardiac remodeling. be either an acute or nonacute injury in terms of clinical 0037 TGFB mediates fibrosis by modulating fibroblast pathology. In any case it involves damage to cardiac tissue proliferation and ECM production, particularly of collagen and typically results in a structural or compensatory and fibronectin. TGFB also promotes the phenotypic trans response. formation of fibroblasts into myofibroblasts characterized by 0.031 AS used herein, “natriuretic peptides” means a expression of C-Smooth muscle actin. Studies have demon composition that includes one or more of an Atrial natri Strated that increased myocardial TGFB expression is asso uretic peptide (ANP), a Brain natriuretic peptide (BNP), or ciated with cardiac hypertrophy and fibrosis. Moreover, a C-type natriuretic peptide (CNP). It is contemplated that functional blockade of TGFB prevents myocardial fibrosis analogues and variants of these peptides be included in the and diastolic dysfunction in pressure overloaded rats, indi definition. Examples of such include anaritide (ANP ana cating that TGFB has a crucial role in the process of logue of different length) or combinations of natriuretic myocardial remodeling, particularly in cardiac fibrosis. peptide including but not limited to ANP/BNP, ANP/CNP, However, the implication of natriuretic peptide(s) in this an BNP/CNP variants. Preferably, natriuretic peptide means process has not been previously explored. BNP (nesiritide). 0038. The present invention is directed to the treatment or 0032) The terms “treating” or “alleviating” refer to both prevention of cardiac remodeling following myocardial therapeutic treatment and prophylactic or preventative mea injury. In a preferred embodiment, the myocardial injury Sures, wherein the object is to prevent or slow down (lessen) comprises an acute myocardial infarction. Preferably the the targeted pathologic condition or disorder. Those in need administration of natriuretic peptide occurs as Soon as of treatment include those already with the disorder as well possible after the injury event. as those prone to have the disorder or those in whom the 0039. In another embodiment, the invention involves the disorder is to be prevented. In the treatment of a fibropro treatment of cardiac dysfunction in a Subject in need thereof US 2006/OO19890 A1 Jan. 26, 2006 comprising the administration of a natriuretic peptide to a 0048. Further details of the invention will be apparent Subject in need thereof wherein Said administration occurs from the Experimental Section as provided below. after Said Subject has undergone myocardial injury. 0040. The manner of administration and formulation of EXPERIMENTAL the natriuretic peptide(s) useful in the invention will depend In vitro on the nature of the condition, the Severity of the condition, the particular Subject to be treated, and the judgment of the Cell Culture practitioner; formulation will depend on mode of adminis 0049. Two lots of primary human cardiac fibroblasts, tration. The peptides of the invention are conveniently derived from an 18-year old Caucasian male (lot 1) and a administered by oral administration by compounding them 56-year old Caucasian male (lot 2), were provided by with Suitable pharmaceutical excipients So as to provide Cambrex Bio Science (Walkersville, Md.). Cells stained tablets, capsules, Syrups, and the like. Suitable formulations positive for C-Smooth muscle actin and Vimenfin antibodies for oral administration may also include minor components corroborating their identity as cardiac fibroblasts and myo Such as buffers, flavoring agents and the like. Typically, the fibroblasts. Both lots were used for the real-time RT-PCR amount of active ingredient in the formulations will be in the Studies, lot 1 was used for the microarray analysis. Cells at range of about 5%-95% of the total formulation, but wide passage 3-5 were cultured in FGM containing 15% FBS. At variation is permitted depending on the carrier. Suitable confluence, cells were split and cultured in 6-well plates for 24 h. Cells were changed to Serum-free medium and treated carriers include Sucrose, pectin, magnesium Stearate, lac with human BNP (American Peptide Company, Sunnyvale, tose, peanut oil, olive oil, water, and the like. Calif.) in the presence or absence of 5 ng/ml of TGFB (R&D 0041. The peptides useful in the invention may also be systems, Minneapolis, Minn.) for 6, 24 and 48 h. BNP and/or administered through Suppositories or other transmucosal TGFB-treated cells were also incubated in the presence of vehicles. Typically, Such formulations will include excipi cGMP-dependent protein kinase (PKG) inhibitor KT5823 (1 ents that facilitate the passage of the compound through the umol/L, Calbiochem, San Diego, Calif.), MAP kinase kinase mucosa Such as pharmaceutically acceptable detergents. (MEK) inhibitor U0126 (0.1-10 umol/L, Sigma, St. Louis, Mo.) or PD98059 (10 umol/L, Sigma) for 48 h. BNP (100 0042. The peptides may also be administered by injec nmol/L) was added into the medium three times a day, Such tion, including intravenous, intramuscular, Subcutaneous, that the total calculated concentrations of exogenous BNP intrarticular or intraperitoneal injection. Preferably the natri were 200 nmol/L, 600 nmol/L, and 900 nmol/L at 6, 24, and uretic peptide(s) are administered intravenously. Typical 48 h, respectively. This dosing protocol was necessary to formulations for Such use are liquid formulations in isotonic maintain the levels of BNP in culture, since two distinct vehicles Such as Hank's Solution or Ringer's Solution. clearance pathways are responsible for the rapid degradation 0.043 Alternative formulations include aerosol inhalants, of natriuretic peptides. Without this treatment regime, it was nasal Sprays, liposomal formulations, Slow-release formula found that BNP was significantly degraded in the cardiac fibroblasts; 50% of added BNP was metabolized within 24 tions, and the like, as are known in the art. has measured by immunoreactive assays and coMP Stimu 0044 Any suitable formulation may be used. A compen lation cell bioassayS. dium of art-known formulations is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Intracellular c0MP Assay Company, Easton, Pa. Reference to this manual is routine in 0050 Cells were cultured in 6-well plates for 24 h, then the art. changed to Serum-free medium, and pre-incubated with 0.1 004.5 The dosages of the peptide(s) of the invention will mmol/L of 3-isobutyl-1-methylxanthine (IBMX) for 1 h depend on a number of factors which will vary from patient before treating with 10-10 mol/L of BNP for 10 min. The to patient. The dose regimen will vary, depending on the medium was aspirated and 0.5 ml of cold PBS was added into each well. Cells were scraped and mixed with 2 conditions being treated and the judgment of the practitio volumes of cold ethanol by vortex. After a 5 min room ner. Further information regarding related formulations and temperature incubation, the precipitate was removed by dosages for brain natriuretic peptide can be found in the centrifugation at 1500xg for 10 min. The Supernatant was package insert or the latest version of Physicians Desk dried by vacuum centrifugation, and levels of c(GMP were Reference (PDR) for nesiritide or the Natrecor(R) product. measured using the cyclic GMPEIAkit (Cayman Chemical, 0046. It should be noted that the peptides useful for the Ann Arbor, Mich.). invention can be administered as individual active ingredi BrdU incorporation ents, or as mixtures of Several different compounds. In addition, the peptide(s) can be used as Single therapeutic 0051 Cells were placed in 96-well plates and cultured for agents or in combination with other therapeutic agents. 24 h before changing to Serum-free medium. Cells were Drugs that could be usefully combined with these com treated with BNP (100 nmol/L, three times a day) in the presence or absence of 5 ng/ml of TGF-B for 24 h. Subse pounds include natural or Synthetic corticosteroids, particu quently, 10umol/L of 5-bromo-2'-deoxyuridine (BrdU) was larly prednisone and its derivatives, monoclonal antibodies added to the cells, and they were cultured for an additional targeting cells of the immune System or genes associated 24 h. BrdU incorporation was detected using the Cell with the development or progression of fibrotic diseases, and Proliferation ELISA kit (Roche, Indianapolis, Ind.). Data Small molecule inhibitors of cell division, protein Synthesis, was analyzed by ANOVA using the Newman-Keuls test to or mRNA transcription or translation, or inhibitors of assess significance. immune cell differentiation or activation. 0047 Asimplicated above, although the peptide(s) of the cDNA Microarray invention may be used in humans, they are also available for 0052 Gene expression profiles were determined from Veterinary use in treating non-human mammalian Subjects. cDNA microarrays containing 8,600 elements derived from US 2006/OO19890 A1 Jan. 26, 2006

clones isolated from normalized cDNA libraries or pur background. Differential expression ratioS were determined chased from ResCien (Invitrogen Life Technologies, Carls as the mean of the two values from dye-Swapped duplicates. bad, Calif.). DNA for spotting was generated by PCR 0055. A statistically significant differential expression amplification using 5'amino-modified primers (BD Bio threshold value was empirically determined according to the sciences Clontech, Palo Alto, Calif.) derived from flanking method of Yang et al. Seven independent self-self-hybrid vector sequences. Amplified DNA was purified in a 96-well izations were performed in which the same RNA sample was format using Qiagen's Qiacquick columns (Valencia, Calif.) labeled with Cy3 dUTP and Cy5 dUTP and hybridized to according to the manufacturer's recommendations. Samples arrays containing 8,448 elements. Only elements that gave a were eluted in Milli-Q purified water, dried to completion Signal greater than 2.0-fold over background in at least one and resuspended in 7 ul of 3xSSC. A fluorescent assay using of the dyes were considered in the analysis. Expression Pico Green (Molecular Probes, Eugene, Oreg.) was ran ratios were converted to logo and normalized to a mean=0. domly performed on 12% of the PCR products to determine Combining data from all hybridizations, the 3 standard the average yield after purification, yields were ~1.5 lug of deviation limit was equivalent to a 1.48 fold change (+/- DNA which corresponds to a concentration of 214 ug/ml. O.563 logo). Of the 45,633 elements analyzed, 0.85% fell Purified DNA was arrayed from 384-well microtiter plates outside this threshold. Therefore, at this standard deviation onto lysine-coated glass slides using an OmniGrid II limit, genes with fold changes greater than 1.48 can be microarrayer (GeneMachines, San Carlos, Calif.). After considered differentially expressed at a 99% confidence printing, DNA was cross-linked to the glass with 65 mioules level for any given hybridization. The percentage of ele UV irradiation and reactive amines were blocked by treat ments that reproducibly fell outside the 3 standard deviation ment with Succinic anhydride. limit between any two duplicates of the seven self-self mRNA Isolation, Labeling, and Hybridizations hybridizations was determined by comparing all 21 pair wise combinations. An average of 18.9 elements +/-15.6 per 0.053 Total RNA was extracted from cells using Qiagen's hybridization duplicated at a fold change of 1.5, correspond RNeasy kit; two wells from a 6 well plate were pooled to yield a total of 4x10 cells per treatment. RNA was amplified ing to a false positive rate of 0.29%. At a fold change of 1.8, using a modified Eberwine protocol that incorporated a an average of 0.71 elements +/-0.97 duplicated, correspond polyA tail into the amplified RNA. Fluorescently-labeled ing to a false positive rate of 0.01%. A 1.8-fold threshold cDNA probes were generated by reverse transcription of 4 value was used to identify differentially expressed genes, ug of RNA with SuperScript II (Invitrogen Life Technolo except in FIG. 3, a 1.5-fold threshold value was used to gies, Carlsbad, Calif.) using anchored dT primers in the designate “weak effects”. presence of Cy3 or Cy5 dUTP (Amersham, Piscataway, Real-Time RT-PCR N.J.). Labeled cDNA probe pairs were precipitated with 0056 Real-time RT-PCR" was performed in a two-step ethanol and purified using Qiaquick columns. Twenty ug manner. cDNA synthesis and real-time detection were car each of poly(A) DNA, yeast tRNA, and human Cot1 DNA ried out in a PTC-100TM Thermal Cycler (MJ Research Inc, (Applied Genetics, Melbourne, Fla.) was added to the elu Waltham, Mass.) and an ABI PrismTM 7700 Sequence Detec ant. The Samples were dried to completion and resuspended tion System (Applied Biosystems, Foster City, Calif.), in 12.5ul 3xSSC, 0.1% SDS. Probes were heated to 95°C. respectively. Random hexamers (Qiagen, Valencia, Calif.) for 5 minutes, applied to the arrays under a 22 mm cover were used to generate cDNA from 200 ng RNA as described slip and allowed to hybridize for at least 16 h at 65 C. The in Applied Biosystems User Bulletin #2. TaqManTM PCR arrays were washed at 55 C. for 10 minutes in 2xSSC, 0.1% Core Reagent Kit or TaqManTM Universal PCR Master Mix SDS, followed by two washes at room temperature in (Applied Biosystems) were used in subsequent PCR reac 1xSSC (10 min) and 0.2xSSC (15 min). Hybridization of tions according to the manufacturer's protocols. Relative each fluorophore was quantified using an AXon GenePix quantitation of gene expression was performed using the 4000A Scanner. relative standard curve method. All real-time RT-PCR reac Microarray Data Analysis tions were performed in triplicate. 0.054 Differential expression values were expressed as 0057 Sequence specific primers and probes were the ratio of the median of background-Subtracted fluorescent designed using Primer Express Version 2 Software (Applied intensity of the experimental RNA to the median of back BioSystems). Sequences of primers and probes can be found ground-subtracted fluorescent intensity of the control RNA. in Table 1 below. Expression levels were normalized to 18S For ratios greater than or equal to 1.0, the ratio was rRNA. The selection of 18S rRNA as an endogenous control expressed as a positive value. For ratioS less than 1.0, the was based on an evaluation of the AC levels (Applied ratio was expressed as the negative reciprocal (i.e., a ratio of Biosystems document # 4308134C) of 6 “housekeeping” 0.5=-2.0). Median ratios were normalized to 1.0 using two genes: Cyclophilin A, 18S, GAPDH, f-actin, B-Glucu pools of 3000 randomly chosen cDNAS in each pool. Six ronidase, and Hypoxanthine Guanine Phosphoribosyl Trans replicates of each of the two pools were printed in 4 evenly ferase. The AC levels of 18S did not differ significantly distributed blocks of the array. Expression data was rejected between treatment conditions; thus, they were expressed at if neither channel produced a signal of at least 2.0-fold over 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 US 2006/OO19890 A1 Jan. 26, 2006

TABLE 1-continued Real-time PCR primers and probes. Western blot analysis

Gene Forward Probe Reverse

Collagen1 5'-GGAATTGGGCTTCGACGTT-3, 5 - 6FAM-TCTGCTGCTGTAAACTCCCTGCATCCC- 5'-TTCAGTTTGGGTTGCTTGTCTGT TAMRA-3' -3'

Fibronectin 5'-AGATCTACCTGTACACCTTGAAT 5'-6FAM-TGTCGTCATGGACGCCTCCA-TAMRA-3' 5'-CATGATACCAGCAAGGAATTGG GACA-3' 3

MP3 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'-GCCCTCGCTTAGATCTGTCTT -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'-TGATGCTGTTGTAGGGGTTTCA muscle actin

Cells were cultured in 6-well plates and treated with BNP Results (100 nM, three times daily) in the presence or absence of 5 cGMP Production in Cardiac Fibroblasts ng/ml TGFB for 48 h. Lysis was induced with 0.2 ml of buffer containing 20 mM Tris-HCL, pH 7.9, 137 mM NaCl, 0.058 To determine if NPRA was expressed in the cul 1% Triton X-100, 5 mM EDTA, 10 mM NaF, 1 mM tured fibroblast cells, c0MP accumulation assays were uti B-glycerophosphate, and protease inhibitor cocktail. The lized. BNP dose-dependently induced intracellular cyclic protein concentration of each lysate was measured using GMP production in cardiac fibroblasts with an EC50 of 50 coomassie protein reagent from PIERCE. Twenty tug of nmol/L. These results are consistent with the report of Cao protein from each Sample was loaded and electrophoresed and Gardner showing NPRA expression in cardiac fibro on 4-12% gradient polyacrylamide gels and electrophoreti blasts. cally transferred to nitrocellulose membranes (Invitrogen, Effects of BNP on TGFB-Induced Fibroblast Proliferation San Diego, Calif.). The membranes were incubated with 0059) To examine the effects of TGFB and BNP on cell rabbit anti-human Collagen 1 antibody (Cortex Biochem, proliferation, BrdU incorporation was measured in cardiac San Leandro, Calif.), HRP-conjugated anti-human fibroblasts treated with TGFB in the presence or absence of Fibronectin antibody, or goat anti-Actin antibody (Santa BNP. TGFB modestly increased (-50%) cardiac fibroblast Cruz Biotehnology, Santa Cruz, Calif.) in TBST buffer proliferation, and BNP inhibited TGFB-induced prolifera containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% tion by ~65% (FIG. 9). Tween-20, and 5% nonfat dried milk at 4 C. for ~16 h. For Effects of BNP on TGFB-Induced Gene Expression ERK phosphorylation, cells were treated with 0.5 umol/L BNP in the presence of 1 umol/L KT5823 or 10 umol/L 0060. In order to determine the effects of BNP on gene U0126 for 15 min; the membranes were incubated with expression profiles induced by TGFT in cardiac fibroblasts, rabbit anti-human phospho-ERK/3 antibody or rabbit anti a microarray analysis was performed. Fluorescently-labeled human ERK/3 antibody (Cell Signaling, Beverly, Mass.). cDNA probes were prepared from pooled mRNAS generated For Secondary antibody detection, membranes were incu from duplicate wells of cells from four groups: unstimulated bated with HRP-conjugated anti-rabbit antibody or anti-goat (control), TGFB-treated, BNP-treated, and co-treated with antibody at room temperature for 1 h and washed 3 times TGFB and BNP for 24 and 48 h (as described above). Arrays with TBST buffer. The blots were soaked in ECL Plus were probed in duplicate for a total of 12 hybridizations (6 reagent for 5 min and exposed to KODAK X-ray film. at each time point): control compared to TGFB-treated, Signals were identified and quantified using a Typhoon TGFB-treated compared to TGFB+BNP-treated, and control Scanner and Densitometer from Amersham BioSciences compared to BNP-treated. (Piscataway, N.J.). Data was analyzed by ANOVA using the 0061. It was observed that BNP had no significant effects Newman-Keuls test to assess significance. on gene expression in unstimulated human cardiac fibro US 2006/OO19890 A1 Jan. 26, 2006 blasts (FIG. 1). In contrast, TGFB induced 394 and 501 gene Microfibril-associated glycoprotein-2 (MAGP2), Matrilin 3 expression changes at 24 and 48 h, respectively. These (MATN3), Fibrillin 1 (FBN1), and Cartilage oligomeric differentially expressed genes represent ~7-8% of the target matrix protein (COMP). Also included in the cluster are genes on the array. Interestingly, BNP had dramatic effects known markers of fibrosis. Such as TIMP3, CTGF, IL11, and on the gene expression changes induced by TGFB (FIG.2). SERPINE1 (PAI-1). Furthermore, the cluster revealed that Approximately, 88% and 85% of TGFB-regulated gene BNP opposed TGFB-induction of myofibroblast markers expression events were opposed by BNP at 24 and 48 h, including C-Smooth muscle actin 2 (ACTA2) and non respectively. These results demonstrate that BNP has strik ingly different effects on gene expression in TGFB Stimu muscle myosin heavy chain (MYH9). lated fibroblasts compared to unstimulated cells. 0064. Many genes involved in cell proliferation were also Gene Expression Clustering regulated by TGFB and were opposed by BNP (FIG. 4B). For example, TGFB induced the expression of positive 0062) To identify different gene expression patterns fol regulators of cell proliferation, including PDGFA, lowing TGFB stimulation, we performed a hierarchical IGFBP10, IGF1, and Parathyroid hormone-like hormone cluster analysis. A visualization of this analysis is shown in (PTHLH). It was also found that TGFB down-regulated both FIG. 3. A complete listing of differentially expressed genes positive and negative regulators of proliferation, Such as, is provided in Table 2. The clustered expression patterns showed temporal effects of TGFB responsive genes (com CDC25B and Cullin 5 (CUL5), respectively. All of these pare A to B). In addition, the dramatic effects of BNP in TGFB-regulated gene events were opposed by BNP opposing TGFB induced up- and down-regulated gene 0065 BNP affected TGFB-induced genes involved in changes were revealed in the clusters (compare A and B to inflammation (FIG. 4C). For example, BNPreversed TGFB C and D). The insignificant effects of BNP on gene expres induction of PTGS2 (COX2), TNF C-induced protein 6 Sion in unstimulated cardiac fibroblast cells were evident in (TNFAIP6), and TNF superfamily, member 4 (TNFSF4) groups E and F. (FIG. 4C and data not shown). TNFAIP6 and TNFSF4 were 0.063 Genes were grouped according to functional cat not included in FIG. 4C, since some of the data points at 48 egories by using a combination of gene expression cluster h did not meet acceptable criteria (see Experimental); at 24 ing and functional annotations. A cluster of genes involved h both genes were elevated -3-fold by TGFB and opposed in fibrosis and ECM production was up-regulated in cells by BNP. TGFB also down-regulated many pro-inflammatory stimulated with TGFB; these genes were down-regulated genes including IL1B, CCR2 (MCP1-R), CXCL1 (GRO1), when treated with BNP (FIG. 4a). This cluster includes CXCL3 (GRO3), and CCL13 (MCP4), which were reversed extracellular matrix components: Collagen 1a2 (COL1A2), by BNP. The significance of these inflammatory changes is Collagen 15A (COL15A), Collagen 7A1 (COL7A1), discussed below.

TABLE 2 Expression data for differentially expressed genes in TGFB-treated human cardiac fibroblasts. Median differential expression values are shown for each hybridization: control vs. TGFB 24 h (column 2); control vs. TGFB 48 h (column 3); TGFB vs. TGFB + BNP 24 h (column 4); TGFB vs. TGFB + BNP 48 h (column 5): control vs. BNP 24 h (column 6); and control vs. BNP 48 h (column 7).

BNP Clone ID 24 h 24 h 24 h 48 h 48 h. 48 h Symbol Name Accession POO777 AO3 2.5 -2.8 .1 .5 -1.6 .1 EST POO777 AO4 8.9 -5.7 3.3 -2.4 EST POO777 A12 2.1 -2.4 - .8 -1.9 -1.1 EST PO1061 EO1 2.7 -3 2.6 -2.8 EST PO1061 B10 -27 2.3 .1 -4 2.4 EST PO1077 AO8 -1.8 3.1 .3 -2.2 .9 No Sequence PO1111 AO8 -1.3 1.4 .3 -1.8 7 EST PO1113. E11 -1.7 18 .1 -1.8 .6 EST PO1111 FO7 -4.5 5.5 3 -5.3 4.2 .1 EST PO1111 AO7 2 -2.7 3 .4 -1.5 EST PO1110 GO3 -1.2 1.5 3 -3.9 2.1 .1 No Sequence PO1108 GO7 4.2 -4.4 -1.1 3.9 -4.5 EST PO1099 GO3 -1.9 1.9 .1 -2.2 .9 .2 EST PO1113 BO3 6.4 -5.1 4.3 -3.7 EST PO1080 A11 4 -3 4.2 -4.1 EST PO1076 EO1 -1.7 18 .1 -1.8 8 EST PO1075 HO9 -3.1 3.6 .4 -2.9 3.2 .4 No Sequence PO1139 D10 3 -2.6 .1 2.1 -2.1 EST PO1132 BO1 -2.1 2 -1.4 3 EST PO1123 HO3 2.2 -2.2 .2 9 -19 .1 EST PO1117 D08 -1.7 1.5 .1 -4.9 2.4 EST PO1115 FO8 -2.2 1.6 - -2.3 7 EST PO1081 FO2 2.4 -1.8 .2 2.4 -2.1 .1 No Sequence PO1087 A12 2.4 -2 2.6 -2.6 EST PO1077 AO2 2.2 -2 .4 -1.3 No Sequence US 2006/OO19890 A1 Jan. 26, 2006

TGF TGF TGF BNP BNP BNP BNP Clone ID 24 h 24 h 24 h. 48 h. 48 h. Symbol Name Accession PO1136 G11 -2 2.5 3 2.5 EST PO1130 BO3 -3.3 3.5 - 5.3 EST PO1124 AO5 -1.2 -1 1.5 EST PO1124 A10 2.1 -2 2. -2.5 EST PO1124 BO4. -1.9 2 3 1.7 EST PO112O GO6 -2.3 2.2 - 2.2 EST PO1117 B11 .8 -2.4 -2 EST PO1116 AO2 -3.1 2.7 2.2 EST PO1088 C10 2.1 -2 -2.1 .1 EST PO1093 CO4 2.6 -2.3 EST PO1095 HO1 -1.8 8 EST PO1099 DO3 .9 -1.8 EST PO1100 AO7 .1 EST PO11 OO DO9 -1.6 6 - EST PO1101 C11 -2.4 .7 - : No Sequence PO1101 E11 -1.4 5 EST PO1103 HO4 -3.2 2.9 EST PO1104 AO9 -1.9 6 No Sequence PO1104 EO3 -2.5 2.3 - 2. EST PO1104 G04 2.5 -2 EST PO1104 G12 -3.7 2.7 -1. 4 EST PO1105 AO5 2.3 -2.3 3 EST PO1105 D09 .8 -1.1 - EST PO1109 AO1 -1.4 .4 .2 alpha-2-macroglobulin NM000014 PO1109 G11 .4 - : ABCG1 ATP-binding cassette, sub NM OO4915 family G (WHITE), member 1 PO1092 EO8 2.3 -2 .2 5 3 ACLY ATP citrate lyase NM 001096 PO1088 CO2 -1.9 8 .2 ACO1 aconitase 1, soluble NM 002197 POO777 GO9 2.6 -2.2 -1.5 ACTA1 actin, alpha 1, skeletal muscle NM OO1100 PO1094 FO)4 2.6 -2.5 -1.4 ACTA2 actin, alpha 2, smooth muscle, NM OO1613 aorta PO1091 G04 .9 -1.6 .1 ACTR3 ARP3 actin-related protein 3 NM O05721 homolog (yeast) PO1096 DO2 -1.3 5 .1 -2.3 2.2 ADAMTS1 a disintegrin-like and NM OO6988 metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 PO1097 D04 .7 -19 - 2.1 -1.8 ADAMTS6 a disintegrin-like and NM O14273 metalloprotease (reprolysin type) with thrombospondin type 1 motif, 6 PO1092 DO3 -6.5 6 -1.1 -6.3 6.5 ADFP adipose differentiation-related NM OO1122 protein PO1070 D09 -5 4.1 3 -9.7 3.8 ADH alcohol dehydrogenase IB NM OOO668 (class I), beta polypeptide PO1134 D11 -1.7 2 3 -3.6 1.6 ADH alcohol dehydrogenase 1C NM OOO669 (class I), gamma polypeptide PO1070 DO5 -1.3 -1.4 .2 -2.2 1.7 ADHS alcohol dehydrogenase 5 NM OOO671 (class III), chi polypeptide PO1094 D10 -2.3 2.5 .1 -2.2 18 ADO RA2B adenosine A2b receptor NM OOO676 PO1124 F09 -1.5 1.6 .1 -1.8 1.9 AHR aryl hydrocarbon receptor NM OO1621 PO1101 BO3 -2.4 1. 2.8 AKAP2 A kinase (PRKA) anchor NM OO72O3 protein 2 PO1120 CO3 -1.9 2 .2 -1.2 1.5 AKR B1 aldo-keto reductase family 1, NM OO1628 member B1 (aldose reductase) PO1134 BO8 -27 2.6 .1 -1.4 1.9 AKR B10 aldo-keto reductase family 1, member B10 (aldose reductase) PO1069 CO1 -2.8 3.1 .2 -2.2 2.6 AKR aldo-keto reductase family 1, NM OO1353 member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3- alpha)-hydroxysteroid dehydrogenase) PO1081, A11 -2.3 3.3 1.6 -2.2 1.9 1.3 AKR1C2 aldo-keto reductase family 1, NM OO1354 member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha US 2006/OO19890 A1 Jan. 26, 2006

TABLE 2-continued Expression data for differentially expressed genes in TGFB-treated human cardiac fibroblasts. Median differential expression values are shown for each hybridization: control vs. TGFB 24 h (column 2); control vs. TGFB 48 h (column 3); TGFB vs. TGFB + BNP 24 h (column 4); TGFB vs. TGFB + 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 hydroxysteroid dehydrogenase, type III) PO1143 D10 -2.8 3.2 1.3 -2.1 2.7 11 AKR1C2 aldo-keto reductase family 1, NM OO1354 member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase, type III) PO1106 C11 -2.3 2.8 -2 2.5 1 AKR1C3 aldo-keto reductase family 1, NM OO3739 member C3 (3-alpha hydroxysteroid dehydrogenase, type II) PO1094 D12 -2.8 3.6 -2.5 1.7 2 ALDH1A3 aldehyde dehydrogenase 1 NM OOO693 amily, member A3 PO1094 EO1 -1.4 8 -2.1 1.6 1 ALDH3A2 aldehyde dehydrogenase 3 NM OOO382 amily, member A2 PO1140 G11 -1.9 2.7 -2.5 18 1 ALDH3A2 aldehyde dehydrogenase 3 NM OOO382 amily, member A2 PO1118 A12 -1.9 .6 -2.6 2.2 ALEX1 ALEX1 protein NM O16608 PO1096 E12 -2.4 2 -2.1 2.2 ANG angiogenin, ribonuclease, NM OO1145 RNase A family, 5 PO1145 EO8 -2 2.3 -2.9 2.6 ANGPT1 angiopoietin 1 NM 001146 PO1091 GO2 -1.2 5 -2.7 2 1 ANGPT2 angiopoietin 2 NM OO1147 PO1094 DO6 -2.1 9 -1.9 3 -11 ANK3 ankyrin 3, node of Ranvier NMOO1149 (ankyrin G) PO1128 AO7 -1.5 8 -2.2 2.3 2 AOX1 aldehyde oxidase 1 NM OO1159 PO1116 HO5 -1.1 .4 -2 8 APELIN apelin; peptide ligand for APJ NM O17413 receptor PO1103 FO6 2.4 -2.4 1.4 -1.5 -11 APG3 autophagy Apg3p/Aut1p-like NM 022488 PO1123 AO7 3.2 -3 1.5 -1.8 APOA1 apolipoprotein A-I NM OOOO39 PO1105 GO6 -2.2 8 -4.5 5.7 APOC1 apolipoprotein C-I NM OO1645 PO1124 GO3 -1.3 .4 -2.4 2 APOE apolipoprotein E NM OOOO41 PO1105 BO2 -1.6 8 -2.9 .9 ARHGAP6 Rho GTPase activating protein 6 NM OO1174 PO1064 GO3 -1.1 3 -2 .6 ARHGEF16 Rho guanine exchange factor NM O14448 (GEF) 16 PO1110 E10 -2 2.1 -2.3 .9 ARHGEF3 Rho guanine nucleotide NM O19555 exchange factor (GEF) 3 PO1142 CO3 -1.6 8 -1.9 7 2 ARH ras homolog gene family, NM 004675 member I PO1138 AO9 .9 -2.2 1.8 -1.9 -1.1 ARL4 ADP-ribosylation factor-like 4 NM OO5738 PO1064 G12 -1.7 8 -1.8 .6 ARNT2 aryl-hydrocarbon receptor NM O14862 nuclear translocator 2 PO1088 HO9 -1.5 7 -1.8 .6 1 ASAH1 N-acylsphingosine NM 004315 amidohydrolase (acid ceramidase) PO1105 FO6 2.9 -2.8 .1 2.1 -2.4 -12. ASNS asparagine synthetase NM OO1673 PO1070 EO6 .8 -1.5 1.6 -1.4 ATF3 activating transcription factor 3 NM OO1674 PO1122 GO7 -1.2 7 -1.8 5 3 AXN2 axin 2 (conductin, axil) NM 004655 PO1115 DO6 -1.4 .6 -2 5 -1.1 B3GALT2 UDP-Gal:betaGlcNAc beta NM OO3783 1,3-galactosyltransferase, polypeptide 2 PO1128 AO8 -1.6 7 -2.4 7 B3GALT3 UDP-Gal:betaGlcNAc beta NM 003781 1,3-galactosyltransferase, polypeptide 3 PO1095 FO6 2.4 -2.2 1.3 -1.5 BA3 brain-specific angiogenesis NM OO1704 inhibitor 3 PO1094 CO2 -1.8 2 -2.4 2.7 BF B-factor, properdin NM OO1710 PO1134 EO2 -1.7 18 -2.2 .6 BFSP1 beaded filament structural NM OO1195 protein 1, filensin PO1081 D08 -1.2 1.7 -3.5 8 2 BIRC1 baculoviral IAP repeat NM OO4536 containing 1 PO1094 BO6 -2.6 2.9 -4 2.5 BMP4 bone morphogenetic protein 4 NM OO12O2 PO1145 AO2 -3.2 2.3 -3.6 3.2 -1.1 BNP2 BCL2fadenovirus E1B 19 kDa NM 004330 interacting protein 2 PO1075 FO5 -1.5 1.5 -1.8 2 2 BRE brain and reproductive organ NM OO4899 expressed (TNFRSF1A modulator) US 2006/OO19890 A1 Jan. 26, 2006 10

TGF TGF BNP BNP TGF BNP BNP Clone ID 24 h 24 h 24 h. 48 h. 48 h. 48 h. Symbol Name Accession PO1124 B10 -1.3 1.5 3 -2.2 1.6 BST1 bone marrow stromal cell NM 004 334 antigen 1 PO1094 BO8 -1.8 1.6 -1.1 -1.2 1.3 biotinidase NM OOOO60 PO1093 E08 -2 1.5 -1.1 -1.9 2.7 complement component 1, r NM OO 733 subcomponent PO1077 E12 -1.4 1.6 .1 -1.8 1.9 complement component 1, s NM OO 734 subcomponent PO1097 GO3 .9 -1.7 - -1.5 chromosome 20 open reading NM O12469 frame 14 PO1140 AO7 2.3 -3.2 - chromosome 20 open reading 158 frame 97 PO1069 EO2 -1.7 1.6 .1 -3.3 3.2 complement component 6 NM OOOO65 PO1077 E10 -3.1 2.9 .1 -8.2 4.7 complement component 7 NM OOO587 PO1099 C10 -1.8 2.1 .2 -2.7 3.5 CA12 carbonic anhydrase XII NM OO 218 PO1117 GO5 -3 2.4 -1.1 -2.2 2.3 CAMK2B calcium/calmodulin-dependent NM OO 22O protein kinase (CaM kinase) II beta PO1114 AO5 -2.7 3.9 .2 -3.5 2.7 CAMK2D calcium/calmodulin-dependent NM OO 221 protein kinase (CaM kinase) II delta PO1080 BO5 -2.3 3 .1 -2.3 2.1 CAMK2D calcium/calmodulin-dependent NM OO 221 protein kinase (CaM kinase) II delta PO1063 EO7 -1.6 2 .2 -1.8 1.6 CASP1 caspase 1, apoptosis-related NMOO 223 cysteine protease (interleukin 1, beta, convertase) PO1093 G08 -2.4 2.3 -1.2 -2.1 2.4 CAV1 caveolin 1, caveolae protein NM OO 753 22 kDa PO1093 E04 1.8 -1.7 -1.1 1.6 -1.9 CBS cystathionine-beta-synthase NM OOOO71 PO1064 DO2 -1.5 .6 -1.3 -2.2 2.8 CCL13 chemokine (C-C motif) ligand NM OO5408 13 PO1072 EO8 -1.3 .4 -1.2 -2.2 3.2 CCL7 chemokine (C-C motif) ligand 7 NM OO6273 PO1127 HO3 1.1 .2 -1.3 2.9 CCL8 chemokine (C-C motif) ligand 8 NM OO5623 PO1070 AO4 -1.4 .9 .2 -3.2 2.4 CCR2 chemokine (C-C motif) NM OOO647 receptor 2 PO1138 BO2 -1.2 3 3 -3.6 1.5 CCRL1 chemokine (C-C motif) NM O16557 receptor-like 1 PO1069 HO9 -1.9 .9 3 -3.6 18 CD36 CD36 antigen (collagen type I NM OOOO72 receptor, thrombospondin receptor) PO1072 EO3 -2.8 2.7 .2 -2.9 2.8 CDC25B cell division cycle 25B NM 004358 PO1093 HO7 2 -4.3 .2 2.1 CDH2 cadherin 2, type 1, N-cadherin NM OO1792 (neuronal) PO1129 EO7 1.7 -1.4 .1 -1.9 CDH4 cadherin 4, type 1, R-cadherin NM OO1794 (retinal) PO1130 HO7 2.1 -2.4 -1.1 1.9 -1.8 CDHS cadherin 5, type 2, VE NM OO1795 cadherin (vascular epithelium) PO1116 HO2 -3.3 2.1 .1 2.4 CDK5 regulatory subunit NM O18249 associated protein 2 PO1102 BO2 -2.1 2.5 -3.4 3.2 CDSN comeodesmosin NM OO1264 PO114O GO2 -1.4 1.3 .1 -2.9 2.4 CEACAMS carcinoembryonic antigen NM OO4363 related cell adhesion molecule 5 PO1094 AO6 -1.6 1.3 3 -4.2 2.9 CEACAMS carcinoembryonic antigen NM 0043.63 related cell adhesion molecule 5 PO1062 GO2 -1.3 1.5 3 -2.9 2.1 CEACAM6 carcinoembryonic antigen NM OO2483 related cell adhesion molecule 6 (non-specific cross reacting antigen) PO1099 BO5 -1.8 18 .1 -2.9 CEACAM7 carcinoembryonic antigen NM OO6890 related cell adhesion molecule 7 PO1090 E04 -1.3 1.6 3 -1.9 18 CEBPD CCAAT/enhancer binding NM 005195 protein (C/EBP), delta PO1070 AO1 -2.6 3.1 - -9.2 9.2 CH3L1 chitinase 3-like 1 (cartilage NM OO1276 glycoprotein-39) PO1125 GO2 -2.9 2 6.2 chitinase 3-like 2 NM 004.OOO US 2006/OO19890 A1 Jan. 26, 2006 11

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 PO1134 F10 8 -6.3 .2 19.5 -8 .1 CILP cartilage intermediate layer NM OO3613 protein, nucleotide pyrophosphohydrolase PO1089 A12 -1.9 2.1 -2.1 2.1 CITED2 Cbp/p300-interacting NM OO6079 transactivator, with Glu/Asp rich carboxy-terminal domain, 2 PO1076 AO7 2.1 -1.8 - 1.4 -1.2 CKAP4 cytoskeleton-associated NM OO6825 protein 4 PO1104 CO9 2.2 -2.4 .1 4.3 -2.8 CKLF chemokine-like factor NM 016326 PO1103 GO5 -1.4 1.6 3 -2.5 1.5 CLDN1 claudin 1 NM 021101 PO1105 DO3 -3 2.7 3 -2.6 CLECSF2 C-type (calcium dependent, NM OO5127 carbohydrate-recognition domain) lectin, superfamily member 2 (activation-induced) PO1064. FO9 2.2 -1.5 .2 1.2 -1.2 CNN1 calponin 1, basic, smooth NM OO1299 muscle PO1090 AO3 -1.1 1.3 .2 -2.2 1.6 CNTNAP1 contactin associated protein 1 NM OO3632 PO1069 FO2 1.2 1.2 .2 3.2 COL15A1 collagen, type XV, alpha 1 NM OO1855 PO1077 E08 1.8 -1.5 1.9 -1.9 COL1A2 collagen, type I, alpha 2 NM OOOO89 PO1093 FO3 1.7 -2.3 1.9 -2.1 COL4A2 collagen, type IV, alpha 2 NM OO1846 PO1105 C12 1.8 -1.5 .4 3.1 -2.3 COL7A1 collagen, type VII, alpha 1 NM OOOO94 (epidermolysis bullosa, dystrophic, dominant and recessive) PO112O GO4 2.7 -2.1 .2 3.8 COL8A2 collagen, type VIII, alpha 2 M60832 PO1084 A12 -4.9 4.7 .1 -9.9 COLEC12 collectin sub-family member NM O30781 12 PO1082 HO6 1.3 -1.3 .2 3.3 COMP cartilage oligomeric matrix NM OOOO95 protein (pseudoachondroplasia, epiphyseal dysplasia 1, multiple) PO1129 C12 1.4 -1.5 3 2.6 -1.6 COMP cartilage oligomeric matrix NM OOOO95 protein (pseudoachondroplasia, epiphyseal dysplasia 1, multiple) PO1076 CO9 -2.2 2.7 .2 -2.1 1.6 COPB coatomer protein complex, NM O16451 subunit beta PO1085. D11 -4 4.2 .1 -7.7 4.1 CPA4 carboxypeptidase A4 NM 016352 PO1104 AO7 -1.9 2 .2 -2.5 2.2 CPD carboxypeptidase D NM OO 304 PO1077 GO1 9 -18 .1 1.7 -1.9 CRABP2 cellular retinoic acid binding NM OO 878 protein 2 PO1095 EO3 -1.8 8 .2 -2.1 CREG cellular repressor of E1A NM OO3851 stimulated genes PO1124 EO1 -2.2 2.1 -2.5 2.2 CREM cAMP responsive element NM OO 881 modulator PO1120 BO1 .8 -1.6 .2 3.9 -3.4 CRLF1 cytokine receptor-like factor 1 NM 004 750 PO1120 D10 -1.5 .9 3 -3.5 2.4 CROT camitine O NM O2 151 octanoyltransferase PO1124 F10 -1.2 3 .2 -1.8 1.7 CRYAA crystallin, alpha A NM OOO394 POO777 AO8 -2 .6 -2.6 2.5 CRYAB crystallin, alpha B NM OO 885 PO1077 E04 -2.1 8 -2.5 2.6 CRYAB crystallin, alpha B NM OO 885 PO1125 B11 -1.8 .2 -1.8 18 CSF1 colony stimulating factor 1 NM OOO757 (macrophage) PO1108 GO5 3.8 -3 2.3 CSPG2 chondroitin sulfate 385 proteoglycan 2 (versican) PO1075 F12 -1.5 .6 CSRP2 cysteine and glycine-rich NM OO 321 protein 2 PO1145 AO3 -2.1 2.4 -3.7 3.4 CST4 cystatin S NM OO 899 POO777 DO3 2.5 -2 1.1 -1.4 CTGF connective tissue growth factor NM OO 901 PO1077 D08 2.6 -3.5 -1.2 18 -2.7 CTGF connective tissue growth factor NM OO 901 PO1069 D11 2 -2.1 .2 1.9 -1.4 CTH cystathionase (cystathionine NM OO 902 gamma-lyase) PO1099 BO1 -1.7 2 1.6 CTNNAL1 catenin (cadherin-associated NM 003798 protein), alpha-like 1 PO1093 G10 -1.4 1.4 -1.8 CTSC cathepsin C NM OO 814 US 2006/OO19890 A1 Jan. 26, 2006 12

TGF TGF TGF BNP BNP TGF BNP BNP Clone ID 24 h. 24 h. 24 48 h. 48 h. 48 h. Symbol Name Accession PO O77 GO3 -1 1.3 -1.8 1.7 CTSH cathepsin H NM 004390 PO O69 H12 -1.5 1.5 -2.3 2.6 CTSK cathepsin K (pycnodysostosis) NM OOO396 PO 093 G09 -2.5 2.1 -2 2.3 CTSL cathepsin L NM OO1912 PO 112 DO2 -1.6 18 -2.9 2.1 CUGBP2 CUG triplet repeat, RNA NM OO6561 binding protein 2 PO 131 G04 -1.3 1.6 -2.2 1.7 CUGBP2 CUG triplet repeat, RNA NM OO6561 binding protein 2 PO 090 HO1 -2 18 -1.5 1.9 CUL5 cullin 5 NM OO3478 PO O85 CO5 -3.8 3.4 -5.5 5 CXCL1 chemokine (C-X-C motif) NM OO1511 ligand 1 (melanoma growth stimulating activity, alpha) PO 093 AO2 -3.7 3.1 -5.8 5.4 CXCL1 chemokine (C-X-C motif) NM OO1511 ligand 1 (melanoma growth stimulating activity, alpha) PO 125 H11 -2.4 -2.3 2.1 CXCL3 chemokine (C-X-C motif) NM OO2O90 ligand 3 PO 136 BO1 -4.5 4.4 -8.4 1O CXCL6 chemokine (C-X-C motif) NM OO2993 ligand 6 (granulocyte chemotactic protein 2) PO O69 DO7 -2.4 2.3 1.7 CYB5 cytochrome b-5 NM OO1914 POO777 A11 -2.5 18 CYR61 cysteine-rich, angiogenic NM OO1554 inducer, 61 POO777 C11 18 -2.5 18 -1.9 CYR61 cysteine-rich, angiogenic NM OO1554 inducer, 61 POO777 C12 -2.6 1.9 -1.9 CYR61 cysteine-rich, angiogenic NM OO1554 inducer, 61 PO 108 B04 2.3 -2.4 1.9 -1.9 CYR61 cysteine-rich, angiogenic NM OO1554 inducer, 61 PO 130 HO3 -2.4 1.9 -1.8 CYR61 cysteine-rich, angiogenic NM OO1554 inducer, 61 PO 100 CO6 2.2 -2.4 18 -1.9 DACT1 dapper homolog 1, antagonist NM O16651 of beta-catenin (xenopus) PO 1.7 -1.4 2.3 DAF decay accelerating factor for NM OOO574 complement (CD55, Cromer blood group system) PO 129 BO4 -2.8 2.5 3.5 DAPK1 death-associated protein NM OO4938 kinase 1 PO 092 GO2 -2.7 2.6 2.8 DAPK1 death-associated protein NM OO4938 kinase 1 PO -1.8 1.9 1.7 DDX38 DEAD/H (Asp-Glu-Ala NM O14003 Asp/His) box polypeptide 38 PO 105. A O -3.7 2.9 5.4 DKK1 dickkopf homolog 1 (Xenopus NM O12242 laevis) PO 113 EO5 -2.4 -2.1 DLC1 deleted in liver cancer 1 NM OO6094 PO 1. 3.3 DPP4 dipeptidylpeptidase 4 (CD26, NM OO1935 adenosine deaminase complexing protein 2) PO 1. 1.5 DPYSL2 dihydropyrimidinase-like 2 NM OO1386 PO -1.9 DSCR1 Down syndrome critical region NM 004.414 gene 1 PO 22 1. EBAF endometrial bleeding NM OO3240 associated factor (left-right determination, factor A: transforming growth factor beta superfamily) PO 23 B -2.3 1.9 ECM2 extracellular matrix protein 2, NM OO1393 female organ and adipocyte specific PO -2.1 1.9 EDG1 endothelial differentiation, NM OO1400 sphingolipid G-protein-coupled receptor, 1 PO -2.4 2.4 EDG2 endothelial differentiation, NM OO1401 lysophosphatidic acid G protein-coupled receptor, 2 PO -2.9 1.9 EDN1 endothelin 1 NM OO1955 PO -2.2 2.3 EFEMP1 EGF-containing fibulin-like NM OO4105 extracellular matrix protein 1 US 2006/OO19890 A1 Jan. 26, 2006 13

TGF TGF TGF BNP BNP TGF BNP BNP Clone ID 24 h. 24 h. 24 48 h 48 h. 48 h. Symbol Name Accession PO -1.4 1.9 .2 -2 2 .1 EFNB3 ephrin-B3 NM OO1406 PO -1.8 1.7 -1.5 1.3 .1 EGR2 early growth response 2 (Krox NM OOO399 20 homolog, Drosophila) PO 121 CO3 2 -1.2 1.5 EHD3 EH-domain containing 3 NM 014600 PO O65 EO2 1.9 -1.6 3.4 -3.3 .1 ELN elastin (supravalvular aortic NM OOO5O1 stenosis, Williams-Beuren syndrome) PO 096 H11 -3.4 3.7 -3.5 3.2 EPAS1 endothelial PAS domain NM OO14 protein 1 PO 102 E11 2.1 -2.5 2.1 EPB41L2 erythrocyte membrane protein NM OO14 31 band 4.1-like 2 PO 104 AO5 -2.3 3.3 -2.2 2.3 EPI64 EBP50-PDZ interactor of 64 kD NM 03.1937 PO 130 HO1 .1 -2.5 3.9 EPOR erythropoietin receptor NM OOO 21 PO O77 AO7 -1.6 2.7 -2.3 2.1 ETV5 ets variant gene 5 (ets-related NM 004.4 54 molecule) PO O97 CO6 -5.9 -15.8 14 EVI2B ecotropic viral integration site 95 2B PO 18 .1 1.3 -1.4 exostoses (multiple) 1 NM OOO 27 PO -1.7 -2.1 1.7 coagulation factor II (thrombin) NM OO1992 receptor PO 128 BO2 18 -1 -1.1 F3 coagulation factor III NM OO1993 (thromboplastin, tissue factor) PO 132 GO3 1.9 1.8 -1.6 FACL3 atty-acid-Coenzyme Aligase, NM 004.4 57 ong-chain 3 PO O96 AO3 18 1.8 -2 FACL3 atty-acid-Coenzyme Aligase, NM 004.4 57 ong-chain 3 PO O83 DO7 2.2 1.4 -1.3 FADS1 atty acid desaturase 1 NM O134 PO O93 BO2 -3.4 3.4 FBLN1 fibulin 1 NM OO1996 PO 123 AO8 3.4 1.6 -1.9 FBLNS fibulin 5 NM OO6329 PO O68 HO9 1.4 2.2 -2 FBN1 fibrillin 1 (Marfan syndrome) NM OOO 38 PO 084 E10 1.9 -1.1 -1.1 FGF18 fibroblast growth factor 18 PO O93 BO3 -4.2 -5.9 5.6 FGF7 fibroblast growth factor 7 (keratinocyte growth factor) PO O92 CO4 -3.2 2.9 -3. 3 FGL2 fibrinogen-like 2 NM OO6682 PO 126 FO6 5.2 -6.5 4.9 FMO2 lavin containing NM OO14 60 monooxygenase 2 PO O78 G11 -1.9 2.1 -3.1 2.2 FMO3 lavin containing NM OO6894 monooxygenase 3 PO O88 F09 -1.8 1.4 -1.5 FOXD1 orkhead box D1 NM 004.4 72 PO 120 BO3 -1.8 1.9 -1.2 1.5 FRA Fos-related antigen NM O24816 PO 138 BO6 -1.8 1.5 -1.4 8 FTHL17 erritin, heavy polypeptide-like NM O31894 17 PO 2.7 -2.1 2.4 -2.5 FUT4 lucosyltransferase 4 (alpha (1,3) fucosyltransferase, myeloid-specific) PO O77 AO5 18 1.5 -1.2 FYN oncogene related to SRC, FGR, YES PO 124 GO1 -1.9 -1.4 1.2 frizzled homolog 7 NM 003507 (Drosophila) PO O83 BO9 3.2 4.2 -3.5 GABARAPL2 GABA(A) receptor-associated NM OO7285 protein-like 2 PO 106 BO5 -1.8 -1.1 2.4 GALT galactose-1-phosphate NM OOO155 uridylyltransferase PO O92 GO7 2.5 1.8 -2.2 GARS glycyl-tRNA synthetase NM 002047 PO O85 DO9 -2.9 -5.3 2.6 GAS1 growth arrest-specific 1 NM OO2O48 PO O63 EO9 -2 1.8 GBP2 guanylate binding protein 2, NM 004120 interferon-inducible PO 123 D12 -1.9 -2.7 2.7 GBP2 guanylate binding protein 2, NM OO4120 interferon-inducible PO 135 CO3 -1.8 -2.7 2.4 GCNT1 glucosaminyl (N-acetyl) NM OO1490 transferase 1, core 2 (beta 1,6-N- acetylglucosaminyltransferase) PO 127 BO1 -2.8 2.2 -29 3.7 GDFS growth differentiation factors 5 NM 000557 (cartilage-derived morphogenetic protein-1) US 2006/OO19890 A1 Jan. 26, 2006 14

TGF TGF TGF BNP TGF BNP BNP Clone ID 24 h. 24 h. 24 48 h 48 h 48 h. Symbol Name Accession PO -1.7 1.7 -1.8 .6 GGA3 golgi associated, gamma NM O14001 adaptin ear containing, ARF binding protein 3 PO O76 HO5 2.4 -2.7 .9 .1 GM2A GM2 ganglioside activator NM 0004-05 protein PO O62 E04 -2.3 1.9 -2.1 .9 - GNPI glucosamine-6-phosphate NM O05471 isomerase PO 138 C10 -2.1 2.2 -2 .9 -1.1 GNPI glucosamine-6-phosphate NM 005471 isomerase PO O74 DO6 3.5 5.7 -36 1. GOLGA4 golgi autoantigen, golgin NM OO2O78 subfamily a, 4 PO O83 CO4 -1.1 1.2 -1.8 5 .1 GOLPH2 golgi phosphoprotein 2 NM O16548 PO 125 G10 18 -1.9 1.6 -1.9 -1.1 GOLPH4 golgi phosphoprotein 4 PO 131 FO8 1.7 -2.3 1.8 -1.6 -1.2 GOT1 glutamic-Oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1) PO 080 AO1 -1.2 1.9 3 -3.6 8 .2 GPM6B glycoprotein M6B NM OO5278 PO O82 EO9 -2.2 2.3 -2.9 GPNMB ycoprotein (transmembrane) NM OO2510 PO O87 G08 2.3 -4.9 4 - GPNMB ycoprotein (transmembrane) NM OO2510 PO 140 E04 -1.8 1.6 -2.5 8 .1 GPRKS G protein-coupled receptor NM OO5308 kinase 5 PO -3.2 18 -1.9 2.9 1. GSTM1 glutathione S-transferase M1 NMOOO561 PO -1.8 1.5 -1.7 2.3 1. GSTM3 glutathione S-transferase M3 NM OOO849 (brain) PO 086 A10 -2.4 1.5 -1.9 2.7 .1 GSTMS glutathione S-transferase M5 NM OOO851 PO O80 CO3 1.7 -1.8 2.1 -1.9 - GTPBP2 GTP binding protein 2 NM O19096 PO 108 AO5 -1.2 1.5 -1.9 7 .1 GYPC glycophorin C (Gerbich blood NM 002101 group) PO 121 BO2 -1.6 1.2 -1.9 .9 .1 HAGE DEAD-box protein NM 018665 PO 133 H11 -1.2 18 -2.1 7 2 HAS2 hyaluronan synthase 2 NM OO5328 PO 101 C10 -1.8 1.6 -1.4 .5 - HEBP1 heme binding protein 1 NM O15987 PO 137 BO2 18 -1.5 1.6 -1.3 HERPUD1 homocysteine-inducible, NM 014685 endoplasmic reticulum stress inducible, ubiquitin-like domain member 1 PO 136 AO5 -2.1 2 -1.8 - HERPUD1 homocysteine-inducible, NM 014685 endoplasmic reticulum stress inducible, ubiquitin-like domain member 1 PO 1.6 -1.1 HEYL hairy/enhancer-of-split related NM 014571 with YRPW motif-like PO 126 BO1 -1.3 1.4 .1 -1.8 .8 - H factor (complement)-like 1 NM 002113 PO O75 H10 -3.6 6.2 -5.3 hepatocyte growth factor NM OOO6O1 (hepapoietin A; scatter factor) PO 110 C10 1.9 -1.6 HMGCR 3-hydroxy-3-methylglutaryl NM OOO859 Coenzyme A reductase PO 112 GO7 -1.7 -1 -1.1 - 3-hydroxy-3-methylglutaryl NM OO2130 Coenzyme A synthase 1 (soluble) PO O64 FO2 2.6 -3.1 3 HNMT histamine N-methyltransferase NM OO6895 PO O78 FO5 -2.1 2.4 -2.1 2.6 1. HPN hepsin (transmembrane NM 002151 protease, serine 1) PO 107 HO6 18 -1.9 1.4 -1.7 -1.3 ARS isoleucine-tRNA synthetase NM OO2161 PO 100 C10 -1.2 1.5 -1.8 1.7 .1 ICOS inducible T-cell co-stimulator NM 012092 PO 124 A06 -1.7 -1.8 1.7 - D2 inhibitor of DNA binding 2, NM 002166 dominant negative helix-loop helix protein PO 18 -1.6 1.6 -1.6 .2 inhibitor of DNA binding 4, NM OO1546 dominant negative helix-loop helix protein PO 088 CO1 -2.4 2.2 -2.5 2.2 - IDH2 isocitrate dehydrogenase 2 NM 002168 (NADP+), mitochondrial PO 130 FO1 4.5 -2.9 1.8 -1.7 IGF1 insulin-like growth factor 1 NM OOO618 (somatomedin C) US 2006/OO19890 A1 Jan. 26, 2006 15

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 PO O63 D10 2.1 1. .2 3.8 -1.9 3 IGF1 insulin-like growth factor 1 NM OOO618 (somatomedin C) POO777 D09 -2.6 2.2 1.1 -2.9 3.2 IGFBP4 insulin-like growth factor NM OO1552 binding protein 4 PO 130 BO2 -11.1 1.2 6.1 -5.4 interleukin 11 NM 000641 PO O88 DO5 2 .2 -1.8 1.4 interleukin 1, beta NM OOO576 PO O63 EO6 33 1.1 -6.7 6.1 interleukin 1 receptor, type I NM OOO877 PO 110 E12 23 1.3 -2.5 1.5 interleukin 1 receptor, type I NM OOO877 PO 145 AO4 2.4 - -4.2 2.7 interleukin 6 signal transducer NM 002184 (gp130, oncostatin M receptor) PO O91 BO3 -1.9 1.9 - -1.3 1.3 IMPA2 inositol(myo)-1 (or 4)- NM O14214 monophosphatase 2 PO O63 EO3 1.7 -1.7 -1.2 2.4 -1.7 INDO indoleamine-pyrrole 2.3 NM 002164 dioxygenase PO O82 FO7 2.1 -2.6 -1.1 2.2 -1.5 INHIBA inhibin, beta A (activin A, NM OO2.192 activin AB alpha polypeptide) PO 130 D09 2.1 -1.7 - 1.7 -1.7 INPP4B inositol polyphosphate-4- NM OO3866 phosphatase, type II, 105 kDa PO -1.7 1.2 1.6 -1.3 INSG1 insulin induced gene 1 NM OO5542 PO -1.7 1.7 - -4.7 3.1 IQGAP2 IQ motif containing GTPase NM OO6633 activating protein 2 PO 2.6 -2.6 2.4 -2.5 interferon-stimulated NM OO6084 transcription factor 3, gamma 48 kDa PO 140 B08 18 -1.7 1.2 -1.8 ITGA11 integrin, alpha 11 NM O12211 PO O88 C11 -1.5 18 1.2 -1.8 1.9 ITGAM integrin, alpha M (complement NM OOO632 component receptor 3, alpha; also known as CD11b (p170), macrophage antigen alpha polypeptide) PO 2.3 -1.8 1.2 2.2 -2.2 JUNEB jun B proto-oncogene PO 1.6 -1.5 1.2 1.9 -1.6 JUP junction plakoglobin PO -1.9 2.1 -1.1 -1.5 1.5 JWA vitamin A responsive; cytoskeleton related PO 22 A09 1.1 3 1.2 -1.9 1.6 KCNE3 potassium voltage-gated NM OO5472 channel, lsk-related family, member 3 PO 13 FO2 -1.8 9 1.2 -2.4 2.3 KHDRBS3 KH domain containing, RNA NM OO6558 binding, signal transduction associated 3 PO O74 BO1 -1.6 .2 KIAAO1O2 KIAA0102 gene product NM O14752 PO 04 A04 -3.2 3.8 - KIAA1049 KIAA1049 protein NM O14972 PO 2O BO2 -1.6 5 1. KF1B kinesin family member 1B NM O15074 PO 088 CO6 -1.6 .6 1.2 KRT4 keratin 4 NM OO2272 PO O85 DO6 -1.8 7 1.2 LAMA4 aminin, alpha 4 NM OO2290 PO 31 HO2 -1.4 .4 LAMC1 aminin, gamma 1 (formerly NM OO2.293 LAMB2) PO 31 H10 -2.4 .8 -1. LCN2 ipocalin 2 (oncogene 24p3) NM 005564 PO OO HO5 -2.8 2.7 1.2 LEPR eptin receptor NM OO2303 PO O88 BO2 -2.3 2.4 LGALS3 ectin, galactoside-binding, NM OO2306 soluble, 3 (galectin 3) PO O81. B11 -3.5 3 -4.6 LHFP ipoma HMGIC fusion partner NM OO578O PO O7 DO6 2.2 -2 1.7 LIMK2 LIM domain kinase 2 NM OO5569 PO O85 GO6 1.2 -1.4 -1. 1.9 LMOf LIM domain only 7 NM OO5358 PO O85 D05 -2.1 2.2 1.2 -3.9 LOC56270 hypothetical protein 628 NM O19613 PO O82 EO1 2.1 -1.5 1.2 18 LOX ysyl oxidase NM OO2317 PO O83 HO2 -1.4 5 1. LPHN2 atrophilin 2 NM O12302 PO 131 D06 -1.6 7 1.2 -2.4 8 LRP4 ow density lipoprotein ABO11540 receptor-related protein 4 PO 18 -1.2 - 2.2 LTBP2 atent transforming growth NM OOO428 actor beta binding protein 2 PO 088 CO4 -2.3 23 1. -4.4 LTF actotransferrin NM OO2343 PO 063 A11 -2.3 2.4 - -4.8 s LUM umican NM OO2345 PO 135 GO5 -2.4 2.4 1.2 -1.7 LY96 ymphocyte antigen 96 NM O15364 PO O85 CO4 -2 18 1.2 MADH3 MAD, mothers against NM OO5902 decapentaplegic homolog 3 (Drosophila) US 2006/OO19890 A1 Jan. 26, 2006 16

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 PO1091 G10 1.8 -1.4 .2 2.2 -2.1 .2 MADHF MAD, mothers against NM OO5904 decapentaplegic homolog 7 (Drosophila) PO1089 CO1 1.2 -1.2 - 18 -1.6 MAGP2 Microfibril-associated NM OO3480 glycoprotein-2 PO1084 A09 1.8 -1.6 .2 1.4 -1.6 MAP3K2 mitogen-activated protein NM OO6609 kinase kinase kinase 2 PO1073 E08 -2 2.4 - -2.3 18 MAP3KS mitogen-activated protein NM OO5923 kinase kinase kinase 5 PO1066 F10 2 -2 .1 1.9 -1.7 MAPK7 mitogen-activated protein NM OO2749 kinase 7 PO1076 B12 .9 -2.1 -1.1 1.7 -1.7 MAPRE2 microtubule-associated NM O14268 protein, RP/EB family, member 2 PO1134 CO4 3.1 -2.1 .1 2.8 -3.3 MATN3 matrilin 3 NM OO2381 PO1145 AO5 -1.7 1.9 - -2.6 2.1 ME1 malic enzyme 1, NADP(+)- NM 002395 dependent, cytosolic PO1072 D11 -3.3 3.7 - -3.5 3.1 MEST mesoderm specific transcript NM OO2402 homolog (mouse) PO1121 FO4 -1.9 2.1 3 -2.1 18 MGC12O3 hypothetical protein MGC1203 PO1068 F12 -2.9 2.8 .1 -2.6 2.4 MGST1 microsomal glutathione S ransferase 1 PO1091 BO6 -1.8 1.6 - -1.5 1.6 MGST2 microsomal glutathione S NM OO2413 ransferase 2 PO1099 HO9 -2.4 2.3 .1 -2.4 MID1 midline 1 (Opitz/BBB NMOOO381 syndrome) PO1062 HO5 -1.4 2.2 3 -2.4 2.4 MME membrane metallo NM OOO902 endopeptidase (neutral endopeptidase, enkephalinase, CALLA, CD10) PO1125 HO8 1. .1 2.6 MMP11 matrix metalloproteinase 11 NM OO5940 (stromelysin 3) PO1072 DO2 2.8 -2.6 -1.3 1.7 MTHFD2 methylene tetrahydrofolate NM OO6636 dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase PO1125 A10 -1.6 1.6 .2 -1.8 MTMR4 myotubularin related protein 4 NM OO4687 PO1130 CO9 1.9 -1.7 3 1.1 MUCDHL mucin and cadherin-like NM O17717 PO1102 A12 1.4 -1.3 .2 2.5 MVK mevalonate kinase (mevalonic NM OOO431 aciduria) PO1133 FO5 1.9 -1.8 1.4 myosin, heavy polypeptide 9, NM OO2473 non-muscle PO1100 BO7 -2 2.4 .1 -5.5 MYOZ2 myOZenin 2 NM O16599 PO1072 CO6 -1.6 1.6 -2.6 NCK1 NCK adaptor protein 1 NM OO6153 PO1086 B12 -1.2 1.4 - -1.8 NCOA3 nuclear receptor coactivator 3 NM OO6534 PO1135 C12 3.3 -3 3 3.1 NEDD9 neural precursor cell NM OO6403 expressed, developmentally down-regulated 9 PO1112 A08 2.5 -2. .2 1.7 NET-6 transmembrane 4 superfamily NM O14399 member tetraspan NET-6 PO1103 EO2 -1.7 2. .2 -2.5 NFA nuclear factor I/A ALO96888 PO1073 EO6 -1.9 1.9 -2.1 NFIB nuclear factor I/B NM OO5596 PO1064 CO2 -1.9 2 .2 -3.3 ND2 nidogen 2 (Osteonidogen) NM OO7361 PO1131 E08 2.3 -1.6 3 5.1 NINJ2 ninjurin 2 NM O16533 PO1072 DO1 2.2 -2.2 .1 2.2 NK4 natural killer cell transcript 4 NM OO4221 PO1121 GO6 -2.2 2. -1.1 -2.5 NOL3 nucleolar protein 3 (apoptosis NM OO3946 repressor with CARD domain) PO1104 CO8 6.9 -6. .1 5.8 NOX4 NADPH oxidase 4 NM O16931 PO1107 D11 -1.7 1.6 - -1.8 NPC2 Niemann-Pick disease, type NM OO6432 C2 PO1132 GO6 2.4 -2 3 1.5 NPR3 natriuretic peptide receptor NM OOO908 C/guanylate cyclase C (atrionatriuretic peptide receptor C) PO1096 FO8 -1.5 1.6 .2 -2.1 neuronal pentraxin II NM OO2523 PO1126 EO7 -1.5 2 .2 1.7 nuclear receptor subfamily 2, NM 021005 group F, member 2 US 2006/OO19890 A1 Jan. 26, 2006 17

TGF TGF TGF BNP TGF BNP BNP Clone ID 24 h. 24 h. 24 48 h. 48 h. 48 h. Symbol Name Accession PO -1.5 1.6 -2.1 1.5 NRCAM neuronal cell adhesion NM OO5010 molecule PO O97 E11 1.9 -1.8 1.6 -1.8 NS1-BP NS1-binding protein NM OO6469 PO 103 CO4 2.4 -2.2 1.3 -1.5 NUDT3 nudix (nucleoside diphosphate NM OO6703 linked moiety X)-type motif 3 PO 2.6 -2.6 2.3 -2.3 ODC1 omithine decarboxylase 1 NM OO2539 PO -1.4 2 -5.7 1.9 OGN Osteoglycin (Osteoinductive NM O14057 factor, mimecan) PO 19 GO7 -2.1 2.2 -2.2 1.6 OSBPL1A Oxysterol binding protein-like NM 018030 1A PO 2.3 -1.6 3.9 OSF-2 osteoblast specific factor 2 NM OO6475 (fasciclin I-like) PO 2.2 -1.6 OSF-2 osteoblast specific factor 2 NM OO6475 (fasciclin I-like) PO 1.5 OXA1L. Oxidase (cytochrome c) NM 005O15 assembly 1-like PO 1.6 OXA1L. Oxidase (cytochrome c) NM 005O15 assembly 1-like PO 1.3 OXTR Oxytocin receptor NM OOO916 PO -1.7 PACE4 paired basic amino acid NM OO2570 cleaving system 4 PO 1.2 PARG1 PTPL1-associated RhoGAP 1 NM OO4815 PO -2.4 PAWR PRKC, apoptosis, WT1, NM OO2583 regulator PO 20 FO4 2.3 papillomavirus regulatory NM 018660 actor PRF PO 1.5 PBP prostatic binding protein NM OO2567 PO -1.2 PCDH1 protocadherin 1 (cadherin-like NM OO2587 1) PO O66 GO5 1.6 PDE1A phosphodiesterase 1A, NM 005O19 C almodulin-dependent PO 128 BO3 -1.7 PDESA hosphodiesterase 5A, c0MP NM OO1083 S pecific PO O87 EO2 3.4 -2.4 PDGFA atelet-derived growth factor NM 0026O7 al pha polypeptide PO O81 FO7 -2.3 2.1 -2.2 PDGFRA atelet-derived growth factor NM OO62O6 receptor, alpha polypeptide PO 142 DO1 -1.1 -1.8 -2.2 PDGFRL platelet-derived growth factor NM OO62O7 receptor-like PO O64 GO2 1.3 -1.1 2.3 PDGFRL platelet-derived growth factor NM OO62O7 receptor-like PO 137 FO4 -1.8 2 PDP pyruvate dehydrogenase NM 018444 phosphatase PO O71 HO7 18 -1.9 PFKP phosphofructokinase, platelet NM OO2627 PO O64 HO7 -1.8 1.7 5 PHF3 PHD finger protein 3 NM O15153 PO 131 G12 1.2 -1 PGB hosphatidylinositol glycan, NM 004.855 C ass B PO O74 HO7 -1.8 1.9 PIK3R1 hosphoinositide-3-kinase, AF279367 regulatory subunit, polypeptide 1 (p85 alpha) PO O68 AO2 -2.4 1.7 .1 PIR Prin PO 112 HO1 18 -1.6 3 PIST PDZ, coiled-coil domain binding partner for the rho amily GTPase TC10 PO 118 HO9 -2.4 2.1 PITPNM phosphatidylinositol transfer NM OO4910 protein, membrane-associated PO 110 GO2 -1.3 1.6 PKIB protein kinase (cAMP NM 032471 dependent, catalytic) inhibitor beta PO 146 A11 1.4 -1.5 -1.9 phospholipase A2, group IVC (cytosolic, calcium independent) PO 124 G10 -2.5 2.5 -1.2 PLA2R1 phospholipase A2 receptor 1, NM OO7366 180 kDa PO 18 -1.7 1.9 -1.1 PLAU plasminogen activator, NM 002658 urokinase PO O64. FO1 -1.8 2.3 -1.7 PLCL1 phospholipase C-like 1 NM OO6226 US 2006/OO19890 A1 Jan. 26, 2006 18

TGF TGF TGF BNP TGF BNP BNP Clone ID 24 h. 24 h. 24 48 h. 48 h. 48 h. Symbol Name Accession PO 118 E04 2.4 -1.8 PLEK2 pleckstrin 2 NM 016445 PO O72 A03 5.2 -5.1 PLN phospholamban NM 002667 PO 084 AO8 2.8 -2.2 PLOD2 procollagen-lysine, 2 NM OOO935 Oxoglutarate 5-dioxygenase (lysine hydroxylase) 2 PO PLP2 proteolipid protein 2 (colonic NM OO2668 epithelium-enriched) PO 30 B04 PMP2 peripheral myelin protein 2 NM 002677 PO PNUTL2 peanut-like 2 (Drosophila) NM 004574 PO PODXL podocalyxin-like NM OO5397 PO : i - POLD3 polymerase (DNA directed), BCO2O587 delta 3 PO 5 1 PP pyrophosphatase (inorganic) NM 021129 PO 3 PPAP2B phosphatidic acid phosphatase NM OO3713 ype 2B PO -2.2 PPARG peroxisome proliferative NM OO5037 activated receptor, gamma PO .1 -5.3 PPL periplakin NM OO2705 PO PPP2R4 protein phosphatase 2A, NM 021131 regulatory subunit B' (PR 53) PO .1 -1.9 5 PRKCM protein kinase C, mu NM OO2742 PO 1.4 PRPS1 phosphoribosyl pyrophosphate NM OO2764 synthetase 1 PO -4.4 PSG1 pregnancy specific beta-1- NM OO6905 glycoprotein 1 PO -5.5 PSG1 pregnancy specific beta-1- NM OO6905 glycoprotein 1 PO -2.6 PSG11 pregnancy specific beta-1- NM OO2785 glycoprotein 11 PO -4.1 PSG4 pregnancy specific beta-1- NM OO2780 glycoprotein 4 PO PTGER4 prostaglandin E receptor 4 NM OOO958 (subtype EP4) PO -2.8 -2.2 PTGIS prostaglandin I2 (prostacyclin) NM OOO961 synthase PO 2.3 -2.8 1.1 PTGS1 prostaglandin-endoperoxide NM OOO962 synthase 1 (prostaglandin G/H synthase and cyclooxygenase) PO -2.6 1.3 PTGS2 prostaglandin-endoperoxide NM OOO963 synthase 2 (prostaglandin G/H synthase and cyclooxygenase) PO 106 GO6 18 -1.5 3.1 PTHLH parathyroid hormone-like NM 002820 OOle PO O71 G12 -1.9 1.4 -3.7 PTN pleiotrophin (heparin binding NM 002825 growth factor 8, neurite growth-promoting factor 1) PO 128 HO8 -2.3 2.4 .1 -1.5 PTTG1 pituitary tumor-transforming 1 NM OO4219 PO O95 AO3 -2.4 2.4 -1.4 PTTG1 pituitary tumor-transforming 1 NM OO4219 PO O97 GO6 -1.7 -2.2 PUS1 pseudouridylate synthase 1 NM O25215 PO O76 CO4 2.5 3.1 QPCT glutaminyl-peptide NM 012413 cyclotransferase (glutaminyl cyclase) PO 129 CO5 -2.1 -1.5 RAB13 RAB13, member RAS NM OO2870 oncogene family PO 115 GO1 -1.8 -1.6 2.2 RAB13 RAB13, member RAS NM 002870 oncogene family PO 110 E09 1.4 18 -1.6 RAI RelA-associated inhibitor NM OO6663 PO 100 EO2 -1.5 -3.3 2.7 RA3 retinoic acid induced 3 NM OO3979 PO O82 AO1 -2.4 -2.6 2.3 RARRES3 retinoic acid receptor NM OO4585 responder (tazarotene induced) 3 PO 117 H10 -1.8 -2.5 RASSFS Ras association (RalGDS/AF NM 031437 6) domain family 5 PO 108 CO7 1.4 2.5 -1.9 RBP1 retinol binding protein 1, NM OO2899 cellular PO 136 CO4 2.6 2.3 -1.6 RGS2 regulator of G-protein NM OO2923 signalling 2, 24 kDa US 2006/OO19890 A1 Jan. 26, 2006 19

TGF TGF TGF BNP BNP TGF BNP BNP Clone ID 24 h. 24 h. 24 48 h. 48 h. 48 h. Symbol Name Accession PO 145 A10 -1.2 1.2 -2 1.6 -1.1 RGS4 regulator of G-protein NM OO5613 signalling 4 PO O90 DO2 -1.3 1.2 -3 1.9 -1.3 RGS4 regulator of G-protein NM OO5613 signalling 4 PO O81 H10 -2.2 1.6 -6.7 4.7 RGSS regulator of G-protein NM OO3617 signalling 5 PO -1.9 1.4 -3.8 3.2 RNASE1 ribonuclease, RNase A family, NM OO2933 1 (pancreatic) PO O88 GO9 -1 1. -1.8 18 RPL5 ribosomal protein L5 NM OOO969 PO 127 E10 18 -1.6 1.7 -1.5 RRAS related RAS viral (r-ras) NM OO6270 oncogene homolog PO 122 BO3 -2 -2.4 3.1 RRP4 homolog of Yeast RRP4 NM O14285 (ribosomal RNA processing 4), 3'-5'-exoribonuclease PO 104 D09 2.1 -1.8 HIF-1 responsive RTP801 NM O19058 PO 121 G04 2.1 4.1 -3.2 .1 RuvB-like 2 (E. coli) NM OO6666 PO O87 BO6 -1.9 2.4 S100 calcium binding protein NM OO2966 A10 (annexin II ligand, calpactin I, light polypeptide (p11)) PO O64 F10 18 -1.5 S100A11 S100 calcium binding protein A11 (calgizZarin) POO777 AO5 -2.3 2.4 S100A4 S100 calcium binding protein NM OO2961 A4 (calcium protein, calvasculin, metastasin, murine placental homolog) POO777 AO6 -2.6 2.7 S100A4 S100 calcium binding protein NM OO2961 A4 (calcium protein, calvasculin, metastasin, murine placental homolog) PO 143 A11 -2.4 2.4 S100A4 S100 calcium binding protein NM OO2961 A4 (calcium protein, calvasculin, metastasin, murine placental homolog) PO 141 FO3 3 3.9 -1.7 3 SAA2 serum amyloid A2 NM 03.0754 PO O61 FO4 -3.1 -2.2 2.8 SAT spermidine?spermine N1 NM OO2970 acetyltransferase PO 124 BO3 -2.9 3.7 -2.1 2.5 SAT spermidine?spermine N1 NM OO2970 acetyltransferase PO 140 G05 -2.1 1.3 -1.3 SCSDL sterol-C5-desaturase (ERG3 NM OO6918 delta-5-desaturase homolog, fungal)-like PO O66 HO4 -2.7 SCD stearoyl-CoA desaturase NM OO5063 (delta-9-desaturase) PO 140 D11 -3.8 3.5 SCD stearoyl-CoA desaturase NM OO5063 (delta-9-desaturase) PO 119 B12 1.9 -3.9 SCDGF-B spinal cord-derived growth NM O25208 factor-B PO O87 A04 1.3 SCG2 secretogranin II (chromogranin NM OO3469 C) PO -1.9 2.8 -2.5 SCRG1 scrapie responsive protein 1 NM OO7281 PO 1.7 .1 -2.6 2.3 SDC4 syndecan 4 (amphiglycan, NM OO2999 ryudocan) PO 1.7 -1.8 1.6 SDCBP syndecan binding protein NM OO5625 (syntenin) PO 8 -1.5 -1.2 SEC23A Sec23 homolog A (S. cerevisiae) NM OO6364 PO -3.6 2.5 SELENEP1 selenium binding protein 1 NM OO394.4 PO -3.2 2.4 5.8 . SELENEP1 selenium binding protein 1 NM OO394.4 PO 1.4 1.5 SEPP1 selenoprotein P. plasma, 1 PO -2.9 -3.3 SERPINE1 serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 PO O90 H11 -1.3 1.2 -1.9 2.1 SFRP1 secreted frizzled-related protein 1 PO O78 FO1 -1.8 2.4 -1.6 1.6 SFRP4 secreted frizzled-related protein 4 US 2006/OO19890 A1 Jan. 26, 2006

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 PO1087 AO6 -2.9 1.9 -1.2 2.2 -1.3 SGNE1 secretory granule, neuroendocrine protein 1 (7B2 protein) PO1106 GO5 18 -1.7 1.3 2.9 -2.2 SKIL SKI-like NM OO5414 PO1102 A06 -1.8 2 3 -3.2 2.8 SLC11A3 solute carrier family 11 NM 014585 (proton-coupled divalent metalion transporters), member 3 PO1105 AO3 1.9 -1.7 11 1.5 -1.4 solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 PO1143 D11 -2.7 2.5 1.3 -2.1 2.8 SLC25A11 solute carrier family 25 NM 003562 (mitochondrial carrier; Oxoglutarate carrier), member 11 PO1111 HO3 1.8 -1.7 1.9 SLC7A11 solute carrier family 7, NM O14331 (cationic amino acid transporter, y-- system) member 11 PO1138 AO8 3 -2.9 - 2.3 SLCFAS solute carrier family 7 (cationic NM OO34.86 amino acid transporter, y+ system), member 5 PO1088 E10 3.1 -2.7 11 2.6 SLCFAS solute carrier family 7 (cationic NM OO3486 amino acid transporter, y+ system), member 5 PO1112 E05 -1.6 1.3 2.2 SLT3 slit homolog 3 (Drosophila) PO1136 FO7 -1.1 1.4 1.2 -2.1 1.9 SLT3 slit homolog 3 (Drosophila) PO1079 GO3 -3.1 3.4 - -3.9 3.5 SNAI2 snail homolog 2 (Drosophila) PO1140 FO7 2.9 -2.6 1.1 2.2 -2.5 SNF1LK SNF1-like kinase PO1083 AO4 -3.2 3.2 -93 SNK serum-inducible kinase PO1085. FO6 -1.2 1.2 1.1 -2.6 1.7 SOD3 superoxide dismutase 3, NM 003 extracellular PO1074 H12 1. 1.1 1.1 -2.6 1.5 SPINT2 serine protease inhibitor, Kunitz type, 2 PO1108 BO2 -2.5 2.6 1.2 -4.2 2.2 SPRY1 sprouty homolog 1, antagonist AFO41037 of FGF signaling (Drosophila) PO1095 FO4 -2.6 2 -1.1 -1.8 18 SORDL sulfide quinone reductase-like NM O21 99 (yeast) PO1128 EO7 1.9 -2 2.6 -2.7 SRPUL sushi-repeat protein NM O144 67 PO1073 BO2 -1.7 1.7 1.2 -2.5 1.9 SRPX sushi-repeat-containing NM OO6307 protein, X chromosome PO1104 F12 -2.1 2.5 1.2 -2.2 2.3 SSBP2 single-stranded DNA binding NM O124 46 protein 2 PO1069 CO6 1.9 -1.3 - 2.7 SSR1 signal sequence receptor, NM 003 44 alpha (translocon-associated protein alpha) PO1130 F10 -1.3 1.6 1.1 -2.3 STC1 stanniocalcin 1 NM 003 55 PO1130 B11 2.1 -2 - 1.7 STCH stress 70 protein chaperone, NM OO6948 microsome-associated, 60 kDa PO1074 EO3 1.7 -1.3 - -1.9 STE sulfotransferase, estrogen preferring PO1127 GO1 -1.4 1.5 1.2 -1.9 STK17B serine/threonine kinase 17b NM OO4226 (apoptosis-inducing) PO1125 C11 -2 .6 - -2.2 STK25 serine/threonine kinase 25 NM OO6374 (STE20 homolog, yeast) PO1076 DO3 -2.7 2.9 1.1 -2.1 STK38 serine/threonine kinase 38 NM OO7271 PO1105 EO3 -2.8 2.7 -1.1 -2.7 STMN1 stathmin 1/oncoprotein 18 NM OO5563 PO1069 A08 -1.5 1.5 1.1 -1.8 STOM stomatin NM 004.099 PO1102 E10 -15 1.6 1.1 -2.3 SVIL supervillin 74 PO1062 HO6 -1.5 2.1 1.2 -2.9 TACSTD2 tumor-associated calcium NM 002353 signal transducer 2 PO1098 E05 1.9 -1.8 1.2 1.2 TAF13 TAF13 RNA polymerase II, NM OO5645 TATA box binding protein (TBP)-associated factor, 18 kDa PO1101 B02 -1.9 1.4 1.1 TCF7L1 transcription factor 7-like 1 (T- NM 031283 cell specific, HMG-box) PO1061 CO1 -1.7 1.6 1.3 transferrin NM OO1063 US 2006/OO19890 A1 Jan. 26, 2006 21

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 PO 144 CO3 -3.4 3.6 1.2 -4 2.9 .1 TFPI issue factor pathway inhibitor NM OO6287 (lipoprotein-associated coagulation inhibitor) PO -1.4 1.6 1.4 -2.3 2.3 .1 issue factor pathway inhibitor 2 NM OO6528 PO -1.4 1.4 1.2 -2.1 1.7 .1 ransforming growth factor, NM OO3238 beta 2. PO -3.5 3.8 1.2 -4.7 4 .2 ransforming growth factor, NM OO3243 beta receptor III (betaglycan, 300 kDa) PO O78 BO4 18 -1.7 - 1.8 -1.9 -1.2 THES2 hrombospondin 2 NM OO3247 PO 124 G04 2.8 -2.5 - 2.4 -3.1 -1. 3 TIMP3 issue inhibitor of NM OOO362 metalloproteinase 3 (Sorsby undus dystrophy, pseudoinflammatory) PO 2.1 -2.4 - 2.6 -3.1 -1.2 TIMP3 issue inhibitor of NM OOO362 metalloproteinase 3 (Sorsby undus dystrophy, pseudoinflammatory) PO 3 -2.5 2.6 - TM4SF1 ransmembrane 4 superfamily NM O14220 member 1 PO -1.6 18 -1.8 1.5 -1.1 TncRNA rophoblast-derived noncoding RNA PO 126 EO9 -1.7 2 -3.7 4.2 .1 TNEAIP2 umor necrosis factor, alpha NM OO6291 induced protein 2 PO -1.5 1.6 - -2.4 1.9 .1 TNFAIP3 umor necrosis factor, alpha NM OO6290 induced protein 3 PO 138 G10 18 -1.7 1.2 2.1 -2 TNFRSF12A umor necrosis factor receptor NM O16639 superfamily, member 12A PO -2.1 3 .4 -2.6 2.5 .2 TNFSF10 umor necrosis factor (ligand) NM OO3810 superfamily, member 10 PO 144 C11 2 -1.9 1.4 .2 TOP2A opoisomerase (DNA) II alpha NM OO1067 170 kDa PO 140 DO3 2.1 -1.7 - 1.2 -1.6 - TTID itin immunoglobulin domain NM OO6790 protein (myotilin) PO -2.9 23 1.1 TXNRD1 hioredoxin reductase 1 NM OO3330 PO O89 DO1 1.7 -3 .1 2.5 -2.3 - UCHL1 ubiquitin carboxyl-terminal NM 004181 esterase L1 (ubiquitin hiolesterase) PO 123 DO7 -1.9 2.4 1.1 UGCG UDP-glucose ceramide NM OO3358 glucosyltransferase PO 1.5 -2.6 1.2 2.4 -1.7 .2 UMPK undine monophosphate kinase NM O12474 PO 2.1 -3.1 1.1 2.4 -1.9 UMPS uridine monophosphate NM OOO373 synthetase (orotate phosphoribosyl transferase and orotidine-5'- decarboxylase) PO1061 BO2 -2.7 2.4 -4.2 2.3 -1.3 VCAM1 vascular cell adhesion NM OO1078 molecule 1 PO1141 CO6 2.7 -1.8 1.3 1.4 -1.2 .2 WISP1 WNT1 inducible signaling NM OO3882 pathway protein 1 POO777 CO9 -1.6 1.8 1.1 -5 4 - WISP2 WNT1 inducible signaling NM 003881 pathway protein 2 POO777 C10 -2.2 2.1 1.1 -5.6 4.4 - WISP2 WNT1 inducible signaling NM OO3881 pathway protein 2 PO1126 HO7 -1.8 1.6 1.1 -3 3.9 - WISP2 WNT1 inducible signaling NM 003881 pathway protein 2 PO1142 D08 3.7 -3 3 3.6 -2.8 .1 X-ray repair complementing defective repair in Chinese hamster cells 4 PO1104 HO7 -1.7 1.7 1.2 -1.9 1.5 .1 Zinc finger protein, multitype 2 NM 012082 PO1064 H12 -1.4 1.5 1.1 -1.8 1.5 - Zinc finger protein 142 (clone NM 005081 pHZ-49) PO1075 EO2 1.5 -1.2 1.1 1.9 -19 ZNF193 zinc finger protein 193 NM OO6299 US 2006/OO19890 A1 Jan. 26, 2006 22

Validation of Microarray by Real-Time RT-PCR and West ACTA2 were also confirmed by real-time RT-PCR (FIG. 6). ern Blot Analyses Additional verification was obtained for the pro-inflamma 0.066 Representative microarray data was validated tory genes COX2 and IL6 at 6, 24, and 48 h (FIG. 6). Again, using real-time RT-PCR and Western analyses. TGFB most likely due to Sensitivity issues, IL6 was not included in induced Collagen 1 mRNA levels in human cardiac fibro FIG. 4C, since it did not exceed the array differential blasts at 6, 24, and 48 h; this induction was blocked by BNP expression threshold value. at all 3 time points (FIG. 5A). Collagen 1 protein synthesis was also induced (-3-fold) at 48 h, and BNP inhibited this 0067. In addition, real-time RT-PCR assays were per stimulation by ~75% (FIG. 5B). BNP also inhibited TGFB formed for 9 genes on primary cultures of human cardiac induced Fibronectin mRNA and protein expression at 48 h fibroblasts from a second independent donor lot of fibro (FIG. 5C,D). These data corroborate the microarray results, blasts (see Table 3). The effects of BNP on TGFB-induced with the exception of Fibronectin, which did not exceed the gene expression in both donors were similar, although donor array differential expression threshold value, most likely due lot 2 was slightly less responsive to TGFB. Taken together, to the lower Sensitivity of the microarray compared to these results confirm the microarray data using independent real-time RT-PCR. The effects of BNP on TGFB stimulation assay methods, as well as, multiple human cardiac fibroblast of pro-fibrotic genes CTGF, PAI-1, TIMP3, IL11, and 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 TGFB TGFB + BNP Time (h) Lot Collagen 1 .O. O.05 1.0 - 0.05 1.9 0.04 1.2 - O.O1 6 .O. O.06 1.1 - 0.13 3.3 - 0.05 1.3 - 0.26 24 .0 - 0.11 1.0 + 0.26 1.5 + 0.09 1.2 - O.O1 24 2 O. O.13 1.2 O.O3 3.8 - 0.38 1.3 O.O3 48 Oi O.2O 10 it O.O1 2.5 it 0.32 1.3 0.18 48 2 Fibronectin O. O.O4 O.9 O.19 11 O.17 1.O. O.29 6 .O. O.21 1.0 + 0.10 1.0 - 0.05 1.O. O.18 24 O. O.19 O.9 O.24 1.O. O.O2 1.O. O.12 24 2 O. O.O4 1.1 O.O4 2.2 - 0.38 1.3 - 0.35 48 O OO1 1.O. O.1 2.O. O.39 1.5 + 0.02 48 2 SERPINE1/PAI-1 O. O.O7 O.7 O.O8 7.3 0.44 1.7 O.37 6 O OO1 O.7 O.O 8.5 + 0.08 O.7 O.10 24 O. O.10 O.7 O.1 2.4 O.O6 1.1 - 0.10 24 2 O. O.22 O.9 O.OO 8.4 1.33 O.9 O.13 48 O. O.17 O.8 O.O3 2.6 0.03 O.9 OO6 48 2 CTGF .O. O.15 0.9 + 0.24 3.5 + 0.08 O.9 O.O3 6 .O. O.28 1.0 + 0.29 3.3 - 0.25 O.7 O.25 24 .O. O.09 1.5 + 0.44 2.2 + 0.16 .5 + 0.04 24 2 .0 0.45 1.4 - 0.13 3.1 - 0.01 1 O.O1 48 O. O.32 1.3 O.12 2.1 - 0.14 O. O.24 48 2 IL11 O. O.2O 11 O.O4 13.3 0.89 2.1 OO6 6 O. O.13 1.2 O.O7 32.3 0.82 .1 + 0.14 24 .O. O.06 1.0 - 0.05 7.7 - 0.81 2.1 - 0.18 24 2 O. O.23 O.7 O.1O 17.6 0.22 O. O.08 48 .O. O.09 0.8 0.09 5.9 0.18 2 - 0.10 48 2 TIMP3 O OO1 O.9 O.11 4 - 0.03 O. O.12 6 O. O.31 1.O. O.12 2.6 0.26 O. O.23 24 O. O.13 O.7 O.O9 .5 + 0.12 3 - 0.14 24 2 O. O.26 O.9 O.OO 3.0 - 0.34 O. O.09 48 O. O.O8 O.6 O.OO 7 O.13 O8 O.O1 48 2 O OO6 O.9 O.O2 3.6 0.27 3 - 0.14 6 O. O.13 O.9 O.21 7 O.14 O8 O.O3 24 O. O.O9 O.9 O.O7 .4 - 0.05 O. O.11 24 2 O. O.13 O.9 O.O3 6 - 0.12 0.9 0.05 48 O. O.17 O.9 O.O6 4 - 0.17 O.9 O.17 48 2 PTGS2/COX-2 O OO1 1.2 - 0.22 9.0 1.49 .8 0.05 6 .O. O.08 1.2 + 0.38 3.5 + 0.67 2 O.19 24 .O. O.07 1.1 - 0.05 4.9 0.36 4 - 0.18 24 2 O. O.10 1.O. O.12 2.2 - 0.12 3 O.O3 48 .O. O.19 1.0 - 0.06 5.4 - 0.92 2 O.O1 48 2 ACTA2 O. O.O3 O.8 O.12 .1 + 0.11 O.9 O.2O 6 O. O.14 O.9 O.11 2.2 O.OO O.9 O.O7 24 .O. O.04 0.9 + 0.25 2.3 - 0.12 ..6+ 0.41 24 2 O. O.17 1.O. O.O3 O. O.19 O. O.21 48 .O. O.05 0.7 - 0.11 2.5 + 0.13 O. O.12 48 2 US 2006/OO19890 A1 Jan. 26, 2006 23

0068. In a related study, a gene microassay profile of rat observed in animal models of myocardial infarction, Sug heart tissue was conducted. The results of this study are gesting that it may be a contributor to matrix remodeling in shown in FIG. 12. Fibrotic and extracellular matrix associ the failing heart. ated genes were stimulated in vivo by L-NAME plus angio 0072 Another hallmark of the fibrotic process is the tensin II. MRNA expression for collagen I, collagen III, and transformation of cardiac fibroblasts to myofibroblasts and fibronectin was markedly reduced by the administration of the induction of pro-fibrotic mediators. Myofibroblasts BNP. acquire contractile properties Similar to Smooth muscle cells. MEK/ERK Pathway Involved in BNP's Anti-Fibrotic Role The results provided above demonstrate that BNP inhibited TGFB-induction of several myofibroblast markers including 0069 Natriuretic peptides were previously shown to ACTA2 and MYH9. BNP also inhibited TGFB pro-fibrotic Stimulate ERK activity in cardiac myocytes and Vascular mediators, such as, CTGF, PAI-1, and IL11. CTGF and endothelial cells. The MEK/ERK pathway has been linked PAI-1 are well-established downstream Signaling genes of to the repression of TGFB/Smad signaling. To determine the TGFB pathway, and IL11 has been associated with tissue whether PKG or ERK signaling is involved in BNP-depen remodeling and fibrosis. IL11 expression in cardiac fibro dent attenuation of TGFB Signaling, cultured cells were blasts also seems to contribute to TGFB-mediated fibrosis. treated with BNP and/or TGFB in the presence of a PKG The use of BNP to suppress this response should result in a inhibitor (KT5823) or two different MEK inhibitors (U0126, protective effect. PD98059). BNP induced ERK phosphorylation was com pletely blocked by KT5823 and U0126, indicating that BNP 0073 Collectively, these effects of BNP on gene expres activates ERK via PKG and MEK signaling cascades (FIG. sion in TGFB-stimulated cells demonstrate a role for BNP in 7a). Both MEK inhibitors (U0126, PD98059) reversed BNP anti-fibrotic processes in cardiac fibroblasts. In Striking inhibition of TGFB-induced Collagen-1 expression analyzed contrast to TGFB-treated cells, BNP had no significant by Western blot (FIG.7b) and real-time RT-PCR (FIG. 7c). effects in unstimulated fibroblasts. This is consistent with the A Similar result was demonstrated for PAI-1 using real-time physiological actions of BNP, working only in opposition to RT-PCR. These findings suggest that the ERK pathway other hormonal Systems Such as the renin-angiotensin-al plays an important role in BNP-dependent inhibition of the dosterone System. fibrotic response induced by TGFB in human cardiac fibro blasts. Changes in Cell Proliferation 0074 The effects of TGFB on cell growth is cell-type Fibrosis and ECM dependent. As provided above, TGFB stimulated cardiac 0070. One of the key features of cardiac fibrosis is the fibroblast proliferation. Whether TGFB has a direct effect on increased deposition of the ECM. The dynamic turnover of cell cycle or an indirect effect through other mechanisms is ECM proteins is controlled by several regulatory mecha unclear. However, cDNA microarray analysis revealed that nisms: de novo biosynthesis of ECM components, pro BNP markedly inhibits the expression of a number of teolytic degradation of ECMs by matrix metalloproteinases TGFB-induced growth factors or growth factor-like genes (MMPs), and inhibition of MMP activities by endogenous including PDGFA, IGF1, FGF18, and IGFBP10 (CYR61). inhibitors, TIMPs. All of these processes have been shown The up-regulation of these genes by TGFB could partially to be profoundly affected by TGFB. The results provided explain the induction of cell proliferation, Suggesting that it herein suggest that TGFB-induced ECM deposition in may be mediated indirectly through the Stimulation of human cardiac fibroblasts occurs largely by increasing ECM growth factor productions. TGFB also induced the expres gene expression, including Fibronectin, COL1A2, COL15A, sion of PTHLH (PTHrP), which has known chronotropic COL7A1, MAGP2, MATN3, FBN1, and COMP. Fibronec and vasodilatory effects. In osteoblast-like cells PTHrP can tin and collagen expression in cardiac fibroblasts has been induce cell proliferation. Interestingly, in the myocardium, well-established in the fibrotic response, however, this is the PTHrP levels are increased in congestive heart failure first report of TGFB induction of other ECM genes including (CHF). MAGP2, MATN3, FBN1 and COMP, further corroborating 0075. The growth inhibitory effects of natriuretic pep TGFB's role in ECM induction. Interestingly, COMP, which tides have previously been reported. Cao and Gardner first is a member of the thrombospondin family, has been shown demonstrated that natriuretic peptides inhibit PDGF, FGF2, to have a direct interaction with Fibronectin, supporting its and mechanical Stretch-induced DNA synthesis in neonatal role in fibrotic processes. We also found Thombospondin 2, rat cardiac fibroblasts. Consistent with these findings, natri which is involved in the activation of latent TGFB' regu uretic peptides and cyclic GMP have been reported to inhibit lated by TGFB in our studies and opposed by BNP (Table 2). cell proliferation induced by angiontensin II, endothelin-1, Also sharing close identity with the latent TGFB family of and norepinephrine in many cell types including cardiac binding proteins is FBN1, a component of extracellular fibroblasts, vascular Smooth muscle cells, endothelial cells, microfibrils. The opposing effects of BNP on these gene and mesangial cells. The results provided herein Suggest an regulatory events, Suggests that BNP modulates cardiac important role for BNP in regulating fibroblast growth fibrosis. during cardiac remodeling. 0071. In addition to the Suppression of TGFB-induced Changes in Inflammatory Genes ECM biosynthesis, BNP may also modulate the degradation of ECM proteins by opposing elevated TIMP3 levels in 0076 Cardiac expression of cytokines is thought to con TGFB-stimulated cells. The TIMP family of proteins is tribute to a decrease in left ventricle contractile performance believed to play Significant roles in controlling extracellular and deleterious remodeling. Although Similar effects have matrix remodeling. Elevation of TIMP3 expression has been been observed with ANP, reported herein for the first time is US 2006/OO19890 A1 Jan. 26, 2006 24 that brain natriuretic peptide blocks TGFB stimulation of (2.68+0.23 vs. 4.74+0.73 ul, p<0.05), ejection fraction Several pro-inflammatory genes including COX2, IL6, (13.6+1.1 vs. 20.4+2.4% p<0.05), and diastolic volume TNFAIP6, and TNFSF4. (19.0+0.9 vs. 22.4+1.1 ul, p<0.05) and stroke work 0077. TGFB has a dual effect in the regulation of inflam (223.0+29.4 vs. 531.5+99.1 mmHul, p<0.05), and decrease matory processes. For example, it increaseS COX2 expres in arterial elastance (6.50+5.7 vs. 42.6+5.1 mmHg/ul, Sion and proStaglandin E2 release in pulmonary artery p<0.01). As shown in FIG. 11, BNP significantly reduced Smooth muscle cells, airway Smooth muscle cells, and the heart/body weigh ratio (0.0039+0.002 vs. 0.0029-0.001, intestinal epithelial cells. On the other hand, TGFB down p<0.05) and as referenced above, abolished the profibrotic regulates the production of MCP-1 and complement com phenotype indicated by decreasing expression of collagen I ponents (C3 and C4) in human proximal tubular epithelial (p<0.01), collagen III (p<0.05) and fibronectin (p<0.05). cells and macrophages. The results provided herein corrobo rates the dual effect of TGFB in the modulation of inflam SUMMARY matory gene expression in cardiac fibroblasts. From these 0082 Along with the endothelin pathway, the renin results, it was found that while TGFB induced some inflam angiotensin and aldosterone System, the fibrosis-promoting matory genes, it down-regulated others, Such as, IL1b, TGFB pathway is important in the pathophysiology of heart MCP1-R, GRO1, GRO3, and MCP4. Both effects are failure. BNP appears to oppose TGFB-regulated gene reversed by BNP. However, in the absence of TGFB stimu expression related to fibrosis and myofibroblast conversion. lation, BNP had no significant effect on the expression of Furthermore, BNP's opposition to the TGFB-stimulated inflammatory genes. It is likely that a balance of pro- and fibrotic response is dependent on the PKG and the MEK/ anti-inflammatory Stimuli is important in the process of ERK pathways. This finding is consistent with the observa cardiac remodeling. tion that BNP deficient mice show increased fibrosis and Signaling Mechanism Underlying BNP's Anti-Fibrotic Role Collagen 1 expression. In addition to BNP's global effects on fibrosis, it may also have effects on other processes, Such 0078 Studies aimed at elucidating the mechanism of as inflammation and proliferation (FIG. 8). These findings BNP's inhibition of a fibrotic response indicate that the ERK support a beneficial role for BNP in the prevention of cardiac Signaling pathway plays an important role. The results fibrosis and the treatment of cardiac diseases. They also provided herein demonstrate that BNP phosphorylates ERK provide the first demonstration that BNP has a direct effect Via PKG-dependent Signaling in primary human cardiac on cardiac fibroblasts to oppose a TGFB-induced fibrotic fibroblasts. Moreover, this activation attenuates the TGFB response, Suggesting that BNP functions as an anti-fibrotic induced fibrotic response as measured by Collagen 1 expres factor in the heart to prevent cardiac remodeling in patho Sion. This is consistent with previous Studies showing that logical conditions. ERK activation is required for both the anti-hypertrophic effect of ANP in cardiac myocytes, and the inhibition of 0083 Independent from the antifibrotic effect, the in vivo TGFB Signaling in mammary and lung epithelial cells. studies as provided herein indicate that BNP may be used to reduce cardiac remodeling and prevent Subsequent heart In Vivo Studies failure. BNP may also be useful as a cardioprotective agent 0079. In a related study, an in vivo model for acute to improve cardiac function post acute myocardial injury myocardial injury was used to explore the effects of BNP. Such as myocardial infarction. Male Sprague Dawley rats ranging in weight from 225 to 250gm were utilized. Acute myocardial injury was induced 0084 All references cited throughout the specification by administration of No-nitro-L-arginine methyl ester are expressly incorporated herein by reference. While the (L-NAME, 40 mg/kg/day)salt (1% NaCl) plus angiotensin II present invention has been described with reference to the (Ang|I, 0.5 mg/kg/day) in the rats. The L-NAME was specific embodiments thereof, it should be understood by administered in drinking water from day 1 to day 14. those skilled in the art that various changes may be made and Angiotensin II was continuously infused Subcutaneously equivalents may be Substituted without departing from the with an osmotic pump from day 11 to day 14. Rat BNP (400 true Spirit and Scope of the invention. In addition, many mg/kg/min) was intravenously infused through an external modifications may be made to adapt a particular situation, infusion pump from day 10 to day 14. material, composition of matter, process, and the like. All Such modifications are within the Scope of the claims 0080 Systolic blood pressure, plasma level of aldoster appended hereto. one, cardiac function heart/body weight ration and gene expression in the heart were analyzed. Systolic blood pres sure was monitored via tail cuff technique with an IITC blood preSSure recording System. Cardiac function was What is claimed is: monitored via a Millar ARIA Pressure Volume Conductance 1. A method for treating cardiac remodeling in a Subject System with an 1.4 F catheter. Gene expression as refer that has undergone myocardial injury, Said method compris enced above with results provided in FIG. 12 were moni ing administering a therapeutically effective amount of tored by RT-PCR with an ABI PrismTM 7700 sequence natriuretic peptide to Said Subject. detection System. 2. A method for treating cardiac dysfunction in a Subject 0081. It was observed that BNP had no effect on systolic that has undergone myocardial injury, Said method compris blood pressure raised by L-NAME+AngI but significantly ing administering a therapeutically effective amount of attenuated aldosterone 1.25.2+0.2 vs. 6.6+0.16 ng/ml, natriuretic peptide to Said Subject. p<0.05). See FIG. 10. As shown in FIG. 13, BNP improved 3. A method for treating cardiac fibrosis in a Subject who cardiac function by Significantly increase in Stroke Volume has undergone myocardial injury, Said method comprising US 2006/OO19890 A1 Jan. 26, 2006 25 administering a therapeutically effective amount of natri Sion of Said genes is altered as a result of TGFB mediated uretic peptide to Said Subject. activation, with brain natriuretic peptide. 4. The method of claims 1 or 2 wherein said natriuretic 7. The method of claim 5 wherein said genes are selected peptide is brain natriuretic peptide. from the group consisting essentially of Collagen1, Collagen 5. A method of inhibiting the production of Collagen 1, Collagen 3 or Fibronectin protein in a subject who has 3, Fibronectin, CTGF, PAI-1, and TIMP3. undergone myocardial injury, Said method comprising 8. A method of inhibiting the transformation of cardiac administering a therapeutically effective amount of brain fibroblast cells into myofibroblast cells in a subject that has natriuretic peptide to Said Subject. undergone myocardial injury, Said method comprising 6. A method of alleviating or reversing the effect of TGFB administering a therapeutically effective amount of brain mediated cell activation in cardiac tissue on the expression natriuretic peptide to Said Subject. of one or more genes associated with fibrosis, comprising contacting one or more cells or tissues in which the expres