Classifications

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  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
  • G01N33/9453—Cardioregulators, e.g. antihypotensives, antiarrhythmics
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4703—Regulators; Modulating activity
  • G01N2333/4704—Inhibitors; Supressors
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4724—Lectins
• • G—PHYSICS
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  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4728—Details alpha-Glycoproteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/321—Arterial hypertension
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/50—Determining the risk of developing a disease
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function

Definitions

• the present invention relates to a method for identifying a subject at risk of developing hypertensive end organ damage, such as congestive heart failure.
• Congestive heart failure HF
• HF congestive heart failure
• a large number of patients die within one to five years after diagnosis.
• an important number of patients progress to develop life threatening complications, other may remain stable for prolonged periods.
• early identification of patients at risk for developing hypertensive end organ damage, such as heart failure may prevent rapid progression, it would be preferable to be able to identify those patients in which heart failure is likely to occur before it actually does so.
• the object of the present invention is to provide a method by which patients can be identified who are at particular risk of developing hypertensive end organ damage, such as heart failure, or who are at particular risk to develop complications of heart failure. After identification, these patients may for example be treated before heart failure or its complications occur, which would be of great clinical importance.
• a method for identifying a subject at risk of developing hypertensive end organ damage comprising the steps of: (a) obtaining a biological sample of said subject ,- (b) determining the level of at least one non-myocytical marker in said sample ,- (c) comparing the level of said marker to a standard level; and (d) determining whether the level of the marker is indicative of a risk for developing hypertensive end organ damage .
• specific markers were identified that can be used to predict which hypertrophied hearts are prone to failure. It is generally known that hypertension causes cardiac hypertrophy, which is one of the most important risk factors for heart failure. However, not all hypertrophied hearts will ultimately fail.
• the present invention is based on the finding that particular non-myocytical genes are abnormally expressed in diseased heart tissue (Example 1 and 2) .
• use is made of non-myocytical markers. That is, markers that are derived from cells other than cardiac myocyte .
• This has the advantage that the method of the invention "probes" other processes than the known myoctic changes that occur in stressed myocytes. This opens the opportunity to not only diagnose heart failure, but also to continuously monitor patients with known heart failure, i.e. monitoring whether adverese non- yocytic processes (e.g. inflammation, scarring etc.) occur that may herald major adverse events.
• adverese non- yocytic processes e.g. inflammation, scarring etc.
• a biological sample is taken from an individual patient. Subsequently, the level of one or more markers in said sample is measured by well-known techniques. Typically, the level is compared with a standard level to determine whether the level of the marker is indicative of the potential of the individual to progress to heart failure. The standard level is based on the level of said marker in healthy subjects. If the level of the marker is elevated compared to the standard level, the subject is at risk for developing CHF or developing complications of heart failure.
• the biological sample may be any sample of body fluid, such as blood, plasma, serum, urine etc., or tissue sample such as a cardiac biopsy. According to a preferred embodiment of the invention, however, the biological sample is a plasma sample derived from peripheral blood.
• the marker is a protein.
• the level of proteins can easily be determined by simple and reliable methods, such as immunological methods using specific antibodies against the proteins.
• the protein is galectin-3, as the level of galectin-3 has been demonstrated to be early and specifically expressed in failure-prone hearts.
• the protein is thrombospondin-2. It has been demonstrated that increased cardiac expression of TSP2 identifies those hypertrophied hearts that are prone to progress to overt heart failure.
• the level of the markers may be determined by a any well-known suitable method.
• the level of the marker is measured by an enzyme-linked immunosorbent assay (ELISA) , thus providing a simple, reproducible and reliable method.
• ELISA enzyme-linked immunosorbent assay
• the present invention further relates to the use of one or more non-myocytical markers for identifying a subject at risk of developing hypertensive end organ damage, such as congestive heart failure.
• Several non-myocytical markers may be used according to the invention.
• the marker is galectin-3, and/or thrombospondin-2.
• the markers identified according to the present invention may further be used in the prevention and/or treatment of hypertensive end organ damage, in particular for the prevention and/or treatment of congestive heart failure.
• the present invention therefore further relates to the use of galectin-3 and/or modulators thereof for the manufacture of a medicament for the prevention and/or treatment of hypertensive end organ damage.
• the invention further relates to the use of thrombospondin-2 and/or modulators thereof for the manufacture of a medicament for the prevention and/or treatment of hypertensive end organ damage .
• Figure 1 is a flow-chart showing the steps for the implementation of previously reported statistical protocols and the comprehensive cutoff points for data mining.
• FIG. 1 shows the results of real time PCR to quantify the expression of mRNA transcripts of four selected genes in myocardial biopsies taken from 10-week old rats, (a) TSP2 was significantly overexpressed in those rats that later progressed to rapid cardiac decompensation compared to those that remained compensated for the study period of 17 weeks, (Jj Osteoactivin expression, ( c) Collagen VI expression, ( d) Expression level of brain natriuretic peptide. The data were normalized to the house keeping gene, cyclophilin.
• Figure 4 is a bar diagram showing the results of the densito etric analysis of myocardial collagen content on day 0 and 48 hours post MI (10 random fields per section) .
• TSP2- null mice failed to mount a reactive fibrosis 48 hours after MI compared to wild-type mice.
• * p ⁇ 0.01, wild-type vs null strains 48 hours post MI; #, p ⁇ 0.01, day 0 vs 48 hours post MI in wild-type mice.
• Figure 5 shows photo- and electron micrographs of the infarcted left ventricular wall. Hae atoxyline/Eosin stained section showing intact matrix around the blood vessel with no evidence of interstitial haemorrhage in wild type mice (a) . Extensive tissue destruction and interstitial bleeding (*) in TSP ⁇ _ mice (Jb) . Electron micrographs from the infarcted left ventricular wall (wild-type strain) (c) .
• FIG. 6 shows the haemodynamic parameters of HF-S, HF-R and ARB treated rats.
• Both HF-S and HF-R animals had left ventricular hypertrophy.
• High fibrosis-score animals had higher LW/B and LVEDP.
• the parameters were measured before the sacrifice.
• N 4 each for HF-S and HF-R and 8 for ARB. *, P ⁇ 0.05 in HF-S vs HF-R and ARB.
• Figure 7 shows the results of left ventricular collagen volume fraction analysis of picrosirius red stained sections of rat myocardium. The bar diagram shows the quantifica ion of LV interstitial collagen. 1, control; 2, HF-R; 3, HF-S; 4, ARB.
• N 4 -6 each group; #, P ⁇ 0.01 vs control; *, P ⁇ 0.05 HF-S vs HF-R; **, P ⁇ 0.05 in HF-R vs SD.
• Figure 8 shows a dot blot of differentially expressed genes in Ren-2 rats. Galectin-3 mRNA level was compared among HF-S, HF-R and ARB treated group of rats. Density and diameter of the dots corresponds directly to the level of gene expression compared to SD controls.
• A Phsopho-imager scanned images from HF-S, HF-R and ARB treated rats respectively. The circled dots represent galectin-3 mRNA expression.
• Thrombospondin-2 increased expression identifies failure- prone cardiac hypertrophy Cardiac hypertrophy increases the risk of heart failure (HF) , but, so far, it has been is difficult to predict which hypertrophied myocardium will progress rapidly to HF. According to the present invention it was reasoned that, apart from hypertrophy-related genes, distinct failure- related genes are expressed before failure is apparent, thus permitting molecular prediction of hypertrophied hearts liable to ail. Cardiac gene expression (12,336 clones) of hypertensive homozygous renin-overexpressing (Ren-2) rats that progressed to HF at 12-14 weeks of age, were compared with expression by littermates that remained compensated for 17 weeks.
• Thrombospondin-2 (TSP2) was selectively overexpressed only in biopsies from rats that later progressed to HF, while brain natriuretic peptide (BNP) was, at this early stage, elevated in all rats.
• TSP2 myocardial infarction
• Ren-2 rats were obtained from the Max- Delbr ⁇ ck-Zentrum fur Molekulare Medizin, Berlin, Germany. 30 male Ren-2 rats on a Sprague Dawley (SD) background and 9 age-matched SD rats as controls were studied. Of 30 Ren-2 rats, 8 were sacrificed at 10-weeks of age and 8 were treated with 0.05 mg/kg/day of candesartan, an angiotensin II receptor type I blocker (ARB) , from 7-13 weeks of age. Of the remaining 14 untreated Ren-2 rats, 6 were sacrificed at 13 weeks upon the development of clinical signs of heart failure and designated as HF-S rats.
• SD Sprague Dawley
• ARB an angiotensin II receptor type I blocker
• Ren-2 rats were closely monitored and were sacrificed at 17 weeks when clinical signs of failure had not yet appeared. These rats were designated as HF-R rats. Hemodynamic parameters were determined before sacrifice and heart, lung and body weight were measured after the sacrifice. The procedure for care and treatment of animals was approved by the institutional animal care committee.
• Biopsies from 10 -week Ren-2 rats A second group of 12 Ren-2 and 4 SD rats were anesthetized and the anterior thorax was shaved at the sternum. The rats were fixed to a hard board on top of a warming pad with the help of self-made loops. A blunt 20- gauge needle was placed in the trachea to serve as a tracheal cannula. The cannula was connected to a volume-cycled rodent respirator (model 683, Harvard Apparatus, South Natick, MA) on room air with a tidal volume of 2.5 to 3 ml and respiratory rate of 80 breaths/min. Further procedures were done with visual help of a micro-dissecting microscope.
• a 5 mm incision at the left 4 th intercostal space was made to access the thorax. After having a clear view of the heart, a biopsy was taken using a custom-made 0.35 mm needle connected to a slowly rotating drill. The whole procedure lasted approximately 15 minutes. Of the 9 Ren-2 rats that survived the operation, 5 developed heart failure between 12-14 weeks of age whereas the remaining four rats stayed compensated until 17 weeks.
• RNA isolation and reverse transcription RNA was isolated from left ventricles with an RNeasy Mini Kit, following the RNeasy Mini Protocol (QIAGEN, Hilden, Germany) , and stored at -80°C. The quality of the extract was measured using the Eukaryote Total RNA nano-assay in a 2100 Bioanalyser (Agilent Technologies, Amstelveen, The Netherlands) .
• RNA was isolated from 10-week rat heart biopsies with the PicoPure RNA Isolation Kit (Arcturus, CA, USA ), according to manufacturer's instructions. The RNA was transcribed into cDNA with reverse transcriptase, using 250 ng of random primers (Invitrogen Life Technologies, Breda, The Netherlands) .
• cDNA microarrays cDNA clones isolated from a normalized rat cDNA library were chosen for analysis on microarrays using an Incyte GEM-2/GEM-3 rat cDNA library (total 12,336 genes). PCR-amplified inserts of each cDNA were printed as high- density arrays on treated glass surfaces. Duplicate hybridizations were performed on these array elements with two SD and six Ren-2 rat myocardial mRNAs at 3 different time points. Log transformation of the values was done in order to homogenize the data, and only differences in expression of >1.7 fold were considered differentially expressed. The protocol for data mining and validation was adopted, as detailed previously (Tan et al . , Proc Natl Acad Sci. 99:
• the dot blots were scanned with a personal fx-phospho imager (Cyclone System Packard, Meriden, CO, USA) . Individual hybridisation signals were identified and quantified densitometrically using Quantity One, Version 4.2.3 software (BioRad, Kunststoff, Germany) . Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) was selected as a housekeeping gene for internal normalization of the blots. Bioirifor atic analysis Bioinformatic analysis of the protein sequences translated from 49 HF-specific candidate genes, selected from microarray analysis and multi-step data-mining strategy, was performed. Based on the annotations of their biological functions, three candidate genes, previously not identified in myocardium, and that encode matrix-related proteins, were chosen for further testing by real time PCR.
• Glyceraldehyde-3 -phosphate dehydrogenase Glyceraldehyde-3 -phosphate dehydrogenase
• Primers, probes and real-time PCR Primers and probes were designed from rat sequences available in GenBankTM using Primer Express Software (PE Applied Biosystems, Foster City, CA, USA). Probes were designed from conserved exon splice sites derived from the Ensembl -Mouse Genome Sequencing Consortium and Ensembl-Human Genome Browser, thus preventing recognition by the assay of any potentially contaminating genomic DNA (Table 1) .
• Optimal PCR conditions were found to be 12.5 ml 2x PCR Master Mix for TaqraanTM assays, with a final concentration of 5 mM MgCl 2 , 300 nM of each primer, 200 nM probe, and 10 ng cDNA-template in a total volume of 25 ml.
• Amplification and detection were carried out using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA). The PCR data were reported relative to the expression level of the housekeeping gene, cyclophilin A.
• mice Myocardial infarction was induced in 22 wild-type (129 SvJ strain) and 16 TSP2- null mutant (TSP2 "/_ ) mice by occluding the left anterior descending coronary artery. Two sham-operated mice were used as controls. These mice were killed, after ether anesthesia, by injecting 1 ml 0.1 M CdCl 2 into the vena cava. The heart was perfusion-fixed with 5% buffered formalin for 10 minutes and immersion-fixed overnight in 10% buffered formalin.
• Tissue specimens of wild- type and TSP2 _ " mice were evaluated using standard electron microscopical techniques -To quantify the extent of fibrosis, computerized planimetry was performed in seven randomly selected fields per section. Each field represented a 400 ⁇ m 2 area. Collagen area was quantified selectively from left ventricular interstitium excluding perivascular and epicardial collagen. Collagen area fraction was calculated as the ratio of area stained by picrosirius-red to total myocardial area per field. The details of the procedure have been reported previously (Cherayil et al . , Proc Natl Acad Sci USA, 87: 7324-7328, 1990; Cleutjens et al . , Am J pathol., 147: 325-338, 1995) .
• Angiotensin II blockade completely prevented the development of cardiac hypertrophy and failure (LV weight/body weight %, 2.52+0.36, dP/dt ⁇ x, 8400 ⁇ 202) when evaluated in sacrificed animals at 13 weeks.
• Microarray revealed 49 genes overexpressed in heart failure susceptible rats.
• Nicotinamide adenine dinucleotide (NAD) trans - hydrogenase was the only gene with reduced expression in failing myocardium. Notably, expression of osteoactivin, TSP2, several pro-collagens and thrombospondin-1 were increased. Many of the identified genes encode proteins with known functions whereas others correspond to genes of unknown function, including novel genes and genes not previously detected in the heart.
• NAD Nicotinamide adenine dinucleotide
• Bioinformatic analysis pointed to three novel cardiac atrix- related, genes Since no information was available as to the function of many of the overexpressed genes in HF, we subjected all the 49 genes to bioinformatic analysis. Initially, we made a broad functional classification of the HF susceptibility genes using GeneFIND (Gene Family Identification Network Design) System (http : //www-nbrf . creorcretown . edu) , which combines several search/alignment tools to provide rapid and accurate gene family. This strategy indicated that most of the overexpressed genes encode matrix-related proteins. Notably, the functions of 3 selected susceptibility genes (osteoactivin, thrombospondin-2 and collagen VI) were not previously reported in the myocardium.
• GeneFIND Gene Family Identification Network Design
• Macroarray showed normalization of HF susceptibility genes by angiotensin II blockade
• RAS renin-angiotensin system
• TSP-2 knock out mice cannot survive acute myocardial infarction
• TSP2 knock out mice In contrast to various rat models of heart failure, there are no carefully documented mouse models that consistently develop heart failure in response to pressure overload. Therefore, we infarcted the anterior myocardium in 22 wild-type and 16 TSP2 "/_ mice to address the biological role of TSP2 in acute myocardial structural damage and consequently, rapid cardiac remodelling. Infarction was not tolerated by TSP2-null mice, since all mice died from cardiac rupture within the first 72 hours after MI . On the other hand, 100% of the wild-type mice that did not succumb to immediate post -operative complications survived (Figure 3) .
• TSP2 is a secreted matricellular glycoprotein whose functions are diverse and incompletely understood. Since no close ortriologues of TSP2 were found in the genomes of Caenorhabc3.itis elegans or Drosophila, it appears that this protein has evolved to cope with the increased complexity of cell-matrix interaction in vertebrates. As evidence for a role of TSP2 in the organization of the extracellular matrix, previous studies in TSP2-null mice have shown that loss of TSP2 expression results in abnormally large collagen fibrils with irregular contours. Furthermore, the skin of TSP2- null mice is fragile and has reduced tensile strength.
• TSP2-null skin fibroblasts are defective in their attachment to a substratum and have increased levels of matrix metalloproteinase-2 (MMP-2) in their culture.
• MMP-2 matrix metalloproteinase-2
• the current study has identified two, apparently contradictory, functions for TSP2 in the myocardium. In chronic hypertension in Ren-2 rats, increased cardiac expression of TSP2 identifies those animals that are prone to heart failure. While this response would appear to indicate that expression of TSP2 is detrimental, it is likely that the response reflects a heightened, previously activated , injury response in rats that later progress to overt failure, in comparison with the response in rats that remained compensated for a prolonged period of time. It is well established that the expression of TSP2 is characteristic of the response to injury in adult animals.
• ⁇ l integrin was among the genes whose expression was clearly increased in the hearts of hypertensive Ren2 rats and was further increased in failing hearts. This finding was substantiated by our recent observation that the stretching of cardiac fibroblasts in vitro increased protein levels of ⁇ l integrin (S. Pokharel and. Y.M. Pinto, unpublished data). It sriould be noted that the picrosirius red staining technique for quantification of collagen relies on the size, alignment, and packing of collagen fibres to show visible polarization of orange-red colour.
• TSP2 functions as a crucial regulator of the integrity of the cardiac matrix. Since increased extracellular matrix formation characterizes both experimental and clinical forms of pressure overload-induced heart failure, the early expression of TSP2 may reflect a matrix response that is crucial in the transition from compensated hypertrophy to heart failure.
• Ren-2 rats were obtained from the Max- Delbr ⁇ ck-Zentrum fur Molekulare Medizin, Berlin, Germany.
• SD Sprague Dawley rats.
• 8 were treated with 0.05 mg/kg/day of candesartan, an angiotensin II receptor type I blocker (ARB), from 7-13 weeks of age.
• ARB angiotensin II receptor type I blocker
• Within 8 untreated Ren-2 rats 4 were sacrificed at 13 weeks upon the development of HF. The remaining 4 Ren-2 rats were monitored and were sacrificed at 17 weeks when signs of clinical failure had not appeared.
• Hemodynamics was taken at 10 weeks and before sacrifice. Heart, lung and body weight were measured after the sacrifice. The procedure for care and treatment of animals was approved by the institutional animal care committee.
• Myocardial biopsies from 10-week Ren-2 rats A second group of 12 Ren-2 and 4 SD rats were anesthetized and a blunt 20-gauge needle was placed in the trachea to serve as a tracheal cannula, which was connected to a volume-cycled rodent respirator (model 683, Harvard Apparatus, South Natick, MA) on room air with a tidal volume of 2.5 to 3 ml and respiratory rate of 80 breaths/min. With the visual help of a micro-dissecting microscope, a 5 mm incision at the left 4 th intercostal space was made to access the thorax. Biopsy was taken using a custom-made 0.35 mm needle.
• cDNA microarrays cDNA clones isolated from a normalized rat cDNA library (total 12,336 genes) were chosen for analysis on microarrays (Incyte Genomics, CA, USA, rat GEM-2/3). PCR amplified inserts of each cDNA were printed as high-density array on glass surfaces. Duplicate hybridizations were performed on these glass chips with two SD and six Ren-2 rat myocardial mRNA at three different time points. The target genes that showed statistically significant (P ⁇ 0.001) changes in expression with at least 2-fold overexpression in HF-S group were reprinted onto a sub-array for further analysis so that the genes were independently assessed four times to improve the level of reliability.
• TGCCCTACGATATGCCCTTGCCTG-3' specific to galectin-3 were designed from sequences available in GenBankTM using Primer Express Software (PE Applied Biosystems, Foster City, CA, USA) .
• RNA isolation, and real time PCR RNA was isolated from rat left ventricle with the RNeasy Mini Kit following the RNeasy Mini Protocol (QIAGEN, Hilden, Germany) and stored at -80°C.
• RNA was isolated from rat heart biopsies with the PicoPure RNA Isolation Kit (Arcturus, CA, USA) according to manufacturer's instructions.
• Optimal PCR conditions were found to be 12.5 ⁇ m 2x PCR Master Mix for TaqmanTM assays with final concentration of 5 mM MgCl 2 , 300 nM of each primer, 200 nM probe and 10 ng cDNA- template in a total volume of 25 ⁇ l .
• ACGACGGCCAGTGAATTGAA-5' primers Each clone was then spotted in duplicates on nylon membrane (macroarray) . The dot blots were scanned with the personal fx-phospho imager (Cyclone System Packard, Meriden, CO, USA) .
• Protein isolation and Western blotting was performed as described previously9.
• Primary antibodies (Galectin-3, Bioreagents; ED-1 and OX-6, a kind gift from Dr. M. de Winther, Department of Molecular Genetics, University of Maastricht, The Netherlands) were diluted 1/1000 in tris- buffer saline with tween-20 (TBS-T) .
• Secondary antibody (horsera ish-peroxidase conjugated IgG, Cell Signaling Technology) was diluted 1/2000 in TBS-T. Protein bands were visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL, USA) according to manufacturer's instructions.
• galectin-3 and accessible binding sites were visualised by a specific anti-galectin-3 monoclonal antibody and biotinylated galectin-3, as described previously (Gabius et al . , Anal Biochem. : 189: 91-94, 1990). As detailed elsewhere (Andre et al . , Chembiochem. 2: 822-830, 2001) galectin-3 was biotinylated under activity- preserving conditions. In confocal laser scanning microscopy, galectin binding sites were detected by FITC-labelled avidin.
• PCNA proliferating nuclear antigen
• Rat cardiac fibroblast proliferation and proline incorporation assays Rat cardiac fibroblasts were isolated from 2-day-old neonatal Sprague-Dawley rats, as described previously (Pokharel et al . , Hypertension, 40: 155-161, 2002). Cells were cultured in Dulbecco's modification of eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS) , along with 1% L-glutamat , 50 U/mL penicillin, and 0.1 g/L streptomycin, and were incubated at 37'C in a humidified 5% C0 2 atmosphere.
• DMEM Dulbecco's modification of eagle's medium
• FBS foetal bovine serum
• Microarray reveals abundance of immune-related genes in HF susceptible rats Firstly, we examined the biological variability in gene expression between HF-S and HF-R groups. The expression levels of most genes between pairs of samples from both groups were highly correlated. We focused on the differentially expressed genes between the failing and non- failing hypertrophied hearts. Log transformation of the values was done and only statistically significant (P ⁇ 0.05) differences in expression levels exceeding the 2-fold threshold were considered to be differentially expressed. Galectin-3 emerged as the most prominently overexpressed gene with more than 5-fold rise in HF rats (Table 3). Of interest, major histocompatibity complex antigen II (MHC-II) and immunoglobulin receptors genes were among these overexpressed genes .
• MHC-II major histocompatibity complex antigen II
• immunoglobulin receptors genes were among these overexpressed genes .
• Macroarray reveals normalization of HF susceptibility genes by angiotensin II blockade
• angiotensin II blockade To validate the differentially expressed genes in HF, we first confirmed the identity of the clones by sequencing and consequently re-spotted these genes onto nylon membrane (macroarray) for repeat hybridization in separate biological samples. This also yielded an overexpression of seven major index genes initially identified by microarray.
• RAS renin- angiotensin system
• Angiotensin I I blockade completely prevented the development of cardiac hypertrophy and failure . On the level of gene expression, it prevented the overexpression of all HF-related candidate genes . Notably, galectin- 3 gene expression was also prevented.
• Galectin-3 binding sites in cardiac fibroblasts Having defined strong expression of galectin-3 in macrophages, we determined whether galectin-3 binds to cardiac fibroblasts.
• In 0.1% Triton-permeabilised cells presence of galectin-3 binding sites resulted in diffuse cytoplasmic as well as perinuclear staining in resting cells ( Figure 10 a) .
• proliferating fibroblasts showed enhanced staining around the nucleus, revealing a mitosis-related alteration in staining profile ( Figure 10 b) . This pattern was independently monitored by confocal microscopy.
• Galectin-3 induced fibroblast proliferation and collagen production Having thus provided evidence for presence of accessible sites in the cardiac fibroblasts, we determined whether galectin-3 stimulates the growth of cardiac fibroblasts. Using recombinant galectin-3, we performed proliferation assays. Galectin-3 was added in different concentrations (0, 10 and 30 ⁇ g/ml) with and without serum enrichment. We observed significant increase in cardiac fibroblast proliferation with 10 and 30 ⁇ g/ml exogenous galectin-3 over 24 hours (galectin-3 at 30 ⁇ g/ml, 347+17.5 counts per minute (cp ) ; galectin-3 at 10 ⁇ g/ml 309+4.8 cpm; control, 145+4.8; p ⁇ O-01).
• galectin-3 is the only chimera- type member of the galectin family. It has a lectin group sharing calcium- independent specificity to ⁇ -galactosides as well as proteins and is located in the phagocytic cups and phagosomes of the macrophages . Besides its anti-apoptotic and growth promoting actions , galectin-3 also regulates monocyte chemotaxis , chemokinesis and modulates the availability of cytokines . Furthermore, recent studies have al so suggested that galectin-3 plays a critical role in phagocytosis by macrophages when cross-linked by Fc ⁇ receptor (FcyR) .
• FcyR Fc ⁇ receptor
• galectin-3 is a macrophage related pro-fibrotic mediator and yet another inflammatory infiltrate cytokine with the potential to influence cardiac remodeling in conditions charaterized by macrophage infiltration.
• An alternative hypothesis on how galectin-3 could add to the progression towards HF emerges from the discovery of galectin-3 as the third receptor for advanced glycosylation end-products (RAGE-3) , that have critical role in collagen cross-linking and myocardial stiffness.
• RAGE-3 advanced glycosylation end-products
• galectin-3 binds to intracellular receptors and induces cardiac fibroblast proliferation and accentuates collagen production.
• galectin-3 is known to specifically interact with intracellular targets besides glycoconjugates .
• galectin-3 binding sites including, Mac-2 binding protein, and laminin.
• Mac-2 binding protein and laminin.
• galectin-3 binding sites it is still not known what induces the rapid perinuclear migration of galectin-3 binding elements in proliferating cells. Whether it is an export of galectin-3 binding sites from the dividing nucleus [ centrifugal migration) or it is a directed cytosolic to nuclear transition (centripetal migration) of these receptors, needs further exploration.
• the current study suggests a key role for immune system activation and galectin-3 production in the progression from left ventricular hypertrophy to HF and demonstrates a link between pro-immune and pro-fibrotic factors.
• galectin-3 preceding HF can reflect the early and aberrant activation of macrophages in hypertrophied failing ventricles.
• Galectin-3 in turn, can relay signals from activated macrophages to cardiac fibroblasts.
• Peripheral detection of galectin-3 can serve as a predictor of HF and therapeutic inhibition of galectin-3 action can become a novel therapeutic target to counteract excess cardiac fibrosis.
• Galectin-3 levels were measured in the serum of patients with cardiovascular disease.
• a commercially available kit to measure galectin-3 by ELISA was employed. The results are summarized in Table 4-6. It was shown that Galectin-3 is significantly elevated in the serum of patients with cardiovascular disease such as heart failure, LVH. Moreover, an upper limit for galectin-3 levels in healthy control subjects was found, which is surpassed in most CHF patients . According to the present invention it has thus for the first time been demonstrated that measurement of galectin-3 in the serum of human subjects reliably distinguishes diseased from non-diseased subjects, and thus provides additional information on non-myocytic disease processes, in conjunction with known myocytic markers (BNP) .
• BNP myocytic markers
• CHF heart failure
• Infl inflammatory vascular disease
• the probes were labelled at the 5' and 3' positions with 6- carboxyfluorescein reporter and 6-carboxytetramethylrhodamine quencher, respectively.
• the position of the primers and probes were annotated according to the sequences derived from GenBank (accession numbers given in parenthesis) . Fwd, forward; Rev, reverse.
• the mean difference is significant at the .05 level.

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