Galectin-3 Marks Activated Macrophages in Failure-Prone Hypertrophied Hearts and Contributes to Cardiac Dysfunction
Background— Inflammatory mechanisms have been proposed to be important in heart failure (HF), and cytokines have been implicated to add to the progression of HF. However, it is unclear whether such mechanisms are already activated when hypertrophied hearts still appear well-compensated and whether such early mechanisms contribute to the development of HF.
Methods and Results— In a comprehensive microarray study, galectin-3 emerged as the most robustly overexpressed gene in failing versus functionally compensated hearts from homozygous transgenic TGRmRen2-27 (Ren-2) rats. Myocardial biopsies obtained at an early stage of hypertrophy before apparent HF showed that expression of galectin-3 was increased specifically in the rats that later rapidly developed HF. Galectin-3 colocalized with activated myocardial macrophages. We found galectin-3-binding sites in rat cardiac fibroblasts and the extracellular matrix. Recombinant galectin-3 induced cardiac fibroblast proliferation, collagen production, and cyclin D1 expression. A 4-week continuous infusion of low-dose galectin-3 into the pericardial sac of healthy Sprague-Dawley rats led to left ventricular dysfunction, with a 3-fold differential increase of collagen I over collagen III. Myocardial galectin-3 expression was increased in aortic stenosis patients with depressed ejection fraction.
Conclusions— This study shows that an early increase in galectin-3 expression identifies failure-prone hypertrophied hearts. Galectin-3, a macrophage-derived mediator, induces cardiac fibroblast proliferation, collagen deposition, and ventricular dysfunction. This implies that HF therapy aimed at inflammatory responses may need to be targeted at the early stages of HF and probably needs to antagonize multiple inflammatory mediators, including galectin-3.
Received April 14, 2004; revision received August 18, 2004; accepted August 23, 2004.
Despite state-of-the art treatment, heart failure (HF) is still a progressive disorder characterized by high morbidity and mortality, suggesting that important pathogenic mechanisms remain active and unmodified by current treatment.1 A growing body of evidence links macrophage activation and fibrosis to the pathogenesis of HF.2,3 Accordingly, there has been increasing interest in developing therapeutic agents with anticytokine properties that might be used as adjunctive therapy in patients with HF. In particular, the growing appreciation of elevated levels of the pleiotropic cytokine tumor necrosis factor (TNF)-α in patients with HF culminated in clinical studies on TNF-α inhibition. However, these trials did not support the use of TNF-α antagonism as a treatment modality of HF.4,5 Therefore, the question arises as to whether inflammatory mechanisms merely reflect a general stress response of an organism in severe HF or whether such inflammatory response already starts early in the pathogenesis of HF and comprises a broader range of cardiotoxic mediators.
Our recent microarray study performed in a rat model of hypertensive HF allowed us to evaluate immunological mediators specific to hypertrophied hearts that have progressed to failure.6 Among a number of candidate genes, we have focused on galectin-3 as the most robustly overexpressed mediator in failing hearts. Galectin-3 is a member of a large family of β-galactoside-binding animal lectins. Macrophages show increased galectin-3 expression at phagocytic cups and phagosomes during the process of phagocytosis.7 Galectin-3 interacts with various ligands located at the extracellular matrix, including laminin, collagen, synexin, and integrins.8,9 The route of import for galectin-3 from extracellular milieu to cytoplasm is shown to be mediated by β1-integrin.9 Extracellular galectin-3 mediates cell migration and cell-cell interactions, whereas intracellular galectin-3 regulates cell cycle and apoptosis.10 Galectin-3 overexpression causes changes in the expression levels of cell cycle regulators, including cyclin D1, and the growth-promoting activity of galectin-3 is dependent predominantly on cyclin D1 promotor activity.11
Here, we report that myocardial galectin-3 expression is already increased at an early time point in rats that later rapidly progressed to HF. Furthermore, recombinant galectin-3 infused into the pericardial sac of healthy Sprague-Dawley (SD) rats induced HF and excess collagen deposition. At the molecular level, galectin-3 increased the expression of cyclin D1 in dividing fibroblasts and failing myocardium. This study demonstrates a novel pathogenic role of macrophage activation and galectin-3 production in the deterioration of cardiac architecture and function.
Characteristics of the Ren-2 Rats
We studied 14 male homozygous Ren-2 rats and 9 age-matched SD rats (Max-Delbruck-Zentrum, Berlin, Germany). Of the 14 Ren-2 rats, 6 were euthanized at 12 to 14 weeks on clinical signs of HF, and the remaining 8 Ren-2 rats were monitored and euthanized at 17 weeks when clinical signs of failure had not appeared. At 10 weeks, none of the Ren-2 rats had developed HF, and at this stage, all the Ren-2 rats had comparable left ventricular (LV) hypertrophy, irrespective of the future consequence of either failure or prolonged compensation. We have reported separately the cDNA microarray experiment (total, 12 336 genes; Incyte Genomics; rat GEM2/3) that showed an increased expression of 48 genes and decreased expression of 14 genes in failing hypertrophied as opposed to compensated hypertrophied hearts.6 The present study is based on the further validation and mechanistic elucidation of the inflammatory and fibrotic responses seen specifically in failing hearts.
Myocardial Biopsies From 10-Week-Old Ren-2 Rats
A total of 12 Ren-2 and 4 SD rats were anesthetized with 2% isoflurane, and a blunt 20-gauge needle was placed in the trachea for intubation, which was connected to a volume-cycled rodent respirator (model 683, Harvard Apparatus) 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 microdissecting microscope, a 5-mm incision at the left fourth intercostal space was made to access the thorax. Three biopsy specimens were taken from the lateral LV myocardium of each rat with a custom-made 0.35-mm needle. Nine of 12 Ren-2 rats survived the procedure and were followed up. Five rats developed HF by 12 to 14 weeks, whereas the remaining 4 rats stayed compensated until the end of the observation period of 18 weeks. The Institutional Animal Care Committee approved the procedure for care and treatment of animals.
Recombinant Galectin-3 Production and Infusion Into the Pericardial Space
Murine recombinant galectin-3 expressed from plasmid prCBP35s was purified by affinity chromatography on lactosylated Sepharose 4B, produced by divinyl sulfone activation. The purity of the obtained protein was studied by gel filtration and 1- and 2-D gel electrophoresis to exclude any contamination or protein modification.12
We infused recombinant galectin-3 into the pericardial cavity of 6 SD rats. Similarly, 6 additional SD rats underwent an identical surgical procedure but received normal saline infusion. Three separate SD rats received biotinylated galectin-3 intrapericardially to locate galectin-3-binding sites in the myocardium. To install the pericardial catheter, rats were anesthetized with subcutaneous pentobarbital sodium (50 mg/kg), and a high midline thoracotomy was performed. A small incision in the pericardial sac was made, and a silicone catheter (diameter, 0.51 mm; Degania Bet) was inserted. The catheter was fixed subcutaneously and connected to an osmotic minipump that was set to pump 0.5 μg galectin-3 per hour for a total duration of 4 weeks. The dose of galectin-3 was calculated on the basis of reported bioactivity, adjusting for the local advantage of pericardial delivery.13
Measurement of Cardiac Geometry and Function
Echocardiography was performed at 0, 14, and 28 days of galectin-3 infusion in rats sedated with 2% isoflurane. Standard views were obtained in 2-D as well as M-mode with a 12-MHz transducer (Hewlett Packard) with ≈220 frames per recording, and the data analysis was made by a blinded observer.
Before the animals were euthanized, hemodynamic parameters were measured. After anesthesia by intraperitoneal injection of urethane (1 g/kg), the right carotid artery was cannulated with a 2F high-fidelity micromanometer (Millar Instruments), and the catheter was advanced into the LV. The output signal from the micromanometer was digitized at 4000 Hz with a MacLab system (ADI Instruments).
RNA Isolation and Real-Time PCR
A complete list for the primers and probes of the galectin-3 gene transcripts is presented as the online supplementary material (Data Addendum 1). RNA was isolated from rat LV with the RNeasy Mini Kit following the RNeasy Mini Protocol (Qiagen) and stored at −80°C. RNA was isolated from rat and human heart biopsies with the PicoPure RNA Isolation Kit (Arcturus) according to the manufacturer’s instructions. Optimal polymerase chain reaction (PCR) conditions were found to be 12.5 μL 2 times PCR Master Mix for Taqman assays with final concentration of 5 mmol/L MgCl2, 300 nmol/L of each primer, 200 nmol/L probe, and 10 ng cDNA template in a total volume of 25 μL.
Protein Isolation and Western Blotting
Protein isolation and Western blotting were performed as described previously.14 Primary antibodies (galectin-3, Bioreagents; collagen-I and collagen-III, Abcam; ED-1 and OX-6, a kind gift from Dr M. de Winther, Department of Molecular Genetics, University of Maastricht, the Netherlands; cyclin D1, Cell Signaling Technologies) were diluted 1/1000 in Tris-buffered saline with Tween-20 (TBS-T). Secondary antibody (horseradish peroxidase-conjugated IgG, Cell Signaling Technology) was diluted 1/2000 in TBS-T. Protein bands were visualized by the enhanced chemiluminescence technique.
In Situ Hybridization, Immunohistochemistry, Galectin-3 Cytochemistry, and Confocal Microscopy
To localize galectin-3 mRNA, we used a nonradioactive in situ hybridization assay. A DIG-labeled oligonucleotide probe was used for the hybridization according to the manufacturer’s instructions (GeneDetect). The extent of specific binding was visualized by application of avidin-biotin-peroxidase complex (ABC) kit reagents (Vector Laboratories). The expression of galectin-3 protein and accessible binding sites were visualized by a specific anti-galectin-3 monoclonal antibody and biotinylated galectin-3, as described previously.12 For confocal laser scanning microscopy experiments, galectin-3-binding sites were detected by FITC-labeled avidin. A Texas Red-labeled secondary antibody was used to visualize immunocytochemically the proliferating cell nuclear antigen (PCNA). Further details on the procedure are available elsewhere.15
Cardiac Fibroblast Proliferation and Proline Incorporation Assays
Rat cardiac fibroblasts were isolated from 2-day-old neonatal SD rats, as described previously.14 Cells were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% FBS, along with 1% l-glutamate, 50 U/mL penicillin, and 0.1 g/L streptomycin and were incubated at 37°C in a humidified 5% CO2 atmosphere. Synchronized cells were treated with murine recombinant galectin-3 (control, 10 μg/mL, and 30 μg/mL) for 24 hours. The number of dividing cells was determined by radiolabeled methyl-[3H]thymidine incorporation (0.5 μCi per well) assay. Collagen production by these cells was measured by [3H]proline uptake after a 72-hour treatment of galectin-3 (control, 10 μg/mL, and 30 μg/mL). The assays were performed in triplicate for fibroblast preparations.
We obtained cardiac biopsies from patients undergoing aortic valve replacement for aortic stenosis. For the study of gene expression in these human biopsies, we selected 5 aortic stenosis patients with cardiac hypertrophy and relatively depressed ejection fraction (<55%) and 17 aortic stenosis subjects with LV hypertrophy and normal or elevated ejection fraction. Patients underwent a detailed cardiovascular assessment by echocardiography before operation (Table 1). The myocardial biopsies were collected and snap-frozen in liquid nitrogen. Informed consent was obtained from the patients, and the institutional Medical Ethical Committee approved the study.
Data are presented as mean±SEM. The paired comparisons were made by unpaired t test. For multiple comparisons, 1-way ANOVA in combination with a Dunnett’s post hoc analysis was made. Analyses were performed by use of the statistical package SPSS 10.0. Probability values of P<0.05 were considered to be statistically significant.
Increased Myocardial Collagen Content in Failing Ren-2 Rats
A computer-assisted densitometric analysis of the picrosirius red-stained sections for the quantification of myocardial collagen revealed a higher degree of interstitial collagen content in the failing Ren-2 rats compared with compensated and wild-type rats (interstitial collagen volume fraction percentage: HF, 7.8±0.38; compensated, 3.8±0.54; wild-type, 2.5±0.3; P<0.05 versus compensated and wild-type).
Microarray Data Revealed an Inflammatory Gene Profile in Failing Hearts
The microarray analysis has been described separately, and the differentially expressed genes are available as supplementary material for that article.6 In this study, we focused on galectin-3, which emerged as the most prominently overexpressed gene with a >5-fold rise in HF rat myocardium compared with compensated hypertrophied myocardium. In addition, major histocompatibility complex antigen II (MHC-II), MHC-associated invariant chain peptide, macrophage mannose receptor, and immunoglobulin receptors genes were among the overexpressed genes.
Western Blotting Showed High Galectin-3 Expression in Failing Myocardium
Given the robust transcriptional increase in galectin-3, we measured its protein levels in the myocardium. Comparable to the results obtained in the microarray, the highest expression level of galectin-3 protein was observed in the same group of animals that had the highest degree of cardiac fibrosis and had developed HF by 12 to 14 weeks (densitometric units: HF, 94.66±9.5; compensated, 35±5.6; controls, 27.2±6.2; P<0.05 versus compensated and controls) (Figure 1a).
Localization of Galectin-3 to Activated Macrophages
We evaluated the source and distribution of galectin-3 in the rat myocardium by in situ hybridization and immunohistochemistry. Importantly, galectin-3 positivity was seen in the areas of fibrosis (Figure 2, a1). Galectin-3-positive areas colocalized with macrophage-specific staining (Figure 2, b1). These macrophages strongly expressed MHC-II antigen as well, indicating an active role of these cells in antigen presentation (Figure 2, c1). These characteristics were present only in HF rats and not in compensated rats (Figure 2, a2, b2, and c2) and wild-type SD rats (Figure 2, a3, b3, and c3). The galectin-3 mRNA expression, as shown by in situ hybridization, localized to the cells infiltrated to the areas of myocardial tissue damage (Figure 2, d1). The normal SD rat myocardium, in contrast, lacked the galectin-3-producing cellular infiltrates (Figure 2, d2).
Galectin-3 Binding Sites in Extracellular Matrix and Cardiac Fibroblasts
We infused biotinylated galectin-3 into the pericardial sac to visualize galectin-3-binding sites in vivo in the myocardium. Galectin-3-binding sites localized predominantly to the myocardial matrix and fibroblasts (Figure 2e). In vitro, galectin-3-binding sites were seen as diffuse cytoplasmic staining in resting cells (Figure 3a). However, proliferating fibroblasts showed enhanced staining around the nucleus, revealing a mitosis-related alteration in staining profile (Figure 3b). We performed similar experiments in isolated cardiomyocytes to localize accessible galectin-3-binding sites. In contrast to cardiac fibroblasts, galectin-3-binding sites were absent from cardiomyocytes (Figure 3c). Confocal microscopy confirmed a compact presence of accessible galectin-3 ligands around the nucleus in proliferating (ie, PCNA-positive) cardiac fibroblasts (Figure 3, d, e, and f), reflecting that in mitotic cells, galectin-3-binding sites migrate to perinuclear areas.16
Galectin-3 Induced Fibroblast Proliferation and Collagen Production In Vitro
Exogenous recombinant galectin-3 (10 and 30 μg/mL) significantly increased cardiac fibroblast proliferation as determined by [3H]thymidine incorporation (galectin-3 at 30 μg/mL, 347±17.5 cpm; galectin-3 at 10 μg/mL, 309±4.8 cpm; control, 145±4.8 cpm; P<0.05 versus 10 μg/mL and control) (Figure 4a). We then monitored the collagen production by cardiac fibroblasts with the addition of exogenous galectin-3 using radioactive proline-incorporation assays. With 30 μg/mL of galectin-3 in the medium, proline incorporation increased by ≈66% (galectin-3 at 30 μg/mL, 1066±56 cpm; control, 707±52.8 cpm; P<0.05). A lower concentration of galectin-3 failed to produce significant effects (galectin-3 at 10 μg/mL, 992±72 cpm; P=0.13) (Figure 4b).
Real-Time PCR Showed High Galectin-3 Expression in Failure-Prone Ren-2 Rat and Dysfunctional Human Hearts
To evaluate the expression of galectin-3 in the myocardium at the stage before Ren-2 rats progressed to HF (ie, 10 weeks of age), we obtained cardiac biopsies in vivo in the spontaneously beating rat heart. Measured by real-time PCR, myocardial expression of galectin-3 gene was increased only in the rats that later progressed to HF (relative expression, 5.8±0.11), whereas it was expressed at relatively lower levels in the rats that subsequently remained compensated (3.4±0.21) and in nontransgenic control rat hearts (2.5±0.047) (Figure 5a). In human myocardial biopsies, there was significantly higher myocardial galectin-3 mRNA expression in hypertrophied hearts with relatively impaired ejection fraction compared with the compensated forms of cardiac hypertrophy (relative expression: failure, 7.08±1.17 versus hypertrophy, 4.60±0.51; P<0.05) (Figure 5b). To measure galectin-3 mRNA expression in nonhypertrophied hearts, we obtained myocardial biopsies from the patients without aortic stenosis who were undergoing cardiac surgery for coronary artery bypass grafting (CABG). Compared with the expression levels observed in aortic stenosis, real-time PCR analysis showed that galectin-3 mRNA expression was lowest in nonhypertrophied hearts from patients undergoing CABG (relative expression, normalized to cyclophilin A, 3.4±0.21, n=5).
Intrapericardial Infusion of Galectin-3 Induced LV Dysfunction and Increased Collagen I/III Ratio
To address whether chronically elevated levels of galectin-3, specifically in the heart, can induce HF and to avoid potential systemic effects of galectin-3, we designed a novel approach of intrapericardial infusion of galectin-3 in healthy rats. At baseline, there was no significant difference in cardiac function between the galectin-3- and placebo-infused rats. Rats infused with galectin-3 for 4 weeks into the pericardial space showed depressed LV ejection fraction, fractional shortening, and the amplitude of the negative slope of dP/dtmax and increased lung weight-to-body weight ratio compared with rats receiving placebo infusion (Table 2). Quantification of collagen content from the LV myocardium showed increased collagen volume fraction in galectin-3-infused rats (Figure 6a). Western blotting revealed a marked increase in collagen I in galectin-3-infused myocardium (densitometric units: galectin-3-infused, 149±13; placebo, 44±6, P<0.01). However, no difference was observed in the level of collagen III (galectin-3-infused, 84±14; placebo, 70±2; P=NS) (Figure 6b). The relative abundance of collagen type I was discernible, because there was 3-fold increase in collagen type I/III ratio (Figure 6c).
Galectin-3 Induced Cyclin D1 Expression In Vivo and In Vitro
We analyzed a potential mediator of the proliferative effects of galectin-3 by measuring the inducible expression of cyclin D1, an important early cell cycle regulator. Failing Ren-2 rats had a higher level of myocardial cyclin D1 expression compared with compensated Ren-2 and wild-type SD rats (densitometric units: HF, 38.7±5.5; compensated, 3.7±1.7; SD, 2.3±0.6; P<0.05 HF versus compensated) (Figure 1, b1). In proliferating cardiac fibroblasts, galectin-3 increased cyclin D1 expression (galectin-3-treated, 4.2±0.57; controls, 1.4±0.41; P<0.05) (Figure 1, b2). In vivo, intrapericardial galectin-3 infusion also led to increased myocardial expression of cyclin D1 (galectin-3-infused, 18.6±1.33; placebo, 0.9±0.05; P<0.05) (Figure 1, b3).
A role for inflammatory mediators in HF has often been shown and is thought to be a rather universal response to the complex local and systemic changes in HF. In contrast to that notion, we now document that increased expression of the macrophage-derived mediator galectin-3 is already apparent in the stage of compensated hypertrophy of failure-prone hearts, before they progress to overt failure. We further demonstrate that galectin-3 induces HF in normal rats. These findings suggest that inflammatory and profibrotic mediators could still be viable therapeutic targets in HF. This supports the idea that an early recognition of failure-prone hearts and intervention with broader-spectrum antiinflammatory agents could have an additional benefit over existing treatment strategies.
Galectin-3 Structure, Functions, and Relevance for Cardiac Remodeling
Identified first as an antigen on the surface of peritoneal macrophages, galectin-3 is the only chimera-type member of the galectin family.17 It belongs to a lectin family sharing the jelly-roll-like folding pattern and calcium-independent specificity to β-galactosides as well as proteins.18,19 In addition to its antiapoptotic and growth-promoting actions, galectin-3 plays a critical role in phagocytosis by macrophages when cross-linked by the Fcγ receptor.7 In agreement with the abundant fibrosis observed in failing ren-2- and galectin-3-infused rats, increased galectin-3 expression has also been shown in a rat model of postradiation pulmonary fibrosis.20 Other studies have shown that galectin-3 expressed by liver analogues of macrophages (ie, Kupffer cells) induce the synthesis of excess fibril-forming collagens in liver.21 Earlier studies have demonstrated the relation between galectin-3 expression and the cell cycle. Nuclear galectin-3 expression is associated with cell proliferation, and this effect is mediated through enhanced cyclin D1 promoter activity.11 Cyclin D1 forms a complex with cyclin-dependent kinases and regulates progression of the early to mid G1 phase of the cell cycle.22 This suggests that galectin-3 can induce cardiac fibroblast proliferation via the activation of cyclin D1, thus allowing a macrophage-derived mediator to affect cardiac fibroblasts.
Kinetics of Galectin-3 Binding Sites in Cardiac Fibroblasts
In line with the profibrotic effects of galectin-3 in vivo, we show that galectin-3 binds to intracellular receptors and induces cardiac fibroblast proliferation and increases collagen production in vitro. Although originally discovered as a carbohydrate-binding protein, galectin-3 is known to specifically interact with intracellular targets in addition to glycoconjugates.23 However, 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)24 or a directed cytosolic-to-nuclear transition (centripetal migration) of these receptors needs further exploration.
Significance of Interstitial Fibrosis and Collagen I Production by Galectin-3
Collagens are essential components of the myocardium, maintaining its structural and functional integrity. In the heart, fibrillar types of collagen form a delicate sheath that interconnects bundles of contractile units. Increased collagen deposition may therefore have a major impact on the diastolic and systolic function of the heart. Whereas collagen III forms an elastic network storing kinetic energy as elastic recoil, collagen I represents a stiff fibrillar protein providing tensile strength.25,26 Only the collagen I and not the collagen III promoter is studded with SP-1-binding sites. Therefore, the differential increase of collagen I over collagen III in galectin-3-infused animals could be explained by the possible differences in the molecular makeup of their promoter sites.27
Conclusions and Implications
The present study, by demonstrating macrophage activation and increased galectin-3 production preceding overt HF, expands on previous publications that have described the possible involvement of inflammatory mechanisms in the advanced stages of HF. Our finding that galectin-3 is overexpressed well before the transition to overt HF suggests the novel concept that already in the compensated phase, some hypertrophied hearts recruit macrophages and proinflammatory mechanisms. We show that exogenous galectin-3 given intrapericardially to healthy hearts over a long term, can induce cardiac dysfunction, which makes it likely that this early recruitment and activation of galectin-3-producing macrophages can drive the progression from compensated hypertrophy toward overt HF. Galectin-3 is the only member of the galectin family with an unusually broad activity including protein-carbohydrate and protein-protein interactions in nuclei, cytoplasm, plasma membrane, and extracellular matrix.8,23 Our in vitro data underscore that this macrophage-derived effector molecule specifically binds to cardiac fibroblasts and induces fibroblast proliferation, also reflected by its ability to upregulate cyclin D1. Relevant for fibrosis, it also induces collagen I production. Failure-prone and dysfunctional rat and human heart specimens all share an increased lectin presence. Therefore, an early recognition of failure-prone hearts and intervention with new antiinflammatory and antifibrotic agents might provide additional benefit over existing treatment strategies. These results shape the concept of considering galectin-3 as a new target for therapeutic intervention at an early stage of compensated hypertrophy in failure-prone hearts.
This study was supported by a VIDI grant (016.036.346) from the Netherlands Organization for Scientific Research (NWO) to Dr Pinto, a grant from the Profileringsfonds from the Academic Hospital Maastricht to Dr Maessen, and a grant from the Mizutani Foundation for Glycoscience to Dr Gabius. We acknowledge the technical assistance of Rudy Duisters and Rick van Leeuwen and thank Dr Bianca Schrans for helping us with in situ hybridization experiments.
The online-only Data Supplement, which contains additional information about Methods and additional figures, can be found with this article at http://www.circulationaha.org.
Yndestad A, Ueland T, Oie E, Florholmen G, Halvorsen B, Attramadal H, Simonsen S, Froland SS, Gullestad L, Christensen G, Damas JK, Aukrust P. Elevated levels of activin A in heart failure: potential role in myocardial remodeling. Circulation. 2004; 109: 1379–1385.
Cingolani OH, Yang XP, Cavasin MA, Carretero OA. Increased systolic performance with diastolic dysfunction in adult spontaneously hypertensive rats. Hypertension. 2003; 41: 249–254.
Mann DL, McMurray JJ, Packer M, Swedberg K, Borer JS, Colucci WS, Djian J, Drexler H, Feldman A, Kober L, Krum H, Liu P, Nieminen M, Tavazzi L, Van Veldhuisen DJ, Waldenstrom A, Warren M, Westheim A, Zannad F, Fleming T. Targeted anticytokine therapy in patients with chronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL). Circulation. 2004; 109: 1594–1602.
Chung ES, Packer M, Lo KH, Fasanmade AA, Willerson JT. Randomized, double-blind, placebo-controlled, pilot trial of infliximab, a chimeric monoclonal antibody to tumor necrosis factor-α, in patients with moderate-to-severe heart failure: results of the anti-TNF Therapy Against Congestive Heart Failure (ATTACH) trial. Circulation. 2003; 107: 3133–3140.
Schroen B, Heymans S, Sharma U, Blankesteijn M, Pokharel S, Cleutjens JP, Porter JG, Evelo CT, Duisters R, Leeuwen RE, Janssen BJ, Debets JJ, Smits JF, Daemen MJ, Crijns HJ, Bornstein P, Pinto YM. Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone forms of cardiac hypertrophy. Circ Res. 2004; 95: 515–522.
Kim HR, Lin HM, Biliran H, Raz A. Cell cycle arrest and inhibition of anoikis by galectin-3 in human breast epithelial cells. Cancer Res. 1999; 59: 4148–4154.
Hermans JJ, van Essen H, Struijker-Boudier HA, Johnson RM, Theeuwes F, Smits JF. Pharmacokinetic advantage of intrapericardially applied substances in the rat. J Pharmacol Exp Ther. 2002; 301: 672–678.
Pokharel S, Rasoul S, Roks AJ, van Leeuwen RE, van Luyn MJ, Deelman LE, Smits JF, Carretero O, van Gilst WH, Pinto YM. N-Acetyl-Ser-Asp-Lys-Pro inhibits phosphorylation of Smad2 in cardiac fibroblasts. Hypertension. 2002; 40: 155–161.
Broers JL, Machiels BM, van Eys GJ, Kuijpers HJ, Manders EM, van Driel R, Ramaekers FC. Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins. J Cell Sci. 1999; 112 (pt 20): 3463–3475.
Bravo R, Macdonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol. 1987; 105: 1549–1554.
Cherayil BJ, Chaitovitz S, Wong C, Pillai S. Molecular cloning of a human macrophage lectin specific for galactose. Proc Natl Acad Sci U S A. 1990; 87: 7324–7328.
Hughes RC. Mac-2: a versatile galactose-binding protein of mammalian tissues. Glycobiology. 1994; 4: 5–12.
Maeda N, Kawada N, Seki S, Arakawa T, Ikeda K, Iwao H, Okuyama H, Hirabayashi J, Kasai K, Yoshizato K. Stimulation of proliferation of rat hepatic stellate cells by galectin-1 and galectin-3 through different intracellular signaling pathways. J Biol Chem. 2003; 278: 18938–18944.
Pauschinger M, Knopf D, Petschauer S, Doerner A, Poller W, Schwimmbeck PL, Kuhl U, Schultheiss HP. Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio. Circulation. 1999; 99: 2750–2756.
Briggs MR, Kadonaga JT, Bell SP, Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986; 234: 47–52.