Title: A Thrombospondin-1 Antagonist of Transforming Growth Factor-β Activation Blocks Cardiomyopathy in Rats with Diabetes and Elevated Angiotensin II
Abstract: In diabetes and hypertension, the induction of increased transforming growth factor-β (TGF-β) activity due to glucose and angiotensin II is a significant factor in the development of fibrosis and organ failure. We showed previously that glucose and angiotensin II induce the latent TGF-β activator thrombospondin-1 (TSP1). Because activation of latent TGF-β is a major means of regulating TGF-β, we addressed the role of TSP1-mediated TGF-β activation in the development of diabetic cardiomyopathy exacerbated by abdominal aortic coarctation in a rat model of type 1 diabetes using a peptide antagonist of TSP1-dependent TGF-β activation. This surgical manipulation elevates initial blood pressure and angiotensin II. The hearts of these rats had increased TSP1, collagen, and TGF-β activity, and cardiac function was diminished. A peptide antagonist of TSP1-dependent TGF-β activation prevented progression of cardiac fibrosis and improved cardiac function by reducing TGF-β activity. These data suggest that TSP1 is a significant mediator of fibrotic complications of diabetes associated with stimulation of the renin-angiotensin system, and further studies to assess the blockade of TSP1-dependent TGF-β activation as a potential antifibrotic therapeutic strategy are warranted. In diabetes and hypertension, the induction of increased transforming growth factor-β (TGF-β) activity due to glucose and angiotensin II is a significant factor in the development of fibrosis and organ failure. We showed previously that glucose and angiotensin II induce the latent TGF-β activator thrombospondin-1 (TSP1). Because activation of latent TGF-β is a major means of regulating TGF-β, we addressed the role of TSP1-mediated TGF-β activation in the development of diabetic cardiomyopathy exacerbated by abdominal aortic coarctation in a rat model of type 1 diabetes using a peptide antagonist of TSP1-dependent TGF-β activation. This surgical manipulation elevates initial blood pressure and angiotensin II. The hearts of these rats had increased TSP1, collagen, and TGF-β activity, and cardiac function was diminished. A peptide antagonist of TSP1-dependent TGF-β activation prevented progression of cardiac fibrosis and improved cardiac function by reducing TGF-β activity. These data suggest that TSP1 is a significant mediator of fibrotic complications of diabetes associated with stimulation of the renin-angiotensin system, and further studies to assess the blockade of TSP1-dependent TGF-β activation as a potential antifibrotic therapeutic strategy are warranted. Diabetic cardiomyopathy is a major cause of congestive heart failure in diabetics and can occur independently of atherosclerosis. 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286: F278-F287Google Scholar In these current studies, we tested the hypothesis that TSP1, through control of TGF-β activation, is a major mediator of the myocardial complications of diabetes with activation of the renin-angiotensin system. We used a peptide antagonist of TSP1, LSKL, to selectively block glucose and angiotensin II-stimulated increases in TGF-β activity as a therapeutic strategy to modify fibrotic disease progression in a rat model of type 1 diabetes with activation of the renin-angiotensin system by surgical manipulation. The peptide antagonist of TSP1-dependent TGF-β activation reduced cardiac fibrosis, myocyte hypertrophy, and improved left ventricular function in these animals. These data support the hypothesis that TSP1 is a key mediator of fibrogenic changes induced by diabetes and suggest that blockade of TSP1-dependent TGF-β activation represents a novel therapeutic strategy to prevent or reverse fibrosis. Both peptides (LSKL and LSAL) were purchased from AnaSpec, Inc., San Jose, CA. Peptides were purified by reversed phase high-performance liquid chromatography and determined to be >98% pure by mass spectrometry. Male Wistar Kyoto rats, 250 to 300 g, were used for all studies (Charles River, Wilmington, MA). Animals were maintained and handled in accordance with approved Institutional Animal Care and Use Committee standards. In experimental animals, hypertension was induced by abdominal aortic coarctation, and diabetes was induced by a single injection of streptozotocin (STZ). Rats were anesthetized by intraperitoneal administration with ketamine (80 to 100 mg/kg) plus xylazine (5 mg/kg). Under sterile conditions, the abdominal cavity was opened via a midline incision, and the abdominal aorta and both renal arteries were exposed. Partial constriction of the abdominal aorta between the renal arteries was made using a small Hemoclip (Weck, Research Triangle Park, NC) set to an internal diameter of 0.45 mm. One week after recovery from surgery, animals were given a single intraperitoneal injection of STZ (52 mg/kg) (Sigma, St. Louis, MO) dissolved in citric acid buffer, pH 4.6. Sham animals underwent the same surgical procedure without placement of the clip and received an injection of citric acid buffer without STZ. Injected animals were fasted for 12 hours, and blood glucose was measured 1 week after injection. Blood glucose measurements were obtained from tail vein samples using a blood glucose meter (Prestige Smart System HDI; Home Diagnostics, Inc., Fort Lauderdale, FL). Glucose levels in the urine were determined with glucose reagent strips (Keto-Diastix; Bayer, Elkhart, IN). Indirect systolic blood pressure was determined by tail-cuff plethysmography in conscious, restrained rats.42Borges GR Oliveira M Salgado HC Fazan Jr, R Myocardial performance in conscious streptozotocin diabetic rats.Cardiovasc Diabetol. 2006; 5: 26Crossref PubMed Scopus (44) Google Scholar Animals were sacrificed 12 weeks after surgery. Experimental and sham animals were randomly placed into the following groups: Sham, Sham + LSKL, Sham + LSAL, diabetic with abdominal aortic coarctation (DAAC), DAAC + LSKL, and DAAC + LSAL (five to eight animals per group). Peptide administration began 6 weeks following induction of experimental or sham procedures. The peptides were solubilized in sterile saline and given to animals by intraperitoneal injection at a dose of 4 mg/kg, three times per week for 6 weeks. Mean arterial pressure was measured in anesthetized animals (ketamine/xylazine, 80:10 mg/kg) using an intravascular pressure transducer (model SPR-294A Millar Micro-Tip catheter transducer; Millar Instruments, Houston, TX) introduced into the aorta via the right carotid artery. Once pressure measurements were completed, echocardiography was performed in the rats using a 15-MHz transducer attached to an Agilent Sonos 5500 echocardiography machine. M-mode measurement was used to determine left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and posterior wall thickness (PW).43Schwarz ER Pollick C Meehan WP Kloner RA Evaluation of cardiac structures and function in small experimental animals: transthoracic, transesophageal, and intraventricular echocardiography to assess contractile function in rat heart.Basic Res Cardiol. 1998; 93: 477-486Crossref PubMed Scopus (34) Google Scholar Doppler tracings of left ventricular outflow at the aortic valve level were obtained to determine ejection time (EjT) and the time of the R-R interval. Left ventricular systolic chamber function was assessed from the rate-corrected velocity of circumferential shortening (VCFr), which was calculated using the equation: (LVEDD − LVESD/LVEDD)/(EjT − RR0.5), where LVEDD is the left ventricular end-diastolic diameter, LVESD is the left ventricular end-systolic diameter, and RR is the time of the R-R interval. Cardiac angiotensin II peptide concentrations were determined by a method previously established that combines solid-phase extraction, high-performance liquid chromatography, and radioimmunoassay.44Meng QC Durand J Chen YF Oparil S Simplified method for quantitation of angiotensin peptides in tissue.J Chromatogr. 1993; 614: 19-25Crossref PubMed Scopus (28) Google Scholar, 45Wei CC Tian B Perry G Meng QC Chen YF Oparil S Dell'Italia LJ Differential ANG II generation in plasma and tissue of mice with decreased expression of the ACE gene.Am J Physiol. 2002; 282: H2254-H2258Google Scholar Radioimmunoassay of relevant peaks revealed detectable levels of angiotensin (Ang) II in all left ventricular (LV) samples examined. Antibodies to Ang II were raised in New Zealand White rabbits immunized against peptide conjugated to poly-l-lysine, as previously described.44Meng QC Durand J Chen YF Oparil S Simplified method for quantitation of angiotensin peptides in tissue.J Chromatogr. 1993; 614: 19-25Crossref PubMed Scopus (28) Google Scholar The sensitivity of the radioimmunoassay for Ang II is 2 pg/ml. For each experimental group, 300 to 430 mg of cardiac tissue was assayed. Serial sections (5 μm) of formalin-fixed, paraffin-embedded tissues were stained with picric acid-Sirius red for quantification of collagen. Images of cross sections of the left ventricle were digitized using a ×10 or ×20 objective of an IMT-2 inverted microscope (Olympus, Tokyo, Japan) equipped with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Images were analyzed using Image Pro Plus (Media Cybernetics, Silver Spring, MD). The percentage of the field area that stained for collagen was determined for 20 to 30 fields for each transmural region (subendomyocardial and subepicardial), and the mean value was calculated for each region. Total collagen content of the left ventricle was quantified biochemically by the hydroxyproline assay.46Woessner Jr, JE The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3409) Google Scholar Specimens were dried and hydrolyzed in 12 N HCl at 120°C overnight. Hydrolysates were neutralized and mixed with chloramine T solution and oxidized for 20 minutes at room temperature. The oxidized product was reacted with p-dimethylaminobenzaldehyde in ethanol and H2SO4 solution at 60°C for 20 to 25 minutes, and the resulting chromophore was quantified spectrophotometrically at 557 nm against a standard curve of known hydroxyproline concentration (3 to 20 μg/ml). Formalin-fixed hearts were embedded in paraffin and sectioned at 5 μm. Slides were heated at 58°C for 1 hour. After removal of paraffin, endogenous peroxidase activity was quenched by 5-minute incubation with 3% H2O2 in H2O. Slides were placed in 0.01 mol/L glycine solution, pH 3, microwaved for 10 minutes, and cooled to room temperature. Slides were then placed in 0.5 mmol/L casein in phosphate-buffered saline, pH 7.4, for 30 minutes. Sections were incubated overnight at 4°C with mouse monoclonal antibody against TSP1 (mAb 133 at 10 μg/ml, purified by our lab in a joint effort with the University of Alabama at Birmingham Hybridoma Core facility).30Schultz-Cherry S Murphy-Ullrich JE Thrombospondin causes activation of latent transforming growth factor-β secreted by endothelial cells by a novel mechanism.J Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (403) Google Scholar For every section, a negative control without anti-TSP1 antibody was processed simultaneously. After three 5-minute washes in Tris-buffered saline (TBS)-T (10 mmol/L Tris-HCl, 0.15 mol/L NaCl, 8 mmol/L sodium azide, and 0.05% Tween 20, pH 8.0), a secondary biotinylated antibody was added for 45 minutes at room temperature. After three 5-minute washes in TBS-T, the avidin biotin-peroxidase complex (Vector Labs, Burlingame, CA) was applied for 30 minutes at room temperature. The color reaction was developed with the diaminobenzidine detection kit (Vector Labs) and counterstained with hematoxylin. For detection of active TGF-β, sections were treated with hyaluronidase (Sigma Chemical Co., St. Louis, MO) at 1 mg/ml in 0.1 mol/L sodium acetate, pH 5.5, containing 0.85% NaCl at 37°C for 30 minutes. After blocking with 5% normal goat serum, a rabbit polyclonal antibody to active TGF-β (LC-1-30; a gift from Dr. Kathy Flanders, National Cancer Institute, Lab of Cell Regulation and Carcinogenesis, National Institutes of Health, Bethesda, MD)47Flanders KC Thompson NL Cissel DS Van Obberghen-Schilling E Baker CC Kass ME Ellingsworth LR Roberts AB Sporn MB Transforming growth factor-β1: histochemical localization with antibodies to different epitopes.J Cell Biol. 1989; 108: 653-660Crossref PubMed Scopus (324) Google Scholar was used in TBS containing 0.1% bovine serum albumin overnight at 4°C. Antibody LC-1-30 recognizes intracellular active TGF-β and has been used to detect active TGF-β in paraffin-embedded tissues.48Barcellos-Hoff MH Ehrhart EJ Kalia M Jirtle R Flanders K Tsang ML Immunohistochemical detection of active transforming growth factor-β in situ using engineered tissue.Am J Pathol. 1995; 147: 1228-1237PubMed Google Scholar Biotinylated goat-anti-rabbit IgG (Vector Labs) was added, followed by avidin peroxidase conjugate. For immunostaining of phosphorylated Smad 2, sections were treated with sodium citrate buffer (10 mmol/L, pH 6.0) at 100°C for 10 minutes. After blocking with 5% bovine serum album