A Thrombospondin- Antagonist of Transforming Growth Factor-ß Activation Blocks Cardiomyopathy in Rats with Diabetes and Elevated Angiotensin II

【摘要】  In diabetes and hypertension, the induction of increased transforming growth factor-ß (TGF-ß) 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-ß activator thrombospondin-1 (TSP1). Because activation of latent TGF-ß is a major means of regulating TGF-ß, we addressed the role of TSP1-mediated TGF-ß 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-ß activation. This surgical manipulation elevates initial blood pressure and angiotensin II. The hearts of these rats had increased TSP1, collagen, and TGF-ß activity, and cardiac function was diminished. A peptide antagonist of TSP1-dependent TGF-ß activation prevented progression of cardiac fibrosis and improved cardiac function by reducing TGF-ß 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-ß activation as a potential antifibrotic therapeutic strategy are warranted.
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Diabetic cardiomyopathy is a major cause of congestive heart failure in diabetics and can occur independently of atherosclerosis. This disease occurs more frequently in diabetics with accompanying hypertension, particularly in African Americans.1,2 Interstitial fibrosis is a major factor underlying the myocardial hypertrophy and diastolic dysfunction that characterize diabetic cardiomyopathy.1-4 The severity of fibrosis is increased in diabetic animals that also have hypertensive disease.3,5 Both hyperglycemia and angiotensin II are critical in the pathogenesis of fibrosis.
Remodeling of the myocardium in diabetes affects both the cardiac myocyte and the cardiac fibroblast.2,6 Alterations in the cardiac myocyte include hypertrophy and altered sarcomere organization. Cardiac fibroblasts exhibit increased proliferation and aberrant remodeling of the extracellular matrix with net accumulation of extracellular matrix in the interstitium and surrounding the coronary arteries. Cardiac fibrosis also contributes to left ventricular hypertrophy and is the major determinant of altered left ventricular compliance leading to systolic and diastolic dysfunction.6,7
The fibrogenic cytokine transforming growth factor (TGF-ß) is known to play a significant role in fibrotic cardiac remodeling, and multiple factors altered in the diabetic condition, including glucose and angiotensin II, stimulate both increased TGF-ß protein expression and increases in TGF-ß bioactivity.8-12,14 The cardiac renin-angiotensin system is up-regulated in diabetes and has been implicated in cardiac fibrosis.6,7,15 Angiotensin II signaling through the AT1 receptor results in cardiac fibroblast proliferation and net accumulation of fibrillar collagen in vitro and cardiac fibrosis in vivo.7 Angiotensin II mediates end-organ fibrosis, independent of hemodynamic effects, through stimulation of TGF-ß.9,11,16-18 Valsartan, an antagonist of the AT1 receptor, attenuates myocardial interstitial fibrosis in diabetic rats by decreasing expression of active TGF-ß in the heart.13 TGF-ß1-neutralizing antibodies and other TGF-ß antagonists such as tranilast reduce cardiac and renal fibrosis in both hypertensive and diabetic animal models.13,19-21 In addition, another AT1 receptor antagonist, losartan, prevents vascular remodeling, leading to aortic aneurysm in a mouse model of Marfan??s syndrome by reducing levels of active TGF-ß.22
The potent fibrogenic action of TGF-ß is regulated at multiple levels, including protein expression and secretion, localization to the extracellular matrix, and modulation of receptor and signaling pathways.23,24 TGF-ß is secreted as a latent complex that must be activated to elicit fibrogenic effects. Activation of the latent complex is a key means of regulation of latent TGF-ß activity.25,26 Several factors can activate latent TGF-ß, including proteases, integrins, latent TGF-ß-binding protein, oxidant modification of the latent complex, and the matricellular protein thrombospondin-1 (TSP1).25-30 TSP1 binds to the latent TGF-ß complex through defined sequences in both the latency-associated peptide and in the mature domain.31-34 TSP1 binding to the latent complex induces a conformational change in the latent TGF-ß complex, rendering it biologically active.25 We identified a conserved sequence (LSKL) in the latency-associated peptide that is necessary for latency and that is recognized by the activating sequence of TSP1.31 A peptide mimetic of this sequence prevents TSP1 binding to the latent complex and acts as an antagonist of TSP1-mediated TGF-ß activation.31,35
TSP1 levels are increased in the hearts of animals with diabetic cardiomyopathy.36 Previous studies from our lab and others showed that both glucose and angiotensin II stimulate increased TSP1 expression, resulting in enhanced TGF-ß activation.37-40 Glucose stimulation increases TSP1 through enhancing USF2-dependent transcription via protein kinase C, extracellular signal-regulated kinase, and p38 mitogen activated protein kinase (MAPK) pathways to overcome nitric oxide-protein kinase G-mediated transcriptional repression.41 Angiotensin II induces TSP1 production in human mesangial cells via p38 MAPK and c-Jun NH2-terminal kinase.39
In these current studies, we tested the hypothesis that TSP1, through control of TGF-ß 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-ß 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-ß 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-ß activation represents a novel therapeutic strategy to prevent or reverse fibrosis.

【关键词】  thrombospondin- antagonist transforming factor-ß activation cardiomyopathy diabetes elevated angiotensin

Materials and Methods

Peptides

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.

Animal Model

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.42 Animals were sacrificed 12 weeks after surgery.

Peptide Administration

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.

Hemodynamic and Echocardiographic Measurements

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).43 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 C LVESD/LVEDD)/(EjT C 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.

Tissue Angiotensin II Assay

Cardiac angiotensin II peptide concentrations were determined by a method previously established that combines solid-phase extraction, high-performance liquid chromatography, and radioimmunoassay.44,45 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.44 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.

Collagen Measurement

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 x10 or x20 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.

Hydroxyproline Content

Total collagen content of the left ventricle was quantified biochemically by the hydroxyproline assay.46 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).

Immunohistochemistry

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).30 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-ß, 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-ß (LC-1-30; a gift from Dr. Kathy Flanders, National Cancer Institute, Lab of Cell Regulation and Carcinogenesis, National Institutes of Health, Bethesda, MD)47 was used in TBS containing 0.1% bovine serum albumin overnight at 4??C. Antibody LC-1-30 recognizes intracellular active TGF-ß and has been used to detect active TGF-ß in paraffin-embedded tissues.48 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 albumin in TBS, a rabbit polyclonal antibody anti-phosphorylated Smad 2 (Cell Signaling, Beverly, MA) was incubated with sections overnight at 4??C. Sections were washed and incubated with biotinylated goat-anti-rabbit IgG (Vector Labs) and then avidin peroxidase conjugate. Sections were counterstained with hematoxylin.

Staining was quantified by image analysis as described for collagen measurement. Ten to 15 fields from the left ventricle were captured, digitized, and analyzed. For TGF-ß and TSP1, staining was quantified by dividing the sum area of the positively stained area by the sum of the reference area. Results are expressed as percent area positive for TGF-ß or TSP1. For quantification of phosphorylated Smad 2, the number of positive (brown) nuclei in 10 to 15 fields in each of three separate sections of left ventricle was counted. Blue-staining nuclei were counted in the same fields. The percent Smad 2-positive nuclei (brown nuclei) was calculated as a percentage of the total nuclei .

Immunoblot Analysis

Immunoblot analysis was performed under reducing conditions as described previously.49 Left ventricular tissues obtained from sham and DAAC animals were homogenized by sonication and lysis in buffer containing 0.1% Triton X-100. After centrifugation at 10,000 x g for 20 minutes at 4??C, the supernatant was analyzed for determination of Smad proteins and for TSP1. Total protein concentration of all samples was measured using the bicinchoninic acid method. Equal protein amounts were loaded and analyzed by immunoblotting on separate gels. Primary antibodies were diluted in TBS-T: rabbit anti-phosphorylated Smad 2 (1:4000) (Cell Signaling), mouse anti-total Smad 2 (1:500; Transduction Laboratories, San Diego, CA), and mouse anti-TSP1 (mAb 133, 10 µg/ml). The specific bands of target proteins were visualized by enhanced chemiluminescence according to the manufacturer??s instructions (Amersham Life Science Inc. Arlington Heights, IL). Bands were quantified using Image Gauge (version 3.41) software (Fuji Photo Film; Tokyo, Japan). Equal loading of protein was confirmed by Coomassie staining of membranes and analysis of actin.

Detection of LSKL Peptide in Body Fluids by Liquid Chromatography-Mass Spectrometry

Sham and DAAC rats (8 to 10 weeks of age) were treated with a single dose (3 mg/kg) of LSKL peptide by intraperitoneal injection 6 weeks after experimental manipulation. Urine was collected for 24 hours, and serum was obtained at 4, 8, 24, 48, and 72 hours following injection of the peptide. Preliminary studies established that LSKL peptide could be detected by liquid chromatography-mass spectrometry when added to processed normal rat urine or serum over a concentration range of 50 nmol/L to 5 µmol/L.50 Urine samples were processed by centrifugation at 2500 rpm for 5 minutes at 4??C to remove cell debris. Serum samples were fractionated using 5MWCO Amicon Ultra4 centrifugal filters (Millipore, Inc., Bedford, MA) with centrifugation for 10 minutes at 3000 rpm at 4??C to remove proteins >5000 d. Processed samples were further fractionated by reverse phase chromatography on a C18 column in Zip tips (Millipore) equilibrated in 0.1% trifluoroacetic acid. Samples (10 µl) were prepared to a final pH of 4 in 0.1% trifluoroacetic acid and added to the equilibrated Zip tips. Unbound material was washed with 0.1% trifluoroacetic acid. Bound proteins were eluted in a 5-µl volume in 0.1% trifluoroacetic acid + 50% acetonitrile. The final sample volume was raised to 25 µl by the addition of Milli-Q water (Millipore). The quantitative measurement of the LSKL peptide was performed using the triple quadrupole capabilities provided by the ABI Sciex 4000Q-trap mass spectrometer (Applied Biosystems, Foster City, CA), which is sensitive in the range of 1 to 10 fmol.

Statistical Analysis

Data (mean ?? SEM) were analyzed using one-way analysis of variance followed by Student Newman-Keuls posthoc analysis for statistical comparisons between groups (InStat software; GraphPad Software Inc., San Diego, CA). A P value <0.05 was considered to be significant.

Results

Characterization of DAAC

Diabetes was confirmed in rats by detection of elevated blood glucose levels 7 to 10 days after injection of streptozotocin. All groups of animals receiving streptozotocin had significant elevations of blood glucose, water consumption, and reduced body weight compared with sham-treated animals (Table 1) . Rats were also subjected to abdominal aortic coarctation, which is a well-established method of inducing hypertension.3,51 At 3 weeks, the diabetic rats with abdominal aortic coarctation (DAAC) had elevated blood pressure as measured by the tail-cuff method52 (Table 2) . However, as the diabetic disease progressed, beginning at 5 to 6 weeks we observed a decrease of mean arterial pressure in the DAAC rats. These observations are consistent with other published studies, which show that streptozotocin produces a lowering of blood pressure in rats.42,52 Furthermore, the decrease in blood pressure is consistent with a decrease in cardiac function as indicated by the abnormal echocardiographic parameters measured at 12 weeks (Table 2) . Despite the lack of measurable elevations in blood pressure, the renin-angiotensin system was activated in these animals, because levels of angiotensin II peptide were elevated in the left ventricles of DAAC rats compared with sham control rats (Figure 1) .

Table 1. Characteristics of DAAC Rats at 12 Weeks

Table 2. Evaluation of Cardiac Function

Figure 1. Angiotensin II peptide is increased in the hearts of DAAC rats. Levels of angiotensin II (ANG II) peptide were measured in tissues from the left ventricles of sham and DAAC rats treated with either saline or LSKL peptide. Sham + saline (n = 6), Sham + LSKL (n = 6), DAAC + saline (n = 2), and DAAC + LSKL (n = 4). Data are expressed as the mean ?? SEM. *P < 0.001 DAAC + LSKL versus sham + saline. The difference between Sham + saline and Sham + LSKL is not statistically significant (P < 0.09) by Mann-Whitney analysis.

Myocardial Fibrosis Occurs in DAAC Animals

At both 6 and 12 weeks after disease induction, significant fibrotic changes were evident in the myocardium as evaluated by image analysis of picric acid-Sirius red-stained sections of the myocardium and by measurement of hydroxyproline content. Picric acid-Sirius red staining was increased in the left ventricular interstitium of DAAC rats compared with sham animals (Figure 2A) . Increased left ventricular fibrosis was present in both endocardium and epicardium (Figure 2B) . Hydroxyproline content in the left ventricle at 12 weeks was increased 2.4-fold in DAAC rats compared with sham animals (Figure 2C) . Interstitial fibrosis was accompanied by myocyte hypertrophy (Table 1) . Perivascular fibrosis is also observed in the DAAC rats (Figure 2A) . Thus, this model replicates the hallmarks of diabetic cardiomyopathy, which include myocardial fibrosis and left ventricular diastolic dysfunction.1-5

Figure 2. Collagen deposition is increased in the hearts of rats with DAAC. A: Picric acid-Sirius red staining of the myocardium. Sections of the left ventricle (left) and cardiac vasculature (right) from sham and DAAC rats at 6 weeks and of DAAC rats at 12 weeks were analyzed for interstitial collagen content by picric acid-Sirius red staining. Original magnification, x10. B: Quantification of increased collagen deposition in the epi- and endocardium of DAAC rats at 12 weeks. Interstitial collagen deposition in both epicardium and endocardium was measured by analysis of picric acid-Sirius red staining as described in Materials and Methods. Sham + saline (n = 8), Sham + LSKL (n = 8), Sham + LSAL (n = 8), DAAC + saline (n = 5), DAAC + LSKL (n = 8), and DAAC + LSAL (n = 5). Data are expressed as the mean percent area staining for collagen ?? SEM. *P < 0.001 versus sham. P < 0.001 versus DAAC. C: Left ventricular hydroxyproline content is increased at 12 weeks in DAAC rats. The hydroxyproline content was measured in the left ventricles of Sham + saline (n = 8), Sham + LSKL (n = 8), Sham + LSAL (n = 8), DAAC + saline (n = 5), DAAC + LSKL (n = 8), and DAAC + LSAL (n = 5) rats. Data are expressed as the mean ?? SEM. *P < 0.001 versus sham group. P < 0.001 versus DAAC.

TSP1 Is Increased in the Hearts of DAAC Animals

We examined whether TSP1 protein is up-regulated in the DAAC model. Immunoblot analysis of left ventricular lysates showed increased TSP1 levels in hearts of DAAC rats (Figure 3A) . Immunohistochemical analysis of hearts from sham animals after 12 weeks of disease induction showed weak staining for TSP1 in cardiac myocytes (Figure 3B) . TSP1 was primarily localized to the endocardium but only weak or absent in the epicardium of the left ventricular sections. In DAAC animals, TSP1 was markedly increased in the left ventricular myocardium compared with sham animals (Figure 3B) . These data establish that TSP1 is elevated in myocardial tissues, which exhibit fibrotic complications because of diabetes and activation of the renin-angiotensin system.

Figure 3. TSP1 is increased in the hearts of DAAC rats. A: Immunoblot of TSP1 in extracts of left ventricle at 12 weeks. Equal amounts of protein extracted from the left ventricles of sham and experimental animals were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% gel), transferred to polyvinylidene difluoride membranes, and immunoblotted with antibody to TSP1 and developed by enhanced chemiluminescence. The bands were quantified by densitometry (n = 4). Data are expressed as the mean ?? SEM. *P < 0.001 versus sham. B: Immunohistochemical staining for TSP1 in left ventricle. Sections were reacted with antibody specific for TSP1, color developed with diaminobenzidine, and counterstained with hematoxylin. The TSP1 immunostaining was quantified by morphometric analysis of 15 to 20 fields in each of three different regions of the left ventricle as described in Materials and Methods. Original magnification, x20. Sham + saline (n = 8), Sham + LSKL (n = 8), Sham + LSAL (n = 8), DAAC + saline (n = 5), DAAC + LSKL (n = 8), and DAAC + LSAL (n = 5). Data are expressed as the mean percent area staining for TSP1 ?? SEM. *P < 0.001 versus sham.

TGF-ß Activity Is Increased in the Hearts of DAAC Animals

Since there is no direct method for quantifying active TGF-ß in tissues, we used three different approaches to determine relative levels of TGF-ß activity. Relative levels of active TGF-ß were assessed in the heart by immunohistochemical analyses of tissues stained with an antibody previously shown to recognize the active form of TGF-ß in formalin-fixed tissues47,48 and by staining for phosphorylated Smad 2 (Figure 4) . In normal animals, staining for active TGF-ß was localized primarily to the myocardium (Figure 4A) , and phosphorylated Smad 2 staining was sparse to absent in the hearts of control sham animals (Figure 4B) . At 12 weeks following disease induction, both active TGF-ß and phosphorylated Smad 2 were increased in the hearts of DAAC animals compared with sham animals (Figure 4, A and B) . In tissues from DAAC animals, phosphorylated Smad 2 was observed in the nuclei of both cardiac myocytes and interstitial fibroblasts, as well as in vascular endothelial and smooth muscle cells (data not shown). To confirm these immunohistochemical analyses, extracts of left ventricular tissues were probed for total and phosphorylated Smad 2 by immunoblotting (Figure 4C) . Total Smad 2 was comparable in all groups, whereas phosphorylated Smad 2 was increased in DAAC hearts compared with sham hearts. Although measurement of TGF-ß activity in tissues by immunohistochemical methods can be problematic, and in some studies, smad phosphorylation by advanced glycation end products is independent of TGF-ß, results from these combined approaches strongly indicate that TGF-ß activity is increased in this disease model.53

Figure 4. TGF-ß signaling is increased in hearts of DAAC rats. A: Immunohistochemical staining for active TGF-ß in the left ventricle 12 weeks after disease induction. Three different regions of the left ventricle were analyzed, and 10 to 15 fields were examined in each region. Sections were reacted with antibody specific for active TGF-ß, color-developed with diaminobenzidine, and counterstained with hematoxylin. The percent area stained for active TGF-ß was quantified as described in Materials and Methods. Original magnification, x20. Sham + saline (n = 8), Sham + LSKL (n = 8), Sham + LSAL (n = 8), DAAC + saline (n = 5), DAAC + LSKL (n = 8), and DAAC + LSAL (n = 5). B: Immunohistochemical staining for nuclear phosphorylated Smad 2 in the left ventricle at 12 weeks. Tissue sections of the left ventricles were immunostained for phosphorylated Smad2, and the percentage of total nuclei stained positive (brown staining) was determined as described in Materials and Methods. Ten to 15 fields were examined in each region, and 80 to 128 nuclei were counted per field. Original magnification, x20. Sham + saline (n = 8), Sham + LSKL (n = 8), Sham + LSAL (n = 8), DAAC + saline (n = 5), DAAC + LSKL (n = 8), and DAAC + LSAL (n = 5). Data are expressed as mean ?? SEM. *P < 0.001 versus sham. P < 0.001 versus DAAC. C: Immunoblot for total and phosphorylated Smad 2 in extracts of the left ventricle at 12 weeks. Equal amounts of protein extracted from the left ventricles of sham and experimental animals were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gels), transferred to polyvinylidene difluoride membranes, and immunoblotted separately with antibodies to phosphorylated Smad 2 (phospho-Smad2) or to Smad 2 (Smad2) and developed by enhanced chemiluminescence. The bands were quantified by densitometry (n = 4).

LSKL Blocking Peptide Improves Cardiac Function in DAAC Rats

To determine whether TSP1 is a key mechanism for TGF-ß activation and fibrogenic progression in this model, a peptide antagonist (LSKL) of TSP1-dependent TGF-ß activation or a control peptide (LSAL) was administered to both sham and DAAC animals. Peptides were administered by intraperitoneal injection, given every other day, starting 6 weeks after aortic coarctation and continuing through week 12. Peptides were detected in the urine of diabetic animals by mass spectrometry 24 hours following intraperitoneal administration of peptides, suggesting that the peptides achieve a systemic distribution (see Supplemental Figure S1 at http://ajp.amjpathol.org).

Animals were evaluated for cardiac function and fibrosis of the heart. Treatment with both control and active peptides had no effect on blood glucose levels in either sham or DAAC animals (Table 1) . Treatment of DAAC rats with LSKL or LSAL peptides had no effect on body weight, blood glucose levels, water intake, LV weight, or LVW/HW (heart weight) and HW/BW (body weight) ratios. This is consistent with studies using anti-TGF-ß-neutralizing antibodies, which showed that blood glucose levels were not affected by blocking TGF-ß.19

Cardiac function was assessed by echocardiographic measurements (Table 2) . DAAC animals showed a slight, but not significant, change in posterior wall thickness compared with sham or to DAAC rats treated with LSKL (Table 2) . The diameter of the left ventricle in diastole (LVEDD) and systole (LVESD) were significantly increased in DAAC rats (P < 0.05) compared with sham rats. These dimensions were decreased in DAAC rats treated with LSKL (P < 0.05) compared with DAAC rats treated with saline or LSAL control peptide. Left ventricular remodeling and function as evaluated by LVEDD/PW ratio and VCFr (rate-corrected velocity of circumferential shortening) were adversely affected (P < 0.05) in DAAC animals compared with sham animals. In contrast, DAAC rats treated with LSKL peptide showed an increase (P < 0.05) in left ventricular function. Treatment with the LSKL peptide, but not the control LSAL peptide, also reduced myocyte hypertrophy (Table 1) .

It is of note that neither active nor control peptide affected left ventricular function, body or heart weight, or blood glucose of sham control animals, indicating that the effect of the LSKL peptide is specific to TGF-ß regulation in the DAAC condition. These data suggest that treatment with the TSP1 antagonist peptide improves cardiac function in DAAC rats.

The LSKL-Blocking Peptide Decreases Cardiac and Perivascular Fibrosis in DAAC Animals

Treatment of DAAC animals with LSKL markedly suppressed collagen deposition in the interstitium of the myocardium as determined by picric acid-Sirius red staining (Figure 5) . No differences in collagen deposition were observed in DAAC animals treated with either saline or the control peptide LSAL (Figure 5A) . Collagen deposition in both the epi- and endocardium was reduced by LSKL treatment (Figure 2B) . Perivascular collagen was also reduced in LSKL-treated animals (Figure 5B) .

Figure 5. LSKL peptide treatment reduces fibrosis in hearts of DAAC rats. Sections of left ventricular myocardium (A) or vasculature (B) from DAAC rats after 12 weeks of disease induction and 6 weeks of treatment with saline, LSKL, or LSAL peptide were analyzed for interstitial collagen content by picric acid-Sirius red staining. Original magnification, x10.

Consistent with the decrease in fibrosis indicated by picric acid-Sirius red staining, hydroxyproline levels in the left ventricle were significantly decreased in LSKL-treated DAAC animals to levels comparable with sham animals (Figure 2C) . Hydroxyproline levels were unaffected in animals treated with LSAL peptide (Figure 2C) . Peptides did not affect hydroxyproline content in hearts of sham animals.

The LSKL Blocking Peptide Decreases TGF-ß Activity and Smad 2 Phosphorylation

The LSKL peptide has been shown to reduce TGF-ß activity in vitro and in vivo.31,35,38,40,54 Therefore, to establish that the reduction in fibrosis in LSKL-treated animals is due to a reduction in active TGF-ß, TGF-ß activity was assessed by immunohistochemistry using an antibody specific for the active form of TGF-ß (antibody LC1-30).47,48 Staining for active TGF-ß was significantly decreased in the myocardium of DAAC animals treated with LSKL peptide, but not in DAAC animals treated with either saline or LSAL control peptide (Figure 4A) . Furthermore, LSKL peptide-treated animals showed a significant decrease in phosphorylated Smad2 in the heart compared with DAAC rats or DAAC animals treated with control (LSAL) peptide (Figure 4, B and C) . LSKL reduced Smad 2 phosphorylation in the left ventricle to a level similar to sham animals, but it had no effect on Smad phosphorylation in the left ventricles of sham animals. Together, these data provide strong evidence that the LSKL peptide is acting to reduce TGF-ß activity by preventing TSP1-dependent TGF-ß activation.

Discussion

Fibrotic end-organ damage is a life-threatening consequence of hypertension and diabetes. Hypertension exacerbates myocardial complications associated with diabetes, resulting in more devastating structural and functional impairments than are caused by either disease.1,2,55 The importance of fibrosis as a major determinant of myocardial performance and disease outcome is increasingly appreciated.

Despite the prevalence and the mortality associated with myocardial fibrosis, there are few effective therapies available that can either halt or reverse fibrotic organ damage. Antagonists of the renin-angiotensin system such as angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists have been used to treat fibrotic complications of diabetes and hypertension.1,15,56-60 Combined treatment with both an angiotensin-converting enzyme inhibitor and an AT1 receptor antagonist showed greater improvement of echocardiographic indices in type 2 diabetics than either therapy alone.61 In addition, simultaneous blockade of Ang II and TGF-ß reduces fibrotic disease considerably more than blockade of either agent alone, suggesting that additional factors contribute to myocardial dysfunction and fibrosis.60 Following myocardial infarction, the incidence of heart failure and mortality rates are approximately twofold higher in patients with diabetes compared with those without diabetes.62 Despite the effectiveness of renin-angiotensin system antagonists, protection is not comparable with nondiabetic patients, and there can be cardiac side effects in a substantial number of patients.63 Given that the presence of both pathologies greatly accelerates the development of cardiac failure, new strategies to prevent and treat cardiac complications in diabetic hypertensive patients are needed.

The major fibrogenic cytokine TGF-ß is a potential therapeutic target, although a certain level of TGF-ß activity is required for tissue homeostasis.64,65 TGF-ß activity is regulated at the level of protein expression and secretion, matrix deposition, and signaling.23,26 However, a major point of control of TGF-ß activity occurs at the level of its conversion from a latent complex to its biologically active form.26 Previously, we identified the matrix glycoprotein TSP1 as the primary regulator of enhanced TGF-ß activation due to glucose and/or angiotensin II by both rat mesangial cells and rat cardiac fibroblasts in vitro.38 TGF-ß activity under normal glucose conditions is unaffected by blocking TSP1-dependent TGF-ß activation, whereas inhibition of TSP1 action completely blocks TGF-ß activity stimulated by high concentrations of glucose, angiotensin II, or both agents in combination.38

In the current study, we used a rat model of diabetes and aortic abdominal coarctation to investigate the role of TSP1-dependent TGF-ß activation in regulation of the excessive TGF-ß activity and fibrosis in this disease. Rats are relatively resistant to atherosclerosis, and thus, alterations in cardiac function following streptozotocin treatment are believed to be due to cardiomyopathy and independent of atherosclerotic complications.66 Hence, this is comparable with human diabetic cardiomyopathy. In this DAAC model, collagen accumulation, TSP1 expression, and TGF-ß activity were all increased. There is also significant evidence of adverse left ventricular remodeling and systolic dysfunction.

Our finding that TSP1 is up-regulated is consistent with a recent report showing increased TSP1 immunostaining in the myocardium and small blood vessels in rats 4 weeks after STZ treatment.36 The mechanism of the increase of TSP1 expression in the heart in these animals has not been established in vivo. Although TGF-ß is a known regulator of TSP1 expression, especially in fibrotic kidney,67-70 it seems that TGF-ß itself is not a major regulator of TSP1 expression in this rat model of diabetes with abdominal aortic coarctation. TSP1 levels in myocardial extracts were unchanged by LSKL peptide treatment in diabetic rats with renin-angiotensin activation. These observations are consistent with data from in vitro studies of rat mesangial cells, which showed that glucose stimulates increased TSP1 transcription through enhancing USF2-dependent transcription via protein kinase C, extracellular signal-regulated kinase, and p38 MAPK pathways to overcome nitric oxide-protein kinase G-mediated transcriptional repression.41 The blocking peptide LSKL had no effect in these in vitro studies, suggesting that under high-glucose conditions, TGF-ß is not a major regulator of TSP1.

Angiotensin II also up-regulates TSP1 in cardiac fibroblasts, endothelial cells, and mesangial cells through AT1 receptors.38,39,71,72 TSP1 up-regulation by angiotensin II in mesangial cells occurs via AT1 receptor signaling through the p38 MAPK and c-Jun NH2-terminal kinase pathways.39 Levels of TSP1 produced by combined stimulation with glucose and angiotensin II suggest that the pathways regulating TSP1 expression are likely parallel.38 Thus in this DAAC model of myocardial fibrosis, it is likely that both glucose and angiotensin II are acting to stimulate TSP1 expression and consequent TGF-ß activation. The involvement of the TSP1-TGF-ß axis in other diseases in which myocardial fibrosis is an endpoint would probably depend on whether the pathogenic stimuli also increase TSP1 expression.

Blockade of TSP1-dependent activation of TGF-ß by a peptide antagonist not only suppresses the development of cardiac fibrosis but also improves left ventricular function and cardiomyocyte hypertrophy. LSKL treatment improved LVEDD, LVEDD/PW, LVESD, and VCFr, indicating that the peptide antagonist prevented adverse left ventricular remodeling and left ventricular systolic dysfunction because of replacement fibrosis and myocyte dropout. Interestingly, normalization of left ventricular function and the reduction in fibrosis and myocyte hypertrophy by LSKL treatment occurred in the absence of changes in LV weight or the LV weight/HW ratio.

The TSP1 peptide antagonist attenuates fibrogenesis through prevention of TGF-ß activation as measured by phosphorylated Smad 2 levels and immunostaining for active TGF-ß levels in tissues. This peptide antagonist had no effect on normal blood glucose levels, blood pressure, or parameters of left ventricular function in sham control animals, suggesting that the peptide is indeed acting at the level of TGF-ß regulation, specifically in response to the pathogenic stimuli of hyperglycemia and angiotensin II.

There are numerous therapeutic strategies under development aimed at targeting TGF-ß, its receptors, signaling intermediates, downstream effectors, and endogenous inhibitors for potential use in the treatment of various types of fibrotic diseases.14,73 Consistent with data from our previous in vitro studies, in the DAAC model the TSP1 antagonist peptide reduced only the excessive, stimulated levels of TGF-ß activity and had no effects on basal levels of activity as measured in immunoblots for phosphorylated Smad 2. Because TGF-ß activity is required for tissue homeostasis,64,65 the ability to selectively target the pathological excess of TGF-ß activity, without abrogating normal homeostatic levels of activity, represents an important therapeutic advantage for any antifibrotic approach that targets TGF-ß. Genetic ablation of TGF-ß, its receptors, or components of the Smad signaling pathway has serious consequences, including developmental defects, systemic inflammation, and increased tumorigenesis.74-76 Although a number of different antagonists are being evaluated for clinical use, their spectrum of side effects remains unclear. Information regarding compromise of homeostatic functions of TGF-ß from animal models or clinical trials with global TGF-ß antagonists is limited.73,77,78 In one systematic study performed in mice with life-long mammary-specific expression of a soluble TGF-ß receptor antagonist, there were no significant differences in immune cell profiles or incidence of neoplasia, except for a slight increase in the CD4 and CD8 memory T-cell populations.78 Although the long-term consequences of inhibiting TSP1-dependent TGF-ß activation remain to be determined, in the present studies, systemic administration of the peptide over 6 weeks did not result in increased inflammation. Despite the lack of any obvious pathology in these present studies, further studies are being directed at determining whether inhibition of TSP1-dependent TGF-ß activation exacerbates the impaired diabetic wound healing or alters immune cell profiles.

This work establishes the importance of TSP1 in regulating TGF-ß activity diabetic and hypertensive fibrotic complications and demonstrates the potential for therapeutics targeted at blocking TGF-ß activation for treatment of fibrotic complications because of angiotensin II and diabetes. It is worth noting that this work suggests that selective targeting of only pathological levels of TGF-ß activity and maintenance of homeostatic levels of TGF-ß might avoid the negative effects of more nonselective inhibition of TGF-ß activity, such as inflammation and carcinogenesis.

Acknowledgements

We thank Wayne E. Bradley for excellent technical assistance, Mingli Liu for excellent animal care, and Dr. Dale Fowler for evaluation of TSP1 staining. We thank Dr. Kathleen Flanders, National Institutes of Health, for the generous gift of the anti-TGF-ß antibody (LC-1-30), and Mr. Ray Moore of the University of Alabama Birmingham Mass Spectrometry facility for assistance with detection of the LSKL peptide.

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作者单位：From the Department of Physiology and Biophysics,* the Department of Pathology and the BioMatrix Engineering and Regenerative Medicine Center, and Department of Medicine, Division of Cardiovascular Medicine, Center for Heart Failure Research, University of Alabama at Birmingham, Birmingham, Alabama