Inhibiting TGF-β Activity Improves Respiratory Function in mdx Mice
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.024
ISSN1525-2191
AutoresCarol A. Nelson, R. Bridge Hunter, Lindsay A. Quigley, Stefan Girgenrath, William Weber, Jennifer A. McCullough, Carol J. Dinardo, Kelly A. Keefe, Lorena Ceci, Nicholas P. Clayton, Alison McVie‐Wylie, Seng H. Cheng, John P. Leonard, Bruce M. Wentworth,
Tópico(s)Chronic Obstructive Pulmonary Disease (COPD) Research
ResumoRespiratory function is the main cause of mortality in patients with Duchenne muscular dystrophy (DMD). Elevated levels of TGF-β play a key role in the pathophysiology of DMD. To determine whether therapeutic attenuation of TGF-β signaling improves respiratory function, mdx mice were treated from 2 weeks of age to 2 months or 9 months of age with either 1D11 (a neutralizing antibody to all three isoforms of TGF-β), losartan (an angiotensin receptor antagonist), or a combination of the two agents. Respiratory function was measured in nonanesthetized mice by plethysmography. The 9-month-old mdx mice had elevated Penh values and decreased breathing frequency, due primarily to decreased inspiratory flow rate. All treatments normalized Penh values and increased peak inspiratory flow, leading to decreased inspiration times and breathing frequency. Additionally, forelimb grip strength was improved after 1D11 treatment at both 2 and 9 months of age, whereas, losartan improved grip strength only at 2 months. Decreased serum creatine kinase levels (significant improvement for all groups), increased diaphragm muscle fiber density, and decreased hydroxyproline levels (significant improvement for 1D11 only) also suggested improved muscle function after treatment. For all endpoints, 1D11 was equivalent or superior to losartan; coadministration of the two agents was not superior to 1D11 alone. In conclusion, TGF-β antagonism may be a useful therapeutic approach for treating DMD patients. Respiratory function is the main cause of mortality in patients with Duchenne muscular dystrophy (DMD). Elevated levels of TGF-β play a key role in the pathophysiology of DMD. To determine whether therapeutic attenuation of TGF-β signaling improves respiratory function, mdx mice were treated from 2 weeks of age to 2 months or 9 months of age with either 1D11 (a neutralizing antibody to all three isoforms of TGF-β), losartan (an angiotensin receptor antagonist), or a combination of the two agents. Respiratory function was measured in nonanesthetized mice by plethysmography. The 9-month-old mdx mice had elevated Penh values and decreased breathing frequency, due primarily to decreased inspiratory flow rate. All treatments normalized Penh values and increased peak inspiratory flow, leading to decreased inspiration times and breathing frequency. Additionally, forelimb grip strength was improved after 1D11 treatment at both 2 and 9 months of age, whereas, losartan improved grip strength only at 2 months. Decreased serum creatine kinase levels (significant improvement for all groups), increased diaphragm muscle fiber density, and decreased hydroxyproline levels (significant improvement for 1D11 only) also suggested improved muscle function after treatment. For all endpoints, 1D11 was equivalent or superior to losartan; coadministration of the two agents was not superior to 1D11 alone. In conclusion, TGF-β antagonism may be a useful therapeutic approach for treating DMD patients. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene leading to a loss of the translated protein.1Koenig M. Hoffman E.P. Bertelson C.J. Monaco A.P. Feener C. Kunkel L.M. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals.Cell. 1987; 50: 509-517Abstract Full Text PDF PubMed Scopus (1960) Google Scholar, 2Hoffman E.P. Brown Jr, R.H. Kunkel L.M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus.Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3714) Google Scholar Dystrophin, a large structural protein, is critical for the assembly of the dystrophin-associated complex, a group of proteins that work in concert to link the actin cytoskeleton to the extracellular matrix of the basal lamina.3Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1202) Google Scholar The dystrophin-associated protein complex lends structural integrity to the sarcolemma and serves as an important scaffold for signaling entities involved in the modulation of cell survival.4Rando T.A. The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies.Muscle Nerve. 2001; 24: 1575-1594Crossref PubMed Scopus (304) Google Scholar, 5Niebroj-Dobosz I. Fidziańska A. Hausmanowa-Petrusewicz I. Controversies about the function of dystrophin in muscle.Folia Neuropathol. 2001; 39: 253-258PubMed Google Scholar In the absence of dystrophin, the associated proteins are dislocated, membranes are more susceptible to microtears, and various signaling pathways are dysregulated, leading to cycles of myofiber degeneration and regeneration. TGF-β, a profibrotic cytokine, is elevated in DMD and is known to play a central role in the cycles of degeneration and regeneration that ultimate lead to the replacement of skeletal muscle with fat and fibrotic tissue in this progressive disease.6Chaudhry S.S. Cain S.A. Morgan A. Dallas S.L. Shuttleworth C.A. Kielty C.M. Fibrillin-1 regulates the bioavailability of TGFbeta1.J Cell Biol. 2007; 176: 355-367Crossref PubMed Scopus (228) Google Scholar Several lines of evidence suggest that lowering TGF-β activity in dystrophic muscle may enhance differentiation and fusion of the precursor satellite cells necessary for muscle regeneration and repair.7Cohn R.D. van Erp C. Habashi J.P. Soleimani A.A. Klein E.C. Lisi M.T. Gamradt M. ap Rhys C.M. Holm T.M. Loeys B.L. Ramirez F. Judge D.P. Ward C.W. Dietz H.C. Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states.Nat Med. 2007; 13 ([Erratum appeared in Nat Med 2007, 13:511]): 204-210Crossref PubMed Scopus (562) Google Scholar Furthermore, TGF-β may promote the differentiation of myogenic cells into fibrotic cells.8Li Y. Foster W. Deasy B.M. Chan Y. Prisk V. Tang Y. Cummins J. Huard J. Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis.Am J Pathol. 2004; 164: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar Thus, therapeutic approaches to inhibit TGF-β may address some of the disease manifestations in DMD and other degenerative myopathies. Respiratory dysfunction is the cause of 80% of the mortality in DMD patients. We studied the effects of administering 1D11 (a neutralizing murine antibody to all three isoforms of TGF-β) on respiratory function, using plethysmography in the mdx mouse, a model of DMD. In addition, we compared antibody treatment to treatment with losartan, an antihypertensive agent that attenuates TGF-β activity by antagonizing angiotensin II receptor type 1 (AT1), and enalapril (an antagonist of the angiotensin-converting enzyme), Short-term studies in which forelimb grip strength was measured in mice dosed from 2 weeks to 2 months of age were used to assess the various treatment modalities. Effective treatment regimes (losartan, 1D11, or a combination of the two agents) were then compared in a long-term study conducted in mice up to 9 months of age, with respiratory function as the key endpoint. Here we demonstrate, for the first time, that TGF-β antagonism normalized respiratory function in the mdx mouse model. Other measured endpoints were also positively affected by drug treatment. In all cases, 1D11 was equivalent to or superior to losartan, and coadministration of the two agents was not superior to 1D11 alone. Furthermore, these agents were well tolerated, with no changes in body weights in any of the test groups at any time point. These findings demonstrate that TGF-β antagonism can improve respiratory function in mdx mice and support its further evaluation as a potential therapeutic for DMD patients. All animal procedures were approved by our institutional review board and were conducted in our animal facility, which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care International. The mice used in this study were male wild-type C57BL/10SnJ and male mdx C57BL/10ScSn-Dmdmdx/J mice (Jackson Laboratories, Bar Harbor, ME) that were housed and bred in our institutional facilities. BALB/c mice were from Charles River Laboratories (Raleigh, NC). Mice were provided with water and chow ad libitum. An outline of all studies and endpoints is given in Supplemental Table S1 (available at http://ajp.amjpathol.org) Treatment was initiated at 2 weeks of age and was continued, without pause, until study termination when the mice reached either 2 or 9 months of age. Administration of 1D11 or control antibody (either 13C4,9Strockbine N.A. Marques L.R. Holmes R.K. O'Brien A.D. Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli.Infect Immun. 1985; 50: 695-700Crossref PubMed Google Scholar a murine anti-Shigella toxin IgG1 antibody produced by Genzyme Corporation, or MOPC 21, an antibody to mineral oil, from Sigma-Aldrich, St. Louis, MO) was by intraperitoneal injection of 5 mg/kg three times per week until 48 hours before the termination of the studies. 1D11 is a murine pan-neutralizing TGF-β IgG1 antibody that neutralizes the active forms of TGF-β1, -β2, and -β3.10Dasch J.R. Pace D.R. Waegell W. Inenaga D. Ellingsworth L. Monoclonal antibodies recognizing transforming growth factor-beta Bioactivity neutralization and transforming growth factor beta 2 affinity purification.J Immunol. 1989; 142: 1536-1541PubMed Google Scholar 1D11 can also be purchased from ATCC (Manassas, VA) and R&D Systems (Minneapolis, MN). Administration of the AT1 angiotensin receptor antagonist losartan (LKT Laboratories, St. Paul, MN) and the angiotensin-converting enzyme inhibitor enalapril (Sigma-Aldrich) was via drinking water at concentrations of 600 mg/L and 200 mg/L, respectively. We conducted two studies in the mdx mouse: a short-term study conducted to 2 months of age and a long-term study conducted to 9 months of age. All mice in the 2-month study were treated from 2 weeks of age to 2 months of age, for a total of 6 weeks of consecutive treatment. All mice in the 9-month study were treated from 2 weeks of age to 9 months of age, for a total of 8.5 months of consecutive treatment. Mice were accessioned into the 2-month study in cohorts, because of limitations on the mouse census in our facilities. Each cohort in the 2-month study included a group of wild-type mice, 1D11-treated mdx mice, and vehicle-treated control mdx mice (n = 8). The first cohort also included a group of mice receiving the control antibody, 13C4 (n = 8). The second cohort included mdx mice treated with either losartan or enalapril (n = 8, each group). The third cohort included a group of mice in which 1D11 (5 mg/kg, three times per week) and losartan (600 mg/L in drinking water) were coadministered, to address whether an additive or synergistic benefit might be observed with combination therapy (n = 9). One group of mice in the third cohort was treated with MOPC 21 containing 1% bovine serum albumin (BSA) in the formulation (n = 10),9Strockbine N.A. Marques L.R. Holmes R.K. O'Brien A.D. Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli.Infect Immun. 1985; 50: 695-700Crossref PubMed Google Scholar to assess immune responsiveness. Terminal biochemical and histological endpoints for 2-month-old mice were made using samples from mice in either the first or the third cohorts of the 2-month studies. In the 2-month study, forelimb grip strength measurements from different cohorts with identical treatment modalities were pooled for statistical analysis. In the 9-month study, mdx mice were dosed with vehicle, losartan, 1D11, or a combination of these agents in a single cohort (n = 9, each group). Wild-type mice were included for an age-matched comparison (n = 9). At the end of the treatment period, one animal had died in each of the vehicle control and wild-type groups, leaving eight mice for evaluation in each of those two groups. No mice were lost in the losartan, 1D11, or combination therapy groups. In both the 2-month and the 9-month studies, body weights were recorded three times per week, and when agents were administered in the drinking water, the water bottles of all groups were weighed to estimate consumption. Quadriceps muscles were homogenized in cell-lysis buffer (Cell Signaling Technology, Danvers, MA), and the clarified cytosol recovered for assay after centrifugation at 10,000 × g. Protein quantities in the cytosol were determined using a bicinchoninic acid kit (Sigma-Aldrich), and 50 μg was used in the enzyme-linked immunosorbent assay (ELISA). Both total TGF-β1 and activated TGF-β1 concentrations were assessed using a mouse TGF-β1 ELISA kit (MB100B; R&D Systems) according to the manufacturer's protocol (n = 6 per group). Muscles were frozen in liquid nitrogen cooled isopentane and then stored at −80°C. Total RNA was isolated with TriReagent (Sigma-Aldrich) and homogenized; particulates were removed by centrifugation. The RNA pellet was dissolved in water and 10 μg RNA of each sample was treated with Ambion DNase TurboDNA (Applied Biosystems, Austin, TX). PCR was performed using TaqMan assays (Applied Biosystems, Foster City, CA). Gene expression analysis was performed using the standard curve method with 18S RNA as an endogenous control. Standard curves were run on each plate. Data were normalized to the mean value for the wild-type control group. The following fluorescently tagged primer sequences were used, all from Applied Biosystems: Mm00450111 for periostin, Mm00442754 for CD4, Mm00441724 for TGF-β1, Mm00436952 for TGF-β2, Mm00436960 for TGF-β3, and Mm001182107_g1 for CD8a. Blood was obtained from 2- and 9-month-old treated mice by retro-orbital collection (n = 8 per group). Serum was shipped on dry ice for creatine kinase determinations (AnaLytics, Gaithersburg, MD). The pharmacokinetic behavior of 1D11 was determined after a single intraperitoneal dose of 5 mg/kg in BALB/c mice. Serum concentrations of 1D11 were determined using a sandwich ELISA, and noncompartmental modeling was performed using WinNonlin software platform version 5.0.1 (Pharsight Products, Phoenix AZ). Serum samples were taken at 6 hours and at 1, 2, 3, 4, 8, and 14 days after the dose (n = 3 mice per time point). In addition, a single serum sample was taken from each mdx mouse treated with 1D11 in the first cohort of the 2-month study (n = 8). To determine the concentration of 1D11 in serum, an ELISA was used. High protein binding polystyrene 96-well plates were coated overnight at 4°C with TGF-β2 (Sigma-Aldrich). The plates were treated with blocking solution (KPL, Gaithersburg, MD) and washed with PBS. A standard curve was prepared using 1:2 serial dilutions ranging from 0.78 ng/mL to 50 ng/mL. Test samples were diluted in PBS containing 0.2% Tween-20, 0.1% BSA, and 0.05% Triton-X-100. Standards, controls, and samples were added to the blocked, coated plates and incubated at 37°C for 1 hour. Goat-anti-mouse IgG (Fc-specific) horseradish peroxidase conjugate was added to each well at a 1:60,000 dilution, incubated for 1 hour, and detected with 3,3′,5,5′-tetramethylbenzidine. Respiratory function was measured in unrestrained mice by barometric plethysmography using a Buxco plethysmograph (Troy, NY), essentially as described by TREAT-NMD (http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.2.002.pdf) and by others.11Lin C.C. Noninvasive method to measure airway obstruction in nonanesthetized allergen-sensitized and challenged mice.Respiration. 2001; 68: 178-185Crossref PubMed Scopus (15) Google Scholar, 12Khurana T.S. Respiratory System Evaluation.TREAT-NMD Neuromuscular Network SOP M.2.2_002. Wellstone Muscular Dystrophy Center, Washington, DC2008Google Scholar Mice were placed in calibrated chambers containing a pneumotachograph that measured pressure differentials within the compartment by a difference in air flow. Mice were allowed to acclimate in chambers for 30 minutes in a dark room before data collection. Data were collected and monitored remotely to minimize variation from environmental stimuli. The inspiration time Ti was defined as the start of inspiration to the end of inspiration and the expiration time Te was defined as the start of expiration to the end of expiration. The relaxation time Tr was defined as the time from the start of expiration to the time when 64% of the total expiratory pressure occurred. Pause and Penh were defined and calculated by the following formulas: Pause = (Te − Tr)/Tr and Penh = (PEP/PIP) × Pause, where PEP is peak expiratory pressure and PIP is peak inspiration pressure. The value of each parameter was collected every minute for 10 minutes and the average was determined. For each test subject, three separate measurements were made on three separate days, and median values were used for statistical analyses. Forelimb grip strength has been studied as a method to monitor muscle function in vivo in various models of muscular dystrophy.13Connolly A.M. Keeling R.M. Mehta S. Pestronk A. Sanes J.R. Three mouse models of muscular dystrophy: the natural history of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin alpha2-deficient mice.Neuromuscular Disord. 2001; 11: 703-712Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Forelimb grip strength was measured using an automated grip-strength meter (Columbus Instruments, Columbus, OH), essentially according to published protocols.14Luca A.D. Use of Grip Strength Meter to Assess the Limb Strength of mdx Mice.TREAT-NMD Neuromuscular Network SOP M.2.2_001. Wellstone Muscular Dystrophy Center, Washington, DC2008Google Scholar Measurements were taken once per week from 5 weeks to 2 months of age in the 2-month study. In the 9-month study, measurements were taken at 7 and 9 months of age. The total peak force generated was determined using a force transducer as the mouse was pulled backward gently from the base of the tail. All measurements were performed in a blinded fashion, to minimize operator influence. Five consecutive measurements were made within 1 minute and were averaged to determine the mean forelimb grip strength. All measurements were performed between 9:00 and 11:00 AM, to minimize diurnal variation. The data were normalized to body weight and expressed as kilogram force per kilogram of body weight. Changes in grip strength were determined by analysis of variance followed by Duncan's multiple comparison test. Soleus and diaphragm muscles were fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 3 to 7 days. All tissues were embedded in paraffin, and 5 μmol/L cross-sections were cut from the center of each muscle. Myogenin immunostaining was performed using a BondMax immunostaining system (Leica Microsystems, Deerfield, IL) including the Bond polymer refine detection kit (DS9800), which contained peroxide block, polymer, diaminobenzidine, and hematoxylin. Primary antibody [mouse anti-rat myogenin clone F5D, X0931 (Dako, Carpinteria, CA)] was incubated for 30 minutes after blocking with rodent block M (RBM961; Biocare Medical, Concord, CA), followed by rabbit anti-mouse (clone M204-3; Epitomics, Burlingame, CA). The positive controls used were rat hearts injected with rat muscle stem cells. The negative controls used were mouse IgG1 (DAK-GO1; Dako) instead of primary antibody and noninjected rat hearts. Entire cross-sections of diaphragm and soleus muscle were scanned at ×20 magnification using a Scanscope XT and Imagscope software v10.10.2028 (Aperio Technologies, Vista, CA). Each tissue was analyzed using a nuclear imaging algorithm (Color deconvolution version 9.0, Aperio Technologies) to quantify the number of myogenin-positive nuclei in the viable regions of tissue, excluding artifacts from the analysis. The nuclear algorithm was then used to digitally capture the intensity of diaminobenzidine staining from 0 (negative) to +3 (moderate positivity) and to quantify the number and intensity of myogenin-positive versus total nuclei in the viable muscle tissue section. Computer-identified myogenin-positive nuclei were manually visualized to confirm the accuracy of the digital algorithm. Muscle tissue was dissected, pinned to squares of closed-cell extruded polystyrene foam (Styrofoam; Dow Corning, Midland, MI), coated with optimal cutting temperature OCT medium, and immediately frozen in liquid nitrogen-chilled isopentane. Embedded muscles were cross-sectioned (10 μmol/L) with a cryostat and the sections were adhered to glass slides. Mounted sections were fixed with 10% buffered formalin for 10 minutes, and washed. Fixed sections were coated with a mixture of wheat germ agglutinin, Alexa Fluor 488 conjugate (Invitrogen, Carlsbad, CA) diluted 1:100, and DAPI (Invitrogen) diluted 1:1000 and incubated for 1 hour at room temperature. Wheat germ agglutinin-stained sarcolemma was photographed at 450 nmol/L and DAPI-stained nuclei at 650 nmol/L. Three random cross-sections from the diaphragm muscles, containing between 1500 and 2000 fibers, were analyzed using MetaMorph software version 6.1 (Universal Imaging Corp Downington, PA) to determine the fiber area, and fiber breadth. The fibers containing central nuclei were counted manually in a blinded fashion. This protocol is similar to the recently published TREAT-NMD SOP,15Dubache-Powell J. Quantitative Determination of Muscle Fiber Diameter (Minimal Feret's Diameter) and Percentage of Centralized Nuclei.TREAT-NMD Neuromuscular Network SOP M.2.2_001. Wellstone Muscular Dystrophy Center, Washington, DC2008Google Scholar except that OCT was used as the embedding medium and the microtome sections were 10 μm instead of 12 μm in thickness. Selected regions were analyzed with morphometry software. The number of fibers analyzed for diaphragm muscle was between 4000 and 5000 for each group.15Dubache-Powell J. Quantitative Determination of Muscle Fiber Diameter (Minimal Feret's Diameter) and Percentage of Centralized Nuclei.TREAT-NMD Neuromuscular Network SOP M.2.2_001. Wellstone Muscular Dystrophy Center, Washington, DC2008Google Scholar For the soleus muscle, three entire cross-sections were processed. The percentage of muscle area and the total number of fibers per unit area were also quantitated. Muscle C2C12 cells (ATCC) were maintained in a humidified incubator at 37°C and 5% CO2 in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM; ATCC #30–2002) supplemented with 10% fetal bovine serum. When cells reached approximately 70% confluency (day 0), the medium was changed to differentiation medium (DMEM plus 2% horse serum; Gibco–Invitrogen, Gaithersburg, MD). TGF-β1 was added on day 0 and day 2. Under control conditions (in the absence of TGF-β), cells were maintained in differentiation medium only. Control and treated cells were collected for RNA, ELISA, or myosin protein expression assays on day 5 after differentiation. After 5 days of TGF-β treatment, C2C12 cells were fixed with ice-cold methanol for 20 minutes at 20°C. Cells were then washed with PBS and permeabilized with 0.5% Triton-X-100 in PBS for 10 minutes at room temperature. Cells were blocked with 10% BSA for 1 hour at 20°C. A mouse anti-myosin heavy chain antibody (MF-20 supernatant; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) was incubated with the cells at 20°C for 1 hour (1:100); cells were then counterstained with a rabbit antibody to GAPDH (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA) (1:100). Cells were washed with PBS, followed by incubation with the secondary antibodies anti-mouse IRDye800 and anti-rabbit IRDye680 (LI-COR Biosciences, Lincoln, NE) for 1 hour at room temperature (1:1000). The LI-COR Odyssey imaging system was used for quantitation of myosin protein expression. The integrated intensity of myosin expression was normalized to that of GAPDH. For images, propidium iodide (1:1000) was used to visualize nuclei and anti-mouse Alexa Fluor 488 (1:1000) was used as secondary antibody in place of anti-mouse IRDye 800, to visualize fused myotubes. Hydroxyproline assays were performed as previously described.16Reddy G.K. Enwemeka C.S. A simplified method for the analysis of hydroxyproline in biological tissues.Clin Biochem. 1996; 29: 225-229Crossref PubMed Scopus (1080) Google Scholar Briefly, muscle samples were hydrolyzed in an autoclave at 120°C for 20 minutes. Autoclaved samples were mixed with chloramine T and allowed to incubate for 25 minutes at room temperature. Ehrlich's aldehyde reagent was added to each sample and incubated at 65°C for 20 minutes to develop the chromophore, which was quantitated against a standard curve of hydroxyproline (2 to 20 μg; Sigma-Aldrich) at 550 nmol/L. Statistical analysis was performed using analysis of variance followed by Dunnett's multiple comparison test against vehicle control or by Tukey's multiple analysis test for comparisons against all combinations of groups, using GraphPad Instat 3.1 software (GraphPad Software, La Jolla, CA). TGF-β1 mRNA levels in the skeletal muscle of mdx mice were elevated threefold to fourfold and that of TGF-β3 1.4-fold to twofold, compared with wild-type mice (Table 1). The steady-state mRNA levels of TGF-β2 were not elevated, as has been reported previously.17Zhou L. Porter J.D. Cheng G. Gong B. Hatala D.A. Merriam A.P. Zhou X. Rafael J.A. Kaminski H.J. Temporal and spatial mRNA expression patterns of TGF-beta1, 2, 3 and TbetaRI, II, III in skeletal muscles of mdx mice.Neuromuscul Disord. 2006; 16: 32-38Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Consistent with the increase in TGF-β1 mRNA levels, total TGF-β1 protein levels were elevated 3.4-fold in quadriceps muscle from mdx mice (221 ± 43 ng/μg protein), compared with wild-type mice (65 ± 10 ng/μg protein). TGF-β exists primarily as the inactive large latent complex that is proteolytically activated in vivo. Levels of active TGF-β1, however, were not detectable in the same samples.Table 1TGF-β Transcript Levels from Skeletal Muscle from mdx MiceMuscleTGF-β1TGF-β2TGF-β3Quadriceps4.0 ± 0.4⁎P < 0.05,0.95 ± 0.08Not doneGastrocnemius4.4 ± 0.7⁎P < 0.05,Not done2.1 ± 0.74⁎⁎P < 0.01, andDiaphragm3.1 ± 0.2⁎⁎P < 0.01, andNot done1.4 ± 0.15⁎⁎⁎P < 0.001, two-way t-test.Data are reported as fold increase over wild type, means ± SE; n = 6 samples per determination from mice 8 weeks of age. P < 0.05, P < 0.01, and P < 0.001, two-way t-test. Open table in a new tab Data are reported as fold increase over wild type, means ± SE; n = 6 samples per determination from mice 8 weeks of age. Body weights of all study mice were determined three times per week. No significant difference in the mean body weight was observed between mice in any treatment group or wild-type controls at any time point. The mean terminal body weights for mice in the short-term and long-term studies are given in Supplemental Table S2 (available at http://ajp.amjpathol.org). No changes in the weight of soleus or tibialis anterior muscles were observed in any groups of 2-month-old mice (data not shown). Terminal body weights at 9 months of age in the treated mice were also not different between groups. However, a 40% increase in the mean weight of the soleus and tibialis anterior muscles from mdx mice was observed, compared with those from wild-type mice at 9 months of age, and this was not affected by treatment. The mdx mice were treated with 5 mg/kg 1D11 by intraperitoneal injection three times per week. To elucidate the relationship between the pharmacokinetics and the pharmacodynamic response, the interim plasma levels of 1D11 were sampled in the first cohort of mice studied (n = 8 per group). Interim plasma levels of 1D11 were 163 ± 11 μg/mL and 254 ± 18 μg/mL, 24 hours after the 10th dose at 6 weeks and the 19th dose at 2 months of age, respectively. In a separate study, the plasma half-life of 1D11 in mice BALB/c mice was determined to be 7.9 days. The concentration of 1D11 needed to neutralize TGF-β1 by 50% in vitro, determined using an A549 cell-based assay, was found to be 0.3 μg/mL (data not shown).18Rapoza M.L. Fu D. Sendak R.A. Development of an in vitro potency assay for therapeutic TGFbeta antagonists: the A549 cell bioassay.J Immunol Methods. 2006; 316: 18-26Crossref PubMed Scopus (15) Google Scholar Thus, the concentration of 1D11 in plasma was approximately 500 times the IC50 value for the neutralization of TGF-β1, suggesting that the dose and dosing regimen chosen were adequate to elicit a maximal systemic pharmacodynamic response in mice. Administration of losartan and enalapril was via drinking water at concentrations of 600 mg/L and 200 mg/L, respectively. These doses have been shown to be efficacious in murine models of kidney disease19Yu L. Border W.A. Anderson I. McCourt M. Huang Y. Noble N.A. Combining TGF-beta inhibition and angiotensin II blockade results in enhanced antifibrotic effect.Kidney Int. 2004; 66: 1774-1784Crossref PubMed Scopus (91) Google Scholar and muscular dystrophy.7Cohn R.D. van Erp C. Habashi J.P. Soleimani A.A. Klein E.C. Lisi M.T. Gamradt M. ap Rhys C.M. Holm T.M. Loeys B.L. Ramirez F. Judge D.P. Ward C.W. Dietz H.C. Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states.Nat Med. 2007; 13 ([Erratum appeared in Nat Med 2007, 13:511]): 204-210Crossref PubMed Scopus (562) Google Scholar, 20Spurney C.F. Sali A. Guerron A.D. Iantorno M. Yu Q. Gordish-Dressman H. Rayavarapu S. van d
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