Thrombospondin-1-deficient mice are not protected from bleomycin-induced pulmonary fibrosis.
Thrombospondin-1 (TSP-1) is an extracellular protein critical to normal lung homeostasis, and is reported to activate latent transforming growth factor-β (TGF-β). Because active TGF-β is causally involved in lung fibrosis after bleomycin challenge, alterations in TSP-1 may be relevant to pulmonary fibrosis. We sought to determine the effects of TSP-1 deficiency on the susceptibility to bleomycin-induced pulmonary fibrosis in a murine model. Age-matched and sex-matched C57BL/6 wild-type (WT) and TSP-1-deficient mice were treated twice weekly for 4 weeks with intraperitoneal bleomycin (0.035 U/g) or PBS, and were allowed to rest 1 week before being killed. Their lungs were inflated with PBS, fixed in formalin, paraffin-embedded, and sectioned. A certified veterinary pathologist blindly scored each slide for inflammation and fibrosis. Lungs were homogenized to obtain RNA and protein for the real-time RT-PCR analysis of connective tissue growth factor (CTGF) and collagen I, and for Western blotting to detect phospho-Smad2, or total Smad2/3, respectively. In response to bleomycin treatment, measures of fibrosis and inflammation, along with CTGF and collagen I mRNA concentrations, were increased in TSP-1-deficient mice compared with WT mice. Notably, Smad 2/3 signaling was of equal strength in WT and TSP-1 knockout mice treated with bleomycin, suggesting that TSP-1 is not required for the activation of TGF-β. These results demonstrate that TSP-1 deficiency does not protect mice from systemic bleomycin challenge, and that TSP-1 deficiency is associated with increased expression of lung collagen and CTGF.
Ezzie
Michael E
ME
Department of Medicine and the Dorothy M. Davis Heart and Lung Research Institute, Columbus, OH 43210, USA.
Piper
Melissa G
MG
Montague
Christine
C
Newland
Christie A
CA
Opalek
Judy M
JM
Baran
Chris
C
Ali
Naeem
N
Brigstock
David
D
Lawler
Jack
J
Marsh
Clay B
CB
D000818:Animals; D001761:Bleomycin; D003094:Collagen / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation / Q000187:drug effects; D011014:Pneumonia / Q000150:complications / Q000378:metabolism / Q000473:pathology; D011658:Pulmonary Fibrosis / Q000150:complications / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D019700:Thrombospondin 1 / Q000172:deficiency* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D019700:Thrombospondin 1; D016212:Transforming Growth Factor beta; D001761:Bleomycin; D055513:Connective Tissue Growth Factor; D003094:Collagen