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Am. J. Respir. Cell Mol. Biol., Volume 24, Number 5, May, 2001 591-598

Fibroblasts from Idiopathic Pulmonary Fibrosis and Normal Lungs Differ in Growth Rate, Apoptosis, and Tissue Inhibitor of Metalloproteinases Expression

Carlos Ramos, Martha Montaño, Jorge García-Alvarez, Víctor Ruiz, Bruce D. Uhal, Moises Selman, and Annie Pardo

Instituto Nacional de Enfermedades Respiratorias, Mexico DF; Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico; and Department of Physiology, Michigan State University, East Lansing, Michigan

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterized by fibroblast proliferation and extracellular matrix accumulation. However, studies on fibroblast growth rate and collagen synthesis have given contradictory results. Here we analyzed fibroblast growth rate by a formazan-based chromogenic assay; fibroblast apoptosis by in situ end labeling (ISEL) and propidium iodide staining; percent of alpha -smooth muscle actin (alpha -SMA) positive cells by fluorescence-activated cell sorter; and alpha 1-(I) collagen, transforming growth factor (TGF)-beta 1, collagenase-1, gelatinases A and B, and tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4 expression by reverse transcriptase/polymerase chain reaction in fibroblasts derived from IPF and control lungs. Growth rate was significantly lower in IPF fibroblasts compared with controls (13.3 ± 38.5% versus 294.6 ± 57%, P < 0.0001 at 13 d). Conversely, a significantly higher percentage of apoptotic cells was observed in IPF-derived fibroblasts (ISEL: 31.9 ± 7.0% versus 15.5 ± 7.6% from controls; P < 0.008). alpha -SMA analysis revealed a significantly higher percentage of myofibroblasts in IPF samples (62.8 ± 25.2% versus 14.8 ± 11.7% from controls; P < 0.01). IPF fibroblasts were characterized by an increase in pro-alpha 1-(I) collagen, TGF-beta 1, gelatinase B, and all TIMPs' gene expression, whereas collagenase-1 and gelatinase A expression showed no differences. These results suggest that fibroblasts from IPF exhibit a profibrotic secretory phenotype, with lower growth rate and increased spontaneous apoptosis.

Abbreviations: alpha -smooth muscle actin, alpha -SMA; base pair(s), bp; complementary DNA, cDNA; competitor GAPDH, cGAPDH; deoxyribonuclease, DNase; extracellular matrix, ECM; fetal bovine serum, FBS; fetal calf serum, FCS; fluorescein isothiocyanate, FITC; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; idiopathic pulmonary fibrosis, IPF; in situ end labeling, ISEL; matrix metalloproteinase, MMP; messenger RNA, mRNA; phosphate-buffered saline, PBS; polymerase chain reaction, PCR; propidium iodide, PI; ribonuclease, RNase; reverse transcriptase, RT; standard deviation, SD; transforming growth factor, TGF; tissue inhibitor of metalloproteinase, TIMP; usual interstitial pneumonia, UIP.




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