Primary culture of lung fibroblasts from hyperoxia-exposed rats and a proliferative characteristics study

Abstract

Lung fibrosis is an ultimate consequence of bronchopulmonary dysplasia (BPD) which shows the excessive proliferation of lung fibroblasts (LFs). To find a better model for studying the role of LFs in hyperoxia-induced lung fibrosis at the cellular level, we isolated LFs from the lung tissue of hyperoxia- and normoxia-exposed rat lungs on postnatal days 7, 14 and 21 for primary culture to study their proliferative behavior. In the present study, the LF predominance was > 95% in our culture method. The LFs isolated from rats exposed to hyperoxia in vivo showed significantly greater proliferation than that from normoxia-exposed rats. Flow cytometry revealed that percentage of LFs in S and G2/M stage increased, and proportion in the G0/G1 stage declined at the same time. A greater presence of myofibroblasts in LFs isolated from rats exposed to hyperoxia compared with those exposed to normoxia. In addition, elevated collagen level, transforming growth factor-β and connective tissue growth factor protein expression in conditioned medium were also found in hyperoxia LFs. These data demonstrate that hyperoxia promotes LFs proliferation, myofibroblast transdifferentiation and collagen synthesis in a time-dependent manner. The primary culture of LFs from hyperoxia-exposed rats is a feasible method for studying the pathogenesis and treatment of lung fibrosis caused by BPD at the cellular level.

Effects of the p16/cyclin D1/CDK4/Rb/E2F1 pathway on aberrant lung fibroblast proliferation in neonatal rats exposed to hyperoxia

“…Our previous study found that LFs isolated from rats exposed to hyperoxia in vivo showed significantly greater proliferation compared with those from rats exposed to normoxia. In addition, using flow cytometry, it was revealed that the percentage of LFs in the S and G 2 /M phases increased, and the proportion of LFs in the G 0 /G 1 phase decreased simultaneously (10). In another study, we confirmed that 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, inhibited hyperoxia-induced LF proliferation by restoring p16 expression (11).…”

“…Excessive LF proliferation is a key process in pulmonary fibrosis, and abnormal regulation of the cell cycle at the G 1 /S phase is necessary for hyperoxia-induced LF overproliferation (10). Our previous study found that exposure to hyperoxia weakened the G 1 /S checkpoint, enabling more LFs to pass this checkpoint and progress from the G 1 phase to the S and G 2 /M phases to complete DNA replication; this phenomenon promotes eventual LF division and proliferation (10).…”

ERK1/2 Signaling Pathway Activated by EGF Promotes Proliferation, Transdifferentiation, and Migration of Cultured Primary Newborn Rat Lung Fibroblasts

“…This results in tissue remodeling, developmental disorders, and interstitial fibrosis that may ultimately lead to the development of BPD [ 14 , 15 ]. This pathology is clearly observed in the immature lung cells of newborn rats that develop BPD [ 16 ]. Using hematoxylin–eosin staining and immunohistochemistry methods, we have found that the immature lungs exposed to long-term hyperoxia result in fibrogenesis and a significantly higher expression of p-ERK protein in both lung tissue and primary cultured fibroblasts of newborn Wistar rats [ 12 ].…”

Fibroblast growth factor 2 acts as an upstream regulator of inhibition of pulmonary fibroblast activation

“…Lungs were isolated from neonatal Sprague–Dawley rats to prepare PFs using the differential adherent properties of fibroblasts as previously described with minor modifications [ 19 ]. Briefly, neonatal rats were euthanized by CO 2 asphyxiation and the lungs were excised under sterile conditions.…”