Lung Fibroblasts Undergo Apoptosis Following Alveolarization

Abstract

In the rat lung, primary saccules are transformed into alveoli from postnatal Days 4 to 13, after which time there is a 20% reduction in the number of lung fibroblasts as the interstitial volume of the alveolar walls decreases. Our objective was to determine whether apoptosis is a factor in the observed decrease in the number of interstitial lung fibroblasts beyond Day 13. We used both histologic and flow cytometric assays to detect in lung fibroblasts the DNA fragmentation and condensation that are characteristic of apoptosis. In addition, we evaluated levels of bcl-2 and BAX messenger RNAs (mRNAs) using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Apoptotic cells were quantitated in glycol methacrylate-embedded sections of neonatal rat lungs using the terminal transferase dUTP-digoxygenin nick end-labeling (TUNEL) method. Although TUNEL-positive interstitial cells were observed in the lungs of rats ranging in age from 10 to 16 d, a dramatic increase in apoptotic cells was seen on Day 17. Although diminished in number, TUNEL-positive cells were still present on Day 28. Hoechst-stained apoptotic bodies were observed in isolated lung cells that were vimentin-positive and factor VIII-negative, which identified the apoptotic cells as fibroblasts as opposed to endothelial cells. Flow cytometric analysis of freshly isolated lung fibroblasts stained with Hoechst 33342 indicated a 24% increase in chromatin condensation in cells from 17-d versus 16-d rats. DNA fragmentation was also quantitated by flow cytometry in freshly isolated fibroblasts labeled with BODIPY-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase. The percentage of lung fibroblasts containing fragmented DNA was 51.4 +/- 13.4 in 17-d, 36.9 +/- 8.6 in 18-d, and 13.8 +/- 5.4 in 19-d rat pups. Finally, evaluation by RT-PCR indicated that on postnatal Day 17, mRNA for bcl-2, which inhibits apoptosis, was decreased to 73.5 +/- 11.4% (P < 0.001) of Day 5 controls; whereas mRNA for BAX, which enhances apoptosis, was increased to 243.0 +/- 102.0% (P < 0.001) of Day 5 values. These results demonstrate that rat lung fibroblasts undergo apoptosis after the completion of alveolarization, and suggest that this decrease in fibroblast number plays an important role in the thinning and remodeling of the alveolar walls of the lung.

Bcl-2 Is a Key Factor for Cardiac Fibroblast Resistance to Programmed Cell Death

“…In addition, lung-derived fibroblasts also lacked Bcl-2 expression. It is again in accordance with results published by others, showing that pulmonary fibroblasts lose Bcl-2 expression during lung maturation (30). Finally, our data pointing to Bcl-2 as a relevant mediator of cardiac fibroblast resistance to hypoxia are consistent with previous reports where endogenous or induced overexpression of Bcl-2 has been involved in the resistance of other cell types, mainly tumoral cells, to hypoxia (47).…”

“…Our data pointed to the blockade of cytochrome c translocation as the main mechanism of cardiac fibroblast resistance to apoptosis. Furthermore, cardiac fibroblasts expressed an easily detectable level of the antiapoptotic protein Bcl-2, which is repressed in many cell types after development (28), including dermal (29) and lung (30) fibroblasts. Here, we provide evidence supporting the notion that the maintenance of Bcl-2 expression in cardiac fibroblasts confers their resistance to mitochondria-dependent apoptosis.…”

Developmental Implications

“…In prototypical models of apoptotic cell death, as p113 decreases due to cleavage by caspase-3, p85 increases proportionately (2). Therefore, some of the p85 content we observed is consistent with previous observations that apoptosis is part of normal development in both fetal (20) and neonatal lung (31)(32)(33). We observed that in RFL p113 content decreased significantly in late gestation, but without a proportional increase in p85 ( Figures 1A and 1B).…”

“…For the entire period, Embryonic Day 16 to adulthood, the decrease in p113 was significant (n ϭ 28, R 2 ϭ 0.27, P Ͻ 0.005). The 85-kD fragment (p85) was also detected ( Figure 1A), consistent with observations that both fetal (20) and neonatal rat lung undergo apoptosis (31)(32)(33). However, the content of p85 did not increase proportionately to the decrease in p113 in late gestation ( Figure 1B); p85 content was relatively constant prenatally (n ϭ 16, R 2 ϭ 0.003) and decreased after birth through adulthood (n ϭ 16, R 2 ϭ 0.86, P Ͻ 0.001).…”

Bronchopulmonary Dysplasia: The Role of Oxidative Stress

“…During organ development, cells must pass through the various cell cycle stages in order to allow for continued remodelling. This process is essential to proper lung development (Bruce et al, 1999;Luyet et al, 2000). Consequently, the redox potential must also cycle continuously (Figure 3).…”