In this study, we have shown that PDGFR-α expression changes dynamically after PNX and that a subpopulation of PDGFR-α–expressing fibroblasts induces α-SMA and differentiate into contractile myofibroblasts. Evidence that FGFR2 and PPAR-γ signaling regulate myofibroblast differentiation was demonstrated in vivo and in vitro. In vivo, α-SMA induction during compensatory regrowth was inhibited by expression of dnFGFR or administration of RZG, whereas treatment with a FGF inhibitor or RZG inhibited contraction of collagen pellets and expression of α-SMA in vitro. Our data suggest that RZG delays activation of the PDGFR-α–positive fibroblasts and that dnFGFR2 promotes expansion of PDGFR-α–positive fibroblasts and differentiation into structural fibroblasts with no contractile function (). Although the relationship between PDGFR-α expression and the myofibroblast phenotype is not clear, our data demonstrate that FGF and PPAR-γ pathways affect the dynamic changes of the myofibroblast and structural fibroblast phenotypes in the alveolar wall during compensatory lung growth. Changes in these fibroblast phenotypes affect septation, and, as a consequence, fractional airspace was increased 21 days after surgery.
Figure 7. Hypothetical model of molecular and cellular changes during reseptation. (A) PDGFR-α–positive cells increase in numbers during realveolarization. Fibroblasts with elevated PDGFR-α expression are located at the ridge of the newly (more ...)
Alveolar septation has been studied during normal development, but most studies on fibroblast differentiation during alveolarization have been descriptive (67
). In PDGFA knockout mice, myofibroblast progenitors fail to spread, which demonstrates an important role of the PDGFR-α–positive cell during alveolar septation (31
). Other researchers have shown that the number of PDGFR-α–GFP–positive cells increases during postnatal alveolarization and that PDGFR-α–GFP expression levels change as alveolarization progresses (50
). In the lung, the lipofibroblast has been implicated in lung development and homeostasis (68
). During lung development, lipofibroblasts reside at the base of the secondary septum and are associated with dim levels of PDGFR-α–GFP, whereas non–lipid-containing fibroblasts express bright levels of PDGFR-α–GFP and are located at the alveolar entry ring (67
). Based on developmental studies, it is well accepted that PDGFA/PDGFR-α signaling and the interstitial fibroblast are important for elastin and extracellular matrix synthesis (31
), which would define a structural phenotype of the PDGF-Rα–positive fibroblast. In this study, we demonstrate that PDGFR-α expression was transiently increased after PNX and that expression of dnFGFR further increased PDGFR-α expression and blocked its down-regulation 7 days after surgery. Although α-SMA expression was inhibited by dnFGFR and RZG, expression of Fabp4 and tenascin C was still increased supporting the hypothesis that the structural role of the interstitial fibroblast is independent of the contractile role. Likewise, an increase in tissue damping and elastance suggest changes of extracellular matrix composition and/or contractile elements in the lung parenchyma as a result of the increase in structural fibroblasts.
Based on published data and our own work, we developed a hypothetical model of reseptation (). Regeneration is induced by mechanical stretch and results in increased numbers of PDGFR-α-positive fibroblasts with dual function: contraction and synthesis of structural proteins. Low levels of PDGFR-α promote the contractile function, and high levels of PDGFR-α promote the structural function. This study demonstrates that loss of the contractile fibroblast results in failure to bud and elongate new septae and increases the number of PDGFR-α–GFP bright cells. Previous studies demonstrating that RZG reversed perinatal hyperoxia and induced myogenic markers and subsequently alveolar simplification demonstrate that too many contractile fibroblasts result in impaired alveologenesis (30
). Recent PNX studies, using 9-month-old mice, demonstrate that although α-SMA protein was transiently increased, fibroblasts had reduced capability for collagen deposition, which also results in partial loss of regenerative capacity (72
). Taken together, these studies suggest that a balance of contractile and structural function is necessary for “normal” regeneration and that a shift toward one or the other results in a delay or block of regeneration.
During lung development, α-SMA is transiently expressed in interstitial fibroblasts, and PDGFR-α–positive cells are more likely to express α-SMA during postnatal alveolarization (19
). Postnatal alveolar myofibroblasts with higher levels of PDGFR-α expression were more resistant to apoptosis, and cytosolic α-SMA expression was associated with bright PDGFR-α–GFP nuclei in PN12 lung fibroblasts (50
). However, in adult lungs, higher levels of PDGFR-α and subsequent decreased apoptosis pose a profibrotic risk. It would therefore be beneficial to maintain low levels of PDGFR-α during regeneration, transiently induce a contractile fibroblast, and then switch to a structural fibroblast. This hypothesis is supported by our findings, which demonstrate that in adult mice α-SMA is rapidly and transiently induced in PDGFR-α–GFPdim
The retinoic acid receptors, retinoic X receptors (RXRs), and PPARγ play an important role in postnatal alveolarization, and retinoic acid treatment in adult animals can stimulate reseptation (37
). Studies have identified binding sites for PPAR-γ/RXR heterodimers in genes involved in fatty acid and lipid metabolism, confirming the role of PPARγ as the master transcriptional regulator of adipogenesis (81
). Real-time PCR analyses demonstrated that RXR-α, RXR-β, and PPAR-γ were down-regulated 3 days after surgery, whereas RZG-treated fibroblasts had increased RXR-α and RXR-β expression. These data suggest that activation of PPAR-γ by RZG stimulates PPAR/RXR heterodimerization, antagonizing myofibroblast differentiation.
During lung development, constitutive inactivation of FGFR3/FGFR4 in all cells induces interstitial α-SMA expression and blocks septation (61
). Real-time PCR analysis on lung fibroblasts after PNX demonstrated that FGFR3 expression was down-regulated before myofibroblast differentiation, suggesting that FGFR3 signaling antagonizes α-SMA, which is consistent with the FGFR3 knock-out data. Treatment with RZG prevented down-regulation of FGFR3 and increased FGFR4 expression, suggesting that FGFR3 and FGFR4 antagonize α-SMA differentiation.
FGF signaling regulates complex epithelial and mesenchymal interactions. During lung development, misexpression of the epithelial FGFR2b isoform in mesenchymal cells established a FGFR2b-FGF10 autocrine feedback loop that inhibited myofibroblast differentiation and reduced fibronectin and elastin deposition (82
). In our experiments, conditional expression of a dnFGFR2b by the epithelium using the Sftpc-rtTA also blocked α-SMA expression in fibroblasts, suggesting that disturbed FGFR2b-FGF10 signaling to the epithelium resulted in an indirect effect on fibroblast differentiation (36
). When we expressed dnFGFR or administered RZG, we did not detect changes in mesenchymal FGF10 expression but detected doubling of epithelial FGF9 expression, which supports the hypothesis that epithelial FGFR2 signaling effects expression of other epithelial genes, which then directly or indirectly regulate fibroblast phenotypes. This hypothesis is supported by the findings that epithelial Shh and Bmp4 expression and mesenchymal Wnt2a expression are up-regulated. During the early pseudoglandular stages of lung development, an epithelial FGF9-mesenchymal FGFR-WNT signaling pathway regulates mesenchyme development (84
). In this study, we find evidence that a FGF9-WNT signaling pathway regulates fibroblast phenotypes during compensatory lung growth. Moreover, we hypothesized that dnFGFR and RZG promote a structural lipofibroblast phenotype over a contractile myofibroblast, which is supported by the findings that inactivation of FGFR2 signaling in mouse adipocytes resulted in increased levels of FGF9 expression and adipocyte hypertrophy (86
Although this study did not identify the progenitor of the PDGFR-α cell, it suggests that PDGFR-α–expressing cells are the progenitor cells of the contractile and structural fibroblasts and that these cells dynamically change their phenotype during compensatory lung growth. The field of mesenchymal cells in the lung is limited by the use of generic markers for fibroblast subpopulations. Developing additional specific markers will facilitate dissection of different mesenchymal subpopulations and allow us to follow rapid changes in fibroblast phenotypes during lung injury and repair. Identifying the molecular regulation of changes in fibroblast phenotypes will help to understand how the lung repairs itself correctly without inducing fibroproliferation or excessive deposition of extracellular matrix that may result in chronic lung disease.