Dynamic regulation of platelet-derived growth factor receptor α expression in alveolar fibroblasts during realveolarization

Abstract

Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α-expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α-GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial-mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation.

Figure 1.

Lung volume and lung mechanics 21 days after left lobe pneumonectomy (PNX). The volume of total lungs and single lobes was determined by fluid displacement and corrected for body weight (43). (A) Mass-specific lung volume (ml/g). The right lung (RL) and total lung (RL+ left lung [LL]) volume of double transgenic mice without surgery (RL: 0.013 ± 0.001; RL+LL: 0.018 ± 0.002) and with sham surgery (RL: 0.014 ± 0.002; RL+LL: 0.019 ± 0.003) was determined (white column). At 21 days after left lung pneumonectomy, the volume of the right lung was determined in PNX animals (PNX: black column: 0.022 ± 0.002) in the absence of fibroblast growth factor (FGF) signaling (PNX + dominant-negative FGF receptor [dnFGFR], light gray column: 0.019 ± 0.004) and after daily injections of rosiglitazone (RZG) (PNX+RZG: dark gray column: 0.019 ± 0.005). *In all groups, mass specific volume of the right lung was significantly increased after surgery and was comparable to right and left lung mass before surgery. (B) Lung histology of SHAM, PNX, PNX+dnFGFR, and PNX+RZG was analyzed by H&E staining 21 days after pneumonectomy. SHAM and PNX animals show normal alveolar tissue. PNX+dnFGFR and PNX+RZG lungs demonstrate alveolar simplification. Scale bar, 50 μm. (C) Fractional airspace of the right lung was assessed by morphometric point intersection analysis. In SHAM-operated mice, the fractional airspace is 59.08 ± 0.25% (white column). In PNX, the fractional airspace is 59.03 ± 0.28% (black column). Compared with PNX lungs, fractional airspace in PNX+dnFGFR lungs is significantly increased to 64.99 ± 0.34% (light gray column). Compared with PNX lungs, fractional airspace in PNX+RZG lungs is significantly increased to 64.00 ± 0.24% (dark gray column). (D) Fractional airspace of the accessory lobe was assessed by morphometric point intersection analysis. In SHAM (white) and PNX (black) mice, the mean fractional airspace of the accessory lobe was not significantly changed (SHAM: 59.32 ± 0.31%; PNX: 59.52 ± 0.48%). In the presence of dnFGFR (light gray) or RZG (dark gray), the mean fractional airspace of the accessory lobe was significantly increased (PNX+dnFGFR: 65.85 ± 0.53%; PNX+RZG: 65.13 ± 0.29%). Comparisons among groups were made by one-way ANOVA using the Bonferroni’s multiple comparison test. Data are presented as mean ± SEM, with P < 0.05 considered significant (n ≥ 3 animals per group). (E and F) Lung mechanics were assessed 21 days after surgery. SHAM (white column), PNX (black column), PNX+dnFGFR (light gray column), and PNX+RZG (dark gray column). Lung mechanics in SHAM-, PNX-, and PNX+RZG-treated mice were comparable. Compared with PNX lungs, expression of dnFGFR after PNX surgery significantly increased tissue damping (E) and tissue elastance (F). Statistical differences were analyzed by Student’s t test (P < 0.05; n = 5 per group).

Figure 2.

Alveolar α smooth muscle action (α-SMA) expression is induced 5 days after PNX. Immunohistochemistry for α-SMA (A–D, red) and NG2 (E–H, red) was performed on lung sections of adult mice 7 days after SHAM (A, E), PNX (B, F), PNX+dnFGFR (C, G), and PNX+RZG (D, H). α-SMA expression was found in peribronchiolar and perivascular fibroblasts in all lungs. Nuclear PDGFR-α–GFP signal could only be found in alveolar fibroblasts (B, arrowhead and insert) and was not found in peribronchiolar or perivascular SMA-positive fibroblasts (A, C, D, arrows). Five days after surgery, α-SMA was induced in PDGFR-α–GFP–positive interstitial fibroblasts (B, arrow). NG2 expression was found in the lung interstitium in all experimental groups and was never colocalized with nuclear PDGFR-α–GFP. Low-magnification inserts in E through H provide overall distribution patterns. Scale bar, 10 μm. Red in A through D: α-SMA. Red in E through H: NG2. Blue: DAPI. Green: PDGFR-α–GFP. B = brochus; V = vessel.

Figure 3.

Primary MLFs generate contractile force in three-dimensional collagen gels. (A) Primary mouse lung fibroblasts (MLFs) were cultured in three-dimensional collagen gels over 7 days in the presence of DMSO (a, b), SU5402 (c), and 100 and 50 mM RZG (d). (B) Gel area size was determined over 7 days of culture. Control pellets significantly contracted over 7 days (Day 0: 1.9 ± 0.01 cm²; Day 3: 1.6 ± 0.08 cm²; Day 5: 0.8 ± 0.15 cm²; Day 7: 0.5 ± 0.09 cm²). In the presence of SU5402, pellets did not contract (Day 0–7: 1.9 cm² ± 0.03). In the presence of 100 mM RZG pellets did not contract (Days 0–7: 1.9 ± 0.07 cm²). In the presence of 50 mM RZG pellets contracted 10% (Day 0: 1.8 ± 0.02 cm²; Day 3: 1.8 ± 0.02 cm²; Day 5: 1.6 ± 0.11 cm²; Day 7: 1.5 ± 0.13 cm²). (C) Immunohistochemistry for α-SMA on sections of collagen pellets after 7 days in culture. Fibroblasts in control pellets stained positive for α-SMA. α-SMA was not detected in fibroblasts cultured in the presence of SU5402 or RZG.

Figure 4.

Number and level of PDGFRα expression transiently increases during compensatory lung growth. Flow cytometry analysis of primary lung fibroblast 3, 5, and 7 days after SHAM, PNX, PNX+dnFGFR, and PNX+RZG surgery. (A) Representative FACS histograms of freshly isolated lung fibroblasts 7 days after surgery. Pseudocolor dot plots. Gates identify nonhematopoietic populations as CD45^neg. Histograms of CD45^neg– and GFP^pos–positive cells gated for GFP and divided into GFP^dim and GFP^bright cells. Line graph of % GFP-positive cells in all CD45^neg cells at SHAM (time point 0), 3, 5, and 7 days after surgery in PNX (solid black line), PNX+dnFGFR (dashed green line), and PNX+RZG (dotted red line) animals. *Changes within treatment groups, analyzed by one-way ANOVA and Neuman-Keuls multiple comparison test. ^#Significant changes between treatments, analyzed by two-way ANOVA and Bonferroni multiple comparison test. Data are presented as means ± SEM, with P < 0.05 considered significant (n ≥ 3 animals). (B) Line graph of % GFP-positive fibroblasts. Compared with SHAM (9.2 ± 0.9%), the percentage of GFP-expressing fibroblasts significantly increased 3 and 5 days after PNX and significantly decreased at 7 days (Day 3: 15.6 ± 1.6%; Day 5: 13.2 ± 2.5%; Day 7: 4.9 ± 0.8%). Compared with SHAM the percentage of GFP expressing cells was significantly increased 5 and 7 days after PNX+dnFGFR intervention (Day 3: 11.0 ± 1.1%; Day 5: 15.9 ± 2.2%; Day 7: 16.0 ± 2.9%). Compared with PNX, the expression of dnFGFR delayed the increase of PDGFR-α–expressing cells and significantly blocked the down-regulation of GFP-expressing cells by 7 days. Compared with SHAM, the percentage of GFP-expressing cells was significantly decreased 7 days after PNX+RZG intervention (Day 3: 8.8 ± 0.9%; Day 5:14.3 ± 2%; Day 7: 6.8 ± 0.6%). Compared with PNX, the increase of GFP-expressing cells was delayed to Day 5 but did not interfere with GFP down-regulation by Day 7. (C) Line graph of % GFP^bright in all GFP-positive fibroblasts. Compared with SHAM (16.5 ± 1.5%), the percentage of GFP^bright–expressing cells significantly increased after PNX to 34.1 ± 6.0% on Day 3, decreased significantly to 11.5 ± 0.8% on Day 5, and reverted to 16.8 ± 3.1% on Day 7. The same significant increase at Day 3 was seen in PNX+dnFGFR (Day 3: 34.5 ± 3.2%); however, the percentage of GFP^bright cells remained significantly increased by 5 days after surgery (Day 5: 31.2 ± 4.0%) but was not significantly changed after 7 days (Day 7: 26.9 ± 3.1%). Compared with SHAM, RZG treatment did not significantly change the percentage of GFP bright cells at any time point (Day 3: 18.3 ± 2.8%; Day 5: 27.0 ± 1.7%; Day 7: 25.2 ± 4.0%). RZG significantly attenuated up-regulation by Day 3 and down-regulation by Day 5 when compared with PNX.

Figure 5.

Expression of dnFGFR and administration of RZG inhibited α-SMA induction in PDGFR-α–GFP^dim cells and increased PDGFR-α–GFP expression per cell. α-SMA and PDFGR-α–GFP expression was analyzed by flow cytometry of freshly isolated CD45^neg and GFP^pos fibroblasts 3, 5, and 7 days after PNX. (A) Representative pseudo color plots 5 days after surgery (SHAM, PNX, PNX+dnFGFR, and PNX+RZG). Plots were subdivided into quadrants representing subpopulations of dim and bright cells (y axis: α-SMA-PE; x axis: PDGFR-α–GFP). Average percentages of cell numbers were plotted in line graphs for GFP^dim/α-SMA^bright (B), GFP^dim/α-SMA^dim (C), and GFP^bright/α-SMA^dim (D). After Sham surgery, PDFGR-α–GFP–positive cells were composed of 72.4 ± 1.6% PDGFRα-GFP^dim/α-SMA^dim, 10.5 ± 0.8% GFP^dim/α-SMA^bright, 16.9 ± 1.6% GFP^bright/α-SMA^dim, and less than 1% GFP^bright/α-SMA^bright. Significant shifts of population distributions were observed 3 days after surgery, peaked at 5 days, and reverted to normal population distribution after 7 days. At 5 days after PNX, the percentage of PDGFR-α–GFP^dim/α-SMA^dim significantly decreased to 40.4 ± 1.9%, whereas the percentage of GFP^dim/α-SMA^bright increased to 44.3 ± 1.3%. The percentage of GFP^bright cells did not change significantly. Expression of dnFGFR after surgery decreased the percentage of PDGFR-α–GFP^dim/α-SMA^dim compared with SHAM-operated mice but significantly less than in PNX-treated mice (52.7 ± 5.4%). Moreover, in the presence of dnFGFR the percentage of GFP^dim/α-SMA^bright (12.2 ± 0.7%) did not increase, whereas the percentage of GFP^bright/α-SMA^dim significantly increased compared with SHAM- and PNX-treated animals (34.3 ± 5.1%). Treatment with RZG after surgery resulted in a reduction of PDGFR-α–GFP^dim/α-SMA^dim cells (57.1 ± 2.7%) and a shift of cells to GFP^bright/α-SMA^dim cells (27.2 ± 4.6%) and not GFP^dim/α-SMA^bright (11.4 ± 1.0%). Data are presented as means ± SEM, with P < 0.05 considered significant (n ≥ 3 animals). *Changes within treatment groups, which were analyzed by one-way ANOVA and Neuman-Keuls multiple comparison test. ^#Significant changes between treatments, which were analyzed by two-way ANOVA and Bonferroni multiple comparison test.

Figure 6.

Dynamic changes in gene expression after PNX. qRT-PCR was performed to determine RNA expression in primary lung fibroblasts or primary lung epithelial cells 3 and 5 days after PNX. (A) Genes expressed in structural fibroblasts: PDGFR-α, acta2, Fabp4, tenascin C. (B) Retinoic acid signaling in fibroblasts: RXR-α, RXR-β, Midkine. (C) FGFR3 and FGFR4 in fibroblasts and epithelial FGF9. (D) Wnt2a in fibroblasts and epithelial β-catenin, Shh, and Bmp4. Gene mRNA levels of SHAM (white bars), PNX (black bars), PNX+dnFGFR (light gray bars), and PNX+RZG (dark gray bars) were compared. Results were normalized to ribosomal L32 mRNA. Results are expressed as the means ± SEM of three to seven animals per group, and differences were analyzed by Students’ t test (*P < 0.05). Only genes and time points with significant changes are shown.

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 forming septum (67). Data from this study demonstrate that, in adult lungs, expression of low levels of PDGFR-α is associated with a contractile myofibroblast. Myofibroblasst induce the tension that is necessary for the budding and elongation of a newly forming septum, and a structural fibroblast supports the newly forming alveolar tissue. (B) Inhibition of FGFR2 signaling or administration of RZG increased PDGFR-α expression per cell, which blocks the differentiation of myofibroblasts and new septae fail to bud or elongate. However, the structural function of the interstitial fibroblast remains, resulting in lungs with alveolar simplification that are stiffer, as demonstrated by increased tissue damping and elastance.