Fibroblast growth factor receptors control epithelial-mesenchymal interactions necessary for alveolar elastogenesis - PubMed (original) (raw)

Fibroblast growth factor receptors control epithelial-mesenchymal interactions necessary for alveolar elastogenesis

Sorachai Srisuma et al. Am J Respir Crit Care Med. 2010.

Abstract

Rationale: The mechanisms contributing to alveolar formation are poorly understood. A better understanding of these processes will improve efforts to ameliorate lung disease of the newborn and promote alveolar repair in the adult. Previous studies have identified impaired alveogenesis in mice bearing compound mutations of fibroblast growth factor (FGF) receptors (FGFRs) 3 and 4, indicating that these receptors cooperatively promote postnatal alveolar formation.

Objectives: To determine the molecular and cellular mechanisms of FGF-mediated alveolar formation.

Methods: Compound FGFR3/FGFR4-deficient mice were assessed for temporal changes in lung growth, airspace morphometry, and genome-wide expression. Observed gene expression changes were validated using quantitative real-time RT-PCR, tissue biochemistry, histochemistry, and ELISA. Autocrine and paracrine regulatory mechanisms were investigated using isolated lung mesenchymal cells and type II pneumocytes.

Measurements and main results: Quantitative analysis of airspace ontogeny confirmed a failure of secondary crest elongation in compound mutant mice. Genome-wide expression profiling identified molecular alterations in these mice involving aberrant expression of numerous extracellular matrix molecules. Biochemical and histochemical analysis confirmed changes in elastic fiber gene expression resulted in temporal increases in elastin deposition with the loss of typical spatial restriction. No abnormalities in elastic fiber gene expression were observed in isolated mesenchymal cells, indicating that abnormal elastogenesis in compound mutant mice is not cell autonomous. Increased expression of paracrine factors, including insulin-like growth factor-1, in freshly-isolated type II pneumocytes indicated that these cells contribute to the observed pathology.

Conclusions: Epithelial/mesenchymal signaling mechanisms appear to contribute to FGFR-dependent alveolar elastogenesis and proper airspace formation.

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Figures

Figure 1.

Figure 1.

Growth characteristics of compound mutant mice. (A) Whole body weights of wild-type, FGFR3/4 compound heterozygous, and compound mutant mice during postnatal growth. Although no differences were noted between genotypes at birth, the weight of compound mutants was significantly less than age-matched control animals by 1 week of age (Postnatal Day [P] 8). (B) Lung weights of wild-type, FGFR3/4 compound heterozygous, and compound mutant mice during postnatal growth. Genotype-specific differences in lung weight were not observed before P8, whereas, at P28, lungs of compound mutants were approximately 50% of age-matched control animals. Data are presented as means (+SEM) (n = 9–12 mice in each group). *P < 0.05 with age-matched wild types. #P < 0.05 with age-matched compound heterozygotes. Open columns = wild type; gray columns = compound heterozygote; solid columns = compound mutant.

Figure 1.

Figure 1.

Growth characteristics of compound mutant mice. (A) Whole body weights of wild-type, FGFR3/4 compound heterozygous, and compound mutant mice during postnatal growth. Although no differences were noted between genotypes at birth, the weight of compound mutants was significantly less than age-matched control animals by 1 week of age (Postnatal Day [P] 8). (B) Lung weights of wild-type, FGFR3/4 compound heterozygous, and compound mutant mice during postnatal growth. Genotype-specific differences in lung weight were not observed before P8, whereas, at P28, lungs of compound mutants were approximately 50% of age-matched control animals. Data are presented as means (+SEM) (n = 9–12 mice in each group). *P < 0.05 with age-matched wild types. #P < 0.05 with age-matched compound heterozygotes. Open columns = wild type; gray columns = compound heterozygote; solid columns = compound mutant.

Figure 2.

Figure 2.

Qualitative and quantitative airspace ontogeny in compound mutant mice. (A_–_F) Shown are representative histology of lungs from control mice (A_–_C) and compound mutant mice (D_–_F) at Postnatal Day (P) 1 (A and D), P8 (B and E), and P28 (C and F). Note that all panels are presented at identical magnification. In control mouse lungs, the formation of secondary crests is evident at P8 (B), whereas minimal secondary crest formation is observed in age-matched compound mutant lungs (E). At P28, a tremendous increase in airspace complexity results from completed alveogenesis in control mice (C), whereas gross simplification resulting from expansion of primitive saccules is observed in compound mutant lungs (F). (G and H) Computer-assisted morphometry analysis quantified the mean linear distance between alveolar walls (chord length [_G_]) and mean size of individual airspaces (airspace area [_H_]) as detailed in M

ethods

. A significant decrease of chord length and airspace area in control mice between birth and P28 results from proper secondary crest initiation and elongation. Compound mutant mice at P8 and P28 display an increase in the size of airspaces over the same time period. Data are presented as means (+SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched wild types; #P < 0.05 with age-matched compound heterozygotes. (A_–_F) Scale bars = 100 μm; open columns = wild type; gray columns = compound heterozygote; solid columns = compound mutant.

Figure 2.

Figure 2.

Qualitative and quantitative airspace ontogeny in compound mutant mice. (A_–_F) Shown are representative histology of lungs from control mice (A_–_C) and compound mutant mice (D_–_F) at Postnatal Day (P) 1 (A and D), P8 (B and E), and P28 (C and F). Note that all panels are presented at identical magnification. In control mouse lungs, the formation of secondary crests is evident at P8 (B), whereas minimal secondary crest formation is observed in age-matched compound mutant lungs (E). At P28, a tremendous increase in airspace complexity results from completed alveogenesis in control mice (C), whereas gross simplification resulting from expansion of primitive saccules is observed in compound mutant lungs (F). (G and H) Computer-assisted morphometry analysis quantified the mean linear distance between alveolar walls (chord length [_G_]) and mean size of individual airspaces (airspace area [_H_]) as detailed in M

ethods

. A significant decrease of chord length and airspace area in control mice between birth and P28 results from proper secondary crest initiation and elongation. Compound mutant mice at P8 and P28 display an increase in the size of airspaces over the same time period. Data are presented as means (+SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched wild types; #P < 0.05 with age-matched compound heterozygotes. (A_–_F) Scale bars = 100 μm; open columns = wild type; gray columns = compound heterozygote; solid columns = compound mutant.

Figure 2.

Figure 2.

Qualitative and quantitative airspace ontogeny in compound mutant mice. (A_–_F) Shown are representative histology of lungs from control mice (A_–_C) and compound mutant mice (D_–_F) at Postnatal Day (P) 1 (A and D), P8 (B and E), and P28 (C and F). Note that all panels are presented at identical magnification. In control mouse lungs, the formation of secondary crests is evident at P8 (B), whereas minimal secondary crest formation is observed in age-matched compound mutant lungs (E). At P28, a tremendous increase in airspace complexity results from completed alveogenesis in control mice (C), whereas gross simplification resulting from expansion of primitive saccules is observed in compound mutant lungs (F). (G and H) Computer-assisted morphometry analysis quantified the mean linear distance between alveolar walls (chord length [_G_]) and mean size of individual airspaces (airspace area [_H_]) as detailed in M

ethods

. A significant decrease of chord length and airspace area in control mice between birth and P28 results from proper secondary crest initiation and elongation. Compound mutant mice at P8 and P28 display an increase in the size of airspaces over the same time period. Data are presented as means (+SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched wild types; #P < 0.05 with age-matched compound heterozygotes. (A_–_F) Scale bars = 100 μm; open columns = wild type; gray columns = compound heterozygote; solid columns = compound mutant.

Figure 3.

Figure 3.

Molecular classification of lung genome-wide expression profiles. Global gene expression profiles were generated from pooled whole-lung RNA samples derived from wild-type (WT), compound heterozygous (HET), and compound mutant (knockout [KO]) mice. Shown are the global relationships between gene expression profiles among all samples, defined by unsupervised hierarchical clustering with Bootstrap analysis, at each time point. No segregation among genotypes was evident at P1 (A). At both P8 (B) and P28 (C), there was clear separation of compound mutant samples with both control groups, with 100% support (n = 3 arrays in each age–genotype group, except n = 2 arrays in WT at P28).

Figure 4.

Figure 4.

Gene discovery in compound mutant lungs. Global gene expression profiles were generated from pooled whole-lung RNA samples derived from wild-type (WT), compound or double heterozygous (dHET) and compound or double mutant (dKO) mice. Differential expression for individual genes was defined separately, at P8 (left) or P28 (right), using a combination of statistical criteria, as described in M

ethods

. Normalized signal intensity measurements are shown for each of the genes identified as significantly differentially expressed (rows) among individual samples (columns), with red indicating a relatively high level of expression, and green representing a relatively low level of expression. Due to space constraints, a limited subset of genes with the greatest evidence for differential expression at P28 is presented; the complete list of genes is provided in Table E1.

Figure 5.

Figure 5.

Coordinated induction of elastic fiber genes in compound mutant lungs. Expression of genes related to elastic fiber formation, assembly, and cross-linking were assessed by quantitative real-time PCR in whole-lung RNA from wild-type, compound heterozygous, and compound mutant mice. The numbers at each bar indicate the magnitude of fold change in compound mutant lungs relative to that in age-matched compound heterozygote lungs. Significant increases in expression for all genes were noted at Postnatal Day (P) 28, coincident with airspace enlargement. Data are presented as means (±SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched control animals. LOX = lysyl oxidase; LOXL = LOX-like; MFAP = microfibrillar-associated protein. Open columns = P1; gray columns = P8; solid columns = P28.

Figure 6.

Figure 6.

Excessive and disorganized elastic fiber accumulation in compound mutant mice. (A) Elastin content, defined as the ratio of desmosine/isodesmosine to total protein, was determined in whole lung tissue derived from wild-type, compound heterozygous, and compound mutant mice. A significant increase in elastin content was observed in compound mutant lungs at all time points studied, as early as Postnatal Day (P) 8. Data are presented as means (+SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched wild types; #P < 0.05 with age-matched compound heterozygotes. Open columns = wild type; gray columns = compound heterozygote; solid columns = compound mutant. (B_–_E) Shown are representative images of control (B and C) and compound mutant (D and E) lungs stained for elastic fibers using a modified Hart's procedure. Elastic fibers are stained black with yellow counterstain. Elastic fibers are rich in vascular and airway structures prior to birth. In control mice, airspace elastic fibers accumulate at the tips of elongating secondary crests beginning before 1 week of age, and are restricted to this location in mature alveoli (red arrows in B and C). Excessive elastic fiber accumulation is apparent by P8 in compound mutant lungs, and appears throughout the wall of enlarged airspaces in the absence of secondary crests at P28 (red arrows in D and E). Relative magnification = 100× (B and D); 400× (C and E).

Figure 6.

Figure 6.

Excessive and disorganized elastic fiber accumulation in compound mutant mice. (A) Elastin content, defined as the ratio of desmosine/isodesmosine to total protein, was determined in whole lung tissue derived from wild-type, compound heterozygous, and compound mutant mice. A significant increase in elastin content was observed in compound mutant lungs at all time points studied, as early as Postnatal Day (P) 8. Data are presented as means (+SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched wild types; #P < 0.05 with age-matched compound heterozygotes. Open columns = wild type; gray columns = compound heterozygote; solid columns = compound mutant. (B_–_E) Shown are representative images of control (B and C) and compound mutant (D and E) lungs stained for elastic fibers using a modified Hart's procedure. Elastic fibers are stained black with yellow counterstain. Elastic fibers are rich in vascular and airway structures prior to birth. In control mice, airspace elastic fibers accumulate at the tips of elongating secondary crests beginning before 1 week of age, and are restricted to this location in mature alveoli (red arrows in B and C). Excessive elastic fiber accumulation is apparent by P8 in compound mutant lungs, and appears throughout the wall of enlarged airspaces in the absence of secondary crests at P28 (red arrows in D and E). Relative magnification = 100× (B and D); 400× (C and E).

Figure 7.

Figure 7.

Normal elastic fiber gene expression in isolated mouse lung fibroblasts. Elastic fiber gene expression was assessed in primary cultures of mouse lung fibroblasts isolated from wild-type and compound mutant mice. Steady-state gene expression was determined for confluent cultures grown under standard conditions (untreated) or after stimulation with the nonspecific fibroblast growth factor (FGF) receptor ligands, FGF1 and FGF2, in the presence of heparin, or with heparin alone as a control. No increases in the expression of Eln, fibrillin 1 or lysyl oxidase-like 1 were observed in untreated compound mutant cells. Furthermore, compound mutant fibroblasts demonstrated normal repression of elastic fiber gene expression upon treatment with exogenous FGFs, or activation of endogenous FGFs with heparin. Data represent the fold change in treated fibroblast relative to wild-type nontreated fibroblast controls. Data are means (+SEM) (n = 3 experiments in each group). *P < 0.05 with treatment-matched wild-type cells. Open columns = control; solid columns = compound mutant.

Figure 7.

Figure 7.

Normal elastic fiber gene expression in isolated mouse lung fibroblasts. Elastic fiber gene expression was assessed in primary cultures of mouse lung fibroblasts isolated from wild-type and compound mutant mice. Steady-state gene expression was determined for confluent cultures grown under standard conditions (untreated) or after stimulation with the nonspecific fibroblast growth factor (FGF) receptor ligands, FGF1 and FGF2, in the presence of heparin, or with heparin alone as a control. No increases in the expression of Eln, fibrillin 1 or lysyl oxidase-like 1 were observed in untreated compound mutant cells. Furthermore, compound mutant fibroblasts demonstrated normal repression of elastic fiber gene expression upon treatment with exogenous FGFs, or activation of endogenous FGFs with heparin. Data represent the fold change in treated fibroblast relative to wild-type nontreated fibroblast controls. Data are means (+SEM) (n = 3 experiments in each group). *P < 0.05 with treatment-matched wild-type cells. Open columns = control; solid columns = compound mutant.

Figure 7.

Figure 7.

Normal elastic fiber gene expression in isolated mouse lung fibroblasts. Elastic fiber gene expression was assessed in primary cultures of mouse lung fibroblasts isolated from wild-type and compound mutant mice. Steady-state gene expression was determined for confluent cultures grown under standard conditions (untreated) or after stimulation with the nonspecific fibroblast growth factor (FGF) receptor ligands, FGF1 and FGF2, in the presence of heparin, or with heparin alone as a control. No increases in the expression of Eln, fibrillin 1 or lysyl oxidase-like 1 were observed in untreated compound mutant cells. Furthermore, compound mutant fibroblasts demonstrated normal repression of elastic fiber gene expression upon treatment with exogenous FGFs, or activation of endogenous FGFs with heparin. Data represent the fold change in treated fibroblast relative to wild-type nontreated fibroblast controls. Data are means (+SEM) (n = 3 experiments in each group). *P < 0.05 with treatment-matched wild-type cells. Open columns = control; solid columns = compound mutant.

Figure 8.

Figure 8.

Abnormal alveolar epithelial cell growth factor expression in compound mutant mice. (A) Expression of growth factors identified as dysregulated in microarray data (insulin-like growth factor [IGF] 1, Igf binding protein [IGFBP] 2, Wnt5a) were assessed by quantitative real-time PCR (qPCR) in whole mouse lung RNA from wild-type and compound mutant mice at Postnatal Day (P) 8. The numbers at each bar indicated the magnitude of fold change in compound mutant lungs relative to that in age-matched control lungs. Data are presented as means (±SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched controls. (B) Expression of epithelial cell paracrine factors, IGF1, and stromal cell–derived factor (SDF) 1α, was assessed by qPCR in freshly isolated type II pneumocyte cell RNA from wild-type and compound mutant mice. Data are presented as means (+SEM) (n = 5–7 cell isolates in each group). *P < 0.05 with age-matched controls. (C) Expression of SDF1α was assessed by ELISA in whole-lung lysates from wild-type and compound mutant mice at P8 and P28. Open columns = control; solid columns = compound mutant. Data are presented as means (+SEM) (n = 8–10 lungs in each group). *P < 0.05 with age-matched wild type.

Figure 8.

Figure 8.

Abnormal alveolar epithelial cell growth factor expression in compound mutant mice. (A) Expression of growth factors identified as dysregulated in microarray data (insulin-like growth factor [IGF] 1, Igf binding protein [IGFBP] 2, Wnt5a) were assessed by quantitative real-time PCR (qPCR) in whole mouse lung RNA from wild-type and compound mutant mice at Postnatal Day (P) 8. The numbers at each bar indicated the magnitude of fold change in compound mutant lungs relative to that in age-matched control lungs. Data are presented as means (±SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched controls. (B) Expression of epithelial cell paracrine factors, IGF1, and stromal cell–derived factor (SDF) 1α, was assessed by qPCR in freshly isolated type II pneumocyte cell RNA from wild-type and compound mutant mice. Data are presented as means (+SEM) (n = 5–7 cell isolates in each group). *P < 0.05 with age-matched controls. (C) Expression of SDF1α was assessed by ELISA in whole-lung lysates from wild-type and compound mutant mice at P8 and P28. Open columns = control; solid columns = compound mutant. Data are presented as means (+SEM) (n = 8–10 lungs in each group). *P < 0.05 with age-matched wild type.

Figure 8.

Figure 8.

Abnormal alveolar epithelial cell growth factor expression in compound mutant mice. (A) Expression of growth factors identified as dysregulated in microarray data (insulin-like growth factor [IGF] 1, Igf binding protein [IGFBP] 2, Wnt5a) were assessed by quantitative real-time PCR (qPCR) in whole mouse lung RNA from wild-type and compound mutant mice at Postnatal Day (P) 8. The numbers at each bar indicated the magnitude of fold change in compound mutant lungs relative to that in age-matched control lungs. Data are presented as means (±SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched controls. (B) Expression of epithelial cell paracrine factors, IGF1, and stromal cell–derived factor (SDF) 1α, was assessed by qPCR in freshly isolated type II pneumocyte cell RNA from wild-type and compound mutant mice. Data are presented as means (+SEM) (n = 5–7 cell isolates in each group). *P < 0.05 with age-matched controls. (C) Expression of SDF1α was assessed by ELISA in whole-lung lysates from wild-type and compound mutant mice at P8 and P28. Open columns = control; solid columns = compound mutant. Data are presented as means (+SEM) (n = 8–10 lungs in each group). *P < 0.05 with age-matched wild type.

Figure 9.

Figure 9.

Increased airspace α-smooth muscle actin (αSMA) expression in compound mutant mice. The amount of elastogenic αSMA-expressing cells in Postnatal Day (P) 8 airspaces of wild-type (A) and compound mutant (C and D) lungs was assessed by immunohistochemistry. Immunoreactivity is observed as red staining with a green counterstain. Nonspecific IgG (B) was used as a staining control. Although similar staining patterns are observed between genotypes in vascular and airway structures, an increase in αSMA staining in the alveolar walls of compound mutant mice is apparent.

Figure 10.

Figure 10.

Decreased airspace cell proliferation in compound mutant mice. Immunohistochemistry for Ki67 was used to assess cell proliferation in airspaces of wild-type (A) and compound mutant (B) lungs at Postnatal Day (P) 8. Immunoreactivity is observed as brown staining for Ki67 with blue counterstained nuclei. Nonspecific IgG was used as a staining control (data not shown). A proliferative index (C) was determined by counting the percentage of Ki67-positive cells (number of Ki67-positive–staining cells relative to the total nuclei per high-power field) in the airspace walls of wild-type(top line) and compound mutant (bottom line) lungs. Although similar proliferation rates are noted between genotypes at birth, a significant reduction in proliferation is noted in P8, P14, and P21 in compound mutant mice. Data are presented as means (±SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched controls.

Figure 10.

Figure 10.

Decreased airspace cell proliferation in compound mutant mice. Immunohistochemistry for Ki67 was used to assess cell proliferation in airspaces of wild-type (A) and compound mutant (B) lungs at Postnatal Day (P) 8. Immunoreactivity is observed as brown staining for Ki67 with blue counterstained nuclei. Nonspecific IgG was used as a staining control (data not shown). A proliferative index (C) was determined by counting the percentage of Ki67-positive cells (number of Ki67-positive–staining cells relative to the total nuclei per high-power field) in the airspace walls of wild-type(top line) and compound mutant (bottom line) lungs. Although similar proliferation rates are noted between genotypes at birth, a significant reduction in proliferation is noted in P8, P14, and P21 in compound mutant mice. Data are presented as means (±SEM) (n = 7–12 mice in each group). *P < 0.05 with age-matched controls.

Figure 11.

Figure 11.

Increased elastin expression in insulin-like growth factor (IGF) 1–treated lung fibroblasts. Elastic fiber gene expression was assessed in primary cultures of mouse lung fibroblasts isolated from wild-type mice. Steady-state gene expression was determined for subconfluent explant cultures grown under standard conditions (Control) or after stimulation with IGF1 (50 ng/ml) for 14 days. Data represent the expression level in IGF1-treated relative to nontreated control animals. Data are means (+SEM) (n = 3 experiments). *P < 0.05.

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