Expression of fibroblast growth factor 9 in normal human lung and idiopathic pulmonary fibrosis

Abstract

The fibroblast growth factor (FGF) family of signaling ligands contributes significantly to lung development and maintenance in the adult. FGF9 is involved in control of epithelial branching and mesenchymal proliferation and expansion in developing lungs. However, its activity and expression in the normal adult lung and by epithelial and interstitial cells in fibroproliferative diseases like idiopathic pulmonary fibrosis (IPF) are unknown. Tissue samples from normal organ donor human lungs and those of a cohort of patients with mild to severe IPF were sectioned and stained for the immunolocalization of FGF9. In normal lungs, FGF9 was confined to smooth muscle surrounding airways, alveolar ducts and sacs, and blood vessels. In addition to these same sites, lungs of IPF patients expressed FGF9 in a population of myofibroblasts within fibroblastic foci, hypertrophic and hyperplastic epithelium of airways and alveoli, and smooth muscle cells surrounding vessels embedded in thickened interstitium. The results demonstrate that FGF9 protein increased in regions of active cellular hyperplasia, metaplasia, and fibrotic expansion of IPF lungs, and in isolated human lung fibroblasts treated with TGF-β1 and/or overexpressing Wnt7B. The cellular distribution and established biologic activity of FGF9 make it a potentially strong candidate for contributing to the progression of IPF.

Keywords: metaplastic epithelium; myofibroblast; smooth muscle cells.

Figure 1.

(A, B) Normal human lung tissue treated for the localization of fibroblast growth factor 9 (FGF9) by immunohistochemistry showed reactivity in smooth muscle (thin arrows) surrounding airways (Air), alveolar ducts and sacs (inset, B), and blood vessels (Ves). Airway and alveolar (Alv) epithelium had no detectable reactivity. (C) Pre-immune serum immunohistochemical controls were free of distinguishing reactivity. (D) In patients with idiopathic pulmonary fibrosis, FGF9 reactivity was found in smooth muscle associated with airways and blood vessels (thick arrowheads) and in myofibroblasts within fibroblastic foci (FF; thin arrows). Bars A, C = 200 µm; bars B, D = 100 µm.

Figure 2.

(A) Western blot analysis of recombinant human fibroblast growth factor 9 (rhFGF9) protein (0.5 ug; 23 kDa, arrow) probed with anti-FGF9 (left panel), or with anti-FGF9 pre-absorbed with an 8-fold excess of the immunizing peptide (rhFGF9) and probed with anti-FGF9 (right panel, arrow). Thermo SuperSignal Molecular Weight Markers, each of which contains an IgG-binding site, run at the stated molecular weight at the left of each panel. (B) FGF9 in human lung fibroblasts (hLF) untreated (1), CMV-LacZ (2), CMV-LacZ + TGF-β1 (3), CMV-Wnt7B (4), and CMV-Wnt7B + TGF-β1 (5). Note that, under our conditions, FGF9 runs at 40 kDa (molecular weight marker on left of panel). (C) Identical western blot as in (B), except that the anti-FGF9 was pre-absorbed with an 8-fold excess of rhFGF9.

Figure 3.

Western blot analysis of isolated human lung fibroblasts (hLF) and human airway smooth muscle cells (hSMC) cultured for 96 hr showing expression of FGF9 at the expected 40 kDa molecular weight.

Figure 4.

(A, B) Immunostaining for Wnt7B is observed in fibroblastic foci (FF) of patients with idiopathic pulmonary fibrosis, with no detectable reactivity in smooth muscle bundles (thick arrow) but light reactivity in pericytes surrounding small vessels (thick arrowheads). (C) A heat-inactivated goat anti-Wnt2 antibody substituted for the Wnt7B antibody was free of distinguishing reactivity in a parallel section, as noted in identifiable FF. (D, E) Parallel sections from the same tissue blocks depicted in (A) through (C), stained for αSMA, identified myofibroblasts within FF (thin arrows) and bundles of smooth muscle (thick arrow) or pericytes (thick arrowheads). (F) Control section in which normal ascites substituted for the primary antibody was free of distinguishing reactivity observed in (D) in nearby regions of parallel sections. FF, though metachromatic with methylene blue, and their complement of myofibroblasts (arrows) are not reactive for αSMA. (G, H) Similarly, FGF9-stained parallel sections had smooth muscle cell bundle reactivity (thick arrows), smooth muscle or pericytes surrounding small vessels (thick arrowheads), and myofibroblasts within FF (thin arrows). (I) Control section in which normal goat serum was substituted for the primary antibody was free of distinguishing reactivity seen with FGF9 antibody in nearby regions of parallel sections, as noted by absence of reactivity in these sites, FF, and their complement of myofibroblasts. Bars A, D, G = 200 µm; bars B, C, E, F, H, I = 100 µm.

Figure 5.

(A–D) Immunostaining for fibroblast growth factor 9 (FGF9) was observed in fibroblastic foci (FF) associated with alveoli (Alv) or interstitium near airways (Air) and indicated that many were associated with high levels of immunoreactive fibroblasts/myofibroblasts (thin arrows). These were often lined with FGF9-positive, hyperplastic/metaplastic epithelium (thin arrowheads), as well as dysplastic airway epithelium (double thin arrowheads). Notably in many cases, smooth muscle cells that appeared in bundles (thick arrows) or dissociated into smaller groupings or single cells (thick arrowheads) were observed in thickened and distorted interstitial regions of idiopathic pulmonary fibrosis lungs. They were identified by their unusually long lengths compared to the shorter myofibroblasts (thin arrows). Additionally, a subpopulation of interstitial blood vessels circumscribed with FGF9-reactive cells (double thick arrowheads), presumed to be either a single layer of smooth muscle or possibly large pericytes, were also αSMA-positive but not reactive for Wnt7B (data not shown). Bars A–D = 100 µm.