Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling

Abstract

There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS.

Keywords: alveolar epithelial cells; hepatocyte growth factor; lung fibroblasts; wound closure.

Fig. 1.

Fibroblasts (FBs) promote wound closure in primary human alveolar epithelial cell (AEC) monolayers. A: phase-contrast pictures were taken of wound areas at 0 and 24 h after wounding to compare wound closure between controls (wounded AEC monolayers without coculture), wounded AEC monolayers cocultured with alveolar macrophages (AMs), or wounded AEC monolayers cocultured with FBs. B: wound closure was analyzed at 24 h after wounding. Each condition has 8 different marked wounds in each well that were analyzed for the degree of wound closure. Values were means ± SE for 3 independent experiments. *P < 0.05.

Fig. 2.

Hepatocyte growth factor (HGF) promotes wound closure in human AEC monolayers. Phase-contrast pictures were taken of marked wound areas at 0 and 24 h after wounding to compare the degree of wound closure in controls (wounded AEC monolayer without additives) and with 9 different growth factors [HGF, human recombinant basic FB growth factor (bFGF), FGF4, growth differentiation factor (GDF)-15, insulin growth factor (IGF)-1, osteoproteogrin (OPG), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), and bone morphogenetic protein 5 (BMP-5)]. The degree of wound closure was analyzed at 24 h after wounding. Each condition has 8 different marked wound areas in each well to analyze for the degree of wound closure. Values were means ± SE for 5 independent experiments. *P < 0.05.

Fig. 3.

FBs promote wound closure though HGF/c-Met signaling. A: phase-contrast pictures were taken from marked wound areas at 0 and 24 h after wounding to compare the degree of wound closure among treatment groups: wounded AEC monolayer without coculture, wounded AEC monolayer cocultured with AMs, with FBs, with FBs + PHA 665752, with FBs + anti-HGF antibody, with HGF (as a positive control) and with FBs + AG 1478. The degree of wound closure was analyzed at 24 h after wounding. Values were means ± SE for 6 independent experiments. *P < 0.05. B: mRNA levels of HGF were measured by real-time PCR. These levels were normalized to the constitutive probe cyclophilin B (CyB). Values are means ± SE. C: HGF protein concentration in coculture media at 48 h after wounding was measured by ELISA. Values were means ± SE for 6 independent experiments. D, left: human AEC monolayers with wounds were cultured without FBs, with FBs, and with FBs + PHA 665752. The AECs were harvested at 2, 6, and 24 h after wounding, and protein levels of phospho-c-Met and c-Met normalized by GAPDH were measured by immunoblotting. This is a representative blot of 3 reproducible experiments. Right: relative intensity of p-c-Met/c-Met at 24 h by Western blotting analyzed by Image J from 3 independent experiments. Values are means ± SE for 3 independent experiments. *P < 0.05.

Fig. 4.

Recombinant HGF promotes cell motility through phosphorylation of c-Met in primary human AEC monolayers. A: phase-contrast pictures were taken from marked wound areas at 0 and 24 h after wounding to compare wound closure among wounded AEC monolayer without HGF, wounded AEC monolayer with HGF, and wounded AEC monolayer with HGF + PHA 665752. The degree of wound closure was analyzed at 24 h after wounding. Values were means ± SE for 3 independent experiments. *P < 0.05. B, left: human AEC monolayers with wounds were cultured without HGF, with HGF, and with HGF + PHA 665752. The AECs were harvested at 15, 60, and 120 min after wounding, and protein levels of phospho-c-Met and c-Met normalized by GAPDH were measured by immunoblotting. This blot is representative of 1 of 4 independent but reproducible experiments. Right: relative intensity of p-c-Met/c-Met at 60 min by Western blotting analyzed by Image J from 4 independent experiments. Values are means ± SE for 3 independent experiments. *P < 0.05. C: cell motility at wound edge in human AEC monolayers with and without HGF was measured by time-lapse microscopy between at 2 h and 20 h after wounding. Values were means ± SE for 3 independent experiments. *P < 0.05.

Fig. 5.

HGF promotes lamellipodia formation and phosphorylation of focal adhesion kinase (FAK). A: human AEC monolayers with wounds were cultured with or without HGF. Immunostaining of scratched AEC monolayer at 2 h after wounding. Red, phalloidin; green, vinculin; blue, DAPI. White area encircled with gray lines indicates lamellipodia area. Magnification: ×50. B: area of lamellipodia at 2 h after wounding was analyzed with Slidebook 5.0 software. Values are means ± SE for 4 independent experiments. *P < 0.05. C: unwounded and wounded AEC monolayers cultured either with or without HGF were harvested at 30 min after wounding and addition of HGF. Protein levels of phospho-c-Met, phospho-FAK, phospho-Src, phospho-Akt, phospho-ERK1/2, and phospho-JNK were normalized by GAPDH measured by immunoblotting (representative blot of 3 independent experiments).

Fig. 6.

HGF restores the delayed wound closure in the elderly. A: phase-contrast pictures were taken from marked wound areas at 0 and 24 h after wounding to compare wound closure in young controls (wounded AEC monolayer from young donors without HGF, young with HGF, elderly controls, and elderly with HGF) (young donors n = 4, elderly donors n = 4). B: wound closure was analyzed at 24 h after wounding. Values are means ± SE for comparison between wounds in AEC monolayers from 4 young and 4 elderly donors. *P < 0.05. C: representative immunostaining of vinculin: green, vinculin; red, phalloidin; and DAPI, blue at the wound edge in scratched AEC monolayer from young (19-yr-old nonsmoker) and elderly (65-yr-old nonsmoker) at 2 h after wounding. White area encircled with gray lines indicates lamellipodia area. Magnification: × 50 (4 different donors/each group). D: areas of lamellipodia at 2 h after wounding were analyzed with Slidebook 5.0 software. Values are means ± SE for comparison between wounds in AEC monolayers from 4 young and 4 elderly donors. *P < 0.05.

Fig. 7.

HGF restores the delayed wound closure due to cyclic stretch (CS). A: phase-contrast pictures were taken from marked wound areas at 0 and 48 h after wounding to compare the degree of wound closure among 4 different groups (wounded AEC monolayers without HGF, with HGF, on CS without HGF, and on CS with HGF). B: degree of wound closure was analyzed at 48 h after wounding. Values are means ± SE for 4 independent experiments. *P < 0.05.