Alteration of marrow cell gene expression, protein production, and engraftment into lung by lung-derived microvesicles: a novel mechanism for phenotype modulation

Abstract

Numerous animal studies have demonstrated that adult marrow-derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that under certain conditions, radiation-injured lung induced expression of pulmonary epithelial cell-specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell-impermeable membrane. Lung-conditioned media had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell-specific RNA-filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung-derived microvesicles and suggest a novel mechanism for marrow cell-directed repair of injured tissue.

Figure 1

Summary of experimental designs. (A): Lung, WBM coculture (three experiments). (B): LCM, WBM coculture (radiation-injured LCM, five experiments; nonirradiated LCM, three experiments). (C): RNase-treated LCM, WBM coculture (two experiments). (D): Transplantation of WBM cocultured with lung or no lung or uncultured WBM into irradiated mice (one experiment). Abbreviations: LCM, lung-conditioned media; WBM, whole bone marrow.

Figure 2

Gene expression in WBM cocultured with lung. Pulmonary epithelial cell marker expression in WBM cocultured with lung for 48 Hr (A) or 7 D (B). Pulmonary epithelial cell marker expression in cocultured WBM for 48 Hr (C) or 7 D (D) (coculture established 5 D postirradiation). (E, F): Expression of other genes in cocultured WBM for 7 days. Abbreviations: CCSP, Clara cell-specific protein; D, days; Hr, hours; L-sel, L-selectin; P-sel, P-selectin; Sp-B, surfactant protein B; Sp-C, surfactant protein C; VCAM, vascular cell adhesion molecule; VEGFR, vascular endothelial growth factor receptor; WBM, whole bone marrow.

Figure 3

Cocultured marrow cell expressing prosurfactant B. (A, C): A prosurfactant protein B (Sp-B)+ (red) WBM cell (solid white arrow); Texas Red/4,6-diamidino-2-phenylindole (DAPI) filters (A) and Texas Red (C). (B): Same cell (solid red arrow) stained with Wright-Giemsa stain. Mouse lung cells were pro-Sp-B+ (open arrows) or – (asterisks). (D): Texas Red/DAPI. (E): Texas Red. Scale bar = 10 µm (two experiments).

Figure 4

Gene expression of WBM cocultured with LCM with/without RNase. (A): Sp-B expression in WBM cocultured with LCM from irradiated or nonirradiated lung for 7 days. (B): Expression of Sp-B and other genes in WBM cocultured with LCM from irradiated lung with or without RNase for 7 days (mean values of two Exps). Abbreviations: Exp, experiment; LCM, lung-conditioned media; PECAM, platelet/endothelial cell adhesion molecule; Sp-B, surfactant protein B; VCAM, vascular cell adhesion molecule; WBM, whole bone marrow.

Figure 5

Isolation and imaging of lung-derived microvesicles. (A): Electron microscopy of the ultracentrifuged lung-conditioned media (LCM) pellet demonstrates numerous 100–250-nm membrane-bound vesicles (top, scale bar = 300 nm; bottom, panels of individual vesicles, scale bar = 100 nm). (B): Fluorescence-activated cell sorting-separated green fluorescent protein+/PKH26+ events (R2). (C): Pulmonary epithelial cell marker expression in LCM and its derived components (one experiment). Abbreviations: Comp, compensated; UCF, ultracentrifuge.

Figure 6

WBM cells cultured with lung-derived microvesicles. (A): Fluorescence-activated cell sorting-separated WBM that consumed GFP+/ PKH26+ particles in culture (R2). Particles were visualized in WBM with fluorescein isothiocyanate (FITC) (C), Texas red (D), 4,6-diamidino-2-phenylindole (E), and all filters (B). Three-dimensional view reveals colocalization of GFP (G) and PKH26 (H). (F): FITC/Texas red filters. Scale bar = 10 µm (one experiment). Abbreviations: CCSP, Clara cell-specific protein; Comp, compensated; GFP, green fluorescent protein; LCM, lung-conditioned media; Sp-B, surfactant protein B; Sp-C, surfactant protein C.

Figure 7

Transplantation results for WBM cocultured with lung. (A): WBM baseline characteristics, transplantation results. (L): GFP+/ prosurfactant protein C (Sp-C)+ cells, percentage of 4,6-diamidino-2-phenylindole (DAPI)+ cells. GFP+/pro-Sp-C+ (solid and dashed white arrows), GFP+/ pro-Sp-C– (asterisk) and GFP–/pro-Sp-C+ (open arrow) cells. (D, G): Fluorescein isothiocyanate (FITC). (C, F): Texas Red. (B, E): Both filters/DAPI. (K): H&E. Three-dimensional view (H–J) reveals colocalization of GFP (J) and pro-Sp-C (I). (H): FITC/Texas Red filters. Scale bar = 20 µm. Abbreviations: GFP, green fluorescent protein; WBM, whole bone marrow.