Adhesion Protein Profile of Lung-Derived Microvesicles

Abstract 4803

We have previously shown that murine bone marrow cells co-cultured opposite murine lung cells, but separated from them by a cell-impermeable membrane, express pulmonary epithelial cell-specific mRNAs, including Surfactants A-D, Aquaporin-5 and Clara Cell Specific Protein. This effect appears to be enhanced when the lungs used in co-culture are harvested from previously-irradiated mice. We have also shown that lung cells release membrane-bound particles called microvesicles which contain lung cell-specific proteins and mRNA. Microvesicles are capable of entering marrow cells in culture and inducing lung-specific mRNA and protein expression in marrow cells that have internalized them (Aliotta JM, et al, Stem Cells, 2007). Currently, it is not known how microvesicles gain entry into marrow cells and if this process involves the binding of specific adhesion proteins present on the marrow cells and/or the surface of microvesicles. Microvesicles derived from sources other than the lung have been described as expressing various adhesion proteins which facilitate their entry into target cells. In order to determine the presence of adhesion proteins on lung-derived microvesicles, we ultracentrifuged (100,000 g) cell-free conditioned media made from the lungs of C57BL/6 mice. Pelleted material was labeled with one of several directly-conjugated antibodies to an adhesion protein. Microvesicles which were positive for the adhesion protein of interest were identified by flow cytometry and quantified relative to its isotype control. Adhesion proteins that were found to be present on lung-derived microvesicles included CD29 (β1 integrin), CD41 (α2b integrin), CD49e (α5 integrin), CD49f (α6 integrin), CD51 (αv integrin), CD54 (ICAM-1), CD61 (β3 integrin), CD62L (L-selectin), CD81 (TAPA-1), CD107a (LAMP-1), CD154 (CD40 ligand), CD184 (CXCR4) and CD309 (VEGFR2). Those not present included CD9 (p24), CD11b (Mac-1), CD18 (β2 integrin), CD31 (PECAM-1), CD49d (α4 integrin), CD62P (P-selectin) and CD162 (PSGL-1). Lung-derived microvesicles isolated from mice that had been exposed to 500 centigrey total body irradiation five days prior to sacrifice were enriched with certain populations of adhesion protein positive microvesicles compared to those isolated from non-irradiated mice. These populations included ICAM-1 (78% increase), TAPA-1 (45% increase), CD40 ligand (108% increase) and VEGFR2 (136% increase) positive microvesicles. We are attempting to determine if lung-derived microvesicle entry into marrow cells is mediated by interactions with these adhesion proteins. These data indicate that adhesion proteins are present on lung-derived microvesicles. It is possible that differences in mRNA expression seen in marrow cells exposed to microvesicles derived from irradiated and non-irradiated lung in be related to qualitative and quantitative differences in those microvesicles.