Lipoprotein (a) Internalization by RAW 264.7 Cells is Associated with Morphological Changes and Accumulation of Lipids Detectable by Two-photon Scanning Microscopy
Maria Valentina Pasquetto
Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy
Alice Santonastaso
Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy
Alessandra Tomaselli
Department of Electrical, Computer and Biomedical Engineering, University of Pavia, Pavia, Italy
Patrizia Vaghi
Centro Grandi Strumenti, University of Pavia, Pavia, Italy
Elton Hasani
Department of Electrical, Computer and Biomedical Engineering, University of Pavia, Pavia, Italy
Luca Tartara
Department of Electrical, Computer and Biomedical Engineering, University of Pavia, Pavia, Italy
Claudia Scotti *
Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy
*Author to whom correspondence should be addressed.
Abstract
Lipoprotein (a) [Lp(a)] is an independent cardiovascular risk factor and its pathogenic mechanism is not completely clear. Lp(a) has been detected in atherosclerotic plaques and macrophages are one of the major cell types involved in atherogenesis. In order to characterize internalization of Lp(a) by RAW 264.7 cells, an established model of mouse macrophages, cells were treated with Lp(a) samples purified from plasma by affinity chromatography, and evaluated by western blotting. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a tetrazolium salt, assay, Lp(a) was found to be non-cytotoxic for the cells at all the concentrations tested (0.0165-1.65 mg/ml). An ELISA performed on the lysate of Lp(a)-treated cells allowed to identify the highest intracellular accumulation of Lp(a) at 72 h treatment. Already at 24 h, however, important morphological alterations were detected upon Oil red and Nile red staining. A three-dimensional reconstruction obtained by two-photon scanning microscopy of the intracellular distribution of Nile red stained structures in treated cells shows preferential uptake of lipids in extra-nuclear regions. These data are useful to clarify the temporal and spacial aspects of intracellular accumulation of Lp(a) in RAW 264.7 cells and pose new bases for future studies on intracellular Lp(a) accumulation.
Keywords: Lipoprotein (a), macrophages, THP-1, internalization, two-photon microscopy, 3D reconstruction.