Nonphysiological overexpression of low-density lipoprotein receptors causes pathological intracellular lipid accumulation and the formation of cholesterol and cholesteryl ester crystals in vitro

Recent therapeutic strategies for the treatment of familial hypercholestero lemia have been based on liver-directed gene transfer of a functional low-d ensity Lipoprotein (LDL) receptor cDNA under control of viral or strong hou sekeeping promoters. Strong viral promoters including cytomegalovirus, Rous sarcoma virus, and simian virus 40 promoters are commonly employed to reac h significant physiological effects. These promoters mediate constitutive a nd nonphysiological overexpression in every transduced cell, while the endo genous LDL receptor expression is controlled by a complex feedback mechanis m based on intracellular cholesterol concentration. To investigate intracel lular consequences of persistent LDL receptor overexpression we constructed a recombinant adenovirus encoding the human LDL receptor under control of the Rous sarcoma virus promoter. The metabolic and morphological effects of LDL receptor expression were characterized by uptake experiments with huma n hepatoma cells using fluorescent and radiolabeled LDL. We observed that l arge amounts of LDL accumulate within LDL receptor transduced cells, which eventually lead to massive intracellular lipid deposition. Kinetic experime nts with LDL-supplemented medium resulted in numerous crystal shaped struct ures in the cytosol of transduced cells as visualized by digital interferen ce contrast optic within 60 min after LDL supplementation. Thin layer chrom atography analyses of cellular lipids suggested these crystalline structure s to be dependent on intracellular cholesterol and cholesterol ester levels , Mock-infected cells showed neither cholesterol lipid accumulation nor cry stal formation. In conclusion, our data demonstrate that nonphysiological o verexpression of the LDL receptor can cause massive lipid accumulation, whi ch cannot be compensated by the hepatoma cell metabolism. This phenomenon m ay result in negative selection of LDL receptor overexpressing cells in vit ro and in vivo.