Reversal of hypercholesterolemia in low density lipoprotein receptor knockout mice by adenovirus-mediated gene transfer of the very low density lipoprotein receptor.
We have used the technique of adenovirus-mediated gene transfer to study the in vivo function of the very low density lipoprotein receptor (VLDLR) in low density lipoprotein receptor (LDLR) knockout mice. We generated a replication-defective adenovirus (AdmVLDLR) containing mouse VLDLR cDNA driven by a cytomegalovirus promoter. Transduction of cultured Hepa (mouse hepatoma) cells and LDLR-deficient CHO-ldlA7 cells in vitro by the virus led to high-level expression of immunoreactive VLDLR proteins with molecular sizes of 143 kDa and 161 kDa. Digestion of the cell extract with the enzymes neuraminidase, N-glycanase, and O-glycanase resulted in the stepwise lowering of the apparent size of the 161-kDa species toward the 143-kDa species. LDLR (-/-) mice fed a 0.2% cholesterol diet were treated with a single intravenous injection of 3 x 10(9) plaque-forming units of AdmVLDLR. Control LDLR (-/-) mice received either phosphate-buffered saline or AdLacZ, a similar adenovirus containing the LacZ cDNA instead of mVLDLR cDNA. Comparison of the plasma lipids in the 3 groups of mice indicates that in the AdmVLDL animals, total cholesterol is reduced by approximately 50% at days 4 and 9 and returned toward control values on day 21. In these animals, there was also a approximately 30% reduction in plasma apolipoprotein (apo) E accompanied by a 90% fall in apoB-100 on day 4 of treatment. By FPLC analysis, the major reduction in plasma cholesterol in the AdmVLDLR animals was accounted for by a marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL) fraction. Plasma VLDL, IDL/LDL, and HDL were isolated from the three groups of animals by ultracentrifugal flotation. In the AdmVLDLR animals, there was substantial loss (approximately 65%) of protein and cholesterol mainly in the IDL/LDL fraction on days 4 and 9. Nondenaturing gradient gel electrophoresis indicates a preferential loss of the IDL peak although the LDL peak was also reduced. When 125I-IDL was administered intravenously into animals on day 4, the AdmVLDLR animals cleared the 125I-IDL at a rate 5-10 times higher than the AdLacZ animals. We conclude that adenovirus-mediated transfer of the VLDLR gene induces high-level hepatic expression of the VLDLR and results in a reversal of the hypercholesterolemia in 0.2% cholesterol diet-fed LDLR (-/-, mice. The VLDLR overexpression appears to greatly enhance the ability of these animals to clear IDL, resulting in a marked lowering of the plasma IDL/LDL. Further testing of the use of the VLDLR gene as a therapeutic gene for the treatment of hypercholesterolemia is warranted.
Kobayashi
K
K
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Oka
K
K
Forte
T
T
Ishida
B
B
Teng
B
B
Ishimura-Oka
K
K
Nakamuta
M
M
Chan
L
L
D000256:Adenoviridae / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002791:Cholesterol, Dietary / Q000008:administration & dosage; D002853:Chromatography, Liquid; D006224:Cricetinae; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006938:Hyperlipoproteinemia Type II / Q000628:therapy*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics*; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D002791:Cholesterol, Dietary; D012333:RNA, Messenger; D011973:Receptors, LDL; C036053:VLDL receptor