Expression of apolipoprotein A-I in porcine brain endothelium in vitro.
Apolipoprotein (apo) A-I is the major protein component of high-density lipoproteins (HDLs), which are responsible for reverse cholesterol transport from peripheral tissues to the liver. A low level of plasma HDL is correlated with susceptibility to atherosclerosis and coronary heart disease. Mammalian apo A-I synthesis has been attributed mainly to liver and intestine. Recently, apo A-I expression has been shown in porcine brain capillaries, suggesting an independent lipid metabolism within the brain. In this study, protein synthesis and secretion were investigated in primary cultures of porcine brain microvascular endothelial cells and compared with those in large vessel endothelium. Active protein synthesis in vitro was demonstrated by metabolic labeling. Cerebral endothelial cells were shown to secrete apo A-I into the culture supernatant, whereas aortic endothelial cells were negative for apo A-I expression. Further studies of transcriptional regulation showed that cerebral endothelium was responsive to apo A-I-inducing agents, such as cholesterol, insulin, and retinoic acid, as previously shown in human hepatoma HepG2 cells. Thus, cultures of porcine cerebral endothelial cells may represent a suitable model for physiological studies of apo A-I-regulation with regard to brain lipid metabolism and blood-brain barrier function. To investigate the interspecies conservation of regulatory elements, 178 bp of the 5' flanking region of the porcine apo A-I gene was cloned using PCR techniques. Alignments of the cDNA, of the deduced apo A-I protein sequence, and of the 5' promoter region with the corresponding genomic sequences of different species show a high degree of similarity between the porcine and the primate apo A-I genes, thus indicating a similar function and possibly common regulatory mechanisms in those species. In contrast, the rodent and avian apolipoprotein A-I promoter sequences differed significantly.
Möckel
B
B
Institut für Biochemie, Technische Hochschule Darmstadt, F.R.G.
Zinke
H
H
Flach
R
R
Weiss
B
B
Weiler-Güttler
H
H
Gassen
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005091:Exons; D005455:Fluorescent Antibody Technique; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013552:Swine; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D018076:DNA, Complementary; D015345:Oligonucleotide Probes