A potential role for sterol 27-hydroxylase in atherogenesis.
OBJECTIVE
27-hydroxycholesterol is the product of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a key enzyme in cholesterol metabolism present in most tissues of the body. 27-hydroxycholesterol increases in abundance with progression of human atherosclerotic lesions, therefore the aim of this study was to determine the pattern of sterol 27-hydroxylase gene expression in normal and diseased arteries and to identify the cell types responsible for its expression.


METHODS
Reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridisation, utilising a sterol 27-hydroxylase cDNA probe, and immunohistochemistry, utilising an antibody to sterol 27-hydroxylase, together with an antibody to smooth muscle cell alpha-actin and an antibody to CD68, a marker for macrophages, were used to study expression of 27-hydroxylase in arterial specimens. In addition, RT-PCR was used to study expression of 27-hydroxylase in cultured macrophages and smooth muscle cells.


RESULTS
Semi-quantitative RT-PCR analysis of normal and atherosclerotic human aortas showed that 27-hydroxylase is constitutively expressed in the normal artery wall, and is substantially up-regulated in atherosclerosis. RT-PCR analysis of 27-hydroxylase expression in vitro demonstrated that macrophages constitutively express high levels throughout their differentiation in culture whilst de-differentiated vascular smooth muscle cells express very low levels. In situ hybridisation revealed that in normal artery and fatty streaks, expression of mRNA for 27-hydroxylase was low in the media, but higher in intimal smooth muscle cells. The macrophages of fatty streaks expressed low or undetectable levels of 27-hydroxylase. However in advanced lesions the highest expression of 27-hydroxylase was detectable in macrophages. Immunohistochemistry demonstrated that high levels of 27-hydroxylase protein occurred in macrophages near the shoulder region of plaques, at the edge of the lipid core.


CONCLUSIONS
27-hydroxylase may constitute a protective mechanism for removing cholesterol from macrophages and smooth muscle cells. Genetic heterogeneity resulting in differences in sterol 27-hydroxylase activity between individuals may affect their ability to deal with accumulated cholesterol in the arterial intima, and hence their relative degree of predisposition to atherosclerosis.
Shanahan
C M
CM
Department of Medicine, University of Cambridge, Box 157, Addenbrooke's Hospital, Hills Road, CB2 2QQ, Cambridge, UK. cs131@mole.bio.cam.ac.uk
Carpenter
K L
KL
Cary
N R
NR
D000199:Actins / Q000276:immunology / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000368:Aged; D000906:Antibodies / Q000032:analysis; D015703:Antigens, CD / Q000276:immunology; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology; D001011:Aorta / Q000201:enzymology / Q000473:pathology; D001161:Arteriosclerosis / Q000201:enzymology* / Q000473:pathology; D015415:Biomarkers; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D053493:Cholestanetriol 26-Monooxygenase; D003331:Coronary Vessels / Q000201:enzymology / Q000473:pathology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D015870:Gene Expression; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism; D017403:In Situ Hybridization; D008264:Macrophages / Q000201:enzymology / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017539:Tunica Intima / Q000201:enzymology / Q000473:pathology; D000097382:CD68 Molecule
D000199:Actins; D000906:Antibodies; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System; D015342:DNA Probes; D018076:DNA, Complementary; D006888:Hydroxycholesterols; D012333:RNA, Messenger; D013250:Steroid Hydroxylases; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C076996:27-hydroxycholesterol; C506831:CYP27A1 protein, human