Shanahan C M, Carpenter K L, Cary N R
Department of Medicine, University of Cambridge, Box 157, Addenbrooke's Hospital, Hills Road, CB2 2QQ, Cambridge, UK.
Atherosclerosis. 2001 Feb 1;154(2):269-76. doi: 10.1016/s0021-9150(00)00473-1.
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.
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.
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.
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.
27-羟胆固醇是线粒体细胞色素P450固醇27-羟化酶的产物，该酶是人体大多数组织中胆固醇代谢的关键酶。随着人类动脉粥样硬化病变的进展，27-羟胆固醇的含量会增加，因此本研究的目的是确定固醇27-羟化酶基因在正常和病变动脉中的表达模式，并鉴定负责其表达的细胞类型。
利用固醇27-羟化酶cDNA探针进行逆转录-聚合酶链反应（RT-PCR）分析和原位杂交，以及利用针对固醇27-羟化酶的抗体、平滑肌细胞α-肌动蛋白抗体和巨噬细胞标志物CD68抗体进行免疫组织化学，用于研究动脉标本中27-羟化酶的表达。此外，RT-PCR用于研究培养的巨噬细胞和平滑肌细胞中27-羟化酶的表达。
对正常和动脉粥样硬化的人主动脉进行半定量RT-PCR分析表明，27-羟化酶在正常动脉壁中组成性表达，在动脉粥样硬化中显著上调。对体外27-羟化酶表达的RT-PCR分析表明，巨噬细胞在整个培养分化过程中组成性表达高水平，而去分化的血管平滑肌细胞表达水平极低。原位杂交显示，在正常动脉和脂肪条纹中，27-羟化酶mRNA在中膜表达较低，但在内膜平滑肌细胞中较高。脂肪条纹中的巨噬细胞表达低水平或无法检测到的27-羟化酶。然而，在晚期病变中，巨噬细胞中可检测到最高水平的27-羟化酶表达。免疫组织化学表明，在斑块肩部区域附近、脂质核心边缘的巨噬细胞中存在高水平的27-羟化酶蛋白。
27-羟化酶可能构成一种从巨噬细胞和平滑肌细胞中清除胆固醇的保护机制。个体之间因基因异质性导致固醇27-羟化酶活性存在差异，这可能会影响他们处理动脉内膜中积累胆固醇的能力，从而影响他们患动脉粥样硬化的相对易感性程度。
