Faculty Bibliography

The enzyme cholesterol 27-hydroxylase, expressed by arterial endothelium and monocytes/macrophages, is one of the first lines of defense against the development of atherosclerosis. By catalyzing the hydroxylation of cholesterol to 27-hydroxycholesterol, which is more soluble in aqueous medium, the enzyme promotes the removal of cholesterol from the arterial wall. Prior studies have suggested that immune reactants play a role in the pathogenesis of atherosclerosis; we report here that immune reactants, IFN-gamma and immune complexes bound to C1q, but not interleukin-1 and tumor necrosis factor, diminish the expression of cholesterol 27-hydroxylase in human aortic endothelial cells, peripheral blood mononuclear cells, monocyte-derived macrophages, and the human monocytoid cell line THP-1. In addition, our studies demonstrate that immune complexes down-regulate cholesterol 27-hydroxylase only after complement fixation via interaction with the 126-kD C1qRp protein on endothelial cells and THP-1 cells. These results are consistent with the prior demonstration that IFN-gamma contributes to the pathogenesis of atherosclerosis and suggest a role for C1q receptors in the atherogenic process. Moreover, these observations suggest that one mechanism by which immune reactants contribute to the development of atherosclerosis is by down-regulating the expression of the enzymes required to maintain cholesterol homeostasis in the arterial wall

Adenosine, acting at its receptors, particularly A(2A) receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A(2A) receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective A(2A) receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A(2A) receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]et hyl)phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-alpha (100 U/ml) but markedly attenuated in cells pretreated with IFN-gamma (100 U/ml). Similarly, IL-1 and TNF-alpha treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-gamma treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-alpha-treated cells but not in control or IFN-gamma-treated cells. Both IL-1 and TNF-alpha increased A(2A) receptor mRNA and protein. In parallel with its effect on A(2A) receptor function, IFN-gamma down-regulated A(2A) receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells

BACKGROUND & AIMS: Cyp 7-/- mice lack a functional cholesterol 7alpha-hydroxylase enzyme and develop cholestasis before up-regulation of 27-hydroxycholesterol 7alpha-hydroxylase activity. Because 7alpha-hydroxylation is not the initial step in this metabolic pathway, we tested the hypothesis that cholesterol 27-hydroxylase is expressed at an earlier step and leads to the production of monohydroxy bile acids. METHODS: Polymerase chain reaction with specific oligonucleotides was used to detect messenger RNA (mRNA) coding for cholesterol 27-hydroxylase in 5-day-old normal and Cyp 7-/- mice. Gas-liquid chromatography-mass spectrometry and reverse isotope dilution were used to identify intermediates in the cholesterol 27-hydroxylase metabolic pathway. Light and electron microscopy were used to evaluate the morphological appearance of the liver. RESULTS: mRNA for cholesterol 27-hydroxylase was identified in the liver and spleen. The monohydroxy bile acids 3beta-hydroxy-5-cholenoate and 3alpha-hydroxy-5beta-cholanoate together with their precursor, 27-hydroxycholesterol, were identified in liver homogenates. Cholestasis, present focally, was manifested as dilated bile canaliculi, partial loss of microvilli, and retention of electron-dense biliary material. CONCLUSIONS: The cholesterol 27-hydroxylase metabolic pathway of bile acid synthesis is expressed in neonatal life. The absence of 7alpha-hydroxylase activities unmasks the cholestatic potential of monohydroxy bile acids. The Cyp 7-/- knockout mouse mimics cholestatic events known to occur in humans and provides a unique opportunity for studying regulatory determinants

Human endothelium obtained from both the aorta and the pulmonary artery has been evaluated for the presence of the messenger RNA coding for the expression of sterol 27-hydroxylase. Unique oligomers were designed to detect the mRNA by reverse transcription followed by the polymerase chain reaction. The amplified product was sequenced and was found to be identical to the published sequence for nucleotides 491 to 802 of the human sterol 27-hydroxylase cDNA. Northern blot analysis confirmed the presence of 27-hydroxylase mRNA in pulmonary artery and aortic endothelium. As part of these studies, enzymatic activity was assayed in cultured arterial endothelium using cholesterol as a substrate and isotope ratio gas-liquid chromatography-mass spectrometry to identify the metabolites, 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, in the medium. Localization of sterol 27-hydroxylase to vascular endothelium indicates intracellular production of the biologically active metabolite 27-hydroxycholesterol

The report of a novel cytochrome P450 enzyme in mouse hippocampus (cyp7b) with close homology to cholesterol 7 alpha-hydroxylase led us to determine the substrate specificity with respect to 27-hydroxycholesterol, known to be a potent inhibitor of cholesterol synthesis. Transfection of 293/T-cells with PcDNA3.1(+)-mcyp7b was followed by metabolism of 2.5 microM 27-hydroxycholesterol to the 7 alpha-hydroxy intermediate, cholest-5-ene,3 beta,7 alpha,27-triol, with complete loss of down-regulation of cholesterol synthesis. Addition of 5 microM and 10 microM concentrations of the triol to HepG2 and CHO cells, respectively, also did not reduce cholesterol synthesis. The contrast between the biologic effect on cholesterol synthesis by these two C27 hydroxysterols and the wide tissue distribution of both cholesterol 27-hydroxylase and hydroxysterol 7 alpha-hydroxylase implies local regulatory effects prior to their further catabolism in the liver to chenodeoxycholic and cholic acids