石井 伊都子

Aggregated LDL (AgLDL) accumulates within the subendothelial space of developing atherosclerotic lesions. We were interested to learn whether endothelial cells can interact with AgLDL. Incubation of endothelial cells with AgLDL resulted in apparent cholesterol retention. Microscopic examination revealed that cholesterol retention resulted mainly from endothelial cell surface attachment of AgLDL. Little AgLDL entered endothelial cells consistent with the small amount of endothelial cell degradation of AgLDL. Although endothelial cell retention of AgLDL was inhibited by LDL, AgLDL retention was not blocked by lactoferrin, C7 anti-LDL receptor monoclonal antibody, or receptor-associated protein, suggesting that LDL receptor family members did not mediate this retention. Surface retention of AgLDL depended on microtubule function and could be regulated by the protein kinase C activator, PMA. Treatment of endothelial cells with PMA either before or during, but not after incubation with AgLDL, inhibited retention of AgLDL. Our findings show that endothelial cells can retain AgLDL but internalize and metabolize little of this AgLDL. Thus, it is unlikely that endothelial cells can transport AgLDL out of atherosclerotic lesions, but it is likely that retention of AgLDL affects endothelial function. Published by Elsevier Inc.

CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrate including endogenous steroids and some drugs that coexist at the active site. To clarify the mechanism of the effect of endogenous steroids on the drug metabolism, the interaction between substrates, nevirapine (NVP) and carbamazepine (CBZ), and endogenous steroids was investigated by theoretical calculations. When the activities of NVP 2-hydroxylation and CBZ 10,11-epoxidation by expressed CYP3A4 were measured in the presence of steroids, NVP 2-hydroxylation was found to be remarkably increased by aldosterone and inhibited by estradiol. CBZ 10,11-epoxidation was increased by androstenedione. Three-dimensional computer modeling has shown that the active site of CYP3A4 is especially large, permitting access of two substrate molecules. The interactions between NVP and aldosterone and between CBZ and androstenedione were estimated by theoretical calculations assuming the substrate and steroids to be present in the active site at the same time. It was shown that NVP or CBZ would be stably fixed close to the oxygen atom at the sixth ligand of heme by interaction with steroids, suggesting that NVP and CBZ may be hydroxylated more easily due to the interaction with steroids. Estradiol was also expected to interact with NVP via a pi/pi interaction between a benzene ring, in which the NVP hydroxylation site is located, and a benzene ring of estradiol, suggested to inhibit the reaction. From these results, interactions between the substrate and endogenous steroids in the active site may change the activity of CYP3A4.

CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrate including endogenous steroids and some drugs that coexist at the active site. To clarify the mechanism of the effect of endogenous steroids on the drug metabolism, the interaction between substrates, nevirapine (NVP) and carbamazepine (CBZ), and endogenous steroids was investigated by theoretical calculations. When the activities of NVP 2-hydroxylation and CBZ 10,11-epoxidation by expressed CYP3A4 were measured in the presence of steroids, NVP 2-hydroxylation was found to be remarkably increased by aldosterone and inhibited by estradiol. CBZ 10,11-epoxidation was increased by androstenedione. Three-dimensional computer modeling has shown that the active site of CYP3A4 is especially large, permitting access of two substrate molecules. The interactions between NVP and aldosterone and between CBZ and androstenedione were estimated by theoretical calculations assuming the substrate and steroids to be present in the active site at the same time. It was shown that NVP or CBZ would be stably fixed close to the oxygen atom at the sixth ligand of heme by interaction with steroids, suggesting that NVP and CBZ may be hydroxylated more easily due to the interaction with steroids. Estradiol was also expected to interact with NVP via a pi/pi interaction between a benzene ring, in which the NVP hydroxylation site is located, and a benzene ring of estradiol, suggested to inhibit the reaction. From these results, interactions between the substrate and endogenous steroids in the active site may change the activity of CYP3A4.

Recently, we reported that several endogenous steroids affect CYP3A4-mediated drug metabolism, using human adult liver microsomes as an enzyme source. Especially, carbamazepine (CBZ) 10,11-epoxidation is activated by androstenedione (AND). In the present studies, we investigated the effects of endogenous steroids on the activity of CBZ 10,11-epoxidation by expressed CYP3A4 and CYP3A7. When expressed CYP3A4 was used as an enzyme source, the addition of AND to the reaction mixture also caused a drastic increase in the activity of CBZ 10,11-epoxidase, and resulted in a change in the kinetics from sigmoid to Michaelis-Menten type. On the other hand, expressed CYP3A7-mediated CBZ 10,11-epoxidation was activated by sulfate conjugate steroids, such as pregnenolone 3-sulfate, 17α-hydroxypregnenolone 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEA-S), whereas the unconjugated form corresponding to these three steroids did not activate the reaction. Especially, DHEA-S was found to be a potent activator of CBZ 10,11-epoxidation by expressed CYP3A7. The kinetic character of CBZ 10,11-epoxidation by CYP3A7 is Michaelis-Menten type regardless of the presence of DHEA-S. The presence of DHEA-S caused a decrease in Km and increase in Vmax for CYP3A7-mediated CBZ 10,11-epoxidation, whereas DHEA-S 16α-hydroxylation was not affected by the coexistence of CBZ. In conclusion, CYP3A4 and CYP3A7-mediated CBZ 10,11-epoxidations are activated by different types of endogenous steroids. This is the first report regarding CYP3A7 cooperativity.

Recently, we reported that several endogenous steroids affect CYP3A4-mediated drug metabolism, using human adult liver microsomes as an enzyme source. Especially, carbamazepine (CBZ) 10,11-epoxidation is activated by androstenedione (AND). In the present studies, we investigated the effects of endogenous steroids on the activity of CBZ 10,11-epoxidation by expressed CYP3A4 and CYP3A7. When expressed CYP3A4 was used as an enzyme source, the addition of AND to the reaction mixture also caused a drastic increase in the activity of CBZ 10,11-epoxidase, and resulted in a change in the kinetics from sigmoid to Michaelis-Menten type. On the other hand, expressed CYP3A7-mediated CBZ 10,11-epoxidation was activated by sulfate conjugate steroids, such as pregnenolone 3-sulfate, 17α-hydroxypregnenolone 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEA-S), whereas the unconjugated form corresponding to these three steroids did not activate the reaction. Especially, DHEA-S was found to be a potent activator of CBZ 10,11-epoxidation by expressed CYP3A7. The kinetic character of CBZ 10,11-epoxidation by CYP3A7 is Michaelis-Menten type regardless of the presence of DHEA-S. The presence of DHEA-S caused a decrease in Km and increase in Vmax for CYP3A7-mediated CBZ 10,11-epoxidation, whereas DHEA-S 16α-hydroxylation was not affected by the coexistence of CBZ. In conclusion, CYP3A4 and CYP3A7-mediated CBZ 10,11-epoxidations are activated by different types of endogenous steroids. This is the first report regarding CYP3A7 cooperativity.

This investigation has elucidated a mechanism for development of macrophage foam cells when macrophages are incubated with native low density lipoprotein (LDL). LDL is believed to be the main source of cholesterol that accumulates in monocyte-derived macrophages within atherosclerotic plaques, but native LDL has not previously been shown to cause substantial cholesterol accumulation when incubated with macrophages. We have found that activation of human monocyte-derived macrophages with phorbol 12-myristate 13-acetate (PMA) stimulates LDL uptake and degradation and acyl-CoAcholesterol acyltransferase-mediated esterification of LDL-derived cholesterol, resulting in massive macrophage cholesterol accumulation that could exceed 400 nmol/mg of cell protein. Cholesterol accumulation showed a biphasic linear LDL concentration dependence with LDL levels as high as 4 mg/ml, similar to LDL levels in artery intima. Protein kinase C mediated the PMA-stimulated macrophage uptake of LDL because the protein kinase C inhibitors, G66983 and GF109203X, inhibited cholesterol accumulation. LDL receptors did not mediate macrophage cholesterol accumulation because accumulation occurred with reductively methylated LDL and in the presence of an anti-LDL receptor-blocking monoclonal antibody. LDL-induced cholesterol accumulation was not inhibited by antioxidants, was not accompanied by increased LDL binding to macrophages, did not depend on the apoB component of LDL, and was not down-regulated by prior cholesterol enrichment of macrophages. We have shown that the mechanism of LDL uptake by macrophages was PAU-stimulated endocytosis of LDL taken up as part of the bulk phase fluid (i.e. fluid phase endocytosis). The amount of LDL taken up with the bulk phase fluid was measured with [H-3]sucrose and accounted for a minimum. of 83% of the LDL cholesterol delivery and accumulation in PMA-activated macrophages. This novel mechanism of macrophage cholesterol accumulation shows that modification of LDL is not necessary for foam cell formation to occur. In addition, the findings direct attention to macrophage fluid phase endocytosis as a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis.

The catalytic properties of CYP3A7 in the metabolism of endogenous and exogenous substrates were compared with those of CYP3A4 and CYP3A5 using COS- 7 expressing enzymes. The highest activities of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3-sulfate (DHEA-S) 16α-hydroxylase were observed in COS-7 cells expressing CYP3A7. In contrast, the activity of testosterone 6β-hydroxylase of CYP3A7 expressed in COS-7 cells was much less than that of CYP3A4 expressed in COS-7 cells. The rate of carbamazepine 10,11-epoxidation was the greatest in COS-7 cells expressing CYP3A4, followed by CYP3A5 and CYP3A7. On the other hand, the formation of reductive metabolite of zonisamide was the highest in COS-7 cells expressing CYP3A4, followed by CYP3A7 and CYP3A5. Furthermore, the addition of triazolam resulted in a decrease in 6β-hydroxylation catalyzed by CYP3A7, but not by CYP3A4, whereas the pretreatment of microsomes with triacetyloleandomycin (TAO) resulted in a decrease in the reaction catalyzed by CYP3A4, but not by CYP3A7. Together with these results, it was suggested that CYP3A7 exerts differential catalytic properties not only in metabolism of endogenous substrates but also in drug metabolism compared to CYP3A4 and CYP3A5.