Atsuyuki Nakata

CD36, a Novel Receptor for Oxidized Low-Density Lipoproteins, Is Highly Expressed on Lipid-Laden Macrophages in Human Atherosclerotic Aorta

CD36 has been reported to be a receptor for oxidized LDL (Ox-LDL). In our previous study, the uptake of Ox-LDL in CD36-deficient macrophages was reduced by approximately 50% compared with that in control macrophages, suggesting an important role of CD36 as a receptor for Ox-LDL in humans. In the current study, we examined the immunohistochemical localization of CD36 in human aorta in comparison with that of scavenger receptor class A type I and type II (SRA). Cryostat sections were made from aortic tissues. For immunohistochemical staining, the following antibodies were used: (1) FA6-152, anti-CD36 antibody, and (2) SRI-2, which recognizes both type I and type II SRAs. Immunohistochemical staining for CD36 and SRA was performed using labeled streptavidin method. In macrophages scattered in aortic walls without atherosclerotic lesions, the expression of CD36 was hardly observed, whereas that of SRA was detected weakly but consistently. In contrast, in atherosclerotic lesions, macrophages around the core region showed a weak immunoreactivity to CD36 and a strong immunoreactivity to SRA. Furthermore, lipid-laden macrophages, which mainly existed in the core region, had a strongly positive immunoreactivity to CD36, but a low or moderate level of immunoreactivity to SRA. The distributions of CD36 and SRA were different from each other, and especially foamed, large-sized macrophages in atherosclerotic plaques tended to more abundantly express CD36 protein. These data demonstrate, for the first time, that the expression of both CD36 and SRA might be differentially regulated in aortic walls, and might play different roles in the formation of foam cells in atherosclerotic lesions.

Oxidized LDL Increases and Interferon-γ Decreases Expression of CD36 in Human Monocyte–Derived Macrophages

CD36 is a glycoprotein with an Mr of 88 kDa that is expressed on platelets, monocytes/macrophages, capillary endothelial cells, and adipocytes. We previously demonstrated that CD36 is involved in the uptake of oxidized low density lipoprotein (OxLDL) by using CD36-deficient macrophages (J Clin Invest. 1995;96:1859). However, the regulation of CD36 expression in human monocyte-derived macrophages has not been fully elucidated. The current study attempted to clarify the effect of OxLDL and cytokines, both of which are present in atherosclerotic lesions and may play an important role in atherogenesis, on the expression of CD36. A cell enzyme-linked immunosorbent assay and flow cytometry were used to detect CD36 protein. A ribonuclease protection assay was used to measure CD36 mRNA in human monocyte-derived macrophages. The expression of CD36 was increased during the differentiation of monocytes to macrophages. Incubation of macrophages with 25 microg/mL OxLDL for 24 hours increased the level of CD36 protein by 56% and that of CD36 mRNA by 58%. Lysophosphatidylcholine did not affect the expression of CD36. The effects of OxLDL were demonstrated in macrophages that had already differentiated to the point where CD36 expression was almost maximal. Interferon-gamma (IFN-gamma) reduced the expression of CD36 in a dose-dependent manner. A concentration of 1000 U/mL IFN-gamma significantly reduced the expression of CD36 protein by 57% and that of CD36 mRNA by 30%. In conclusion, CD36 may be important in the formation of foam cells by induction through its ligand (OxLDL). Moreover, some local factors, such as IFN-gamma, may suppress CD36 expression on macrophages in human atherosclerotic lesions.

CD36 mediates long-chain fatty acid transport in human myocardium: Complete myocardial accumulation defect of radiolabeled long-chain fatty acid analog in subjects with CD36 deficiency

Long-chain fatty acids (LCFA) are the major energy substrate for heart and their oxidation is important for achieving maximal cardiac work. However, the mechanism of uptake of LCFA by myocardium has not been clarified. We previously reported that bovine myocardial LCFA transporter has a sequence homology to human CD36. Clinically, total defect of myocardial uptake of radiolabeled long-chain fatty acid analog [123I-BMIPP: Iodine-123 15-(p-iodophenyl)-(R,S)-methylpentadecanoic acid] has been reported in some restricted cases, but the etiology has not been clarified. In the present study, we analyzed CD36 expression and CD36 gene in subjects who showed total lack of myocardial 123I-BMIPP accumulation, and, vice versa, evaluated myocardial 123I-BMIPP uptake in subjects with CD36 deficiency. Four unrelated subjects were evaluated; Two were found to have negative myocardial LCFA accumulation by 123I-BMIPP scintigraphy, after which the expression of CD36 on their platelets and monocytes was analyzed. Remaining two subjects were identified as CD36 deficiency by screening, then 123I-BMIPP scintigraphy was performed. Expression of CD36 on platelets and monocytes was measured by flow cytometric analysis. The molecular defects responsible for CD36 deficiency was detected by allele-specific restriction enzyme analysis. CD36 expression was totally deficient in all 4 subjects on both platelets and monocytes. Two subjects were homozygous for a 478C→T mutation. One was heterozygous for the dinucleotide deletion of exon V and single nucleotide insertion of exon X, and remaining one was considered to be heterozygous for the dinucleotide deletion of exon V and an unknown gene abnormality. All cases demonstrated a completely negative accumulation of myocardial LCFA despite of normal myocardial perfusion, which was evaluated by thallium scintigraphy. In addition, all cases demonstrated apparently normal hepatic LCFA accumulation Thus, these findings suggested that CD36 acts as a major myocardial specific LCFA transporter in humans.

Localization of Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Human Coronary Arteries Possible Roles of HB-EGF in the Formation of Coronary Atherosclerosis

BACKGROUND Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a newly identified member of the EGF family. Our previous in vitro studies showed that HB-EGF is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs), suggesting the role of HB-EGF in the pathogenesis of atherosclerosis. The purposes of the present study were to investigate the localization of HB-EGF in both normal and atherosclerotic human coronary arteries and to elucidate the possible roles of this growth factor in the formation of atherosclerotic lesions. METHODS AND RESULTS The immunohistochemical localization of HB-EGF, SMCs, macrophages, and EGF receptors (EGFRs) was examined in human coronary arteries obtained at autopsy. The medial SMCs of coronary arteries in neonates, infants, and children consistently synthesized HB-EGF protein. In normal adults, however, the relative number of HB-EGF-positive medial SMCs decreased gradually with age after about 30 years of age. In nonatherosclerotic coronary arteries with diffuse intimal thickening, SMCs of the intima, especially those located in the area of the medial side of the intima, were strongly positive for HB-EGF protein. In atherosclerotic plaques of coronary arteries with eccentric intimal thickening, both SMCs and macrophages in and around the core lesions, in addition to the intimal and medial SMCs located adjacent to the plaque, produced HB-EGF protein. A strong immunostaining of EGFRs was observed in these SMCs, suggesting a close association of HB-EGF and EGFR expression. CONCLUSIONS These data suggest that HB-EGF might play important roles in the migration of SMCs from the media to the intima, the proliferation of intimal SMCs, and the interaction between SMCs and macrophages in the process of coronary atherogenesis.

Immunohistochemical localization of Betacellulin, a member of epidermal growth factor family, in atherosclerotic plaques of human aorta.

Betacellulin (BTC), a new member of the EGF family, has been reported to be a potent mitogen for rat vascular smooth muscle cells (SMCs). BTC mRNA is known to be expressed in several human organs. However, the localization of BTC in human vascular tissues has not yet been clarified. We investigated whether or not BTC protein is involved in the pathogenesis of human atherosclerosis. Recombinant human BTC showed a mitogenic activity on cultured human aortic SMCs by measuring [3H]thymidine incorporation. The immunohistochemical localization of BTC, SMCs, macrophages, EGF receptors and ErbB4 was examined in autopsied human aortas. BTC was detected in both intimal and medial SMCs of the aortic wall. The percentage of BTC-positive medial SMCs in early types of atherosclerotic lesions decreased with age, but in adult, it was significantly higher in advanced types than in early types of atherosclerotic lesions. BTC-positive SMCs were predominantly localized in the medial side of the intima. Furthermore, numerous BTC-positive SMCs and macrophages were observed around the core lesion of atherosclerotic plaques. Receptors for BTC, EGF receptor and ErbB4, were expressed on SMCs, suggesting that BTC is associated with EGF receptor family-mediated signaling. BTC is produced in human aortic tissue and might play important roles in atherogenesis.

Localization of CD9, an Enhancer Protein for Proheparin-Binding Epidermal Growth Factor–Like Growth Factor, in Human Atherosclerotic Plaques: Possible Involvement of Juxtacrine Growth Mechanism on Smooth Muscle Cell Proliferation

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF family, has a potent mitogenic activity for vascular smooth muscle cells (SMCs). We previously reported that HB-EGF is involved in atherogenesis of human aorta and coronary arteries. ProHB-EGF (the membrane-anchored form of HB-EGF) has also been demonstrated to possess a mitogenic activity, which is approximately 30-fold increased when coexpressed with CD9 in mouse L cells. Thus, in the process of atherogenesis, CD9 may be involved in the proliferation of SMCs. We immunohistochemically investigated the localization of CD9 and proHB-EGF in the human aorta and coronary arteries. In normal aorta and coronary arteries, CD9 immunostaining was virtually negative, whereas proHB-EGF immunostaining was positive, especially in the arteries of babies. In contrast, in atherosclerotic lesions, some intimal SMCs were strongly positive for CD9 and proHB-EGF immunostaining. The juxtacrine growth activities of human aortic SMCs were inhibited in vitro by adding neutralization antibodies for CD9 or adding the specific inhibitor of HB-EGF. Besides, coexpressed CD9 and proHB-EGF cells markedly incorporated [(3)H]thymidine into the SMCs. CD9 is localized immunohistochemically in the SMCs of the atherosclerotic aorta and coronary arteries. CD9, when coexpressed with proHB-EGF, enhances proHB-EGF activities for SMC growth in a so-called juxtacrine manner in vitro and may be involved in atherogenesis.

Effects of pravastatin on plasma and urinary mevalonate concentrations in subjects with familial hypercholesterolaemia: a comparison of morning and evening administration

In order to determine whether there is a difference in the effect of the hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor pravastatin on cholesterol synthesis between the morning and the evening, we studied the 24-h profile of mevalonate in plasma and urine in 11 subjects with heterozygous familial hypercholesterolaemia. In study 1, eight subjects with familial hypercholesterolaemia took pravastatin (20 mg) once in the morning, and another 20-mg dose in the evening after a 1-week wash-out period. In study 2, five subjects with familial hypercholesterolaemia took pravastatin (20 mg per day) in the morning on 3 consecutive days and on 3 days in the evening after a 1 day wash-out. Plasma mevalonate concentrations were reduced at 9 h and 5 h after pravastatin administration in the morning and the evening, respectively. Urinary mevalonate excretion was significantly reduced at 4–8 h after pravastatin administration in the morning (51 vs 19 nmol · h−1) and at 4–16 h after pravastatin administration in the evening (56 vs 27 nmol · h−1). Daily urinary mevalonate excretion was equally and significantly reduced by pravastatin in the morning or evening. In conclusion, we found that morning and evening administration of pravastatin caused equal reductions in plasma and urinary mevalonate concentrations.

Acetylcholine-Induced Coronary Microvascular Vasospasm in a Patient with Angina Pectoris and Normal Coronary Angiogram: A Case Report

We demonstrated a continuous intracoronary infusion of acetylcholine-induced marked decrease of coronary blood flow estimated by intracoronary Doppler flow wire without significant epicardial coronary narrowing. This case can be called a patient with microvas cular vasospastic angina.