Hiroharu Kataoka

Expression of Lectinlike Oxidized Low-Density Lipoprotein Receptor-1 in Human Atherosclerotic Lesions

BACKGROUND Oxidized LDL (Ox-LDL) seems to play key roles in atherogenesis. Lectinlike Ox-LDL receptor-1 (LOX-1) is a recently identified cell-surface receptor for Ox-LDL. The relationship of this novel receptor for Ox-LDL to atherogenesis, however, has not yet been clarified. In this study, we explored the expression of LOX-1 in the atherosclerotic lesions of human carotid arteries. METHODS AND RESULTS Using carotid endarterectomy specimens obtained from 21 patients and 2 samples of normal human aortas, we examined LOX-1 expression by reverse transcription-polymerase chain reaction and immunohistochemistry. In aortas without atherosclerosis, LOX-1 expression was undetectable by immunohistochemistry and negligible by reverse transcription-polymerase chain reaction. In carotid arteries, luminal endothelial cells covering early atherosclerotic lesions were more frequently positive for LOX-1 expression than those in advanced atherosclerotic lesions. Endothelial cells in the intimal neovasculature of advanced lesions also expressed LOX-1. In addition, macrophages and smooth muscle cells in the intima of advanced atherosclerotic plaques were positive for LOX-1 expression. CONCLUSIONS LOX-1 may play important roles in Ox-LDL uptake and subsequent functional alteration in the luminal endothelium in early atherosclerotic lesions and in intimal neovascular endothelial cells in advanced plaques. Furthermore, LOX-1 may also be involved in Ox-LDL uptake and subsequent foam cell transformation in macrophages and smooth muscle cells in the atherosclerotic intima.

Molecular Cloning of a Novel Scavenger Receptor for Oxidized Low Density Lipoprotein, SR-PSOX, on Macrophages

Receptor-mediated endocytosis of oxidized low density lipoprotein (OxLDL) by macrophages has been implicated in foam cell transformation in the process of atherogenesis. Although several scavenger receptor molecules, including class A scavenger receptors and CD36, have been identified as OxLDL receptors on macrophages, additional molecules on macrophages may also be involved in the recognition of OxLDL. From a cDNA library of phorbol 12-myristate 13-acetate-stimulated THP-1 cells, we isolated a cDNA encoding a novel protein designated SR-PSOX (scavenger receptor that bindsphosphatidylserine and oxidized lipoprotein), which acts as a receptor for OxLDL. SR-PSOX was a type I membrane protein consisting of 254 amino acids, expression of which was shown on human and murine macrophages with a molecular mass of 30 kDa. SR-PSOX could specifically bind with high affinity, internalize, and degrade OxLDL. The recognition of OxLDL was blocked by polyinosinic acid and dextran sulfate but not by acetylated low density lipoprotein. Taken together, SR-PSOX is a novel class of molecule belonging to the scavenger receptor family, which may play important roles in pathophysiology including atherogenesis.

Macrophage-Derived Matrix Metalloproteinase-2 and -9 Promote the Progression of Cerebral Aneurysms in Rats

Background and Purpose— Mechanisms of initiation, progression and rupture of cerebral aneurysms have not yet been fully understood despite its clinical significance. Matrix metalloproteinases (MMPs) are a family of proteinases which are involved in the remodeling of vascular walls. In the present study, we investigated the significance of MMPs in the progression of cerebral aneurysms. Methods— Cerebral aneurysms were experimentally induced in 7-week-old male Sprague-Dawley rats. MMP-2 and MMP-9 expression was examined by immunohistochemistry and RT-PCR. Gelatinase activity in aneurysmal walls was assessed by in situ zymography. A selective inhibitor for MMP-2, -9 and -12, tolylsam, was used to examine the effect of inhibition of MMP-2 and MMP-9. Results— Macrophages infiltrated in arterial walls of experimentally induced rat cerebral aneurysms and expressed MMP-2 and -9. Macrophage infiltration and MMP expression was increased with the progression of aneurysms. Gelatinase activity attributable to MMP-2 and MMP-9 increased in arterial walls of rat cerebral aneurysms. Furthermore, tolylsam reduced the ratio of advanced aneurysms in our rat model. Conclusions— These data suggest that macrophage-derived MMP-2 and -9 may play an important role in the progression of cerebral aneurysms. The findings of this study will shed a new light into the pathogenesis of cerebral aneurysms and highlight the importance of inflammatory response causing the degeneration of extracellular matrix in the process of this disease.

Increased Expression of Lectinlike Oxidized Low Density Lipoprotein Receptor-1 in Initial Atherosclerotic Lesions of Watanabe Heritable Hyperlipidemic Rabbits

A novel lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) was recently identified in bovine aortic endothelial cells. It is strongly suggested to have a potential role in the initiation and development of atherosclerosis. In this study, we have isolated cDNA clones encoding the rabbit homologue of LOX-1 by screening a rabbit placenta cDNA library. In amino acid sequence and domain structure organization, the rabbit LOX-1 is highly conserved with the human counterpart. Transfection of rabbit LOX-1 cDNA to HEK-293 cells confers on them the activity to bind and internalize oxidized low density lipoprotein. Rabbit LOX-1 was identified as a 45-kDa protein by Western blot analysis with a specific monoclonal antibody. Notably, analyses by reverse transcription-polymerase chain reaction and Western blot revealed that LOX-1 was accumulated in 8-week-old Watanabe heritable hyperlipidemic rabbit aortas compared with normal rabbit aortas. Immunostaining confirmed that the augmented expression of LOX-1 was primarily localized within the intima at the earliest stages of atherogenesis. The most prominent staining was in the endothelial cells of lesions. Furthermore, the distinctive staining of LOX-1 was identified in the endothelium of non-lesion areas of Watanabe heritable hyperlipidemic rabbit aortas. Taken together, these findings support the possibility that LOX-1 might be involved in the initiation of atherosclerosis.

Oxidized LDL Modulates Bax/Bcl-2 Through the Lectinlike Ox-LDL Receptor-1 in Vascular Smooth Muscle Cells

Oxidized low density lipoprotein (Ox-LDL) induces apoptosis in vascular smooth muscle cells (VSMCs), which may increase atherosclerotic plaque instability. In this study, we examined the molecular mechanisms causing the Ox-LDL–induced apoptosis in VSMCs, especially focusing on the involvement of Bax/Bcl-2 and the lectinlike Ox-LDL receptor-1 (LOX-1). In cultured bovine aortic smooth muscle cells (BASMCs), Ox-LDL at high concentrations (>60 &mgr;g/mL) induced cell death as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. DNA fragmentation was increased in BASMCs treated with high concentrations of Ox-LDL, indicating that the Ox-LDL–induced cell death in VSMCs was apoptosis. Ox-LDL upregulated LOX-1 expression through phosphorylation of extracellular signal–regulated kinase in BASMCs, and a neutralizing anti–LOX-1 monoclonal antibody, which can block LOX-1–mediated cellular uptake of Ox-LDL, prevented the Ox-LDL–induced apoptosis in BASMCs. This antibody also suppressed the increase in the Bax to Bcl-2 ratio induced by Ox-LDL in BASMCs. Furthermore, LOX-1 expression was well colocalized with Bax expression in the rupture-prone shoulder areas of human atherosclerotic plaques in vivo. LOX-1 may play an important role in Ox-LDL–induced apoptosis in VSMCs by modulating the Bax to Bcl-2 ratio. These molecular mechanisms may be involved in destabilization and rupture of atherosclerotic plaques.

Expression of lectin-like oxidized low density lipoprotein receptor-1 in human and murine macrophages: upregulated expression by TNF-α

Uptake of oxidized low density lipoprotein (Ox‐LDL) and subsequent foam cell transformation have been implicated in early atherogenesis. Although multiple molecules, including class A and B scavenger receptors, have been identified as Ox‐LDL receptors, additional receptors may also be involved in this process. Here, we provide evidence that lectin‐like Ox‐LDL receptor‐1 (LOX‐1), a novel Ox‐LDL receptor initially identified in vascular endothelial cells, is also expressed in macrophages in humans and mice. Expression of LOX‐1 can be induced after macrophage‐like differentiation in vitro in human peripheral blood monocytes and the related cell line THP‐1 cells. Furthermore, LOX‐1 expression can also be detected in resident peritoneal macrophages, and can be upregulated by an inflammatory cytokine TNF‐α. These results suggest that LOX‐1 in macrophages may play an important role in Ox‐LDL uptake and subsequent foam cell formation in this cell type.

NF-κB Is a Key Mediator of Cerebral Aneurysm Formation

Background— Subarachnoid hemorrhage caused by the rupture of cerebral aneurysm (CA) remains a life-threatening disease despite recent diagnostic and therapeutic advancements. Recent studies strongly suggest the active participation of macrophage-mediated chronic inflammatory response in the pathogenesis of CA. We examined the role of nuclear factor-&kgr;B (NF-&kgr;B) in the pathogenesis of CA formation in this study. Methods and Results— In experimentally induced CAs in rats, NF-&kgr;B was activated in cerebral arterial walls in the early stage of aneurysm formation with upregulated expression of downstream genes. NF-&kgr;B p50 subunit–deficient mice showed a decreased incidence of CA formation with less macrophage infiltration into the arterial wall. NF-&kgr;B decoy oligodeoxynucleotide also prevented CA formation when it was administered at the early stage of aneurysm formation in rats. Macrophage infiltration and expression of downstream genes were dramatically inhibited by NF-&kgr;B decoy oligodeoxynucleotide. In human CA walls, NF-&kgr;B also was activated, especially in the intima. Conclusions— Our data indicate that NF-&kgr;B plays a crucial role as a key regulator in the initiation of CA development by inducing some inflammatory genes related to macrophage recruitment and activation. NF-&kgr;B may represent a therapeutic target of a novel medical treatment for CA.

Identification of Soluble Forms of Lectin-Like Oxidized LDL Receptor-1

Lectin-like oxidized LDL receptor-1 (LOX-1) is a type II membrane protein belonging to the C-type lectin family molecules, which can act as a cell-surface endocytosis receptor for atherogenic oxidized LDL. In this study, we show that soluble forms of LOX-1 are present in conditioned media of cultured bovine aortic endothelial cells (BAECs) and CHO-K1 cells stably transfected with LOX-1 cDNA. Immunoblot analysis of conditioned media from TNF-alpha-activated BAECs and CHO-K1 cells stably expressing LOX-1 revealed that soluble LOX-1 has an approximate molecular mass of 35 kDa. In TNF-alpha-activated BAECs, cell-surface expression of LOX-1 precedes soluble LOX-1 production. Cell-surface biotinylation followed by immunoprecipitation and immunoblotting showed that soluble LOX-1 in cell-conditioned media is derived from LOX-1 expressed on the cell surface. Production of soluble LOX-1 was inhibited by PMSF, suggesting that PMSF-sensitive proteases may be involved in this process. Purification of soluble LOX-1 by high-performance liquid chromatography and N-terminal amino acid sequencing of soluble LOX-1 identified the 2 cleavage sites between Arg(86)-Ser(87) and Lys(89)-Ser(90), which were located in the membrane proximal extracellular domain of LOX-1. The data demonstrate that cell-surface LOX-1 can be cleaved at 2 different sites and transformed into soluble forms. Further studies may explore therapeutic and diagnostic applications of soluble LOX-1 in atherosclerotic diseases.

Impact of Monocyte Chemoattractant Protein-1 Deficiency on Cerebral Aneurysm Formation

Background and Purpose— Recent studies have suggested that chronic inflammation actively participates in cerebral aneurysm (CA) formation. Macrophages accumulate in CA walls and express proinflammatory genes promoting CA progression, but the molecular mechanisms of monocyte/macrophage recruitment into CA walls remain to be elucidated. Methods— Monocyte chemoattractant protein-1 (MCP-1) expression in experimentally induced CAs was assessed by immunohistochemistry and Western blotting. The role of MCP-1 in CA formation was examined by MCP-1−/− mice and a plasmid DNA encoding a dominant negative mutant of MCP-1 (7ND). MCP-1 expression in human CAs was examined by immunohistochemistry. Results— MCP-1 expression was upregulated in aneurysmal walls at the early stage of CA formation. MCP-1−/− mice exhibited a significant decrease of CA formation and macrophage accumulation with decreased expression of matrix metalloproteinase-2, -9, and inducible nitric oxide synthase. Immunohistochemistry for the DNA binding form of nuclear factor-kappa B showed nuclear factor-kappa B activation in MCP-1-expressing cells. Blockade of MCP-1 activity by 7ND resulted in the inhibition of CA progression in rats. In human CAs, MCP-1 was also expressed in CA walls. Conclusions— These data suggest that MCP-1 plays a crucial role in CA formation as a major chemoattractant for monocyte/macrophage. MCP-1 expression in CA walls is induced through nuclear factor-kappa B activation. MCP-1 may be a novel therapeutic target of medical treatment preventing CA progression.

Simvastatin Suppresses the Progression of Experimentally Induced Cerebral Aneurysms in Rats

Background and Purpose— The pathophysiology of cerebral aneurysms (CAs) is linked to chronic inflammation and degradation of extracellular matrix in vascular walls. Because statins have protective effects on various vascular diseases independent of their lipid-lowering effects, we investigated the effect of simvastatin on CA progression. Methods— CAs were induced in Sprague-Dawley rats with or without oral administration of simvastatin. The size and media thickness of CAs was evaluated 3 months after aneurysm induction. Expression of macrophage chemoattractant protein-1, vascular cell adhesion molecule-1, endothelial nitric oxide synthase, interleukin-1&bgr;, inducible nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 in aneurysmal walls was examined by reverse transcriptase–polymerase chain reaction and immunohistochemistry. To examine whether simvastatin has a suppressive effect on preexisting CAs, simvastatin administration started at 1 month after aneurysm induction. Results— Rats treated with simvastatin exhibited a significant increase in media thickness and a significant reduction in aneurysmal size compared with control rats. Treatment with simvastatin resulted in reduced expression of macrophage chemoattractant protein-1 and vascular cell adhesion molecule-1, increased expression of endothelial nitric oxide synthase, and reduced the number of macrophage infiltration. In quantitative polymerase chain reaction and immunohistochemistry, simvastatin significantly inhibited upregulated expression of interleukin-1&bgr;, inducible nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 associated with CA progression. Gelatin zymography revealed decreased activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 in aneurysmal walls by simvastatin treatment. Simvastatin also effectively inhibited aneurysm enlargement and thinning of the media of preexisting CAs. Conclusions— Treatment with simvastatin suppresses the development of CAs by inhibiting inflammatory reactions in aneurysmal walls. Simvastatin also has a preventive effect on the progression of preexisting CAs. Simvastatin is a promising candidate of a novel medical treatment for the prevention of CA progression.