Masafumi Morimoto

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.

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.

Mouse model of cerebral aneurysm: Experimental induction by renal hypertension and local hemodynamic changes

Background and Purpose— Rupture of cerebral aneurysm (CA) is the major cause of subarachnoid hemorrhage. Molecular mechanisms of this disease, however, remain unknown. To make possible genetic analysis of CA formation with genetically altered mice, we have successfully established a mouse model of saccular CA that recapitulates the essential features of human saccular CA. Methods— In C57black/6 male mice, various stages of CAs were experimentally induced at the right anterior cerebral artery–olfactory artery bifurcations by ligations of left common carotid arteries and posterior branches of bilateral renal arteries with high salt diet. Both light and electron microscopic studies were performed with the longitudinal sections of anterior cerebral artery–olfactory artery bifurcations. Results— In the treated group, various aneurysmal changes were detected in 14 of 18 mice. On the other hand, in the control group, no aneurysmal changes were found in 15 mice. In microscopic studies, aneurysmal changes were shown to include mainly fragmentation of internal elastic lamina, thinning of the smooth muscle cell layer, and degeneration of adventitial tissue, which were very similar to critical changes in human saccular CA. Conclusions— This mouse model of CA will be useful for studying the effects of complex determinants on CA formation and makes it possible to understand the pathogenesis of CA at the molecular level.

Efficacy of direct revascularization in adult Moyamoya disease: haemodynamic evaluation by positron emission tomography.

Summary To evaluate the efficacy of direct cerebrovascular reconstruction to prevent intracranial bleeding from the point of view of haemodynamic status, we performed positron emission tomography (PET) studies in 5 adult patients with Moyamoya disease before and after superficial temporal artery to middle cerebral artery (STA-MCA) anastomosis. Regional cerebral blood flow (rCBF), regional cerebral metabolic rate of oxygen (rCMRO2) and regional oxygen extraction fraction (rOEF) in the MCA territories and regional cerebral blood volume (rCBV) in the striatum were measured before and after STA-MCA anastomosis. Correlation between the change of these PET parameters and post-operative decreased opacification of Moyamoya vessels were analyzed. Pre-operatively, significant elevation of rCBV were observed in the basal ganglia as well as significant reduction of rCBF and elevation of rOEF with reduction of rCMRO2 in the MCA territories, indicating “misery” perfusion in the cerebral hemisphere and blood pooling in the Moyamoya vessels under increased haemodynamic stress. Post-operative PET study showed improvement of misery perfusion and reduction of rCBV in the basal ganglia. Reduction of rCBV in the basal ganglia generally compatible with decreasing Moyamoya vessels on angiographic findings. Our results suggests that direct bypass surgery could have a potential both for decreasing haemodynamic stress on Moyamoya vessels and to improve misery perfusion in the hemisphere.

Mechanism of intracranial rebleeding in Moyamoya disease

Intracranial hemorrhage is the major catastrophic event in the natural course of Moyamoya disease, and outcome of the patients with rebleeding is very poor. However, the mechanism underlying intracranial rebleeding is not well elucidated. We retrospectively analyzed 15 patients who bled two times or more among 46 bled patients with Moyamoya disease. The results indicated that there were two different types in the manner of rebleeding. One group consisted of seven cases, which bled two times or more at the same site than the original bleeding site. In four of these seven cases, a ruptured aneurysm was identified at the distal part of collateral vessel or on the major vessel. In the other three cases, no source of bleeding was identified. In all of these cases, rebleeding occurred within 2 months after the initial insult except for one case. Another group consisted of eight cases, which bled repeatedly but at different sites from the initial bleeding site. In any of these cases, neither aneurysms nor other vascular abnormalities were identified. In all of these cases, rebleeding occurred more than 2 months after the initial bleeding. The present result indicated that intracranial bleeding might occur as a result of rupture of a tiny aneurysm at the periphery of collateral vessels. These aneurysms may be blown out after initial bleeding. When they persist after the event, they may rupture again in a fairly short interval. In other cases, bleeding occur at different sites from the initial site. They are considered to be a result of ruptured weak Moyamoya vessels which are forced to act as collateral pathways and are under unusually increased hemodynamic stress.

Lysophosphatidylcholine induces early growth response factor-1 expression and activates the core promoter of PDGF-A chain in vascular endothelial cells

Abstract—Lysophosphatidylcholine (lyso-PC), a polar phospholipid that is increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to transcriptionally induce the expression of endothelial genes relevant to atherogenesis. In cultured bovine aortic endothelial cells (BAECs), we show that lyso-PC induces the expression of early growth response factor (Egr)-1 and thereby activates the proximal promoter of the platelet-derived growth factor (PDGF)-A chain located 55 to 71 bp upstream from the transcription start site, which has been shown to be crucial for PDGF-A chain expression induced by fluid shear stress and fibroblast growth factor-1. Northern blot analyses showed that lyso-PC (10 to 20 &mgr;mol/L) transiently (30 minutes to 1 hour) induced expression of Egr-1 mRNA. Induced expression of Egr-1 mRNA, which was associated with increased amounts of Egr-1 protein in nuclei, preceded PDGF-A chain mRNA induction in lyso-PC-activated BAECs. Nuclear runoff assay revealed that lyso-PC stimulates transcription of the Egr-1 gene. Transient transfection of the oligonucleotide corresponding to the proximal promoter of the PDGF-A chain (oligo A) linked to the luciferase reporter gene revealed that lyso-PC can activate the core promoter of the PDGF-A chain by 5-fold. Insertion of a guanine at 3 sites in the oligo A abolished the lyso-PC-induced increases in luciferase activities. Electrophoretic mobility shift assay with use of radiolabeled oligo A showed a lyso-PC-inducible shift band, which was suppressed by excess amounts of unlabeled oligo A or an anti-Egr-1 antibody. In addition, lyso-PC-induced Egr-1 expression was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase-1 (MEK1), suggesting that lyso-PC-induced expression of Egr-1 depends on the MEK1/extracellular signal-regulated kinase pathway. Taken together, transcriptional activation of Egr-1-dependent genes by this atherogenic lipid may be a key regulator of atherogenesis.

Peroxisome Proliferator‐Activated Receptor α Ligands Increase Lectin‐Like Oxidized Low Density Lipoprotein Receptor‐1 Expression in Vascular Endothelial Cells

Abstract: Lectin‐like oxidized low density lipoprotein receptor‐1 (LOX‐1) is a receptor for atherogenic oxidized LDL expressed in vascular endothelial cells, smooth muscle cells, and macrophages. Peroxiosme proliferator‐activated receptors (PPARs) include subfamilies PPARα and PPARγ, which are expressed in endothelial cells and involved in transcriptional regulation of a large number of genes that affect lipoprotein and fatty acid metabolism as well as inflammation. We therefore explored the effects of PPARα and PPARγ ligands on expression of LOX‐1. Treatment of cultured bovine aortic endothelial cells (BAECs) with PPARα ligands (fenofibrate and WY14643) remarkably upregulated LOX‐1 expression, which was detected by western and northern blot analyses. LOX‐1 promoter‐reporter gene assay showed that PPARα ligands activate transcription of the LOX‐1 gene. PPARγ ligands, in contrast, did not significantly affect LOX‐1 expression in BAECs. PPARα appears to be a key regulator in promoting LOX‐1 expression in BAECs. Upregulated expression of LOX‐1 may modulate the catabolism of oxidized LDL and atherosclerotic progression.