Yoshiteru Tada

Critical Roles of Macrophages in the Formation of Intracranial Aneurysm

Background and Purpose— Abnormal vascular remodeling triggered by hemodynamic stresses and inflammation is believed to be a key process in the pathophysiology of intracranial aneurysms. Numerous studies have shown infiltration of inflammatory cells, especially macrophages, into intracranial aneurysmal walls in humans. Using a mouse model of intracranial aneurysms, we tested whether macrophages play critical roles in the formation of intracranial aneurysms. Methods— Intracranial aneurysms were induced in adult male mice using a combination of a single injection of elastase into the cerebrospinal fluid and angiotensin II-induced hypertension. Aneurysm formation was assessed 3 weeks later. Roles of macrophages were assessed using clodronate liposome-induced macrophage depletion. In addition, the incidence of aneurysms was assessed in mice lacking monocyte chemotactic protein-1 (CCL2) and mice lacking matrix metalloproteinase-12 (macrophage elastase). Results— Intracranial aneurysms in this model showed leukocyte infiltration into the aneurysmal wall, the majority of the leukocytes being macrophages. Mice with macrophage depletion had a significantly reduced incidence of aneurysms compared with control mice (1 of 10 versus 6 of 10; P<0.05). Similarly, there was a reduced incidence of aneurysms in mice lacking monocyte chemotactic protein-1 compared with the incidence of aneurysms in wild-type mice (2 of 10 versus 14 of 20, P<0.05). There was no difference in the incidence of aneurysms between mice lacking matrix metalloproteinase-12 and wild-type mice. Conclusions— These data suggest critical roles of macrophages and proper macrophage functions in the formation of intracranial aneurysms in this model.

Edaravone, a Free Radical Scavenger, Inhibits MMP-9–Related Brain Hemorrhage in Rats Treated With Tissue Plasminogen Activator

Background and Purpose— Intracerebral hemorrhage, induced by recombinant tissue plasminogen activator (rtPA) in ischemic stroke, is attributable to the increased activity of matrix metalloproteinase-9 (MMP-9). Patients with acute infarct benefit from the neuroprotective drug edaravone, a free radical scavenger. We examined the mechanisms by which edaravone may help to suppress rtPA-induced brain hemorrhage. Methods— Male Wistar rats weighing 250 to 280 g were subjected to 3-hour transient middle cerebral artery occlusion (MCAO) and divided randomly into 3 groups. Immediately after reperfusion, 1 group was intravenously injected with 10 mg/kg rtPA, another with rtPA plus 3 mg/kg edaravone, and the 3rd group received no treatment. We assessed the hemorrhage volume and the activity of MMP-9 in the brain 24 hours postischemia. We also studied the activity of MMP-9, its mRNA expression, and nuclear factor-kappa B (NF-&kgr;B) activity in rtPA-stimulated human microvascular endothelial cells (HBECs). Results— The degree of hemorrhage and the level of endothelial cell–derived MMP-9 were elevated in rats treated with rtPA alone and attenuated in rats treated with rtPA plus edaravone. In rtPA-stimulated HBECs, edaravone suppressed the activity and mRNA expression of MMP-9 in a dose-dependent manner. Edaravone also inhibited NF-&kgr;B activation. Conclusions— We demonstrate that edaravone inhibits rtPA-induced cerebral hemorrhage in the ischemic brain of rats via the inhibition of MMP-9 expression in vivo, which is substantiated by inhibition of MMP-9 expression and NF-&kgr;B activation in HBECs. Edaravone may render thrombolytic therapy safer for the administration of rtPA in patients with ischemic stroke.

Endothelial damage due to impaired nitric oxide bioavailability triggers cerebral aneurysm formation in female rats.

Objective Epidemiological data indicate a high incidence of cerebral aneurysms in postmenopausal women. To elucidate the pathogenesis of cerebral aneurysms, we focused on the contribution of endothelial damage in rats. Methods We induced estradiol deficiency by oophorectomy (OVX), hypertension, or both, and hemodynamic stress in 7-week-old female Sprague–Dawley rats. They were then given hormone-replacement therapy with 17β-estradiol or an angiotensin II type 1 receptor blocker (ARB). The effects of estradiol, angiotensin II type 1 receptor blocker, or both on cultured endothelial cells were also examined. Results The number of anomalously shaped endothelial cells was higher in OVX than hypertensive rats (P < 0.05). Rats subjected to hypertension and OVX exhibited a marked increase in the incidence of saccular cerebral aneurysms. Estradiol or angiotensin II type 1 receptor blocker treatment reduced this incidence (P < 0.05). The endothelial nitric oxide synthase (eNOS) mRNA level in the intracranial artery of OVX and hypertensive and OVX rats was low (P < 0.05). Immunohistochemically, the expression of eNOS and estrogen receptor alpha (ERα) in the vascular wall of hypertensive and OVX rats was decreased; angiotensin II and the nicotinamide adenine dinucleotide phosphate oxidase subunits nicotinamide adenine dinucleotide phosphate oxidase 4 and p22phox were strongly expressed in cerebral aneurysms. In the absence of estradiol, eNOS was downregulated and nicotinamide adenine dinucleotide phosphate oxidase expression was increased in endothelial cells; angiotensin II augmented these phenomena. The regulation of eNOS was mediated by ERα. These results suggest that estrogen deficiency induces endothelial dysfunction and reactive oxygen species generation, triggering endothelial damage that leads to cerebral aneurysms and that hypertension is an additional risk factor. Conclusion A therapy targeted at the endothelium and management of hypertension may help to prevent cerebral aneurysms.

Reduction of endothelial tight junction proteins is related to cerebral aneurysm formation in rats.

Objective The formation of cerebral aneurysms is associated with endothelial damage and macrophage migration. Hypothesizing that the opening of tight junctions due to the disappearance of the tight junction proteins occludin and zona occludens-1 (ZO-1) in damaged endothelia allows macrophage migration, leading to cerebral aneurysm formation, we investigated the role of tight junction proteins. Methods The vascular wall of female rats subjected to hypertension, oophorectomy (OVX), and hemodynamic stress to induce cerebral aneurysms was evaluated morphologically, immunohistochemically, and by quantitative RT-PCR. We also assessed the regulation of tight junction proteins in human brain endothelial cells (HBECs). Results In the very early stage before aneurysm formation, the expression of occludin and ZO-1 was reduced in injured endothelial cell junctions exhibiting gaps. In the course of aneurysmal progression their reduction progressed and was correlated with macrophage migration. In hypertension along with OVX rats we observed an increase in angiotensin II and the degradation molecules matrix metalloproteinase-9 (MMP-9), nicotinamide-adenine dinucleotide phosphate oxidases and monocyte chemoattractant protein-1. The mineralocorticoid receptor blocker eplerenone increased occludin and ZO-1 expression; this was associated with a reduction in angiotensin II and the degradation molecules and resulted in the inhibition of macrophage exudation and aneurysm formation. In HBECs, occludin and ZO-1 downregulation by angiotensin II and estrogen deficiency was reversed by eplerenone, the MMP inhibitor SB3CT, and apocynin. Our results suggest that macrophage migration is associated with the reduction in tight junction proteins induced by the degradation molecules. Conclusion In rats, the destruction of tight junctions may facilitate macrophage migration and cerebral-aneurysm formation.

Roles of Hypertension in the Rupture of Intracranial Aneurysms

Background and Purpose— Systemic hypertension has long been considered a risk factor of aneurysmal rupture. However, a causal link between systemic hypertension and the development of aneurysmal rupture has not been established. In this study, using a mouse model of intracranial aneurysm rupture, we examined the roles of systemic hypertension in the development of aneurysmal rupture. Methods— Aneurysms were induced by a combination of deoxycorticosterone acetate (DOCA)-salt and a single injection of elastase into the cerebrospinal fluid in mice. Antihypertensive treatment was started 6 days after aneurysm induction. Aneurysmal rupture was detected by neurological symptoms and confirmed by the presence of intracranial aneurysm with subarachnoid hemorrhage. Hydralazine (direct vasodilator) or discontinuation of DOCA-salt treatment was used to assess the roles of systemic hypertension. Captopril (angiotensin-converting enzyme inhibitor) or losartan (angiotensin II type 1 receptor antagonist) was used to assess the roles of the local renin–angiotensin system in the vascular wall. Results— Normalization of blood pressure by hydralazine significantly reduced the incidence of ruptured aneurysms and the rupture rate. There was a dose-dependent relationship between reduction of blood pressure and prevention of aneurysmal rupture. Captopril and losartan were able to reduce rupture rate without affecting systemic hypertension induced by DOCA-salt treatment. Conclusions— Normalization of blood pressure after aneurysm formation prevented aneurysmal rupture in mice. In addition, we found that the inhibition of the local renin–angiotensin system independent from the reduction of blood pressure can prevent aneurysmal rupture.

Role of Mineralocorticoid Receptor on Experimental Cerebral Aneurysms in Rats

Activation of the renin-angiotensin (Ang)-aldosterone system is involved in the pathology of vascular diseases. Although the blockade of the mineralocorticoid receptor protects against vascular diseases, its role in cerebral aneurysms remains to be elucidated. We treated female rats subjected to renal hypertension, increased hemodynamic stress, and estrogen deficiency for 3 months with the mineralocorticoid receptor blocker eplerenone (30 or 100 mg/kg per day) or vehicle (vehicle control). Eplerenone reduced the incidence of cerebral aneurysms and saline intake without lowering of the blood pressure. In the aneurysmal wall, the production of Ang II and nitrotyrosine was increased. The mRNA levels of Ang-converting enzyme 1 and NADPH oxidase subunits NOX4, Rac1, monocyte chemoattractant protein 1, and matrix metalloproteinase 9 were increased. Eplerenone brought about a reduction in these molecules, suggesting that mineralocorticoid receptor blockade suppresses cerebral aneurysm formation by inhibiting oxidative stress, inflammatory factors, local renin-Ang system activation, and saline intake. Other female rats implanted with pellets of the mineralocorticoid receptor agonist deoxycorticosterone acetate manifested a high incidence of cerebral aneurysm formation and the upregulation of molecules related to oxidative stress, inflammatory factors, and the local renin-Ang system; their saline intake was increased. We demonstrate that mineralocorticoid receptor activation at least partly contributes to the pathogenesis of cerebral aneurysms.

Protective mechanisms of the angiotensin II type 1 receptor blocker candesartan against cerebral ischemia: in-vivo and in-vitro studies.

Background Angiotensin II type 1 (AT1) receptor blockers decrease ischemia by mechanisms dependent on and independent of arterial blood pressure in hypertensive rats and AT1-R knockout mice, respectively. However, the detailed mechanisms underlying the effects of AT1 receptor blockers remain unclear. Aims To elucidate the systemic and focal effects of AT1 receptor blockers against cerebral ischemia in in-vivo and in-vitro studies. Methods Normotensive Wistar rats were treated for 2 weeks with 0.5 or 1 mg/kg candesartan cilexetil and then subjected to 2-h middle cerebral artery occlusion–reperfusion. Human umbilical endothelial cells were stimulated with the active form of candesartan and angiotensin II in the absence and presence of an angiotensin II type 2 (AT2) receptor antagonist. Results In candesartan-pretreated hypotensive and nonhypotensive rats, blood pressure was moderately increased during middle cerebral artery occlusion and fell gradually to the baseline after the reperfusion; it remained elevated in the control even after the reperfusion occlusion. Candesartan treatment resulted in a decrease in the cortical infarct volume and oxidative damage, the hypoxic status was improved, and the expression of repair-associated and growth-associated proteins in the cortical penumbra was augmented. Candesartan also increased the eNOS mRNA level and the lumen size of the middle cerebral artery. In human umbilical endothelial cells, candesartan increased the eNOS protein level AT2-R dependently, inhibited the expression of nicotinamide adenine dinucleotide phosphate oxidase subunits and angiotensin II-induced intracellular reactive oxygen species and nitric oxide, and promoted the extracellular release of nitric oxide, suggesting that it augmented the bioavailability of nitric oxide. Conclusion Among the mechanisms candesartan exerts in its protection against cerebral ischemia, restoration of endothelial function may represent an attractive therapeutic goal to address cerebral ischemia.

Statins Promote the Growth of Experimentally Induced Cerebral Aneurysms in Estrogen-Deficient Rats

Background and Purpose— The pathogenesis of cerebral aneurysms is linked to inflammation, degradation of the extracellular matrix, and vascular wall apoptosis. Statins exert pleiotropic effects on the vasculature, independent of their cholesterol-lowering properties. To explore the detailed pathogenesis of cerebral aneurysms, we examined their progression in a rat model and studied whether statins prevent their initiation and growth. Methods— Cerebral aneurysms were induced in female rats subjected to hypertension, increased hemodynamic stress, and estrogen deficiency. The development of aneurysm was assessed morphologically on corrosion casts. The effects of pravastatin (5, 25, or 50 mg/kg per day) and of simvastatin (5 mg/kg per day) on their aneurysms were studied. Human brain endothelial cells were also used to determine the effects of pravastatin. Results— Pravastatin (5 mg/kg per day) reduced endothelial damage and inhibited aneurysm formation; there was an association with increased endothelial nitric oxide synthase (eNOS) levels and a decrease in human brain endothelial cell adhesion molecules. Unexpectedly, 25 mg/kg per day and 50 mg/kg per day pravastatin and 5 mg/kg per day simvastatin promoted aneurysmal growth, and high-dose pravastatin induced aneurysmal rupture. The deleterious effects exerted by these statins were associated with an increase in apoptotic caspase-3 levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, suggesting that statins exert bidirectional effects. Conclusions— Our results provide the first evidence that cerebral aneurysm growth is partly associated with apoptosis and issue a warning that statins exert bidirectional effects on cerebral aneurysms. Additional intensive research is necessary to understand better their mechanisms and to identify patients in whom the administration of statins may elicit deleterious effects.

Ibudilast inhibits cerebral aneurysms by down-regulating inflammation-related molecules in the vascular wall of rats.

OBJECTIVEPhosphodiesterase-4 (PDE-4) is a cyclic adenosine monophosphate–specific enzyme involved in various inflammatory diseases. We studied its role in and the effect of ibudilast, which predominantly blocks PDE-4, on rat cerebral aneurysms. METHODSCerebral aneurysms were induced at the anterior cerebral artery–olfactory artery bifurcation of female rats subjected to hypertension, increased hemodynamic stress, and estrogen deficiency. The effect of ibudilast (30 or 60 mg/kg/d for 3 months) on their cerebral aneurysms was studied by morphological and immunohistochemical assessment and quantitative real-time polymerase chain reaction assay. In our in vitro study, we grew endothelial cells stimulated by angiotensin II under estrogen-free conditions and examined the effect of ibudilast on PDE-4 activation and the cyclic adenosine monophosphate level. RESULTSMorphological evaluation using vascular corrosion casts showed ibudilast significantly suppressed cerebral aneurysms in a dose-dependent manner. In rats with induced cerebral aneurysms, the gene and protein expression of PDE-4 was high, and endothelial leukocyte adhesion molecules (P-selectin, intracellular adhesion molecule 1, and vascular adhesion molecule 1), matrix metalloproteinase-9, and tumor necrosis α were expressed. Macrophage migration was also increased. Treatment with ibudilast down-regulated these molecules, suppressed macrophage migration into the aneurysm wall, and inhibited PDE-4 activation and the elevation of cyclic adenosine monophosphate in endothelial cells. CONCLUSIONThese results suggest that blocking of PDE4 is associated with the reduction of inflammation-related molecules and macrophage migration, thereby reducing the progression of cerebral aneurysms. It may represent a new conservative therapy to treat patients with cerebral aneurysms.

Estrogen Protects Against Intracranial Aneurysm Rupture in Ovariectomized Mice

Clinical observations suggest that postmenopausal women have a higher incidence of aneurysmal rupture than premenopausal women. We hypothesize that a relative deficiency in estrogen may increase the risks of aneurysmal growth and subarachnoid hemorrhage in postmenopausal women. We assessed the effects of estrogen and selective estrogen receptor subtype agonists on the development of aneurysmal rupture in ovariectomized female mice. We used an intracranial aneurysm mouse model that recapitulates the key features of human intracranial aneurysms, including spontaneous rupture. Ten- to 12-week-old ovariectomized female mice received treatment with estrogen, nonselective estrogen receptor antagonist, estrogen receptor-&agr; agonist, or estrogen receptor-&bgr; agonist starting 6 days after aneurysm induction so that the treatments affected the development of aneurysmal rupture without affecting aneurysmal formation. Estrogen significantly reduced the incidence of ruptured aneurysms and rupture rates in ovariectomized mice. Nonselective estrogen receptor antagonist abolished the protective effect of estrogen. Although estrogen receptor-&agr; agonist did not affect the incidence of ruptured aneurysms or rupture rates, estrogen receptor-&bgr; agonist prevented aneurysmal rupture without affecting the formation of aneurysms. The protective role of estrogen receptor-&bgr; agonist was abolished by the inhibition of nitric oxide synthase. We showed that estrogen prevented aneurysmal rupture in ovariectomized female mice. The protective effect of estrogen seemed to occur through the activation of estrogen receptor-&bgr;, a predominant subtype of estrogen receptor in human intracranial aneurysms and cerebral arteries.