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Background: Subarachnoid hemorrhage due to the rupture of intracranial aneurysms is catastrophic. Its prevention requires a detailed understanding of its pathogenesis. Using our newly established estrogen-deficient rat aneurysm model, we found that estrogen receptors (ERs) were down-regulated, matrix metallopoteinase-9 and the tissue inhibitor of metalloproteinase-2 (MMP9/TIMP2) were imbalanced, and interleukin-1β (IL-1β) was increased in the vulnerable aneurysmal wall. As NLRP3 inflammasome activates the production of caspase1-dependent IL-1β, we focused on its role in the elicitation and rupture of intracranial aneurysms. We hypothesized that NLPR3 is mediated by ERs and associated with vascular vulnerability. Methods: To induce aneurysms, 10-week-old female Sprague-Dawley rats were subjected to hemodynamic changes and hypertension (HT). They were then randomized into two groups of 19 rats each. Group 1 was ovariectomized (HT-OVX + ), group 2 was intact (HT-OVX - ). Sham-operated rats were the control. Vascular smooth muscle cells from human brain were treated or not treated with ER-α and ER-β agonists. Results: During 12-week observation, aneurysms ruptured in 47% of the HT-OVX + and 16% of the HT-OVX - rats (p < 0.05). Immunohistochemically, the expression of ERα but not of ERβ was decreased, while the expression of the inflammasome components NLRP3, caspase1, IL-1β, of MMP-9, and the mRNA level of NLRP3, IL-1β and MMP-9 was increased in HT-OVX + rats. In human smooth muscle cells subjected to estrogen deficiency, the expression of NLRP3 was increased by angiotensin II and abrogated by the ER-α agonist but not the ER-β agonist. These findings suggest that in intracranial aneurysms prone to rupture, the activation of NLRP3, mediated by a decrease in ERα, plays a part. Conclusion: For a better understanding of the relationship between the rupture of intracranial aneurysms and the activation of inflammasomes under estrogen-deficient conditions, additional studies are underway.

Background and Purpose— Inflammation is emerging as a key component of the pathophysiology of intracranial aneurysms. Peroxisome proliferator–activated receptor-γ (PPARγ) is a nuclear hormone receptor of which activation modulates various aspects of inflammation. Methods— Using a mouse model of intracranial aneurysm, we examined the potential roles of PPARγ in the development of rupture of intracranial aneurysm. Results— A PPARγ agonist, pioglitazone, significantly reduced the incidence of ruptured aneurysms and the rupture rate without affecting the total incidence aneurysm (unruptured aneurysms and ruptured aneurysms). PPARγ antagonist (GW9662) abolished the protective effect of pioglitazone. The protective effect of pioglitazone was absent in mice lacking macrophage PPARγ. Pioglitazone treatment reduced the mRNA levels of inflammatory cytokines (monocyte chemoattractant factor-1, interleukin-1, and interleukin-6) that are primarily produced by macrophages in the cerebral arteries. Pioglitazone treatment reduced the infiltration of M1 macrophage into the cerebral arteries and the macrophage M1/M2 ratio. Depletion of macrophages significantly reduced the rupture rate. Conclusions— Our data showed that the activation of macrophage PPARγ protects against the development of aneurysmal rupture. PPARγ in inflammatory cells may be a potential therapeutic target for the prevention of aneurysmal rupture.

Background and Purpose— Inflammatory cells play a significant role in secondary injury after ischemic stroke. Recent studies have suggested that a lack of autophagy in myeloid cells causes augmented proinflammatory cytokine release and prolonged inflammation after tissue injury. In this study, we investigated the roles of myeloid cell autophagy in ischemic brain injury. Methods— Focal cerebral ischemia was induced via transient middle cerebral artery occlusion in mice with autophagy-deficient myeloid lineage cells (Atg5flox/flox LysMCre+) and in their littermate controls (Atg5flox/flox). Infarct volume, neurological function, inflammatory cell infiltration, and proinflammatory cytokine expression levels were evaluated. Results— Mice lacking autophagy in myeloid lineage cells had a lower survival rate for 14 days than control mice (20% versus 70%; P Conclusions— These data suggest that the lack of myeloid cell autophagy aggravates secondary injury by augmenting and prolonging inflammation after ischemic stroke without affecting the initial injury.

Background: Nicotine is one of main chemicals of tobacco smoke and promotes atherosclerosis and stroke. Tobacco smoke is considered an independent risk factor for intracranial aneurysm formation, growth, and rupture. There are mainly 5 subtypes of nicotine receptors. Roles of alpha7 nicotinic acetylcholine receptor (α7nAChR) in inflammation and vascular remodeling are diverse and context-dependent. Notably, endothelial α7nAChR is considered to mediate nicotine-induced inflammation. Activation of endothelial α7nAChR by nicotine may promote aneurysm rupture by increasing the aneurysm wall inflammation. Using a mouse model of intracranial aneurysm, we examined effects of nicotine in aneurysm rupture. Moreover we investigated potential roles of α7nAChR stimulation by nicotine in the pathophysiology of intracranial aneurysms. Methods: Intracranial aneurysms were induced by a combination of elastase injection into the cerebrospinal fluid and deoxycorticosteron acetate-salt (DOCA-salt) hypertension in male mice. Mice were treated with (1) nicotine (5 mg/kg/day, n=25); (2) saline sc (n=22) for three weeks after aneurysm induction. To investigate the effect of α7nAChR, mice were treated with (1) saline sc + saline ip (n=11); (2) saline sc + α7nAChR antagonist (Methyllycaconitine, MLA 5mg/kg/day) ip (n=13); (3) nicotine (5 mg/kg/day, sc, 28 days) + saline ip (n=18); (4) nicotine sc + MLA ip (n=18). Results: Nicotine alone significantly increased aneurysmal rupture compared with saline treatment (89% vs 46%, p=0.009). While α7nAChR antagonist did not affect the incidence of aneurysm or rupture rates, the α7nAChR antagonist significantly reduced the deleterious effect of nicotine as indicated by the reduction of the rupture rates (41% vs 100%: nicotine sc + MLA ip group vs nicotine sc + saline ip group, p=0.027). Conclusion: Our data indicate the promotion of aneurysm rupture by nicotine may be mediated by its stimulation of alpha7nAChR.

Introduction: Neutrophil extracellular traps (NETs) are originally described as proinflammatory, antimicrobial structures consisting of extracellular chromatin decorated with granular and cytoplasmic proteins. The formation of NETs requires peptidylarginine deiminase (PAD) 4. Recent studies revealed that NETs can play an important role in promoting inflammation in vascular diseases. Inflammation is emerging as a key component of the pathophysiology of intracranial aneurysm. In both human and mouse studies, activated neutrophils were shown to be associated with formation and rupture of intracranial aneurysms. Therefore, we hypothesized that the formation of NETs by PAD4 promotes the development of aneurysm rupture. We tested this hypothesis utilizing both pharmacological and genetic approaches in mice. Methods: We used 8- to 10-week-old male C57BL/6J mice, global PAD4 knock out (KO) and granulocyte-specific PAD4 KO mice. Aneurysm was induced by a combination of elastase injection and hypertension as we previously describes. We used deoxycorticosterone acetate (DOCA)-salt hypertension. A single dose of elastase was injected into the cerebrospinal fluid using a stereotaxic approach. Results: Cl-amidine, an inhibitor for PAD4, significantly decreased the rupture rates (92 vs 36%, P < 0.01). At the same time, Cl-amidine decreased the number of neutrophils and the formation of NETs analyzed by Ly-6G staining and citrullinated histone H3 staining, respectively. In the inhibitor treated mice, the messenger ribonucleic acid (RNA) level of ICAM-1, MCP-1 and TNFα in the cerebral arteries were lower than that of vehicle control treated mice. Furthermore, the rupture rates were decreased in the global PAD4 KO mice and the granulocyte-specific PAD4 KO mice than in the corresponding wild type mice groups. Finally, inhibition of the formation of NETs by DNase treatment also reduced the rupture rates. Conclusions: Our data indicate that the formation of NETs by granulocyte PAD4 promotes the development of aneurysm rupture. NETs may be represented a potential therapeutic target for the prevention of aneurysm rupture.

Introduction: Subarachnoid hemorrhage (SAH) is a catastrophic event with high morbidity and a poor prognosis. To prevent SAH, its pathogenesis must be understood. Dental infection may play a part in the pathophysiology of intracranial aneurysms. In our newly established rat model of aneurysms, the vascular inflammatory response was associated with their rupture. Therefore we hypothesized that the inflammatory response exacerbated by periodontal pathogens affects experimental cerebral aneurysm rupture. Methods: Aneurysms were induced in 10-week-old female Sprague-Dawley rats by eliciting estrogen deficiency, renal hypertension, and hemodynamic stress. Two weeks later they were divided into 2 groups; group 1 (n=13) was treated with Porphyromonas gingivalis lipopolysaccharide (LPS), group 2 (n=17) was the saline control. Both groups were intraperitoneally injected once a week. Results: During the 90-day observation period, 7 group 1 (54%) and 6 group 2 rats (35%) suffered aneurysmal rupture. The incidence of rupture within 60 days was significantly higher in group 1 than group 2 (38% vs 6%, pin vitro studies, IL-1β mRNA was increased in vascular smooth muscle cells exposed to LPS. These results suggest that LPS enhances the rupture of intracranial aneurysms via the promotion of local and systemic pro-inflammatory responses. Conclusion: Our study first documents that in rats, Porphyromonas gingivalis LPS exacerbates vascular inflammation and enhances the rupture of intracranial aneurysms.

Background: There are no pharmacological therapies for patients with unruptured cerebral aneurysms. Elsewhere we showed that the mineralocorticoid receptor antagonist eplerenone prevented the formation of cerebral aneurysms in ovariectomized rats. This pilot study was designed to evaluate whether it can be used to prevent the growth and rupture of cerebral aneurysms in humans with hypertension. Methods: Between August 2011 and May 2014, 82 patients with 89 aneurysms were enrolled in an open-label uncontrolled clinical trial. Of these, 81 patients (88 unruptured aneurysms) were followed for a mean of 21.2 months (153.7 aneurysm-years). All patients had a history of hypertension and were treated with eplerenone (20 - 100 mg per day). The primary endpoints were rupture and enlargement of the intracranial aneurysms. Results: The mean aneurysmal size was 4.6 ± 2.6 mm. During follow-up period that included 153.7 aneurysm-years, 3 aneurysms enlarged and one large thrombosed aneurysm rupture. The annual rupture was 0.65% overall and 13.16% for aneurysms larger than 10 mm. No aneurysms smaller than 9 mm ruptured. The annual rate for reaching the primary endpoint was 2.60% overall. It was 1.37% for aneurysms smaller than 9 mm and thus seems to be lower than in other studies. Eplerenone had no protective effects in aneurysms larger than 10 mm. In the course of our study, 12 patients (14.8%) permanently discontinued taking the eplerenone. Conclusions: Our observations suggest that it is clinically feasible to prevent growth and rupture of cerebral aneurysms smaller than 9 mm by treatment with eplerenone. Protocols for large clinical trial are needed to assess the possible long-term clinical benefits the drug may confer on patients with unruptured cerebral aneurysms.

Background and Purpose: Peroxisome proliferator-activated receptor gamma (PPARγ) plays a role in regulating not only lipid and glucose metabolism but also atherosclerosis and inflammation which are associated with intracranial aneurysmal rupture. It has been suggested that PPARγ is present in various kinds of cell types including macrophages which we reported to contribute to intracranial aneurysm formation. In addition, macrophage PPARγ regulates inflammatory cytokines negatively. Therefore we hypothesized that macrophage PPARγ is protective against intracranial aneurysmal rupture. We tested this hypothesis by using our own established mouse model of intracranial aneurysm. Methods: Intracranial aneurysms were induced in male mice using a combination of a single injection of elastase into the cerebrospinal fluid and the deoxycorticosterone acetate (DOCA) salt hypertension. Six days after aneurysm induction, we started 2-week treatment with vehicle or PPARγ agonist (pioglitazone:PGZ) or clodronate liposome. PPARγ antagonist (GW9662) was added to PGZ treatment. We induced aneurysms to macrophage-specific PPARγ knockout mice and treated them with vehicle or PGZ with the same protocol mentioned above. Aneurysmal rupture was detected by neurological symptoms and confirmed by the presence of intracranial aneurysms with subarachnoid hemorrhage. Results: Both PGZ and depletion of macrophage by clodronate liposome reduced the incidence of ruptured aneurysms and rupture rate. PGZ treatment decreased the inflammatory cytokines that were increased in cerebral arteries of mice with aneurysm induction compared to mice without aneurysm induction. To examine whether the macrophage PPARγ is associated with intracranial aneurysmal rupture, we administered PGZ to macrophage-specific PPARγ knock out mice. PGZ did not have protective effect against aneurysmal rupture in macrophage-specific PPARγ knock out mice although it exerted protective effects in wild type mice. Conclusion: PPARγ activation is protective against intracranial aneurysmal rupture by suppressing inflammatory cytokines and macrophage PPARγ is associated with this protective effects.

Introduction: The pathogenesis of cerebral aneurysms is multifactorial. Hyperhomocysteinemia (HHcy) leads to endothelial and platelet deterioration and may be associated with the formation of cerebral aneurysms. We tested the hypothesis that HHcy plays a role in the growth and/or rupture of cerebral aneurysms. Methods: We exposed 13-week-old female Sprague-Dawley rats fed a high-salt diet to estrogen deficiency, hemodynamic stress, and induced hypertension. To induce HHcy we added methionine to their drinking water. Results: In methionine induced rats the aneurysms the total rupture rate was higher than in rats not treated with methionine. One fourth of the aneurysms arising at the anterior communicating (Acom) artery, and half of the aneurysms at the posterior half of the circle of Willis, but no aneurysms at the anterior cerebral artery-olfactory artery (ACA-OA) bifurcation ruptured. Immunohistochemically, the infiltration of M1 macrophages was increased at the Acom artery in methionine induced rats and the messenger ribonucleic acid level of matrix metalloproteinase (MMP)-9, the ratio of MMP-9 to tissue inhibitor of metalloprotease (TIMP)2, and the interleukin-6 level were higher at the Acom artery than the ACA-OA bifurcation. Treatment with folic acid abolished the exacerbated effects due to HHcy, suggesting that the propensity of cerebral aneurysms at the Acom artery to rupture is attributable to disequilibrium between the degradation molecules and their inhibitors and to macrophage infiltration. Conclusion: HHcy may be associated with vulnerability of the cerebral aneurysmal wall to rupture in hypertensive ovariectomized rats.