Hemodynamic Force as a Potential Regulator of Inflammation-Mediated Focal Growth of Saccular Aneurysms in a Rat Model.

Past studies have elucidated the crucial role of macrophage-mediated inflammation in the growth of intracranial aneurysms (IAs), but the contributions of hemodynamics are unclear. Considering the size of the arteries, we induced de novo aneurysms at the bifurcations created by end-to-side anastomoses with the bilateral common carotid arteries in rats. Sequential morphological data of induced aneurysms were acquired by magnetic resonance angiography. Computational fluid dynamics analyses and macrophage imaging by ferumoxytol were performed. Using this model, we found that de novo saccular aneurysms with a median size of 3.2 mm were induced in 20/45 (44%) of animals. These aneurysms mimicked human IAs both in morphology and pathology. We detected the focal growth of induced aneurysms between the 10th and 17th day after the anastomosis. The regional maps of hemodynamic parameters demonstrated the area exposed to low wall shear stress (WSS) and high oscillatory shear index (OSI) colocalized with the regions of growth. WSS values were significantly lower in the growing regions than in ones without growth. Macrophage imaging showed colocalization of macrophage infiltration with the growing regions. This experimental model demonstrates the potential contribution of low WSS and high OSI to the macrophage-mediated growth of saccular aneurysms.

Involvement of neutrophils in machineries underlying the rupture of intracranial aneurysms in rats.

Subarachnoid hemorrhage due to rupture of an intracranial aneurysm has a quite poor prognosis after the onset of symptoms, despite the modern technical advances. Thus, the mechanisms underlying the rupture of lesions should be clarified. To this end, we obtained gene expression profile data and identified the neutrophil-related enriched terms in rupture-prone lesions using Gene Ontology analysis. Next, to validate the role of neutrophils in the rupture of lesions, granulocyte-colony stimulating factor (G-CSF) was administered to a rat model, in which more than half of induced lesions spontaneously ruptured, leading to subarachnoid hemorrhage. As a result, G-CSF treatment not only increased the number of infiltrating neutrophils, but also significantly facilitated the rupture of lesions. To clarify the mechanisms of how neutrophils facilitate this rupture, we used HL-60 cell line and found an enhanced collagenolytic activity, corresponding to matrix metalloproteinase 9 (MMP9), upon inflammatory stimuli. The immunohistochemical analyses revealed the accumulation of neutrophils around the site of rupture and the production of MMP9 from these cells in situ. Consistently, the collagenolytic activity of MMP9 could be detected in the lysate of ruptured lesions. These results suggest the crucial role of neutrophils to the rupture of intracranial aneurysms; implying neutrophils as a therapeutic or diagnostic target candidate.

The Bilateral Ovariectomy in a Female Animal Exacerbates the Pathogenesis of an Intracranial Aneurysm.

Considering the poor outcome of subarachnoid hemorrhage (SAH) due to the rupture of intracranial aneurysms (IA), mechanisms underlying the pathogenesis of IAs, especially the rupture of lesions, should be clarified. In the present study, a rat model of IAs in which induced lesions spontaneously ruptured resulting in SAH was used. In this model, the combination of the female sex and the bilateral ovariectomy increased the incidence of SAH, similar to epidemiological evidence in human cases. Importantly, unruptured IA lesions induced in female animals with bilateral ovariectomy were histopathologically similar to ruptured ones in the presence of vasa vasorum and the accumulation of abundant inflammatory cells, suggesting the exacerbation of the disease. The post-stenotic dilatation of the carotid artery was disturbed by the bilateral ovariectomy in female rats, which was restored by hormone replacement therapy. The in vivo study thus suggested the protective effect of estrogen from the ovary on endothelial cells loaded by wall shear stress. -estradiol or dihydrotestosterone also suppressed the lipopolysaccharide-induced expression of pro-inflammatory genes in cultured macrophages and neutrophils. The results of the present study have thus provided new insights about the process regulating the progression of the disease.

Dedifferentiation of smooth muscle cells in intracranial aneurysms and its potential contribution to the pathogenesis.

Smooth muscle cells (SMCs) are the major type of cells constituting arterial walls and play a role to maintain stiffness via producing extracellular matrix. Here, the loss and degenerative changes of SMCs become the major histopathological features of an intracranial aneurysm (IA), a major cause of subarachnoid hemorrhage. Considering the important role of SMCs and the loss of this type of cells in IA lesions, we in the present study subjected rats to IA models and examined how SMCs behave during disease progression. We found that, at the neck portion of IAs, SMCs accumulated underneath the internal elastic lamina according to disease progression and formed the intimal hyperplasia. As these SMCs were positive for a dedifferentiation marker, myosin heavy chain 10, and contained abundant mitochondria and rough endoplasmic reticulum, SMCs at the intimal hyperplasia were dedifferentiated and activated. Furthermore, dedifferentiated SMCs expressed some pro-inflammatory factors, suggesting the role in the formation of inflammatory microenvironment to promote the disease. Intriguingly, some SMCs at the intimal hyperplasia were positive for CD68 and contained lipid depositions, indicating similarity with atherosclerosis. We next examined a potential factor mediating dedifferentiation and recruitment of SMCs. Platelet derived growth factor (PDGF)-BB was expressed in endothelial cells at the neck portion of lesions where high wall shear stress (WSS) was loaded. PDGF-BB facilitated migration of SMCs across matrigel-coated pores in a transwell system, promoted dedifferentiation of SMCs and induced expression of pro-inflammatory genes in these cells in vitro. Because, in a stenosis model of rats, PDGF-BB expression was expressed in endothelial cells loaded in high WSS regions, and SMCs present nearby were dedifferentiated, hence a correlation existed between high WSS, PDGFB and dedifferentiation in vivo. In conclusion, dedifferentiated SMCs presumably by PDGF-BB produced from high WSS-loaded endothelial cells accumulate in the intimal hyperplasia to form inflammatory microenvironment leading to the progression of the disease.

Eicosapentaenoic acid prevents the progression of intracranial aneurysms in rats.

BACKGROUND: As subarachnoid hemorrhage due to rupture of an intracranial aneurysm (IA) has quite a poor outcome despite of an intensive medical care, development of a novel treatment targeting unruptured IAs based on the correct understanding of pathogenesis is mandatory for social health. METHODS: Using previously obtained gene expression profile data from surgically resected unruptured human IA lesions, we selected G-protein coupled receptor 120 (GPR120) as a gene whose expression is significantly higher in lesions than that in control arterial walls. To corroborate a contribution of GPR120 signaling to the pathophysiology, we used an animal model of IAs and examine the effect of a GPR120 agonist on the progression of the disease. IA lesion was induced in rats through an increase of hemodynamic stress achieved by a one-sided carotid ligation and induced hypervolemia. Eicosapentaenoic acid (EPA) was used as an agonist for GPR120 in this study and its effect on the size of IAs, the thinning of media, and infiltration of macrophages in lesions were examined. RESULT: EPA administered significantly suppressed the size of IAs and the degenerative changes in the media in rats. EPA treatment also inhibited infiltration of macrophages, a hallmark of inflammatory responses in lesions. In in vitro experiments using RAW264.7 cells, pre-treatment of EPA partially suppressed lipopolysaccharide-induced activation of nuclear factor-kappa B and also the transcriptional induction of monocyte chemoattractant protein 1 (MCP-1), a major chemoattractant for macrophages to accumulate in lesions. As a selective agonist of GPR120, TUG-891, could reproduce the effect of EPA in RAW264.7 cells, EPA presumably acted on this receptor to suppress inflammatory responses. Consistently, EPA remarkably suppressed MCP-1 expression in lesions, suggesting the in vivo relevance of in vitro studies. CONCLUSIONS: These results combined together suggest the potential of the medical therapy targeting GPR120 or using EPA to prevent the progression of IAs.

High-Fat Diet Intake Promotes the Enlargement and Degenerative Changes in the Media of Intracranial Aneurysms in Rats.

Subarachnoid hemorrhage due to rupture of intracranial aneurysms is a life-threatening disease. Although some previous reports have demonstrated an association between lipid accumulation and degenerative changes in aneurysmal walls in humans, epidemiological studies have failed to identify dyslipidemia as a risk factor for intracranial aneurysms. Thus, we examined whether an increase in serum cholesterol levels facilitates the progression of intracranial aneurysms in a rat model. Rats were given a high-fat diet (HFD) and subjected to an intracranial aneurysm model. The HFD elevated their serum cholesterol levels. The intracranial aneurysms induced at the anterior cerebral artery-olfactory artery bifurcation were significantly larger in the high-fat group than in the normal-chow group. Histological analysis demonstrated that the loss of medial smooth muscle layers was exacerbated in the high-fat group and indicated the presence of macrophage-derived foam cells in the lesions. In in vitro experiments, the expression levels of the pro-inflammatory genes induced by LPS in RAW264.7-derived foam cells were significantly higher than those in RAW264.7 cells. The combination of these results suggests that increased serum cholesterol levels facilitate degenerative changes in the media and the progression of intracranial aneurysms presumably through foam cell transformation.

Macrophage Imaging of Intracranial Aneurysms.

Considered with a poor outcome of subarachnoid hemorrhage due to rupture of intracranial aneurysms (IAs), treatment interventions to prevent rupture of the lesions are mandatory for social health. As treatment option is limited to surgical manipulations, like microsurgical clipping, endovascular coiling or deployment of flow diverter, and these surgical interventions have a potential risk of complications in nature, a proper selection of rupture-prone IAs among ones incidentally found is essential. Today, a rupture risk in each case is estimated by several factors like patient characteristics and morphological ones of each lesion. However, unfortunately, an IA without treatment sometimes unexpectedly ruptures resulting in a devastating outcome or an IA surgically treated is turned out to have a thick wall. To achieve more efficient treatment interventions, the development of a novel diagnostic modality is required. Here, mainly through the accumulation of experimental findings, the crucial contribution of macrophage-mediated chronic inflammatory responses to IA progression have been revealed, making macrophage being a promising target for a diagnosis. If we could non-invasively visualize accumulation of macrophages in lesions, this imaging technique 'macrophage imaging' may enable a qualitative evaluation of IAs to stratify rupture-prone 'dangerous' lesions among many stable ones. Thereby, a development of macrophage imaging makes an indication of surgical interventions being more accurate and also greatly facilitates a development of a novel medical therapy if used as a surrogate marker.

Intracranial Aneurysm as a Macrophage-mediated Inflammatory Disease.

Subarachnoid hemorrhage (SAH) is mainly attributable to the rupture of intracranial aneurysms (IAs). Although the outcome of SAH is considerably poor in spite of the recent intensive medical care, mechanisms regulating the progression of IAs or triggering rupture remain to be clarified, making the development of effective preemptive medicine to prevent SAH difficult. However, a series of recent studies have been expanding our understanding of the pathogenesis of IAs. These studies have suggested the crucial role of macrophage-mediated chronic inflammation in the pathogenesis of IAs. In histopathological analyses of IA lesions in humans and induced in animal models, the number of macrophages infiltrating in lesions is positively correlated with enlargement or rupture of IAs. In animal models, a genetic deletion or an inhibition of monocyte chemotactic protein-1, a major chemoattractant for macrophages, or a pharmacological depletion of macrophages consistently suppresses the development and progression of IAs. Furthermore, a macrophage-specific deletion of Ptger2 (gene for prostaglandin E receptor subtype 2) or a macrophage-specific expression of a mutated form of IκBα which inhibits nuclear translocation of nuclear factor κB significantly suppress the development of IAs, supporting the role of macrophages and the inflammatory signaling functioning there in the pathogenesis of IAs. The development of drug therapies suppressing macrophage-mediated inflammatory responses in situ can thus be a potential strategy in the pre-emptive medicine targeting SAH. In this manuscript, we summarize the experimental evidences about the pathogenesis of IAs focused on inflammatory responses and propose the definition of IAs as a macrophage-mediated inflammatory disease.

KRAS G12D or G12V Mutation in Human Brain Arteriovenous Malformations.

BACKGROUND: Brain arteriovenous malformations (BAVMs) are vascular malformations composed of tangles of abnormally developed vasculature without capillaries. Abnormal shunting of arteries and veins is formed, resulting in high-pressure vascular channels, which potentially lead to rupture. BAVMs are generally considered a congenital disorder. But clinical evidence regarding involution, regrowth, and de novo formation argue against the static condition of this disease. Recently, the presence of the somatic activating KRAS mutations in more than half of BAVM cases was reported, suggesting the role of KRAS function in the pathogenesis. METHODS: KRAS mutation in codon35 (G→A, G12D; G→T, G12V) was examined by a digital polymerase chain reaction analysis using genome purified from paraffin-embedded slides of human BAVMs. We also examined protein expression of KRAS G12D in lesions to corroborate results from digital polymerase chain reaction analysis. RESULTS: We detected codon35 G→A mutation in 15 (39.5%) among 38 samples and codon35 G→T mutation in 10 (27.0%) among 37 samples we could assess mutations. There were no samples positive for both codon35 G→A and G→T mutation. The ratio of codon35 G→A mutation ranged from 0.60% to 12.28% and that of G→T was from 1.20% to 8.99%. We next examined protein expression of KRAS G12D in BAVM lesions in immunohistochemistry. A KRAS G12D mutant was detected mainly in endothelial cells of dilated vessels in lesions. CONCLUSIONS: KRAS mutations in codon35 were detected in about two thirds of specimens examined. KRAS function may actively contribute to the pathobiology of BAVM and can become a therapeutic target.