Human Aneurysms Research Articles

Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-β-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-β-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-β-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Correction to: Collagen Turnover in Relation to Risk Factors and Hemodynamics in Human Intracranial Aneurysms.

Correction to: Collagen Turnover in Relation to Risk Factors and Hemodynamics in Human Intracranial Aneurysms.

Interleukin-6 Promotes Murine Estrogen Deficiency-Associated Cerebral Aneurysm Rupture.

Estrogen deficiency is associated with cerebral aneurysm rupture, but the precise mechanism is unknown. To test the hypothesis that IL-6 is required for the increase in aneurysm rupture rate observed in estrogen-deficient mice. We analyzed IL-6 expression in human cerebral aneurysms. We induced cerebral aneurysms in estrogen-deficient female C57BL/6 mice that had undergone 4-vinylcyclohexene diepoxide (VCD) treatment or bilateral ovariectomy (OVE). Mice were blindly randomized to selective IL-6 inhibition (IL-6 receptor [IL-6R] neutralizing antibody, n=25) or control (isotype-matched IgG, n=28). Murine cerebral arteries at the circle of Willis were assessed for aneurysm rupture and macrophage infiltration. IL-6 is expressed in human cerebral aneurysms, but not in control arteries. Serum IL-6 is elevated in ovariectomized female mice compared to sham control (14.3±1.7 pg/mL vs 7.4±1.5 pg/mL, P=.008). Selective IL-6R inhibition suppressed cerebral aneurysm rupture in estrogen-deficient mice compared with control (VCD: 31.6% vs 70.0%, P=.026; OVE: 28.6% vs 65.2%, P=.019). IL-6R inhibition had no effect on formation or rupture rate in wild-type mice. IL-6R neutralizing antibody significantly reduced macrophage infiltration at the circle of Willis (1.9±0.2 vs 5.7±0.6 cells/2500 μm2; n=8 vs n=15; P<.001). IL-6 is increased in the serum of estrogen-deficient mice and appears to play a role in promoting murine estrogen deficiency-associated cerebral aneurysm rupture via enhanced macrophage infiltration at the circle of Willis. Inhibition of IL-6 signaling via IL-6 receptor neutralizing antibody inhibits aneurysm rupture in estrogen-deficient mice. IL-6 receptor inhibition had no effect on aneurysm formation or rupture in wild-type animals.

Persistence of Intraluminal Thrombus Makes Saccular Aneurysm More Biologically Active than Fusiform in an Experimental Rat Model

Introduction: Saccular aneurysms are thought to have a worse prognosis than fusiform aneurysms in humans, due to hemodynamic reasons. However, data comparing hemodynamic and biology in saccular and fusiform aneurysms are lacking. The main objective was to evaluate the impact of aneurysm morphology on intra-luminal thrombus (ILT) formation and activity. Methods: Forty Lewis rats were ran­domly divided into 2 groups of 20: “saccular” (Group A) and “fusiform” (Group B) aneurysms. Decellularized thoracic aortas from guinea pigs were xenografted to create saccular or fusiform aneurysms. Final imaging evaluation of the aneurysms was carried out during the third week, by quantitative Doppler ultrasound and magnetic resonance imaging. Assays of myeloperoxidase (MPO), platelet factor 4 (PF4), advanced oxidation protein products (AOPPs) iron and matrix metallopeptidase-9 (MMP-9) were performed as biological criteria. Results: Quantitatively, saccular aneurysms are characterized by a more thicker ILT, lower inflow velocities and more important relative backflow velocities as compared to fusiform aneurysms. Compared to fusiform, saccular aneurysms released significantly more MPO (p = 0.004), PF4 (p = 0.02), AOPPs (p < 0.002), iron (p < 0.0001) and MMP-9 (p < 0.04). Conclusion: Experimental saccular and fusiform aneurysms show differential specific hemodynamics, which seem to impact the histology and the biology of the ILT in each type of aneurysm.

Collagen Turnover in Relation to Risk Factors and Hemodynamics in Human Intracranial Aneurysms

Background and Purpose- Determinants for molecular and structural instability, that is, impending growth or rupture, of intracranial aneurysms (IAs) remain uncertain. To elucidate this, we endeavored to estimate the actual turnover rates of the main molecular constituent in human IA (collagen) on the basis of radiocarbon (14C) birth dating in relation to IA hemodynamics. Methods- Collagen turnover rates in excised human IA samples were calculated using mathematical modeling of 14C birth dating data of collagen in relation to risk factors and histological markers for collagen maturity/turnover in selected IA. Hemodynamics were simulated using image-based computational fluid dynamics. Correlation, logistic regression, and receiver operating characteristic analyses were performed. Results- Collagen turnover rates were estimated in 46 IA (43 patients); computational fluid dynamics could be performed in 20 IA (20 patients). The mean collagen turnover rate (γ) constituted 126% (±1% error) per year. For patients with arterial hypertension, γ was greater than 2600% annually, whereas γ was distinctly lower with 32% (±1% error) per year for patients without risk factors, such as smoking and hypertension. There was a distinct association between histological presence of rather immature collagen in human IA and the presence of modifiable risk factors. Spatial-temporal averaged wall shear stress predicted rapid collagen turnover (odds ratio, 1.6 [95% CI, 1.0-2.7]). Receiver operating characteristic analysis demonstrated a good test accuracy (area under the curve, 0.798 [95% CI, 0.598-0.998]) for average wall shear stress with a threshold ≥4.9 Pa for rapid collagen turnover. Conclusions- Our data indicate that turnover rates and stability of collagen in human IA are strongly associated with the presence of modifiable risk factors and aneurysmal hemodynamics. These findings underline the importance of strict risk factor modification in patients with unruptured IA. Future should include more detailed risk factor data to establish a more causal understanding of hemodynamics and the rupture risk of individual IA.

A two-miRNA signature (miR-21 and miR-92) in peripheral whole blood as a potential biomarker for diagnosis of human cerebral aneurysms.

microRNAs (miRs) have been reported as blood-based noninvasive indicators for the diagnosis of various diseases. However, the utility of whole blood-based miRs in the diagnosis of intracranial aneurysm (IA) is still not clear. The present study aimed to examine miR expression profiling in the peripheral whole blood of IA patients and healthy controls. Seventy-three IA patients, including 34 unruptured and 39 ruptured, and 28 healthy subjects, were recruited for diagnostic analysis. microRNA (miR) expression profiling in whole blood from healthy controls and IA patients was evaluated using miRNA microarray assay. RT-qPCR was used to evaluate miR expression. Receiver operating characteristics (ROC) curves and the area under the ROC curves (AUC) were used to calculate the diagnostic power of miRs in whole blood of IA. We observed significantly higher miR-21 and miR-92 expression levels in aneurysmal tissues and whole blood of IA patients as compared to healthy subjects. miR-21 expression level was significantly positively correlated with miR-92 in IA tissues and whole blood of IA patients. ROC analysis revealed that miR-21 (AUC = 0.843, sensitivity = 0.849, specificity = 0.750) and miR-92 (AUC = 0.892, sensitivity = 0.945, specificity = 0.786) were promising in diagnosis of IA with high detectability. Intriguingly, miR-21 combined with miR-92 markedly improved the diagnostic power of IA (AUC = 0.920, sensitivity = 1.000, specificity = 0.786). miR-21 combined with miR-92 could be considered as a potential biomarker for IA screening.

RETRACTED: Structural diversity and anticancer activity on human myocardial aneurysm cells of two Cu(II)-coordination polymers

This article has been retracted, and the online PDF has been watermarked “RETRACTION”. The retraction notice is available at http://doi.org/10.3233/MGC-220954.

Zero-/two-dimensional coordination complexes based on the substituted imidazo[1,2-a]pyridine ligands: structural diversity and antitumor activity on human myocardial aneurysm cells

In the current study, two similar substituted imidazo[1,2-α]pyridine ligands with different coordination groups were facilely synthesized, and were further used for construction of two novel Cu(II)-based coordination complexes with the chemical formulae of Cu(L1)2(H2O)4 (1, HL1 = (E)−4-(3-(2-chloroimidazo[1,2-a]pyridin-3-yl)acryloyl)benzoic acid) and [Cu(L2)2]n (2, HL2 = (E)−4-(2-chloroimidazo[1,2-α]pyridin-3-yl)−2-oxobut-3-enoic acid) via a one pot solvothermal reaction. In addition, their inhibitory effect on human myocardial aneurysm cells were evaluated. Firstly, to explore the inhibitory effect of complexes on cancer cell viability and proliferation, the Cell Counting Kit-8 (CCK-8) assay was performed. Next, to evaluate the influence of the compounds on the cell migration and invasion ability, the transwell assay was conducted. In addition to this, for the cancer cell apoptosis evaluation after compounds treatment, the flow cytometry was carried out in this research.

Inactivation of cysteine 674 in the SERCA2 accelerates experimental aortic aneurysm

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is vital to maintain intracellular calcium homeostasis. SERCA2 cysteine 674 (C674) is highly conservative and its irreversible oxidation is upregulated in human and mouse aortic aneurysms, especially in smooth muscle cells (SMCs). The contribution of SERCA2 and its redox C674 in the development of aortic aneurysm remains enigmatic. Objective: Our goal was to investigate the contribution of inactivation of C674 to the development of aortic aneurysm and the mechanisms involved. Approach and results: Using SERCA2 C674S knock-in (SKI) mouse line, in which half of C674 was substituted by serine 674 (S674) to represent partial irreversible oxidation of C674 in aortic aneurysm, we found that in aortic SMCs the replacement of C674 by S674 resulted in SMC phenotypic modulation. In SKI SMCs, the increased intracellular calcium activated calcium-dependent calcineurin, which promoted the nuclear translocation of nuclear factor of activated T-lymphocytes (NFAT) and nuclear factor kappa-B (NFκB), while inhibition of calcineurin blocked SMC phenotypic modulation. Besides, the replacement of C674 by S674 accelerated angiotensin II-induced aortic aneurysm. Conclusions: Our results indicate that the inactivation of C674 by causing the accumulation of intracellular calcium to activate calcineurin-mediated NFAT/NFκB pathways, resulted in SMC phenotypic modulation to accelerate aortic aneurysm, which highlights the importance of C674 redox state in the development of aortic aneurysms.

Ex vivo expansion of regulatory T cells from abdominal aortic aneurysm patients inhibits aneurysm in humanized murine model

ObjectiveAbdominal aortic aneurysm (AAA) is a chronic inflammatory disease. Studies of human aneurysm tissue demonstrate dense inflammatory cell infiltrates with CD4+ T cells predominating. Regulatory T cells (Tregs) play an important role in inhibiting pro-inflammatory T cell proliferation, therefore, limiting collateral tissue destruction. The aim of this study was to investigate whether ex vivo augmentation of human Tregs attenuates aneurysm formation in humanized murine model of AAA. MethodsCirculating Treg population in AAA patients and age- and gender-matched controls were determined by real-time polymerase chain reaction and flow cytometry. To create humanized murine model of AAA, irradiated Rag1-deficient (Rag1-/-) mice, without mature T lymphocytes, at 7 weeks of age were given 5 × 106 of human CD4+ T cells intraperitoneally. Then the mice underwent CaCl2 aneurysm induction. Aortic diameters were measured before and at 6 weeks after aneurysm induction. Aortic tissue was collected for histology and protein extraction. Verhoeff-Van Gieson stain was used for staining elastic fiber. CD4+ T cells in the aortic tissue were detected by immunohistochemical staining. ResultsIn human peripheral blood mononuclear cells, the proportion of Tregs are decreased in AAA patients compared with matched control patients with significant vascular disease. We first validated the role of Tregs in the CaCl2 model of AAA. To determine the role of human T cells in AAA formation, Rag1-/- mice, resistant to CaCl2-aneurysm induction, were transplanted with human CD4+ T cells. Human CD4+ T cells were able to drive aneurysm formation in Rag1-/- mice. We show that ex vivo augmentation of human Tregs by interleukin-2 resulted in decreased aneurysm progression. ConclusionsThese data suggest that the ex vivo expansion of human Tregs may be a potential therapeutic strategy for inhibiting progression of AAA.

A Pilot Study on Biaxial Mechanical, Collagen Microstructural, and Morphological Characterizations of a Resected Human Intracranial Aneurysm Tissue

Intracranial aneurysms (ICAs) are focal dilatations that imply a weakening of the brain artery. Incidental rupture of an ICA is increasingly responsible for significant mortality and morbidity in the American's aging population. Previous studies have quantified the pressure-volume characteristics, uniaxial mechanical properties, and morphological features of the human aneurysms. In this pilot study, for the first time, we comprehensively quantified the mechanical, collagen fiber microstructural, and morphological properties of a resected human posterior inferior cerebellar artery aneurysm. The tissue from the dome of a right posterior inferior cerebral aneurysm was first mechanically characterized using biaxial tension and stress relaxation tests. Then, the load-dependent collagen fiber architecture of the aneurysm tissue was quantified using an in-house polarized spatial frequency domain imaging system. Finally, optical coherence tomography and histological procedures were used to quantify the tissue's microstructural morphology. Mechanically, the tissue was shown to exhibit hysteresis, a nonlinear stress-strain response, and material anisotropy. Moreover, the unloaded collagen fiber architecture of the tissue was predominantly aligned with the testing Y-direction and rotated towards the X-direction under increasing equibiaxial loading. Furthermore, our histological analysis showed a considerable damage to the morphological integrity of the tissue, including the lack of elastin, intimal delamination, and calcium deposition. This pilot study has provided new insights into the multiscale properties of aneurysm tissues. The method presented herein, together with the novel results, can be used to develop accurate constitutive models to use in computational models of the aneurysm hemodynamics, growth, and remodeling.

Effect of collagen fibres and elastic lamellae content on the mechanical behaviour of abdominal aortic aneurysms

The main purpose of this study was a detailed analysis of the mechanical and structural characteristics of human abdominal aneurysms in comparison with normal abdominal aortae and determination of the correlations between their mechanical behaviour and the microstructural content. Various mechanical properties, i.e., mechanical failure properties, elastic moduli, inflection point coordinates, index of anisotropy and incompressibility were determined under uniaxial loading conditions in the circumferential and axial directions. Constitutive parameters were derived from the commonly used constitutive model proposed by Holzapfel et al. [9]. The microstructural arrangement was examined by histological staining supported by scanning electron microscopy analysis. The content of collagen fibres and elastic lamellae was tested in relation to mechanical properties and constitutive parameters. Significant differences were found in the microstructural arrangement and layer composition of the aneurysmal specimens, compared to the normal aorta group. The mechanical properties and constitutive parameters of the aneurysmal specimens were significantly altered, indicating a weakening of the load-bearing properties of the walls of the aneurysms. A comparative analysis discovered significant correlations between structural composition and mechanical parameters, in particular with respect to the number of collagen fibres and failure stress, which can be important for clinical evaluation of abdominal aortic aneurysm (AAA) rupture. Changes in the content of collagen fibres and elastic lamellae correlate with mechanical and constitutive parameters, indicating AAA severity.

Divergent effects of canonical and non-canonical TGF-β signalling on mixed contractile-synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms.

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF‐β driven via Smad (canonical) and ERK (non‐canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF‐β‐driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF‐β‐dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF‐β‐dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a ‘mixed’ contractile‐synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.

Comparative Study of Hemodynamics and Pathological Consequences of Saccular Versus Fusiform Experimental Aortic Aneurysms

Introduction - Saccular aneurysms are thought to have a worst evolution than fusiform aneurysms in humans, probably for hemodynamic reasons (1,2). Some authors advocate earlier aggressive therapy towards saccular aneurysms to prevent death from rupture (3). However, data is lacking regarding comparison of saccular versus fusiform aneurysms. The aim of this study was to compare the differential hemodynamics and pathological consequences of fusiform versus saccular aneurysms in rats. Methods - Forty Lewis rats, at 9 weeks of age, were prospectively randomized in two groups of twenty individuals each: “saccular” (Group A) and “fusiform” groups (Group B). Decellularized thoracic aortas from guinea pigs were xenografted to the abdominal aorta in either an end-to-end (fusiform) or side-to-end (saccular) anastomosis manner (4). Main objective was to evaluate hemodynamic outcomes through duplex-scan and magnetic resonance imaging (MRI), one week and three weeks after surgery. Rats were sacrificed 4 weeks after surgery in order to analyze aneurysms histology, dosage of matrix metallopeptidase-9 (MMP-9)/matrix metallopeptidase-2 (MMP-2), plasmin/anti-plasmin complex in the plasma and in conditioned media. Results - All rats displayed a patent aortic aneurysm at one and three weeks. At three weeks, duplex-scan showed a mean growth of 34.7±9.1 % in group A, compared to the first week. Similar results were found in group B with a mean growth of 33.2±9.8 % (p=not significant). A complete flow recirculation was found in saccular aneurysms unlike fusiform aneurysms where recirculation was incomplete (persistent longitudinal flow). These results were confirmed by MRI examination. Corresponding histology showed an organized intraluminal thrombus (ILT) as a background cup in group A whereas it was partially developed on one side in group B (Figure 1). The ILT from saccular aneurysms was enriched in iron coming from lysis of Red Blood Cells as compared to fusiform aneurysms. Similarly, calcifications were more important in group A as compared to group B, corresponding to more tissue retention of free DNA, potentially released by dying neutrophils. Conclusion - Saccular and fusiform aneurysms show a similar growth over time but differential specific hemodynamics impact the form, the aspect and the biology of ILT in each type of aneurysm. In particular, ILT remains more active in saccular aneurysms whereas fusiform aneurysms display a healing process through fibrosis. The active ILT in saccular aneurysms maintain an inflammatory environment by recruiting neutrophils and might be a risk factor to their worst evolution in time compared to fusiform aneurysms.

Selective and marked decrease of complement receptor C5aR2 in human thoracic aortic aneurysms: a dysregulation with potential inflammatory effects

ObjectiveThe aetiology of thoracic aortic aneurysm (TAA) is largely unknown, but inflammation is likely to play a central role in the pathogenesis. In this present study, we aim to investigate...

Protease Activity in Vascular Disease.

Protease Activity in Vascular Disease.

Time-dependent and site-dependent morphological changes in rupture-prone arteries: ovariectomized rat intracranial aneurysm model.

The pathogenesis of intracranial aneurysm rupture remains unclear. Because it is difficult to study the time course of human aneurysms and most unruptured aneurysms are stable, animal models are used to investigate the characteristics of intracranial aneurysms. The authors have newly established a rat intracranial aneurysm rupture model that features site-specific ruptured and unruptured aneurysms. In the present study the authors examined the time course of changes in the vascular morphology to clarify the mechanisms leading to rupture. Ten-week-old female Sprague-Dawley rats were subjected to hemodynamic changes, hypertension, and ovariectomy. Morphological changes in rupture-prone intracranial arteries were examined under a scanning electron microscope and the association with vascular degradation molecules was investigated. At 2-6 weeks after aneurysm induction, morphological changes and rupture were mainly observed at the posterior cerebral artery; at 7-12 weeks they were seen at the anterior Willis circle including the anterior communicating artery. No aneurysms at the anterior cerebral artery-olfactory artery bifurcation ruptured, suggesting that the inception of morphological changes is site dependent. On week 6, the messenger RNA level of matrix metalloproteinase-9, interleukin-1β, and the ratio of matrix metalloproteinase-9 to the tissue inhibitor of metalloproteinase-2 was significantly higher at the posterior cerebral artery, but not at the anterior communicating artery, of rats with aneurysms than in sham-operated rats. These findings suggest that aneurysm rupture is attributable to significant morphological changes and an increase in degradation molecules. Time-dependent and site-dependent morphological changes and the level of degradation molecules may be indicative of the vulnerability of aneurysms to rupture.

Aneurysm Architecture: First in vivo Imaging of Human Cerebral Aneurysms with Extravascular Optical Coherence Tomography

Objective: The present study was conducted to explore the value of 3-dimensional microscope integrated extravascular optical coherence tomography (OCT) as the first suitable intraoperative imaging modality of cerebral aneurysm (CA) and parent vessel wall morphology. Methods: Incidental CAs (n = 16) of the anterior circulation with indication for microsurgical clipping were scanned. Results: Analysis revealed that intraoperative OCT achieved to delineate the microstructural composition of the parent vessel in all cases and the CA wall in 68.8%. Clinical relevant characteristics such as thickness, calcification, residual tunica media, and atherosclerotic plaque of CA wall could be demonstrated with high image quality approaching the spatial resolution of histopathology. Conclusion: Our findings demonstrate that intraoperative OCT may hold promise as an additional imaging tool during neurovascular procedures.

Blockade of the Platelet-Driven CXCL7-CXCR1/2 Pathway Prevents Cerebral Aneurysm Formation

Abstract INTRODUCTION Platelet aggregation is intimately associated with vascular inflammation and is commonly seen on routine histology studies of cerebral aneurysms. Platelets, when activated, have been shown to augment neutrophil response and the proinflammatory cascade. Antiplatelet therapy, especially via ADP-GPIIb/IIIa antagonism, has inherent anti-inflammatory actions that could be of therapeutic interest in prevention of aneurysm formation, but could also interfere with healing of coiled aneurysms. Platelet-neutrophil complexes have been found to aggravate atherosclerosis through a positive feedback loop involving CXCL1, CXCR1/2, and CXCL7. We propose that treatment with GPIIb/IIIa antagonists or anti-CXCR1/2 receptor blockers could be used to prevent aneurysm formation. METHODS First, we induced cerebral aneurysm formation in a previously described intracranial aneurysm model via carotid artery ligation, hypertension, and stereotactic elastase injection in C57BL/6 mice, and analyzed vessels for lesion and thrombus formation. Raybiotech cytokines arrays were used to analyze 96 cytokines in induced aneurysms. Cerebral aneurysm formation was then studied in animals treated with 1:3 DMSO:PBS (control), clopidogrel, or anti-CXCR1/2 small molecule inhibitor. RESULTS CD31 + platelet aggregates are a common feature in human and mouse cerebral aneurysm specimens. Mice treated with 1 mg/kg clopidogrel develop significantly less aneurysms than controls (0% vs 57%, n = 6 and 7 respectively, P = .049). Semi-quantitative analysis of 96 different cytokines using Raybiotech arrays shows increased protein expression of CXCL7 in murine cerebral aneurysms when compared to controls. Treatment with clopidogrel results in reciprocal decrease in detected CXCL7. Targeting CXCL7-CXCR1/2 axis with 10 mg/kg reparixin (CXCR1/2 antagonist) tends decrease aneurysm formation. (14% vs 57%, n = 6 and 7, P = .13). CONCLUSION Small molecule inhibitors targeting platelet CXCL7-CXCR1/2 inflammatory axis can be used to prevent murine cerebral aneurysm formation.

RNA sequencing analysis revealed the induction of CCL3 expression in human intracranial aneurysms

Intracranial aneurysm (IA) is a socially important disease as a major cause of subarachnoid hemorrhage. Recent experimental studies mainly using animal models have revealed a crucial role of macrophage-mediated chronic inflammatory responses in its pathogenesis. However, as findings from comprehensive analysis of unruptured human IAs are limited, factors regulating progression and rupture of IAs in humans remain unclear. Using surgically dissected human unruptured IA lesions and control arterial walls, gene expression profiles were obtained by RNA sequence analysis. RNA sequencing analysis was done with read count about 60~100 million which yielded 6~10 billion bases per sample. 79 over-expressed and 329 under-expressed genes in IA lesions were identified. Through Gene Ontology analysis, ‘chemokine activity’, ‘defense response’ and ‘extracellular region’ were picked up as over-represented terms which included CCL3 and CCL4 in common. Among these genes, quantitative RT-PCR analysis using another set of samples reproduced the above result. Finally, increase of CCL3 protein compared with that in control arterial walls was clarified in IA lesions. Findings of the present study again highlight importance of macrophage recruitment via CCL3 in the pathogenesis of IA progression.