Intracranial Aneurysm Tissues Research Articles

The SNHG12/microRNA-15b-5p/MYLK axis regulates vascular smooth muscle cell phenotype to affect intracranial aneurysm formation

ObjectiveThis research was dedicated to investigating the impact of the SNHG12/microRNA (miR)-15b-5p/MYLK axis on the modulation of vascular smooth muscle cell (VSMC) phenotype and the formation of intracranial aneurysm (IA). MethodsSNHG12, miR-15b-5p and MYLK expression in IA tissue samples from IA patients were tested by RT-qPCR and western blot. Human aortic vascular smooth muscle cells (VSMCs) were cultivated with H2O2 to mimic IA-like conditions in vitro, and the cell proliferation and apoptosis were measured by MTT assay and Annexin V/PI staining. IA mouse models were established by induction with systemic hypertension combined with elastase injection. The blood pressure in the tail artery of mice in each group was assessed and the pathological changes in arterial tissues were observed by HE staining and TUNEL staining. The expression of TNF-α and IL-1β, MCP-1, iNOS, caspase-3, and caspase-9 in the arterial tissues were tested by RT-qPCR and ELISA. The relationship among SNHG12, miR-15b-5p and MYLK was verified by bioinformatics, RIP, RNA pull-down, and luciferase reporter assays. ResultsThe expression levels of MYLK and SNHG12 were down-regulated and that of miR-15b-5p was up-regulated in IA tissues and H2O2-treated human aortic VSMCs. Overexpressed MYLK or SNHG12 mitigated the decrease in proliferation and increase in apoptosis of VSMCs caused by H2O2 induction, and overexpression of miR-15b-5p exacerbated the decrease in proliferation and increase in apoptosis of VSMCs caused by H2O2 induction. Overexpression of miR-15b-5p reversed the H2O2-treated VSMC phenotypic changes caused by SNHG12 up-regulation, and overexpression of MYLK reversed the H2O2-treated VSMC phenotypic changes caused by up-regulation of miR-15b-5p. Overexpression of SNHG12 reduced blood pressure and ameliorated arterial histopathological damage and VSMC apoptosis in IA mice. The mechanical analysis uncovered that SNHG12 acted as an endogenous RNA that competed with miR-15b-5p, thus modulating the suppression of its endogenous target, MYLK. ConclusionDecreased expression of SNHG12 in IA may contribute to the increasing VSMC apoptosis via increasing miR-15b-5p expression and subsequently decreasing MYLK expression. These findings provide potential new strategies for the clinical treatment of IA.

Aberrant expression and regulatory role of histone deacetylase 9 in vascular endothelial cell injury in intracranial aneurysm.

Intracranial aneurysm (IA) is one of the most challenging cerebrovascular lesions for clinicians. The aim of this study was to investigate the abnormal expression and role of histone deacetylase 9 (HDAC9) in IA-associated injury of vascular endothelial cells (VECs). First, IA tissue and normal arterial tissue were collected and VECs were isolated from IA patients. The expression levels of HDAC9, microRNA (miR)-34a-5p, and vascular endothelial growth factor-A (VEGFA) were determined. Cell viability, proliferation, apoptosis, and migration were assessed by Cell Counting Kit-8 (CCK-8) assay, EdU staining, TUNEL staining, and transwell assay. The binding of miR-34a-5p to VEGFA was analyzed by the dual-luciferase assay, and the accumulation of HDAC9 and lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) on the miR-34a-5p promoter was detected by the chromatin immunoprecipitation assay. The results showed that HDAC9 and VEGFA were increased and miR-34a-5p was decreased in IA tissues and cells. Silencing of HDAC9 inhibited apoptosis and increased viability, proliferation, and migration of VECs, whereas overexpression of HDAC9 exerted the opposite functions. HDAC9 accumulated at the miR-34a-5p promoter to decrease miR-34a-5p expression by reducing H3 locus-specific acetylation and further promoted VEGFA expression. Knockdown of miR-34a-5p or VEGFA overexpression reversed the protective role of HDAC9 silencing in VECs injury. In conclusion, our study suggests that HDAC9 may be a therapeutic target for IA.

Axl promotes intracranial aneurysm rupture by regulating macrophage polarization toward M1 via STAT1/HIF-1α.

Macrophage infiltration and polarization are crucial for the pathogenesis of intracranial aneurysm (IA) rupture. Axl, a receptor tyrosine kinase, is involved in inflammation and efferocytosis in multiple organs. Upregulated soluble Axl in cerebrospinal fluid (CSF) and plasma is correlated with intracranial aneurysm rupture. This study aimed to investigate the role of Axl in IA rupture and macrophage polarization. Male C57BL/6J mice were used to induce IA. The level of Axl from control vessels and unruptured and ruptured IA samples was detected. In addition, the relationship between Axl and macrophages was confirmed. The pathway of Axl-mediated macrophage polarization was explored after IA induction in vivo and in bone marrow-derived macrophages (BMDMs) stimulated by LPS/IFN-γ in vitro. The animals were randomized into three groups and treated intraperitoneally with the vehicle, selective AXL antagonist R428, and recombinant mouse growth arrest-specific 6 (rmGas6) for 21 consecutive days. Then, we evaluated the influence of Axl on IA rupture by administrating R428 to inhibit or rmGas6 to activate the Axl receptor in vivo. Compared with that in normal vessels, Axl expression was significantly upregulated in unruptured IA samples. The ruptured IA tissue exhibited significantly higher expression of Axl than the unruptured IA tissue. Axl and F4/80 were coexpressed in IA tissue and LPS/IFN-γ-stimulated BMDMs. R428 treatment significantly reduced the rate of M1-like macrophage infiltration and IA rupture. In contrast, rmGas6 treatment promoted M1 macrophage infiltration and IA rupture. Mechanistically, R428 inhibited the phosphorylation of Axl and STAT1 and the expression of hypoxia-inducible factor-1α (HIF-1α) and decreased the levels of IL-1β, NOS2, and MMP9 in LPS/IFN-γ-stimulated BMDMs. rmGas6 promoted the phosphorylation of Axl and STAT1 and the expression of HIF-1α. In addition, STAT1 knockdown abolished Axl-mediated M1 macrophage polarization. The inhibition of Axl reduced macrophage polarization toward the M1 phenotype via the STAT1/HIF-1α signaling pathway and prevented IA rupture in mice. This finding suggests that pharmacological inhibition of Axl might be used to prevent the progression and rupture of IA.

Interruption of TRPC6-NFATC1 signaling inhibits NADPH oxidase 4 and VSMCs phenotypic switch in intracranial aneurysm

Intracranial aneurysm (IA) is a frequent cerebrovascular disorder with unclear pathogenesis. The vascular smooth muscle cells (VSMCs) phenotypic switch is essential for IA formation. It has been reported that Ca2+ overload and excessive reactive oxygen species (ROS) are involved in VSMCs phenotypic switch. The transient receptor potential canonical 6 (TRPC6) and NADPH oxidase 4 (NOX4) are the main pathway to participate in Ca2+ overload and ROS production in VSMCs. Ca2+ overload can activate calcineurin (CN), leading to nuclear factor of activated T cell (NFAT) dephosphorylation to regulate the target gene's transcription. We hypothesized that activation of TRPC6-NFATC1 signaling may upregulate NOX4 and involve in VSMCs phenotypic switch contributing to the progression of IA. Our results showed that the expressions of NOX4, p22phox, p47phox, TRPC6, CN and NFATC1 were significantly increased, and VSMCs underwent a significant phenotypic switch in IA tissue and cellular specimens. The VIVIT (NFATC1 inhibitor) and BI-749327 (TRPC6 inhibitor) treatment reduced the expressions of NOX4, p22phox and p47phox and the production of ROS, and significantly improved VSMCs phenotypic switch in IA rats and cells. Consistent results were obtained from IA Trpc6 knockout (Trpc6-/-) mice. Furthermore, the results also revealed that NFATC1 could regulate NOX4 transcription by binding to its promoter. Our findings reveal that interrupting the TRPC6-NFATC1 signaling inhibits NOX4 and improves VSMCs phenotypic switch in IA, and regulating Ca2+ homeostasis may be an important therapeutic strategy for IA.

Serum Interleukin-1 Levels Are Associated with Intracranial Aneurysm Instability.

Serum interleukin-1 (IL-1) are possibly indicative of the inflammation in the intracranial aneurysm (IA) wall. This study aimed to investigate whether IL-1 could discriminate the unstable IAs (ruptured intracranial aneurysms (RIAs) and symptomatic unruptured intracranial aneurysms (UIAs)) from stable, asymptomatic UIAs. IA tissues and blood samples from 35 RIA patients and 35 UIA patients were collected between January 2017 and June 2020 as the derivation cohort. Blood samples from 211 patients with UIAs were collected between January 2021 and June 2022 as the validation cohort (including 63 symptomatic UIAs). Blood samples from 35 non-cerebral-edema meningioma patients (non-inflammatory control) and 19 patients with unknown-cause subarachnoid hemorrhage (hemorrhagic control) were also collected. IL-1β and IL-1.ra (IL-1 receptor antagonist) were measured in serum and IA tissues, and the IL-1 ratio was calculated as log10 (IL-1.ra/IL-1β). Based on the derivation cohort, multivariate logistic analysis showed that IL-1β (odds ratio, 1.48, P = 0.001) and IL-1.ra (odds ratio, 0.74, P = 0.005) were associated with RIAs. The IL-1 ratio showed an excellent diagnostic accuracy for RIAs (c-statistic, 0.91). Histological analysis confirmed the significant correlation of IL-1 between serum and aneurysm tissues. IL-1 ratio could discriminate UIAs from non-inflammatory controls (c-statistic, 0.84), and RIAs from hemorrhagic controls (c-statistic, 0.95). Based on the validation cohort, the combination of IL-1 ratio and PHASES score had better diagnostic accuracy for symptomatic UIAs than PHASES score alone (c-statistic, 0.88 vs 0.80, P < 0.001). Serum IL-1 levels correlate with aneurysm tissue IL-1 levels and unstable aneurysm status, and could serve as a potential biomarker for IA instability.

Integrated analysis identifies the IL6/JAK/STAT signaling pathway and the estrogen response pathway associated with the pathogenesis of intracranial aneurysms.

We intended to identify the potential key biomarker and pathways that correlated with infiltrating immune cells during the pathogenesis of intracranial aneurysms (IA), to develop a diagnostic model, and to predict therapeutic drugs. Three datasets containing intracranial aneurysm tissue samples and normal artery control samples from Gene Expression Omnibus (GEO) were included. Gene-set variation analysis(GSVA) and gene set enrichment analysis (GSEA) were conducted to find the significant differentially expressed pathways in IA formation. The least absolute shrinkage and selection operator (LASSO) regression and the multivariate logistic regression analysis were performed to identify the characteristic genes in the IL6/JAK/STAT signaling pathway (ISP) and the estrogen response pathway (ERP). A diagnostic model was constructed. xCell was used to identify immune cell types in IA pathogenesis. We used the weighted gene co-expression network analysis (WGCNA) algorithm to explore the correlations between the key modules and the four traits. Potential therapeutic drugs were investigated in Enrichr and Drugbank database. The ISP is significant positively correlated with IA onset. The biological function of the ISP is positively correlated with that of the ERP, and is significantly associated with immune cells activities. CSF2RB, FAS, IL6, PTPN1, STAT2, TGFB1 of the ISP gene set and ALDH3A2, COX6C, IGSF1, KRT18, MICB, NPY1R of the ERP gene set were proved to be the characteristic genes. The STAT2 gene can be the potential biomarker of IA onset. The immune score of IA samples was significantly higher than the controls. The STAT2 gene expression is associated with infiltration of immune cells. The WGCNA results were consistent with our finds. Acetaminophen can be a potential therapeutic drug for IA targeting STAT2. We identified that the ISP was one of the most significant positively correlated pathways in IA onset, and it was activated in this process concordant with the ERP and immune responses. Except for beneficial effects, complex and multiple roles of estrogen may be involved in IA formation. STAT2 could be a potential biomarker and a promising therapeutic target of IA pathogenesis.

Dexmedetomidine alleviates inflammatory response and oxidative stress injury of vascular smooth muscle cell via α2AR/GSK-3β/MKP-1/NRF2 axis in intracranial aneurysm

Vascular smooth muscle cell (VSMC) phenotypic modulation regulates the initiation and progression of intracranial aneurysm (IA). Dexmedetomidine (DEX) is suggested to play neuroprotective roles in patients with craniocerebral injury. Therefore, we investigated the biological functions of DEX and its mechanisms against IA formation and progression in the current study. The rat primary VSMCs were isolated from Sprague–Dawley rats. IA and superficial temporal artery (STA) tissue samples were obtained from patients with IA. Flow cytometry was conducted to identify the characteristics of isolated VSMCs. Hydrogen peroxide (H2O2) was used to mimic IA-like conditions in vitro. Cell viability was detected using CCK-8 assays. Wound healing and Transwell assays were performed to detect cell motility. ROS production was determined by immunofluorescence using DCFH-DA probes. Western blotting and RT-qPCR were carried out to measure gene expression levels. Inflammation responses were determined by measuring inflammatory cytokines. Immunohistochemistry staining was conducted to measure α2-adrenergic receptor levels in tissue samples. DEX alleviated the H2O2-induced cytotoxicity, attenuated the promoting effects of H2O2 on cell malignancy, and protected VSMCs against H2O2-induced oxidative damage and inflammation response. DEX regulated the GSK-3β/MKP-1/NRF2 pathway via the α2AR. DEX alleviates the inflammatory responses and oxidative damage of VSMCs by regulating the GSK-3β/MKP-1/NRF2 pathway via the α2AR in IA.

Bioinformatics analysis reveals potential biomarkers associated with the occurrence of intracranial aneurysms

To better understand the molecular mechanisms of intracranial aneurysm (IA) pathogenesis, we used gene coexpression networks to identify hub genes and functional pathways associated with IA onset. Two Gene Expression Omnibus (GEO) datasets encompassing intracranial aneurysm tissue samples and cerebral artery control samples were included. To discover functional pathways and potential biomarkers, weighted gene coexpression network analysis was employed. Next, single-gene gene set enrichment analysis was employed to investigate the putative biological roles of the chosen genes. We also used receiver operating characteristic analysis to confirm the diagnostic results. Finally, we used a rat model to confirm the hub genes in the module of interest. The module of interest, which was designated the green module and included 115 hub genes, was the key module that was most strongly and negatively associated with IA formation. According to gene set variation analysis results, 15 immune-related pathways were significantly activated in the IA group, whereas 7 metabolic pathways were suppressed. In two GEO datasets, SLC2A12 could distinguish IAs from control samples. Twenty-nine hub genes in the green module might be biomarkers for the occurrence of cerebral aneurysms. SLC2A12 expression was significantly downregulated in both human and rat IA tissue. In the present study, we identified 115 hub genes related to the pathogenesis of IA onset and deduced their potential roles in various molecular pathways; this new information may contribute to the diagnosis and treatment of IAs. By external validation, the SLC2A12 gene may play an important role. The molecular function of SLC2A12 in the process of IA occurrence can be further studied in a rat model.

The roles and diagnostic value of miRNA-1246 in the serum of patients with intracranial aneurysms

BackgroundInflammatory response is one of the important factors affecting the formation of intracranial aneurysm. miR-1246 is involved in the regulation of several inflammatory diseases; however, its expression levels and the mechanism of action in intracranial aneurysms remain further investigated.MethodsBioinformatics was used to analyze the levels of micro-RNAs (miRNAs) in the serum of intracranial aneurysm patients as well as in the intracranial aneurysm tissues downloaded from the GEO RNA-seq database. Blood samples were collected pre-operatively from patients with intracranial aneurysms as well as from healthy volunteers, and miRNA-1246 expression levels were detected using quantitative reverse transcriptase polymerase chain reaction. Meanwhile, the diagnostic value of miR-1246 for intracranial aneurysm was explored using the receiver operating characteristic (ROC) curve.Principle findings and resultsSerum levels of miR-1246 were elevated in intracranial aneurysm patients. Bioinformatics studies revealed that the target genes of miR-1246, TP53, glycogen synthetase kinase (GSK), and transcription factor YY1 may play important roles in the development of intracranial aneurysms. miR-1246 is involved in inflammatory response, lipid, and atherosclerotic signaling pathways.Conclusions and significanceHigh level of miR-1246 is found in the serum of patients with intracranial aneurysms and may serve as a diagnostic or/and treatment marker for intracranial aneurysms.

Circular RNA circ_0021001 regulates miR-148b-3p/GREM1 axis to modulate proliferation and apoptosis of vascular smooth muscle cells.

Intracranial aneurysm (IA) is an abnormal expression in the intracranial arteries, which is related to the growth and apoptosis of vascular smooth muscle cells (VSMCs). Circular RNA (circRNA) circ_0021001 (also named circARFIP2) has been identified to mediate the regulation of VSMCs proliferation. However, the molecular mechanism of circ_0021001 involved in VSMC dysfunction in IA is poorly defined.The expression levels of circ_0021001, microRNA-148b-3p (miR-148b-3p), and Gremlin 1 (GREM1) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, proliferation, cell cycle progression, and apoptosis were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Protein levels of proliferating cell nuclear antigen (PCNA), p21, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and GREM1 were examined by western blot assay. The binding relationship between miR-148b-3p and circ_0021001 or GREM1 was predicted by StarBase and then verified using a dual-luciferase reporter assay.The expression levels of circ_0021001 and GREM1 were increased, and that of miR-148b-3p was decreased in IA tissues and HUASMCs. Moreover, the downregulation of circ_0021001 could repress proliferation ability and induce apoptosis of HUASMCs. The mechanical analysis uncovered that circ_0021001 served as a sponge of miR-148b-3p to regulate GREM1 expression.Circ_0021001 silencing could suppress cell growth and induce apoptosis of HUASMCs partially through modulating the miR-148b-3p/GREM1, presented circ_0021001 as a promising therapeutic target for IA.

Genome-wide linkage analysis combined with genome sequencing in large families with intracranial aneurysms

Rupture of an intracranial aneurysm (IA) leads to aneurysmal subarachnoid haemorrhage (ASAH), a severe type of stroke. Some rare variants that cause IA in families have been identified, but still, the majority of genetic causes, as well as the biological mechanisms of IA development and rupture, remain unknown. We aimed to identify rare, damaging variants for IA in three large Dutch families with multiple affected members with IA (N = 9, 11, and 6). By combining linkage analysis and genome sequencing (GS), we identified six rare and damaging variants for which all cases within one of the families were heterozygous. These variants were p.Tyr87Cys in SYCP1, p.Phe1077Leu in FMNL2, p.Thr754Lys in TBC1D2, p.Arg321His in ZNF782, p.Arg979Trp in CCDC180, and p.Val125Met in NCBP1. None of the variants showed association with IA status in a large cohort of 937 patients from the general IA patient population and 1046 controls. Gene expression in IA and cerebral artery tissue further prioritized FMNL2 and TBC1D2 as potential important players in IA pathophysiology. Further studies are needed to characterize the functional consequences of the identified variants and their role in the biological mechanisms of IA.

Potential Role of the Chemotaxis System in Formation and Progression of Intracranial Aneurysms Through Weighted Gene Co-Expression Network Analysis.

BackgroundIntracranial aneurysm (IA) is the most common and is the main cause of spontaneous subarachnoid hemorrhage (SAH). The underlying molecular mechanisms for preventing IA progression have not been fully identified. Our research aimed to identify the key genes and critical pathways of IA through gene co-expression networks.MethodsGene Expression Omnibus (GEO) datasets GSE13353, GSE54083 and GSE75436 were used in the study. The genetic data were analyzed by weighted gene co-expression network analysis (WGCNA). Then the clinically significant modules were identified and the differentially expressed genes (DEGs) with the genes were intersected in these modules. GO (gene ontology) and KEGG (Kyoto Gene and Genomic Encyclopedia) were used for gene enrichment analysis to determine the function or pathway. In addition, the composition of immune cells was analyzed by CIBERSORT algorithm. Finally, the hub genes and key genes were identified by GSE122897.ResultsA total of 266 DEGs and two modules with clinical significance were identified. The inflammatory response and immune response were identified by GO and KEGG. CCR5, CCL4, CCL20, and FPR3 were the key genes in the module correlated with IA. The proportions of infiltrating immune cells in IA and normal tissues were different, especially in terms of macrophages and mast cells.ConclusionThe chemotactic system has been identified as a key pathway of IA, and interacting macrophages may regulate this pathological process.

Identification and Validation of TREM2 in Intracranial Aneurysms.

Background: Intracranial aneurysm (IA) is a cerebrovascular disease that seriously endangers human heath and life. However, the pathogenesis of IA has not been clarified. Objective: In this study, we explored the role of the triggering receptor expressed on myeloid cells-2 (TREM2) gene to explore a novel mechanism underlying IA. Methods: First, we verified the role of the candidate gene, TREM2 in a modified mouse model of IA. Second, we verified elevated expression of TREM2 using the Gene Expression Omnibus (GEO) database (GSE54083 and GSE75436) and developed protein interaction (PPI) network analysis using the top one hundred DEGs from GSE75436 dataset. Finally, we predicted a likely mechanism by which TREM2 is involved in the pathology of IA using single-gene Gene Set Enrichment Analysis (GSEA). Results: The expression of TREM2 and inflammatory factors was significantly increased in the modified mouse IA model, and showed a positive correlation. Elevated expression of TREM2 was also found in IA patients tissues from the GSE54083 and GSE75436 data sets. PPI network analyses suggested that the DEGs were involved in a variety of inflammatory processes. The GSEA results suggest that TREM2 may participate in IA progression by regulating macrophage function. Conclusion: TREM2 is highly expressed in both human and mouse IA tissues, and may participate in IA progression by regulating macrophage function and inflammatory factor expression. The molecular mechanism of TREM2 involvement in the IA process can be further studied using our modified mouse IA model.

RNA Sequencing Data from Human Intracranial Aneurysm Tissue Reveals a Complex Inflammatory Environment Associated with Rupture.

Intracranial aneurysm (IA) rupture leads to deadly subarachnoid hemorrhages. However, the mechanisms leading to rupture remain poorly understood. Altered gene expression within IA tissue is linked to the pathobiology of aneurysm development and progression. Here, we analyzed expression patterns of control tissue samples and compared them to those of unruptured and ruptured IA tissue samples using data from the Gene Expression Omnibus (GEO). FASTQ files for 21 ruptured IAs, 21 unruptured IAs, and 16 control tissue samples were accessed from the GEO database. DESeq2 was used for differential expression analysis in three comparisons: unruptured IA versus control, ruptured IA versus control, and ruptured versus unruptured IA. Genes that were differentially expressed in multiple comparisons were evaluated to find those progressively increasing/decreasing from control to unruptured to ruptured. Significance was tested by either analysis of variance/Gabriel or Brown-Forsythe/Games Howell (p<0.05 was considered significant). We used additional RNA sequencing and proteomics datasets to evaluate if our differentially expressed genes (DEGs) were present in other studies. Bioinformatics analyses were performed with g:Profiler and Ingenuity Pathway Analysis. In total, we identified 1768 DEGs, of which 318 were found in multiple comparisons. Unruptured versus control reflected vascular remodeling processes, while ruptured versus control reflected inflammatory responses and cell activation/signaling. When comparing ruptured to unruptured IAs, we found massive activation of inflammation, inflammatory responses, and leukocyte responses. Of the 318 genes in multiple comparisons, 127 were found to be significant in the multi-cohort correlation analysis. Those that progressively increased (70 genes) were associated with immune system processes, while those that progressively decreased (38 genes) did not return any gene ontology terms. Many of our DEGs were also found in the other IA tissue sequencing studies. We found unruptured IAs relate more to remodeling processes, while ruptured IAs reflect more inflammatory and immune responses.

The Potential Role of hsa_circ_0005505 in the Rupture of Human Intracranial Aneurysm.

Objective: Recently, abundant number of studies have revealed many functions of circular RNAs in multiple diseases, however, the role of circular RNA in the rupture of human intracranial aneurysm is still unknown. This study aims to explore the potential functions of circular RNA in the rupture of human intracranial aneurysms. Methods: The differentially expressed circular RNAs between un-ruptured intracranial aneurysms (n = 5) and ruptured intracranial aneurysms (n = 5) were analyzed with the Arraystar human circRNAs microarray. Quantitative real-time PCR (qPCR) was used to verify the results of the circRNA microarray. The role of circular RNA in intracranial aneurysm rupture was assessed in vitro. MTT assay, CCK-8 assay, Caspase3/7 assay, assay of cell apoptosis and Celigo wound healing was conducted to evaluate the relationship between circular RNA and the rupture of human intracranial aneurysms. Results: A total of 13,175 circRNA genes were detected. Among them 63 circRNAs upregulated and 54 circRNAs downregulated significantly in ruptured intracranial aneurysms compared with un-ruptured intracranial aneurysms (p < 0.05 Fold Change > 1.5). Five upregulated circRNAs were selected for further study (hsa_circ_0001947, hsa_circ_0043001, hsa_circ_0064557, hsa_circ_0058514, hsa_circ_0005505). The results of qPCR showed only hsa_circ_0005505 significantly upregulated (p < 0.05). The expression of hsa_circ_0005505 was higher in ruptured intracranial aneurysm tissues. And our in vitro data showed that hsa_circRNA_005505 promotes the proliferation, migration and suppresses the apoptosis of vascular smooth muscle cell. Conclusion: This study revealed an important role of hsa_circ_0005505 in the proliferation, migration and apoptosis of vascular smooth muscle cell, and indicated that hsa_circ_0005505 may associate with the pathological process of intracranial aneurysms.

Epigenetic landscapes of intracranial aneurysm risk haplotypes implicate enhancer function of endothelial cells and fibroblasts in dysregulated gene expression

BackgroundGenome-wide association studies have identified many single nucleotide polymorphisms (SNPs) associated with increased risk for intracranial aneurysm (IA). However, how such variants affect gene expression within IA is poorly understood. We used publicly-available ChIP-Seq data to study chromatin landscapes surrounding risk loci to determine whether IA-associated SNPs affect functional elements that regulate gene expression in cell types comprising IA tissue.MethodsWe mapped 16 significant IA-associated SNPs to linkage disequilibrium (LD) blocks within human genome. Using ChIP-Seq data, we examined these regions for presence of H3K4me1, H3K27ac, and H3K9ac histone marks (typically associated with latent/active enhancers). This analysis was conducted in several cell types that are present in IA tissue (endothelial cells, smooth muscle cells, fibroblasts, macrophages, monocytes, neutrophils, T cells, B cells, NK cells). In cell types with significant histone enrichment, we used HiC data to investigate topologically associated domains (TADs) encompassing the LD blocks to identify genes that may be affected by IA-associated variants. Bioinformatics were performed to determine the biological significance of these genes. Genes within HiC-defined TADs were also compared to differentially expressed genes from RNA-seq/microarray studies of IA tissues.ResultsWe found that endothelial cells and fibroblasts, rather than smooth muscle or immune cells, have significant enrichment for enhancer marks on IA risk haplotypes (p < 0.05). Bioinformatics demonstrated that genes within TADs subsuming these regions are associated with structural extracellular matrix components and enzymatic activity. The majority of histone marked TADs (83% fibroblasts [IMR90], 77% HUVEC) encompassed at least one differentially expressed gene from IA tissue studies.ConclusionsThese findings provide evidence that genetic variants associated with IA risk act on endothelial cells and fibroblasts. There is strong circumstantial evidence that this may be mediated through altered enhancer function, as genes in TADs encompassing enhancer marks have also been shown to be differentially expressed in IA tissue. These genes are largely related to organization and regulation of the extracellular matrix. This study builds upon our previous (Poppenberg et al., BMC Med Genomics, 2019) by including a more diverse set of data from additional cell types and by identifying potential affected genes (i.e. those in TADs).

Bioinformatic Identification of Differentially Expressed Genes and Pathways in Intracranial Aneurysm

Background: Intracranial Aneurysm (IA) is a serious disease with high mortality and high morbidity rates, but the pathophysiological mechanisms of IA remain unclear. This study aimed to identify the Differentially Expressed Genes (DEGs) between IA tissues and Superficial Temporal Artery (STA) tissues using bioinformatic analysis. Methods: To investigate the key genes that are important for IAs, we analyzed microarray datasets (GSE75436) from the Gene Expression Omnibus (GEO) database, including 15 IA samples and 15 normal STA samples. First, we used the GEO2R tool to screen for DEGs (P-value&lt;0.01 and |log2 FC| ≥2) between IA and STA tissues. Subsequently, the Database for Annotation, Visualization, and Integrated Discover software was used to perform function and pathway enrichment analyses. Finally, protein-protein interaction network analysis was performed using the Search Tool for Retrieval of Interacting Genes and Cytoscape software. Real-Time Quantitative Polymerase Chain Reaction (RT-QPCR) was performed to prove our assumption. Results: A total of 829 DEGs, of which 399 were upregulated and 430 were downregulated, were identified. The upregulated genes were mostly associated with Staphylococcus aureus infection, amoebiasis, rheumatoid arthritis, phagocytosis, and tuberculosis. The downregulated genes were mainly involved in vascular smooth muscle contraction, calcium signaling, histidine metabolism, cGMP-PKG signaling, and cAMP signaling. From the DEGs, five genes were selected as hub genes on the basis of the connection degree, which is one of 12 calculation methods from a plugin of Cytoscape called cytoHubba. The PCR results demonstrated that the expression levels of the top five hub genes, namely, Tumor Necrosis Factor (TNF), interleukin 8 (IL-8), Protein Tyrosine Phosphatase Receptor Type C (PTPRC), interleukin 1β (IL-1β), and Toll-like receptor 4 (TLR 4), were significantly higher in the IA samples than in the STA samples. Conclusion: TNF showed higher expression in the IA samples than in the STA samples. Thus, this gene may be involved in the occurrence and development of IA. The immune response and inflammation play important roles in the progression of IA. However, the specific pathophysiological mechanism needs further study.

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 human aneurysms. In this pilot study, for the first time, we comprehensively quantified the mechanical, collagen fiber microstructural, and morphological properties of one 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 lack of elastin, intimal thickening, and calcium deposition. This new unified characterization framework can be extended to better understand the mechanics-microstructure interrelationship of aneurysm tissues at different time points of the formation or growth. Such specimen-specific information is anticipated to provide valuable insight that may improve our current understanding of aneurysm growth and rupture potential.

Histone deacetylase 9 inhibition upregulates microRNA-92a to repress the progression of intracranial aneurysm via silencing Bcl-2-like protein 11

Objective Histone deacetylases (HDACs) have been revealed to be involved in cerebrovascular diseases, while the role of HDAC9 in intracranial aneurysm (IA) remains seldom studied. We aim to explore the role of the HDAC9/microRNA-92a (miR-92a)/Bcl-2-like protein 11 (BCL2L11) axis in IA progression. Methods Expression of HDAC9, miR-92a and BCL2L11 in IA tissues was assessed. IA rat models were established by ligation of left renal artery and common carotid artery, and the rats were respectively injected with relative plasmid vectors and/or oligonucleotides. The blood pressure was measured to estimate the IA degree, and the pathological changes were observed. The expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF) was detected, and the levels of inflammatory factors were evaluated. Expression of apoptosis-related proteins, HDAC9, miR-92a and BCL2L11 was assessed. Results HDAC9 and BCL2L11 were upregulated while miR-92a was downregulated in IA clinical samples and rat models. HDAC9 inhibition or miR-92a elevation improved pathological changes and repressed apoptosis and expression of MMP-2, MMP-9, VEGF and inflammatory factors in vascular tissues from IA rats. Oppositely, HDAC9 overexpression or miR-92a reduction had contrary effects. miR-92a downregulation reversed the effect of silenced HDAC9 on IA rats. Conclusion HDAC9 inhibition upregulates miR-92a to repress the progression of IA via silencing BCL2L11.

Sustained expression of MCP-1 induced low wall shear stress loading in conjunction with turbulent flow on endothelial cells of intracranial aneurysm.

Shear stress was reported to regulate the expression of AC007362, but its underlying mechanisms remain to be explored. In this study, to isolate endothelial cells of blood vessels, unruptured and ruptured intracranial aneurysm (IA) tissues were collected from IA patients. Subsequently, quantitative real‐time PCR (qRT‐PCR), Western blot and luciferase assay were performed to investigate the relationships between AC007362, miRNAs‐493 and monocyte chemoattractant protein‐1 (MCP‐1) in human umbilical vein endothelial cells (HUVECs) exposed to shear stress. Reduced representation bisulphite sequencing (RRBS) was performed to assess the level of DNA methylation in AC007362 promoter. Accordingly, AC007362 and MCP‐1 were significantly up‐regulated while miR‐493 was significantly down‐regulated in HUVECs exposed to shear stress. AC007362 could suppress the miR‐493 expression and elevate the MCP‐1 expression, and miR‐493 was shown to respectively target AC007362 and MCP‐1. Moreover, shear stress in HUVECs led to the down‐regulated DNA methyltransferase 1 (DNMT1), as well as the decreased DNA methylation level of AC007362 promoter. Similar results were also observed in ruptured IA tissues when compared with unruptured IA tissues. In conclusion, this study presented a deep insight into the operation of the regulatory network of AC007362, miR‐493 and MCP‐1 upon shear stress. Under shear stress, the expression of AC007362 was enhanced by the inhibited promoter DNA methylation, while the expression of MCP‐1 was enhanced by sponging the expression of miR‐493.