Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2021.11869 MMR-0-0-11869 Articles Involvement of TLR2/4-MyD88-NF-κB signaling pathway in the pathogenesis of intracranial aneurysm ZhangXuezhi WanYilv FengJiugeng LiMeihua JiangZhiqun Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China Correspondence to: Dr Zhiqun Jiang, Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, 17 Yongwai Zheng Street, Nanchang, Jiangxi 330006, P.R. China, E-mail: jiangzhiqun123@126.com 04 2021 26 01 2021 23 4 230 22032020 07122020 Copyright © 2021, Spandidos Publications 2021

Toll-like receptor (TLR) 2/4 serves an important regulatory role in nerve tissue injury. However, the downstream and potential mechanisms remain to be elucidated. The present study was designed to investigate the roles of the TLR2/4-major myeloid differentiation response gene 88 (MyD88)-NF-κB signaling pathway in the development of intracranial aneurysm. The expression of TLR2, TLR4 and MyD88 in the blood of normal controls and patients with intracranial aneurysm were detected by quantitative PCR and ELISA. Human brain vascular smooth muscle cells were treated by Angiotensin II (Ang II) to evaluate the involvement of TLR2/4-MyD88-NF-κB signaling pathway in the process. The in vitro experiment was divided into four groups: The control group, an Ang II group, an Ang II + small interfering (si)RNA control group and an Ang II + TLR2-group. Cell viability, migration, apoptosis and expression of TLR2, TLR4, MyD88, NF-κB and phosphorylated (p-)p65 expression were detected. The results demonstrated that the expression of TLR2, TLR4, MyD88 and NF-κB at mRNA and protein levels in patients with intracranial aneurysm was significantly higher compared with corresponding protein in normal controls (P<0.05). In vitro experiments demonstrated that Ang II treatment increased the cell proliferation and migration rate but reduced the apoptotic rate compared with the control (P<0.05). The expression of TLR2, TLR4, MyD88, NF-κB and p-p65 was significantly increased in the Ang II group (vs. control; P<0.05). By contrast, TLR2-short interfering RNA reduced the cell proliferation and migration rate, and reduced the expression of TLR2, TLR4, MyD88, NF-κB and p-p65 (vs. Ang II + short interfering RNA control; P<0.05). In conclusion, the data of the present study indicated that the TLR2/4-MyD88-NF-κB signaling pathway is involved in the pathogenesis of intracranial aneurysm.

 intracranial aneurysm Toll-like receptor 2/4 major myeloid differentiation response gene 88 NF-κB Introduction

Intracranial aneurysm is one of the common disorders in clinical neuroscience with an incidence of approximately 3.2% worldwide (1). The incidence of intracranial aneurysm in the female population is at ≤12.9%, significantly higher compared with the male population (2). Notably, intracranial aneurysm is a non-ignorable factor for subarachnoid hemorrhage following cerebral thrombosis and hypertensive cerebral hemorrhage in cerebrovascular accidents (3,4). Indeed, >85% of spontaneous subarachnoid hemorrhage is caused by rupture of aneurysms (3,4). Therefore, it is of great clinical significance to explore the pathogenesis of intracranial aneurysms and find effective therapeutic targets.

There are many types of Toll-like receptor (TLRs) subfamilies. TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are found on the cell surface and can be activated by a variety of extracellular ligands, while TLR3, TLR7, TLR8 and TLR9 are intracellularly expressed and generally recognized by nucleic acid structure (5). TLRs serve important roles in the inflammatory response of central nervous system (6). TLR2 and TLR4 expression increases in patients with ischemic stroke and TLR4 knockdown attenuates brain edema, inflammation and damage after intracerebral hemorrhage (ICH) (7). In addition, TLR2 promotes inflammatory damage after ICH (8). Hemoglobin can induce the formation of TLR2/TLR4 heterodimer after ICH, which can amplify the harmful effects of TLR2 and TLR4 in the brain (9).

The major myeloid differentiation response gene 88 (MyD88) is an important adaptor that is downstream of TLR2 and TLR4 (10). Activated MyD88 can promote the phosphorylation and nuclear translocation of NF-κB, thus upregulating the expression of pro-inflammatory factors and matrix metalloproteinases and aggravating tissue damage (11–13). Therefore, TLR2/4 and its downstream signaling pathway serve an important regulatory role in nerve tissue injury. It has also been reported that TLR4 is upregulated during cerebral aneurysm formation in endothelial cells (14,15). How TLR2/4 and their downstream signaling pathway is involved in the pathogenesis of intracranial aneurysm remains to be elucidated.

The present study focused on the association between the TLR2/4-MyD88-NF-κB signaling pathway and intracranial aneurysm. The expression of the TLR2/4-MyD88-NF-κB signaling pathway was detected in the serum from intracranial aneurysm patients. Through an in vitro model, the involvement of the TLR2/4-MyD88-NF-κB signaling pathway in the pathogenesis of intracranial aneurysm was investigated.

 Materials and methods <sec> <title>Patients

Venous blood (5 ml; upper extremity) was collected from patients with intracranial aneurysm (n=222) and age- and sex-matched normal controls (n=200). The inclusion criteria were as follows: Intracranial aneurysm diagnosed by digital subtraction angiography following initial screening of magnetic resonance angiogram or computerized tomography angiography; ruptured aneurysm; Hunt and Hess grade 1–3 (16); and patients receiving endovascular embolization. The exclusion criteria included: Delayed neurological deficits; Glasgow Coma Scale score (17) decreased by ≥2 points; with or without cerebral infarction unrelated to aneurysm treatment or other causes; neurogenic pulmonary edema, hydrocephalus and epilepsy. In total, 222 patients (50% male; age: 45–65 years) were enrolled and 200 normal controls (50% male; age: 44–65 years) were recruited from the Health Examination Center of the First Affiliated Hospital of Nanchang University between January 1, 2018 and May 1, 2019. Written informed consents were obtained from the participants. All experimental procedures were approved by the Ethics Committee of Nanchang University (approval no. NCU-20201213).

 Reverse transcription-quantitative (RT-q)PCR

Total RNA from blood cells (3×105 cells/ml) was extracted using a TRIzol® assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The purity of RNA was determined using a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) based on the optical density (OD): OD280/260. Thereafter, RNA was transcribed into cDNA according to the instructions of the reverse transcription kit (cat. no. 639522; Takara Biotechnology Co., Ltd.). qPCR was used to detect the expression level of the targeted genes using the synthesized cDNA as templates. The primers (5′→3′) are listed in Table I. The PCR was processed in a 20 µl reaction system including 1 µl cDNA, 2 µl primers, 10 µl 2X ULtraSYBR Mixture (CoWin Biosciences) and 7 µl dH2O with thermocycling as follows: Denaturing 10 sec at 95°C, annealing 30 sec at 58°C and extension 30 sec at 72°C for 40 cycles. Relevant expression of MyD88, TLR2 and TLR4 was normalized to GAPDH using the 2−ΔΔCq method (18). Experiments were repeated 6 times.

 ELISA

TLR2 (cat. no. ab131556; Abcam), TLR4 (cat. no. MBS2702401; MyBioSource, Inc.), MyD88 (cat. no. ab171341; Abcam) and NF-κB (cat. no. ab133112; Abcam) levels in serum were detected by ELISA method following the instructions of the assay kits.

 Experimental groups

Human brain vascular smooth muscle cells (BVSMCs) were purchased from Shanghai Maisha Biotechnology Co., Ltd, and cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin in 5% CO2 at 37°C. The experiments were divided into four groups: A control group, an angiotensin II (Ang II) group, an Ang II + small interfering (si)RNA control group and an Ang II + TLR2-siRNA group. The plasmids were transfected when cell confluence reached 80%. The transfection solution included 125 µl optiMEM, 5 µl Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) and 2.5 µg pLV Puro vector (cat. no. VL3103; Beijing Inovogen Tech. Co., Ltd.) or 1 µg siRNA, and the mixture was incubated at room temperature for 5 min. The mixture was added into the corresponding well (final concentration of plasmid: 1 µg/ml). The culture medium (medium containing 20% FBS) was refreshed 6 h following transfection. Following 24 h of transfection, BVSMCs were treated with Ang II to induce cell remodeling as previously described (19). The sequences of three TLR2 siRNAs and negative control (NC) were synthesized by Universal Biological Systems (Anhui) Co., Ltd. and were as follows: TLR2 siRNA-1, 5′-GCUGACAUCCAAUGGAAUUAAUUCCAUUGGAUGUCAGC-3′; TLR2 siRNA-2, 5′-GGGACUUCAUUCCUGGCAAUUGCCAGGAAUGAAGUCCC-3′; TLR2 siRNA-3, 5′-GCAAGCUGCGGAAGAUAAUAUUAUCUUCCGCAGCUUGC-3′; and NC, 5′-UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT-3′.

Following treatment for 48 h, cell remodeling of BVSMCs was detected based on cell proliferation, migration and apoptosis. The expressions of TLR2, TLR4, MyD88, NF-κB and p-p65 were detected by RT-qPCR and western blotting.

 Cell Counting Kit-8 (CCK-8) assay

Medium (10 µl) with CCK-8 (Gibco; Thermo Fisher Scientific, Inc.) was added into each well following drug treatment or transfection. After an additional 4-h incubation in a CO2 incubator at 37°C, the absorbance at 570 nm was recorded by a microplate reader (Thermo Fisher Scientific, Inc.). Cell proliferation was calculated based on OD values.

 Flow cytometry

The cells in each group were collected after digestion by trypsin (Gibco; Thermo Fisher Scientific, Inc.). Thereafter, the cells were incubated with Annexin V-FITC and propidium iodide using an Annexin V-FITC apoptosis detection kit (cat. no. C1062; Beyotime Institute of Biotechnology) for 30 min in the dark at room temperature. Apoptosis was detected using an Accuri C6 flow cytometer (BD Biosciences) within 1 h. Data were analyzed using FlowJo software (version 7.6; FlowJo, LLC). The apoptotic rate was calculated based on the percentages of early apoptotic cells (EA) and late apoptotic cells (LA). The equation was as follows: Apoptotic rate (%) = [(Number of EA + Number of LA)/Total number of EA and LA] ×100.

 Cell migration

Cells were seeded into a 6-well plate (1×106 cells/well) and following 24 h of incubation at 37°C, a uniform line was made across the center of the well using a 200-µl pipette tip. Following 24 or 48 h incubation in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS in a CO2 incubator at 37°C, the images were captured under a light microscope (magnification, ×200; Olympus Corporation). The migration speed was calculated based on the following formula: Cell mobility (µm/h) = [Width(1)-width(2)]/72 h. Width(1) represented the width measured immediately (0 h) after drawing the line. Width(2) represented the width measured 24 or 48 h after drawing the line.

 Western blotting

Protein was extracted by a protein isolation kit (cat. no. 28-9425-44; ReadyPrep) which was purchased from Cytiva. The concentration of the protein was quantified with a bicinchoninic acid protein assay kit. Thereafter, 25 µg protein was separated via 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were then transferred onto PVDF membranes and blocked in 5% milk (2 h at room temperature). The following primary antibodies were incubated with the membranes overnight at 4°C: Rabbit polyclonal anti-TLR2 (1:500; cat. no. ab213676, Abcam); rabbit polyclonal anti-TLR4 (1:1,000; cat. no. bs-20594R, BIOSS); rabbit polyclonal anti-MyD88 (1:1,000; cat. no. bs-1047R, BIOSS); rabbit polyclonal anti-NF-κB p65 (1:1,000; cat. no. bs-0465R, BIOSS); rabbit polyclonal anti-p-p65 (1:1,000; AF2006, affinity). The secondary antibody (1:2,000; ZB-2305; OriGene Technologies, Inc.) was co-incubated for 2 h at room temperature. Protein bands were visualized using ECL solution (cat. no. SW2010-1; Beijing Solarbio Science & Technology Co., Ltd.). Densitometric analysis was performed using Quantity One software (version 4.6; Bio-Rad Laboratories, Inc.).

 Statistical analyses

Data in the present study were presented as the mean ± standard deviation and analyzed using SPSS version 17.0 (SPSS, Inc.). All data were in normal distribution and unpaired t-test or one-way analysis of variance followed by the Bonferroni's test was employed in the data analysis. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Expression of TLR2, TLR4, MyD88 and NF-κB in the serum of patients with intracranial aneurysms

The expression of TLR2, TLR4 and MyD88 mRNA in patients with intracranial aneurysm was significantly higher compared with normal controls (Fig. 1A; P<0.05). The expression levels of TLR2, TLR4, MyD88 (Fig. 1B) and NF-κB p65 (Fig. 1C) in patients with intracranial aneurysm were significantly higher compared with normal control (Fig. 1B and C; P<0.05).

 TLR2-siRNA attenuates Ang II-induced cell proliferation of BVSMCs

As shown in Fig. 2A and B, 10−7 M Ang II facilitated cell proliferation after administration for 24 and 48 h compared with control group. Thus, this concentration of Ang II was selected to produce the remodeling model of BVSMCs. As shown in Fig. 2C, the cell proliferation increased significantly 24 h after Ang II administration. Therefore, 10−7 M Ang II was selected to treat the cells for 24 h.

TLR2 expression in the TLR2 siRNA-1 group decreased significantly (Fig. 3; P<0.05 vs. control). Therefore, TLR2 siRNA-1 was selected in the subsequent experiments.

Compared with the control group, the proliferation of Ang II group increased significantly (P<0.05; Fig. 4). By contrast, the cell proliferation of Ang II + TLR2 siRNA group decreased significantly (P<0.05 vs. Ang II + siRNA NC group).

 TLR2-siRNA attenuates Ang II-induced cell migration

Compared with control group, the cell migration rate of Ang II group increased significantly (P<0.05). By contrast, the cell migration rate of Ang II + TLR2 siRNA group decreased significantly compared with Ang II + siRNA NC group (P<0.05; Fig. 5).

 TLR2-siRNA attenuates Ang II-induced decrease of apoptosis of BVSMCs

Compared with the control group, the apoptosis rate of Ang II group decreased significantly (P<0.05). By contrast, the apoptosis rate of Ang II + TLR2 siRNA group increased significantly (Fig. 6; P<0.05 vs. Ang II + siRNA NC group).

 TLR2-siRNA reduced Ang II-induced expression of TLR2, TLR4 and MyD88

Compared with control group, TLR2, TLR4 and MyD88 expression at mRNA and protein levels in Ang II group increased significantly (P<0.05). Compared with Ang II + siRNA NC group, TLR2, TLR4 and MyD88 expression in Ang II + TLR2 siRNA group decreased significantly (P<0.05; Fig. 7).

 TLR2-siRNA attenuates Ang II-induced expression of NF-κB p65 and p-p65 of BVSMCs

Compared with control group, the expression of NF-κB p65, p-p65 and p-p65/p65 in the Ang II group was significantly higher (P<0.05; Fig. 8). Compared with Ang II + siRNA NC group, the expression of NF-κB p65, p-p65 and p-p65/p65 in Ang II + TLR2 siRNA group was significantly lower (P<0.05; Fig. 8).

 Discussion

The present study reported that the expression of TLR2/4-MyD88-NF-κB was promoted in patients with intracranial aneurysm. In vitro experiments revealed that Ang II-induced remodeling of BVSMCs was ameliorated by TLR2 silencing. Clinical evidence and results from basic experiments revealed that TLR2/4-MyD88-NF-κB signaling pathway was potentially involved in the development of intracranial aneurysm.

The pathogenesis of intracranial aneurysm remains to be fully elucidated, although genetic, environmental, estrogen level, hypertension, smoking and alcohol are closely related to the occurrence and development of intracranial aneurysm (20). Ang II can cause vascular endothelial cell remodeling and induce vascular tissue dysfunction (21,22). Ang II serves an important role in myocardial remodeling by promoting the growth of cardiac fibroblasts and expression of extracellular matrix protein (23). It has been shown that Ang II stimulates the secretion of aldosterone in the adrenal cortex to regulate vasomotion, promotes the inflammatory response and oxidative stress of blood vessels and leads to the occurrence of vascular remodeling by acting on the type 1 receptor of angiotensin (24). Ang II stimulation can also promote the proliferation and migration of vascular smooth muscle (25). Thus, Ang II-stimulated BVSMCs were used to investigate the mechanisms underlying the proliferation and migration of vascular smooth muscle (25).

TLR2 and TLR4 are associated with the pathogenesis of vascular inflammation, atherosclerosis and diabetes (26–28). These receptors share a common downstream signaling pathway and serve a synergistic role in the activation of pre-inflammatory response (29,30). TLR2 and TLR4 are highly expressed in macrophages and endothelial cells in human atherosclerotic plaques, both of which promote the proliferation of vascular smooth muscle cells in animal atherosclerotic models (31,32). By knocking down the expression of TLR2, it was found that the effects of Ang II stimulation on human BVSMCs were blocked. These data suggested that TLR2 was involved in Ang II-induced cell remodeling.

As an inflammatory transcription factor, NF-κB is involved in many cell activities, including cell proliferation, stress response, immune response, apoptosis and inflammation (33,34). The activity of NF-κB in aneurysms is markedly increased (35,36), suggesting that NF-κB signaling pathway serves an important role in the occurrence and development of aneurysms. TLRs recognize the ligands and send signals to the intracellular domain through leucine rich repetitive sequences in the extracellular domain, and induce the corresponding signal conversion cascade reaction (37). Its signal transduction mechanism may include MyD88-dependent and independent mechanisms, which eventually lead to the activation of NF-κB (37). Using clinical samples, in the present study it was found that the expression levels of TLR2, TLR4, MyD88 and NF-κB p65 protein in patients with intracranial aneurysm were higher compared with normal control. These results also indicated that the TLR2/4/NF-κB signaling pathway served important roles in intracranial aneurysm.

The in vitro data of the present study demonstrated that the expression of TLR2, TLR4, MyD88, NF-κB p65 and p-p65 in BVSMCs stimulated by Ang II increased, but were downregulated by TLR2 silencing. Ang II can activate TLR4 and increase its expression (38). MyD88 can independently regulate cell activation and movement, and the TLR4/MyD88 signaling pathway is involved in Ang II-induced apoptosis (39). Ang II pretreatment results in upregulation of NF-κB and p-p65 expression (40). NF-κB is considered to be a typical pro-inflammatory molecule, regulating the transcription of a number of inflammatory cytokines (41). The results of the present study indicated that the TLR2/4-MyD88-NF-κB signaling pathway can be activated by Ang II treatment.

There remained some limitations to the present study. First, more clinical data ought to be collected to verify that the TLR2/4-MyD88-NF-κB signaling pathway is specific for the occurrence or development of intracranial aneurysm, but not subarachnoid hemorrhage. Subarachnoid hemorrhage can be caused by a ruptured aneurysm, arteriovenous malformation, or head injury (3,4). The present study did not distinguish subarachnoid hemorrhage patients from intracranial aneurysm patients. Future research should investigate the influence of subarachnoid hemorrhage caused by other factors on this signaling pathway. Second, Ang II treatment could affect the biological activities of BVSMCs, including the proliferation, apoptosis and migration. The TLR2/4-MyD88-NF-κB signaling pathway is an important pathway for inflammatory response (40). Whether inflammatory reaction takes part in the process deserves future study. Finally, 10% FBS was used in the cell migration assay. FBS may have also promoted the migration of the cells, which may cause bias or reduce the ability to observe the full effect of the knockdown of TLR2.

In conclusion, TLR2, TLR4, MyD88 and NF-κB p65 protein expression increased in patients with aneurysms, and the occurrence of aneurysms was related to the TLR2/4-MyD88-NF-κB signaling pathway. Interference of TLR2/4-MyD88-NF-κB signaling pathway will affect aneurysms.

 Acknowledgements

Not applicable.

 Funding

No funding was received.

 Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Authors' contributions

XZ, YW, JF and ML performed the experiments and analyzed the data. XZ and ZJ designed the study, wrote the manuscript and confirmed the authenticity of all the raw data. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

Written informed consents were obtained from the participants. All experimental procedures were approved by the Ethics Committee of Nanchang University (approval no. NCU-20201213).

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests

 References JabbarliRDingerTFDarkwah OppongMPierscianekDDammannPWredeKHKaierKKöhrmannMForstingMKleinschnitzCSureURisk factors for and clinical consequences of multiple intracranial aneurysms: A systematic review and meta-analysisStroke49848855201810.1161/STROKEAHA.117.02034229511128 LatherHDGornikHLOlinJWGuXHeidtSTKimESHKadian-DodovDSharmaAGrayBJaffMRPrevalence of intracranial aneurysm in women with fibromuscular dysplasia: A report from the US registry for fibromuscular dysplasiaJAMA Neurol7410811087201710.1001/jamaneurol.2017.133328715558 BrownRDJrBroderickJPUnruptured intracranial aneurysms: Epidemiology, natural history, management options, and familial screeningLancet Neurol13393404201410.1016/S1474-4422(14)70015-824646873 KheyKMWHuardAMahmoudSHInflammatory pathways following subarachnoid hemorrhageCell Mol Neurobiol40675693202010.1007/s10571-019-00767-431808009 TakedaKAkiraSToll-like receptorsCurr Protoc Immunol10914 12 1114 12 10201510.1002/0471142735.im1412s109 KielianTToll-like receptors in central nervous system glial inflammation and homeostasisJ Neurosci Res83711730200610.1002/jnr.2076716541438 FangHChenJLinSWangPWangYXiongXYangQCD36-mediated hematoma absorption following intracerebral hemorrhage: Negative regulation by TLR4 signalingJ Immunol19259845992201410.4049/jimmunol.140005424808360 GoeringJPopeMRFlemingSDTLR2 regulates complement-mediated inflammation induced by blood loss during hemorrhageShock453339201610.1097/SHK.000000000000047726679472 WangYCZhouYFangHLinSWangPFXiongRPChenJXiongXYLvFLLiangQLYangQWToll-like receptor 2/4 heterodimer mediates inflammatory injury in intracerebral hemorrhageAnn Neurol75876889201410.1002/ana.2415924752976 RamsteadAGRobisonABlackwellAJeromeMFreedmanBLubickKJHedgesJFJutilaMARoles of toll-like receptor 2 (TLR2), TLR4, and MyD88 during pulmonary Coxiella burnetii infectionInfect Immun84940949201610.1128/IAI.00898-1526787722 SuQLvXSunYYeZKongBQinZRole of TLR4/MyD88/NF-κB signaling pathway in coronary microembolization-induced myocardial injury prevented and treated with nicorandilBiomed Pharmacother106776784201810.1016/j.biopha.2018.07.01429990871 GuoJLiangWLiJLongJKnockdown of FSTL1 inhibits oxLDL-induced inflammation responses through the TLR4/MyD88/NF-κB and MAPK pathwayBiochem Biophys Res Commun47815281533201610.1016/j.bbrc.2016.08.13827569284 ChenFZhuXSunZMaYAstilbin inhibits high glucose-induced inflammation and extracellular matrix accumulation by suppressing the TLR4/MyD88/NF-κB pathway in rat glomerular mesangial cellsFront Pharmacol91187201810.3389/fphar.2018.0118730459606 AokiTNishimuraMIshibashiRKataokaHTakagiYHashimotoNToll-like receptor 4 expression during cerebral aneurysm formation. Laboratory investigationJ Neurosurg113851858201010.3171/2009.9.JNS0932919852543 OkadaTSuzukiHToll-like receptor 4 as a possible therapeutic target for delayed brain injuries after aneurysmal subarachnoid hemorrhageNeural Regen Res12193196201710.4103/1673-5374.20079528400792 BracardSLebedinskyAAnxionnatRNetoJMAudibertGLongYPicardLEndovascular treatment of Hunt and Hess grade IV and V aneurymsAJNR Am J Neuroradiol23953957200212063223 CatapanoJSZeoliTFrisoliFABurkhardtJKLawtonMTLong-term independence in older patients with aneurysmal subarachnoid hemorrhage in the Barrow Ruptured Aneurysm Trial (BRAT)World NeurosurgS1878-S8750(20)32524-9, Dec 1, 2020 (Epub ahead of print). doi: 10.1016/j.wneu.2020.11.139 LivakKJSchmittgenTDAnalysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) methodMethods25402408200110.1006/meth.2001.126211846609 YaghiniFASongCYLavrentyevENGhafoorHUFangXREstesAMCampbellWBMalikKUAngiotensin II-induced vascular smooth muscle cell migration and growth are mediated by cytochrome P450 1B1-dependent superoxide generationHypertension5514611467201010.1161/HYPERTENSIONAHA.110.15002920439821 SathyanSKoshyLVSrinivasLEaswerHVPremkumarSNairSBhattacharyaRNAlapattJPBanerjeeMPathogenesis of intracranial aneurysm is mediated by proinflammatory cytokine TNFA and IFNG and through stochastic regulation of IL10 and TGFB1 by comorbid factorsJ Neuroinflammation12135201510.1186/s12974-015-0354-026198819 MaAWangDAnYFangWZhuHComparative transcriptomic analysis of mice liver treated with different AMPK activators in a mice model of atherosclerosisOncotarget81659416604201710.18632/oncotarget.1502728178661 FuCChenBJinXLiuXWangFGuoRChenZZhengHWangLZhangYPuerarin protects endothelial progenitor cells from damage of angiotensin II via activation of ERK1/2Nrf2 signaling pathwayMol Med Rep1738773883201829359784 ZhangZZChengYWJinHYChangQShangQHXuYLChenLXXuRSongBZhongJCThe sirtuin 6 prevents angiotensin II-mediated myocardial fibrosis and injury by targeting AMPK-ACE2 signalingOncotarget87230272314201710.18632/oncotarget.2030529069788 AllenAMZhuoJMendelsohnFALocalization and function of angiotensin AT1 receptorsAm J Hypertens13S31S38200010.1016/S0895-7061(99)00249-6 YuSChenYChenSYeNLiYSunYKlotho inhibits proliferation and migration of angiotensin II-induced vascular smooth muscle cells (VSMCs) by modulating NF-κB p65, akt, and extracellular signal regulated kinase (ERK) signaling activitiesMed Sci Monit2448514860201810.12659/MSM.90803830004089 AllamRScherbaumCRDarisipudiMNMulaySRHägeleHLichtnekertJHagemannJHRupanagudiKVRyuMSchwarzenbergerCHistones from dying renal cells aggravate kidney injury via TLR2 and TLR4J Am Soc Nephrol2313751388201210.1681/ASN.201111107722677551 BielinskiSJHallJLPankowJSBoerwinkleEMatijevic-AleksicNHeMChamblessLFolsomARGenetic variants in TLR2 and TLR4 are associated with markers of monocyte activation: The atherosclerosis risk in communities MRI studyHum Genet129655662201110.1007/s00439-011-0962-421298446 LiuYYinHZhaoMLuQTLR2 and TLR4 in autoimmune diseases: A comprehensive reviewClin Rev Allergy Immunol47136147201410.1007/s12016-014-8445-824352680 PengCWangHZhangWJJieSHTongQXLuMJYangDLInhibitory effect of miR-125b on hepatitis C virus core protein-induced TLR2/MyD88 signaling in THP-1 cellsWorld J Gastroenterol2243544361201610.3748/wjg.v22.i17.435427158204 LeeITLinCCHsuCKWuMYChoRLYangCMResveratrol inhibits staphylococcus aureus-induced TLR2/MyD88/NF-κB-dependent VCAM-1 expression in human lung epithelial cellsClin Sci (Lond)127375390201410.1042/CS2013081624617573 HeMIchinoseTYoshidaYArashidaniKYoshidaSTakanoHSunGShibamotoTUrban PM2.5 exacerbates allergic inflammation in the murine lung via a TLR2/TLR4/MyD88-signaling pathwaySci Rep711027201710.1038/s41598-017-11471-y28887522 KollgaardTEnevoldCBendtzenKHansenPRGivskovMHolmstrupPNielsenCHCholesterol crystals enhance TLR2- and TLR4-mediated pro-inflammatory cytokine responses of monocytes to the proatherogenic oral bacterium Porphyromonas gingivalisPLoS One12e0172773201710.1371/journal.pone.017277328235036 SenRBaltimoreDMultiple nuclear factors interact with the immunoglobulin enhancer sequencesCell 1986. 46: 705–716J Immunol17774857496200617114415 SongZShenFZhangZWuSZhuGCalpain inhibition ameliorates depression-like behaviors by reducing inflammation and promoting synaptic protein expression in the hippocampusNeuropharmacology174108175202010.1016/j.neuropharm.2020.10817532492450 WeiHMaoQLiuLXuYChenJJiangRYinLFanYChoppMDongJZhangJChanges and function of circulating endothelial progenitor cells in patients with cerebral aneurysmJ Neurosci Res8918221828201110.1002/jnr.2269621793037 AokiTFrosenJFukudaMBandoKShioiGTsujiKOllikainenENozakiKLaakkonenJNarumiyaSProstaglandin E2-EP2-NF-κB signaling in macrophages as a potential therapeutic target for intracranial aneurysmsSci Signal10eaah6037201710.1126/scisignal.aah603728174280 LiHBLiXHuoCJSuQGuoJYuanZYZhuGQShiXLLiuJJKangYMTLR4/MyD88/NF-κB signaling and PPAR-ү within the paraventricular nucleus are involved in the effects of telmisartan in hypertensionToxicol Appl Pharmacol30593102201610.1016/j.taap.2016.06.01427292124 JiYYWangZDLiuJTLiuNInhibitory effect of fenofibrate on angiotensin II-induced toll-like receptor 4 expression, myeloperoxidase activity and expression in RAW264.7 cellsYao Xue Xue Bao444624672009(In Chinese)19618719 GaoWWangHZhangLCaoYBaoJZLiuZXWangLSYangQLuXRetinol-binding protein 4 induces cardiomyocyte hypertrophy by activating TLR4/MyD88 pathwayEndocrinology15722822293201610.1210/en.2015-202227100622 YangJJiangHChenSSChenJXuSKLiWQWangJCCBP knockdown inhibits angiotensin II-induced vascular smooth muscle cells proliferation through downregulating NF-κB transcriptional activityMol Cell Biochem3405562201010.1007/s11010-010-0400-220157765 ZhangHDingJFanQLiuSTRPC6 up-regulation in Ang II-induced podocyte apoptosis might result from ERK activation and NF-kappaB translocationExp Biol Med (Maywood)23410291036200910.3181/0901-RM-1119546355

Expression of TLR2, TLR4, MyD88 and NF-κB p65 is promoted in patients with intracranial aneurysms. (A) TLR2, TLR4 and MyD88 expression at mRNA level; (B) TLR2, TLR4 and MyD88 expression at protein level; and (C) NF-κB p65 protein level. *P<0.05 vs. normal control (normal control: n=200; patients: n=222). TLR, Toll-like receptor; MyD88, major myeloid differentiation response gene 88.

Ang II promotes the cell proliferation of human brain vascular smooth muscle cells. (A) Ang II treatment for 24 h; (B) Ang II treatment for 48 h; and (C) Treatment by 10−7 M Ang II for 24 or 48 h. *P<0.05 vs. control group, #P<0.05 vs. 24 h group (n=6). Ang II, Angiotensin II.

TLR2-siRNA reduces TLR2 expression in human brain vascular smooth muscle cells. (A) TLR2 expression at mRNA level and (B) TLR2 expression at protein level. *P<0.05 vs. NC (n=6). TLR, Toll-like receptor; si, small interfering; NC, negative control.

TLR2-siRNA attenuates Ang II-induced cell proliferation. Compared with, *P<0.05 vs. control group; #P<0.05 vs. Ang II + siRNA-NC (n=6). TLR, Toll-like receptor; si, small interfering; Ang II, Angiotensin II; NC, negative control.

TLR2-siRNA attenuates Ang II-induced cell migration. Upper panel: Representative images of the cells (scale bar, 100-µm); lower panel: Quantification data of cell migration rate. *P<0.05 vs. control group; #P<0.05 vs. Ang II + siRNA-NC (n=6). TLR, Toll-like receptor; si, small interfering; Ang II, Angiotensin II; NC, negative control.

TLR2-siRNA attenuates Ang II-induced decrease of apoptosis. Left panel: Representative images of flow cytometry; right panel: Quantification data of apoptosis. *P<0.05 vs. control group; #P<0.05 vs. Ang II + siRNA-NC (n=6). TLR, Toll-like receptor; si, small interfering; Ang II, Angiotensin II; NC, negative control.

TLR2-siRNA attenuates Ang II-induced expression of TLR2, TLR4 and MyD88. (A) mRNA expression of TLR2, TLR4 and MyD88. (B) Protein level of TLR2, TLR4 and MyD88. *P<0.05 vs. control group; #P<0.05 vs. Ang II + siRNA-NC (n=6). TLR, Toll-like receptor; si, small interfering; Ang II, Angiotensin II; MyD88, major myeloid differentiation response gene 88; NC, negative control.

TLR2-siRNA attenuates Ang II-induced expression of NF-κB p65, p-p65 and p-p65/p65. *P<0.05 vs. control group; #P<0.05 vs. Ang II + siRNA-NC (n=6). TLR, Toll-like receptor; si, small interfering; Ang II, Angiotensin II; p-, phosphorylated; NC, negative control.

Primer sequences of MyD88, TLR2 and TLR4.

Genes Sequence (5′→3′) Primer length (bp) Product length (bp) Annealing temperature (°C)
MyD88 F CAGCGACATCCAGTTTGTGC 20 153 59.7
MyD88 R GGCCTTCTAGCCAACCTCTT 20
TLR2 F ATGCTGCCATTCTCATTCTTC 21 100 57.8
TLR2 R TCCAGGTAGGTCTTGGTGTTC 21
TLR4 F GACCTGTCCCTGAACCCTAT 20 136 57.6
TLR4 R CTAAACCAGCCAGACCTTGA 20
GAPDH F CAATGACCCCTTCATTGACC 20 106 57.2
GAPDH R GAGAAGCTTCCCGTTCTCAG 20

MyD88, major myeloid differentiation response gene 88; TLR, Toll-like receptor; F, forward; R, reverse.