Brain Vascular Smooth Muscle Cells Research Articles

Medin Induces Pro-Inflammatory Activation of Human Brain Vascular Smooth Muscle Cells.

Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. VSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 µM) without or with small molecule nuclear factor-κB (NFκB) inhibitor RO106-9920 (10 µM) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). Medin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0±12.8x, 20.2±4.1x and 8.7±3.1x, respectively, medin 5 µM versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. Medin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NFκB. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.

Exploring the roles and therapeutic implications of melatonin-mediated KLF6 in the development of intracranial aneurysm

Background Intracranial aneurysm (IA) is a cerebrovascular disease with a high mortality rate due to ruptured subarachnoid hemorrhage. While Krüppel-like factor 6 (KLF6) dysregulation has been implicated in cancer and cardiovascular diseases, its role in IA remains unclear. Materials and methods The GSE122897 and GSE15629 datasets were downloaded from the Gene Expression Omnibus database. Immune cell infiltration and hypoxia analysis were performed to explore the effects of KLF6 on IA. Weighted gene co-expression network analysis was used to identify hub genes related to KLF6 expression for subsequent analyses. Hypoxia-related genes were identified. Drug prediction was performed for IA. Samples from healthy individuals and patients with IA were collected to detect the expression of endothelin-1 (ET-1), vascular hematoma factor (vWF), and KLF6. A model of H2O2-induced human brain vascular smooth muscle cells (HBVSMC) injury was constructed to explore the effects of KLF6 and melatonin to treat IA. Results T cells CD4 memory resting and monocytes were significantly different in the KLF6 high and low expression groups. Four hypoxia-related gene sets were significantly enriched in the KLF6 high-expression group. Six hypoxia-related hub genes were obtained, which were significantly associated with KLF6. Drug prediction showed that melatonin may be a potential drug for IA. The levels of ET-1, vWF, and KLF6 were significantly upregulated in patients with IA. KLF6 exacerbates H2O2-induced injury in HBVSMC, ameliorated by melatonin. Conclusion KLF6 may be a potential target for IA treatment, with melatonin-mediated KLF6 effects playing a crucial role in the development of IA.

Identification and Verification of the Oxidative Stress-Related Signature Markers for Intracranial Aneurysm-Applied Bioinformatics.

Intracranial aneurysm (IA) is a localized abnormal dilation of the cerebral vascular wall, the degeneration of which is closely related to high oxidative stress. Clinical information and RNA-seq data from five public datasets were downloaded from the Gene Expression Omnibus (GEO). Using the "GSVA" package, enrichment analysis was performed on the gene sets of the oxidative stress, reactive oxygen species (ROS), metabolism, and inflammatory pathways retrieved from the MsigDB and Kyoto encyclopedia of genes and genomes (KEGG) databases. Weighted gene co-expression network analysis (WGCNA) was conducted using the "WGCNA" package, followed by using the "limma" R package to select differentially expressed genes (DEGs). Key genes were determined by applying three machine learning algorithms (random forest, Lasso, and SVM-RFE). The expression levels of the key genes were verified by the quantitative real-time polymerase chain reaction (qRT-PCR) in IA. Finally, ESTIMATE and CIBERPSORT algorithms were used for immune infiltration analysis. The enrichment score of the oxidative stress, ROS, metabolism, and inflammatory pathways was calculated, and we found that these pathways were significantly activated in IA samples with higher immune infiltration. The intersection between the blue module related to oxidative stress (610 genes identified by WGCNA) and 380 upregulated DEGs contained a total of 209 key genes, which were further processed by machine learning algorithms to obtain four crucial diagnostic markers (FLVCR2, SDSL, TBC1D2, and SLC31A1) for IA. These key genes are highly expressed in human brain vascular smooth muscle cells. The expressions of the four markers were significantly positively correlated with the abnormal activation phenotypes of oxidative stress, the ROS and glucometabolic pathways, and suppressive immune infiltration. This study employed WGCNA combined with three machine learning algorithms to identify four oxidative stress-related signature markers for IA, providing novel insights into the clinical management of IA patients.

Amyloid Beta Oligomers Activate Death Receptors and Mitochondria-Mediated Apoptotic Pathways in Cerebral Vascular Smooth Muscle Cells; Protective Effects of Carbonic Anhydrase Inhibitors.

Amyloid beta (Aβ) deposition within the brain vasculature is an early hallmark of Alzheimer's disease (AD), which triggers loss of brain vascular smooth muscle cells (BVSMCs) in cerebral arteries, via poorly understood mechanisms, altering cerebral blood flow, brain waste clearance, and promoting cognitive impairment. We have previously shown that, in brain endothelial cells (ECs), vasculotropic Aβ species induce apoptosis through death receptors (DRs) DR4 and DR5 and mitochondria-mediated mechanisms, while FDA-approved carbonic anhydrase inhibitors (CAIs) prevent mitochondria-mediated EC apoptosis in vitro and in vivo. In this study, we analyzed Aβ-induced extrinsic and intrinsic (DR- and mitochondria-mediated) apoptotic pathways in BVSMC, aiming to unveil new therapeutic targets to prevent BVSMC stress and death. We show that both apoptotic pathways are activated in BVSMCs by oligomeric Aβ42 and Aβ40-Q22 (AβQ22) and mitochondrial respiration is severely impaired. Importantly, the CAIs methazolamide (MTZ) and acetazolamide (ATZ) prevent the pro-apoptotic effects in BVSMCs, while reducing caspase 3 activation and Aβ deposition in the arterial walls of TgSwDI animals, a murine model of cerebral amyloid angiopathy (CAA). This study reveals new molecular targets and a promising therapeutic strategy against BVSMC dysfunction in AD, CAA, and ARIA (amyloid-related imaging abnormalities) complications of recently FDA-approved anti-Aβ antibodies.

Secreted protein acidic and rich in cysteine (SPARC) induces apoptosis of human brain vascular smooth muscle cells through regulating HK2 in intracranial aneurysm

BackgroundVascular smooth muscle cell (VSMC) dysfunction is one of the crucial pathologic processes in the development of intracranial aneurysm (IA). Secreted protein acidic and rich in cysteine (SPARC), a multifunctional glycoprotein, is overexpressed in many tumor, but its underlying mechanism in vascular disease has not been elucidated. The aim of this study is to evaluate the potential function of SPARC in IA generation and regulation of mitochondrial function in VSMC.MethodsHuman brain vascular smooth muscle cells were treated with recombinant SPARC to detect apoptosis-related markers. The downstream targets affecting mitochondrial dysfunction after SPARC treatment were explored by transcriptome sequencing and bioinformatics analysis, and verified using by immunohistochemistry and western blot. Further in vitro experiments verified the role of downstream targets in regulating VSMC mitochondrial function.ResultsSecreted protein acidic and rich in cysteine (SPARC) expression was associated with the risk of IA rupture. SPARC induces mitochondrial pathway apoptosis in human brain VSMC. We screened 40 differentially expressed genes related to mitochondrial function after SPARC treatment. Hexokinase 2 (HK2) was identified as a downstream target of mitochondrial pathway apoptosis in VSMC induced by SPARC. In addition, immunohistochemical results confirmed that the difference between SPARC and HK2 expression is located mainly in the smooth muscle layer of IA. Overexpression of HK2 reversed the SPARC-induced increase in apoptosis and mitochondrial damage in VSMC.ConclusionSecreted protein acidic and rich in cysteine (SPARC) regulated mitochondrial function in VSMC and induced apoptosis through HK2, which plays an important role in the formation and rupture of IA. Targeting SPARC may be a novel strategy to delay the development of intracranial aneurysms.

Abstract P1018: Peptidyl-prolyl Cis-trans Isomerase-like 4 Is A Novel Regulator Of Smooth Muscle Cell Remodeling

Introduction: Smooth muscle cell (SMC) remodeling is a complex and multifaceted process that plays an essential role in the maintenance and function of various organ systems in the body. In response to physiological and pathological stimuli, smooth muscle cells exhibit remarkable plasticity, undergoing morphological and functional changes that can impact vascular tone. Dysregulation of SMC remodeling can contribute to the development and progression of numerous pathological conditions, such as hypertension and atherosclerosis. However, the role of SMC remodeling in intracranial aneurysm (IA) formation has yet to be explored. Objective-Method: In this study, we aimed to elucidate the role of PPIL4, an IA-implicated gene, in SMC remodeling using a targeted experimental approach. We performed immunostaining and bulk RNA sequencing (RNA-seq) in human primary brain vascular smooth muscle cells (HPBVSMC) treated with lentiviral shRNA and open-reading-frame (ORF) particles to individually downregulate and overexpress PPIL4, respectively. Results: We found that PPIL4 downregulation results in significant upregulation of the genes encoding contractile proteins, including ACTA2, CNN1, TNNT1, and TNNT2, in HPBVSMC and leads to a significantly increased cell size, favoring a contractile SMC phenotype. Conversely, overexpressing PPIL4 using ORF-lentiviral particles caused downregulation of contractile proteins, increased expression of MMP1 and MMP3, and promoted a synthetic phenotype in SMCs. Furthermore, PPIL4 overexpression caused cell elongation and spindle-like cell formation in HPBVSMC. Conclusion: In conclusion, our findings highlight the critical role of PPIL4 in modulating SMC remodeling, suggesting a potential mechanistic link between PPIL4 dysregulation, SMC remodeling, and IA formation, which warrants further investigation.

Methamphetamine and Designer Stimulants Modulate Tonic Human Cerebrovascular Smooth Muscle Contractility: Relevance to Drug-Induced Neurovascular Stress.

To avoid criminal prosecution, clandestine chemists produce designer stimulants that mimic the pharmacological and psychoactive effects of conventional stimulants, such as methamphetamine. Following persistent or high-dose exposure, both acute vasoconstriction and loss of vascular homeostasis are reported dangers of conventional stimulants, and designer stimulants may pose even greater dangers. To compare the effects of a conventional stimulant and two designer stimulants on vascular contraction, this study examined the direct effects of 1,3-benzodioxolylbutanamine (BDB) and N-butylpentylone in comparison to methamphetamine on the function of human brain vascular smooth muscle cells (HBVSMCs). HBVSMCs suspended in collagen gels were exposed to varying concentrations of each drug, and the degree of constriction was assessed over one week. The MTT assay was used to measure the impact of the three drugs on the cellular metabolic activity as a marker of cellular toxicity. The highest concentration tested of either methamphetamine or N-butylpentylone produced a loss of HBVSMC contractility and impaired cellular metabolism. BDB showed a similar pattern of effects, but, uniquely, it also induced vasoconstrictive effects at substantially lower concentrations. Each drug produced direct effects on HBVSMC contraction that may be a mechanism by which the cardiovascular system is damaged following high-dose or persistent exposure, and this could be exacerbated by any sympathomimetic effects of these compounds in whole organisms. BDB appears to impact HBVSMC function in ways distinct from methamphetamine and N-butylpentylone, which may present unique dangers.

CircHECTD1 Promotes the Proliferation and Migration of Human Brain Vascular Smooth Muscle Cells via Interacting with KHDRBS3 to Stabilize EZH2 mRNA Expression.

The objective of this paper is to explore the role of circHECTD1 in vascular smooth muscle cells (VSMCs) and atherosclerosis (AS). VSMCs were treated with platelet-derived growth factor-BB (PDGF-BB) in vitro, and the level of circHECTD1 was determined using qRT-PCR. Cell proliferation, migration, and invasion were analyzed using CCK8 and transwell assays. Cell apoptosis and cell cycle were analyzed using flow cytometry. The binding interaction between circHECTD1 and KHDRBS3 or EZH2 was investigated using the RIP, RNA pull-down. CircHECTD1 was upregulated in PDGF-BB-induced VSMCs with a dose-dependent and time-dependent manner. Knockdown of circHECTD1 suppressed VSMCsproliferation and migration and enhanced cell apoptosis in VSMCs, while circHECTD1 overexpression yielded opposite effects. Mechanistically, circHECTD1 could interact with KHDRBS3, thus enhanced the stability of EZH2 mRNA and increased EZH2 protein level. In addition, silencing EZH2 in VSMCs reversed the proliferation-enhancing effect of circHECTD1 overexpression. Our findings provided providing a potential prognostic and therapy biomarker for AS.

MiR-140 promotes the progression of intracranial aneurysms by targeting BCL2L2.

To investigate the role of miR-140/BCL2L2 axis on the formation of intracranial aneurysms. The expression of miR-140 in the serum of patients with intracranial aneurysms and healthy volunteers was detected. CCK-8 assay and Annexin V-FITC/PI double staining flow cytometry were used to evaluate the effect of miR-140 knockdown on the proliferation and apoptosis of human brain vascular smooth muscle cells (HBVSMCs). Meanwhile, the relationship between miR-140 and BCL2L2 was examined. MiR-140 was found to be upregulation in intracranial aneurysm patients. MiR-140 knock-out significantly inhibited the apoptosis of HBVSMCs and promoted cell proliferation. BCL2L2 was a direct target gene of miR-140 and suppressed its expression. Knockdown of miR-140 alleviates the development of intracranial aneurysms. MiR-140/BCL2L2 axis promotes the progression of intracranial aneurysms by regulating apoptosis of HBVSMCs. Therefore, miR-140 is a potential therapeutic target for intracranial aneurysms.

DNA methylation of the MAP3K10 gene may participate in the development of intracranial aneurysm

BackgroundThe goal of this study was to explore the association between mitogen-activated protein kinase kinase kinase 10 (MAP3K10) methylation and blood lipid levels and intracranial aneurysm (IA) risk. Materials and methodsA total of 96 age- and sex-matched investigators participated in the assessment of MAP3K10 methylation. Fourteen CpG sites of the MAP3K10 gene were selected for methylated-pyrosequencing. Human brain vascular smooth muscle cell was used to assess the regulatory role of DNA methylation in MAP3K10 gene transcription. ResultsMAP3K10 mean methylation was positively correlated with triglyceride (TG, r = 0.388; p = 0.007) in men, but negatively correlated with TG (r = -0.434; p = 0.002) in women. MAP3K10 methylation in patients with IA was significantly lower than that in controls (p < 0.05), and this phenomenon was more significant in women (12 CpG sites presented significance at p < 0.05). MAP3K10 methylation might be a potential predictor of the risk of IA (CpG1, AUC = 0.81, p < 0.001; mean methylation, AUC = 0.69, p = 0.001). The predictive value was also more significant in women (CpG1: AUC = 0.86, p < 0.001; mean methylation: AUC = 0.73, p = 0.006). No significant association was found between DNA methylation and MAP3K10 gene transcription in vitro experiment. ConclusionPatients with IA had lower MAP3K10 methylation levels than healthy controls. MAP3K10 methylation may be a potential predictor of IA risk, particularly in women.

Circ_FOXO3 regulates KLF6 through sponge adsorption of miR-122-5p to repress H2O2-induced HBVSMC proliferation, thus promoting IA development in vitro model.

The phenotypic transformation of human brain vascular smooth muscle cells (HBVSMC) is widely involved in the appearance and progression of intracranial aneurysms (IA). Aneurysm (IA) Circular RNA circ_FOXO3 functions pivotally in vascular diseases and tumors, but its regulatory role as well as its molecular mechanism in IA is still uncertain. This research was to explore how circ_FOXO3 works and its mechanism in vitro model of HBVSMC IA induced by H2O2. Thirty-eight patients with IA and their normal tissues were clinically collected. Examination of endothelin-1, vascular hematoma factor, circ_FOXO3, microRNA (miR)-122-5p and KLF6 and the correlation of circ_FOXO3 with clinical case information were ensured. Establishment of an in vitro IA model was through HBVSMC induced by H2O2 and transfection with circ_FOXO3, miR-122-5p and KLF6 related plasmids was to figure out their roles in cell growth. The relationship among circ_FOXO3, miR-122-5p with KLF6 was detected. Up-regulated circ_FOXO3 and KLF6 and reduced miR-122-5p were in IA tissues; Circ_FOXO3 was associated with smoking history, Hunt-Hess grading and endothelial injury degree. Repressive circ_FOXO3 or KLF6 and strengthening miR-122-5p facilitated H2O2-induced proliferation and repressed HBVSMC apoptosis, while elevation of circ_FOXO3 or depressive miR-122-5p was opposite. circ_FOXO3 bound to miR-122-5p, whose target was KLF6, which participated in controlling IA by mediating the circ_FOXO3/miR-122-5p axis. In summary, the findings suggest that circ_FOXO3 suppresses H2O2-induced proliferation of HBVSMC but promotes apoptosis via modulation of miR-122-5p/KLF6 axis. Targeted therapy of circ_FOXO3/miR-122-5p/KLF6 axis is supposed to be a promising treatment approach for IA patients.

ATF4 promotes brain vascular smooth muscle cells proliferation, invasion and migration by targeting miR-552-SKI axis

BackgroundStudies have indicated vascular smooth muscle cells (VSMCs) played a crucial role in atherosclerosis and microRNAs (miRNAs) played key roles in biological functions of VSMCs. Whereas, the potential function and mechanism of miR-552 in VSMCs remains unclear. Our aim was to explore the role of miR-552 on VSMCs and underlying mechanism.Material/MethodsMTT assay and transwell assay were used to measure the proliferation, invasion, and migration of human brain VSMCs (HBVSMCs) and mice VSMCs (mVSMCs), respectively. Bioinformatics tools and luciferase assay were adopted to verify the association between miR-552 and SKI. Rescue experiments were employed to assess the interaction of miR-552 and SKI in modulating biological functions in HBVSMCs and mVSMCs. The expression level of transcription factors (TFs)was measured via qRT-PCR assay. The effect of ATF4 on miR-552 and SKI expression was tested by qRT-PCR or western blot assay. Finally, chromatin immunoprecipitation (ChIP) assay and JASPAR databases were used to analyze the regulatory linkage between ATF4 and miR-552.ResultsWe found that miR-552 was upregulated in HBVSMCs treated with PDGF-bb and miR-552 overexpression could promote proliferation, invasion, and migration of HBVSMCs and mVSMCs, whereas, miR-552 knockdown had the opposite impact. In addition, we also found that SKI was a direct target of miR-552, which reversed miR-552-mediated proliferation, invasion, and migration in HBVSMCs and mVSMCs. Furthermore, we also discovered that miR-552 overexpression promoted the effects of ATF4 elevation on proliferation, migration and invasion of HBVSMCs and mVSMCs, but, miR-552 decline had the opposite impact.ConclusionsATF4-miR-552-SKI axis played critical roles in the proliferation and migration of HBVSMCs and mVSMCs, which were closely involved in atherosclerosis (AS). Therefore, our findings might offer a novel therapeutic target for AS.

AT2 Receptor Stimulation Inhibits Vascular Smooth Muscle Cell Senescence Induced by Angiotensin II and Hyperglycemia.

Hyperglycemia has been widely reported to induce vascular senescence. We have previously demonstrated that angiotensin II (Ang II) could promote brain vascular smooth muscle cell (VSMC) senescence, and its type 2 (AT2) receptor deletion could enhance VSMC senescence. Therefore, we examined the possible cross-talk between Ang II and hyperglycemia on VSMC senescence, and the roles of AT2 receptor agonist, compound 21 (C21) on it. Aortic VSMCs were prepared from adult male mice and stimulated with Ang II and/or high glucose (Glu) and/or C21 and/or an autophagy inhibitor, 3-methyladenine (3-MA), and/or an autophagy agonist, rapamycin (RAP) for the indicated times. Cellular senescence, oxidative stress, and protein expressions were evaluated. Combination treatment with Ang II and Glu synergistically increased the proportion of VSMC senescent area compared with control group and each treatment alone, which was almost completely attenuated by C21 treatment. Moreover, combination treatment induced significant changes in the levels of superoxide anion, the expressions of p21 and pRb, and the ratio of LC3B II/I expression, which were also significantly attenuated by C21 treatment. The proportion of VSMC senescent area and the levels of superoxide anion by combination treatment were increased after 3-MA treatment, and the proportion of senescent area and the expressions of p21 and pRb were decreased after RAP treatment, both of which were further attenuated by C21 treatment. Ang II and hyperglycemia synergistically promoted VSMC senescence, at least partly through the participation by autophagy, oxidative stress, and p21-pRb pathway, which could be inhibited by C21.

Differential Expression Profile of Sphingosine‐1‐Phosphate Receptors in Human Brain Vascular Smooth Muscle and Endothelial Cells Following Hypoxia Plus Glucose Deprivation

Sphingosine‐1‐phosphate receptors (S1PRs) and their signaling pathways play an important role in mediating vascular health and function. Upon ligand activation, S1PRs 1‐5 couple with diverse heterotrimeric G‐protein subunits (Gαi, Gαq/11, Gα12/13) initiating multimodal downstream signaling pathways which result in various physiological outcomes in the vasculature, including cell proliferation and migration, barrier integrity preservation or loss, contraction, and inflammation. Specifically, S1PR2 activation has been linked to endothelial activation, barrier integrity loss, and inflammation, whereas S1PR1 activation contributes to barrier integrity preservation, vasodilation, and anti‐inflammatory properties. Although the role of S1PRs during pathophysiological conditions such as acute ischemic stroke is under current investigation, S1PR expression in the brain vasculature following ischemic injury has not yet been investigated. Therefore, the present study characterized the expression profile of S1PRs 1‐5 in human brain microvascular endothelial cells (ECs) and brain vascular smooth muscle cells (VSMCs) following hypoxia plus glucose deprivation (HGD). Male ECs and VSMCs were exposed to either HGD (1% O2) or normoxia (21% O2) for 3h. Semi‐quantitative RT‐PCR was used to examine gene expression and concomitantly, protein levels were assessed with standard western blot and/or FIJI‐mediated ICC analysis. We hypothesized that ECs and VSMCs differentially express S1PRs 1‐5 both basally and during ischemia‐like conditions. At the mRNA level, S1PRs 1‐5 gene expression was observed in VSMCs with S1PR4 and 5 being just above the level of detection. S1PRs 1‐4 expression was detected in ECs with S1PR4 having the lowest expression. During normoxic conditions, we observed that S1PRs 1 and 3 mRNA levels were most prominently expressed in both the ECs and VSMCs. In ECs, HGD exposure did not alter the gene expression of S1PRs 1‐3. At the protein level, we observed that HGD increased S1PR1 levels, however S1PR2 and 3 protein levels were unaltered. In VSMCs, HGD increased both mRNA and protein levels of S1PR2 but did not alter S1PRs 1 and 3 mRNA expression and protein levels. In conclusion, acute exposure to HGD appears to differentially regulate S1PRs in VSMCs and ECs at the transcriptional and translational level, respectively. In VSMCs, HGD increased S1PR2 gene expression and protein levels, whereas in ECs, HGD increased S1PR1 protein levels. The differential expression in S1PRs both basally and following HGD exposure may suggest differential signaling mechanisms within the two brain vascular cell types. Furthermore, differential expression of S1PR1 and 2 highlight the potential beneficial and/or detrimental involvement of S1PRs in the pathophysiology of ischemic injury within the cerebrovasculature. Further investigation will be necessary to determine the signaling role of S1PRs and their impact on cerebrovascular cell function following acute ischemic injury in the brain.

OxLDL Mediates Brain Vascular Smooth Muscle Cell LOX‐1 Splicing, Oxidative Stress, and Inflammation Following Ischemic‐like Injury

A critical risk factor known to mediate vascular injury and found to be clinically correlated with acute ischemic stroke is elevated oxidized low‐density lipoprotein (OxLDL) levels. OxLDL, via the lectin‐like OxLDL receptor 1 (LOX‐1), has been shown to alter vascular wall function in part by mediating mechanisms associated with oxidative stress and inflammation. LOX‐1 is characterized by three alternative splice variants. Variant (V)1 corresponds to the full‐length protein that is able to bind OxLDL; whereas, this binding activity is disrupted in V2 and V3. LOX‐1 splice variant characterization as well as mediators of inflammation and oxidative stress in the cerebrovasculature has yet to be addressed following OxLDL exposure alone or in the context of acute ischemic injury. Therefore, we investigated the expression profile of LOX‐1 (V1, 2, 3), proinflammatory mediators (PTGS2, IL‐1β), and oxidative stress mediator (SOD1) in primary male human brain vascular smooth muscle cells (VSMCs) following an in vitro acute ischemic injury (HGD; hypoxia plus glucose depravation) ± OxLDL. We hypothesized that HGD would alter LOX‐1 variant expression as well as induce a proinflammatory and oxidative stress response that would be exacerbated by OxLDL via LOX‐1 receptor. VSMCs were preconditioned with human OxLDL (50 and 25μg/dL; 12h) or vehicle. Next VSMCs were treated with BI‐0115 (selective LOX‐1 inhibitor; 10 μM) or vehicle (&lt;0.1% DMSO) for 0.5h prior to being exposed to normoxia (21% O2) or HGD (1% O2) for 6h in the continued presence of OxLDL or vehicle. LOX‐1 variants were visualized using semi‐quantitative RT‐PCR analysis. PTGS2, IL‐1β, and SOD1 gene expression were assessed using quantitative real time‐PCR. COX‐2 glycosylation (active form) was measured via western blotting and used as a functional proinflammatory outcome. VSMCs exhibited a differential expression profile of the LOX‐1 variants under normoxia: V1 &gt; V3 &gt; V2. OxLDL alone increased V1 expression, had no effect on V2 levels, and increased V3 expression in a dose dependent manner. Following HGD, we observed a decrease in V1 expression with no change in V2 or V3 in comparison to normoxia. HGD plus OxLDL increased V1 and V2 expression; however, V2 expression was OxLDL dose dependent. Similarly, V3 expression was increased, however only at OxLDL 50 μg/dL in comparison to HGD alone. We next observed that HGD increased PTGS2, IL‐1β, and SOD1 mRNA expression, while OxLDL alone increased IL‐1β and SOD1 in a dose and LOX‐1 receptor dependent manner. Paradoxically HGD plus OxLDL decreased levels of IL‐1β and SOD1 in a LOX‐1 dependent manner. Moreover, LOX‐1 inhibition as well as OxLDL alone decreased levels of PTGS2 compared to HGD plus vehicles. Finally, we observed that HGD and OxLDL individually increased COX‐2 glycosylation, suggesting enzyme activation. In conclusion, the beneficial down‐regulation of LOX‐1 V1 by HGD was attenuated by the presence of OxLDL, suggesting that the combinatory effect predisposes the cerebrovasculature to an increased OxLDL/LOX‐1 response during acute ischemic stroke. Furthermore, LOX‐1 inhibition and the resulting attenuation of OxLDL‐mediated increases in VSMC IL‐1β and SOD1 transcription suggests that LOX‐1 may be a potential therapeutic target for patients with a higher risk of stroke.

STAT3 inhibitor mitigates cerebral amyloid angiopathy and parenchymal amyloid plaques while improving cognitive functions and brain networks

Previous reports indicate a potential role for signal transducer and activator of transcription 3 (STAT3) in amyloid-β (Aβ) processing and neuritic plaque pathogenesis. In the present study, the impact of STAT3 inhibition on cognition, cerebrovascular function, amyloid pathology, oxidative stress, and neuroinflammation was studied using in vitro and in vivo models of Alzheimer’s disease (AD)-related pathology. For in vitro experiments, human brain vascular smooth muscle cells (HBVSMC) and human brain microvascular endothelial cells (HBMEC) were used, and these cultured cells were exposed to Aβ peptides followed by measurement of activated forms of STAT3 expression and reactive oxygen species (ROS) generation. Further, 6 months old 5XFAD/APOE4 (5XE4) mice and age-matched negative littermates were used for in vivo experiments. These mice were treated with STAT3 specific inhibitor, LLL-12 for 2 months followed by neurobehavioral and histopathological assessment. In vitro experiments showed exposure of cerebrovascular cells to Aβ peptides upregulated activated forms of STAT3 and produced STAT3-mediated vascular oxidative stress. 5XE4 mice treated with the STAT3-specific inhibitor (LLL-12) improved cognitive functions and functional connectivity and augmented cerebral blood flow. These functional improvements were associated with a reduction in neuritic plaques, cerebral amyloid angiopathy (CAA), oxidative stress, and neuroinflammation. Reduction in amyloid precursor protein (APP) processing and attenuation of oxidative modification of lipoprotein receptor related protein-1 (LRP-1) were identified as potential underlying mechanisms. These results demonstrate the broad impact of STAT3 on cognitive functions, parenchymal and vascular amyloid pathology and highlight the therapeutic potential of STAT3 specific inhibition for treatment of AD and CAA.

Engineering a 3D human intracranial aneurysm model using liquid-assisted injection molding and tuned hydrogels

Physiologically relevant intracranial aneurysm (IA) models are crucially required to facilitate testing treatment options for IA. Herein, we report the development of a new in vitro tissue-engineered platform, which recapitulates the microenvironment, structure, and cellular complexity of native human IA. A new modified liquid-assisted injection molding technique was developed to fabricate a three-dimensional hollow IA model with clinically relevant IA dimensions within a mechanically tuned Gelatin Methacryloyl (GelMA) hydrogel. An endothelium lining was created inside the IA model by culturing human umbilical vein endothelial cells over pre-cultured human brain vascular smooth muscle cells. These cellularized IA models were subjected to medium perfusion at flow rates between 6.3 and 15.75 mL/min for inducing biomimetic vessel wall shear stress (10–25 dyn/cm2) to the cells for ten days. Both cell types maintained their secretome profiles and showed more than 96% viability, demonstrating the biocompatibility of the hydrogel during perfusion cell culture at such flow rates. Based on the characterized viscoelastic properties of the GelMA hydrogel and with the aid of a fluid-structure interaction model, the capability of the IA model in predicting the response of the IA to different fluid flow profiles was mathematically shown. With physiologically relevant behavior, our developed in vitro human IA model could allow researchers to better understand the pathophysiology and treatment of IA. Statement of significanceA three-dimensional intracranial aneurysm (IA) tissue model recapitulating the microenvironment, structure, and cellular complexity of native human IA was developed. • An endothelium lining was created inside the IA model over pre-cultured human brain vascular smooth muscle cells over at least 10-day successful culture. • The cells maintained their secretome profiles, demonstrating the biocompatibility of hydrogel during a long-term perfusion cell culture. • The IA model showed its capability in predicting the response of IA to different fluid flow profiles. • The cells in the vessel region behaved differently from cells in the aneurysm region due to alteration in hemodynamic shear stress. • The IA model could allow researchers to better understand the pathophysiology and treatment options of IA.

CircRNA DOCK1 Regulates miR-409-3p/MCL1 Axis to Modulate Proliferation and Apoptosis of Human Brain Vascular Smooth Muscle Cells.

BackgroundIntracranial aneurysm is an abnormal expansion in the intracranial arteries, which is associated with growth and apoptosis of vascular smooth muscle cells. Circular RNAs (circRNAs) have implicated in the progression of intracranial aneurysms. The purpose of this paper is to study the function and mechanism of circRNA dedicator of cytokinesis 1 (circ_DOCK1) in regulating proliferation and apoptosis of human brain vascular smooth muscle cells (HBVSMCs).MethodsHBVSMCs were exposed to hydrogen peroxide (H2O2). Cell proliferation and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry, respectively. Circ_DOCK1, microRNA (miR)-409-3p, and myeloid cell leukemia sequence 1 (MCL1) levels were examined by quantitative reverse transcription polymerase chain reaction or western blotting. The target association was assessed by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation assays.ResultsExposure to H2O2 decreased proliferation and increased apoptosis of HBVSMCs. Circ_DOCK1 expression was reduced in H2O2-treated HBVSMCs. Circ_DOCK1 overexpression rescued H2O2-caused reduction of proliferation and PCNA expression and attenuated H2O2-induced apoptosis and expression of Bcl-2, Bax, and cleaved PARP. MiR-409-3p was targeted by circ_DOCK1 and upregulated in H2O2-treated HBVSMCs. MiR-409-3p upregulation mitigated the role of circ_DOCK1 in proliferation and apoptosis of H2O2-treated HBVSMCs. MCL1 was targeted via miR-409-3p and downregulated via H2O2 treatment. Circ_DOCK1 overexpression enhanced MCL1 expression via modulating miR-409-3p. MiR-409-3p knockdown weakened H2O2-induced proliferation reduction and apoptosis promotion via regulating MCL1.ConclusionCirc_DOCK1 overexpression mitigated H2O2-caused proliferation inhibition and apoptosis promotion in HBVSMCs by modulating miR-409-3p/MCL1 axis.

Integrative analysis of the human brain mural cell transcriptome

Brain mural cells, including pericytes and vascular smooth muscle cells, are important for vascular development, blood-brain barrier function, and neurovascular coupling, but the molecular characteristics of human brain mural cells are incompletely characterized. Single cell RNA-sequencing (scRNA-seq) is increasingly being applied to assess cellular diversity in the human brain, but the scarcity of mural cells in whole brain samples has limited their molecular profiling. Here, we leverage the combined power of multiple independent human brain scRNA-seq datasets to build a transcriptomic database of human brain mural cells. We use this combined dataset to determine human-mouse species differences in mural cell transcriptomes, culture-induced dedifferentiation of human brain pericytes, and human mural cell organotypicity, with several key findings validated by RNA fluorescence in situ hybridization. Together, this work improves knowledge regarding the molecular constituents of human brain mural cells, serves as a resource for hypothesis generation in understanding brain mural cell function, and will facilitate comparative evaluation of animal and in vitro models.

Circular RNA circDUS2 Is a Potential Biomarker for Intracranial Aneurysm.

Background: CircRNAs have been found to play a crucial role in the pathological process of various kinds of diseases. However, the role of circRNAs in the formation and rupture of intracranial aneurysm is still unknown.Methods: Differentially expressed circRNAs profiles between superficial temporal arteries (n = 5) and intracranial aneurysms (n = 5) were analyzed using the Arraystar human circRNAs microarray. Quantitative real-time PCR was utilized to validate the differential expression of circDUS2. Fluorescence in situ hybridization (FISH) was meant for the location of circDUS2 in human brain vascular smooth muscle cell (HBVSMC). Structural analysis was used to speculate on the function of circDUS2.Results: Five hundred forty-three upregulated and 397 downregulated significantly in intracranial aneurysm as compared to superficial temporal arteries. Quantitative real-time PCR verified the elevated expression of the upregulated circDUS2. The FISH test revealed that circDUS2 is located in the cytoplasm of brain vascular smooth muscle cells.Conclusion: This study showed differential expression data of circRNAs between superficial temporal artery and intracranial aneurysm and revealed that circDUS2 is a potential molecular marker for intracranial aneurysm.