Brain Microvascular Endothelial Cells Research Articles

Modulation of the Blood-Brain Barrier by Sigma-1R Activation.

Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood-brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood-brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.

Hyperglycaemia perturbs blood-brain barrier integrity through its effects on endothelial cell characteristics and function

ABSTRACT Breakdown of blood-brain barrier (BBB) represents a key pathology in hyperglycemia-mediated cerebrovascular damage after an ischemic stroke. As changes in the level and nature of vasoactive agents released by endothelial cells (ECs) may contribute to BBB dysfunction, this study first explored the specific impact of hyperglycemia on EC characteristics and secretome. It then assessed whether secretome obtained from ECs subjected to normoglycaemia or hyperglycemia might regulate pericytic cytokine profile differently. Using a triple cell culture model of human BBB, composed of brain microvascular EC (BMEC), astrocytes and pericytes, this study showed that exposure to hyperglycemia (25 mM D-glucose) for 72 h impaired the BBB integrity and function as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux of sodium fluorescein. Dissolution of zonula occludens-1, a tight junction protein, and appearance of stress fibers appeared to play a key role in this pathology. Despite elevations in angiogenin, endothelin-1, interleukin-8 and basic fibroblast growth factor levels and a decrease in placental growth factor levels in BMEC subjected to hyperglycemia vs normoglycaemia (5.5 mM D-glucose), tubulogenic capacity of BMECs remained similar in both settings. Similarly, pericytes subjected to secretome obtained from hyperglycemic BMEC released higher quantities of macrophage migration inhibitory factor and serpin and lower quantities of monocyte chemoattractant protein-1, intercellular adhesion molecule, interleukin-6 and interleukin-8. Taken together these findings indicate the complexity of the mechanisms leading to BBB disruption in hyperglycemic settings and emphasize the importance of endothelial cell-pericyte axis in the development of novel therapeutic strategies.

Calcium and IL-6 regulate the anterograde trafficking and plasma membrane residence of the iron exporter ferroportin to modulate iron efflux

Iron is an essential element for proper cell functioning, but unbalanced levels can cause cell death. Iron metabolism is controlled at the blood-tissue barriers provided by microvascular endothelial cells. Dysregulated iron metabolism at these barriers is a factor in both neurodegenerative and cardiovascular diseases. Mammalian iron efflux is mediated by the iron efflux transporter ferroportin (Fpn). Inflammation is a factor in many diseases and correlates with increased tissue iron accumulation. Evidence suggests treatment with interleukin 6 (IL-6) increases intracellular calcium levels and calcium is known to play an important role in protein trafficking. We have shown that calcium increases plasma membrane localization of the iron uptake proteins ZIP8 and ZIP14, but if and how calcium modulates Fpn trafficking is unknown. In this article, we examined the effects of IL-6 and calcium on Fpn localization to the plasma membrane. In HEK cells expressing a doxycycline-inducible GFP-tagged Fpn, calcium increased Fpn-GFP membrane presence by 2 h, while IL-6 increased membrane-localized Fpn-GFP by 3 h. Calcium pretreatment increased Fpn-GFP mediated 55Fe efflux from cells. Endoplasmic reticulum calcium stores were shown to be important for Fpn-GFP localization and iron efflux. Use of calmodulin pathway inhibitors showed that calcium signaling is important for IL-6-induced Fpn relocalization. Studies in brain microvascular endothelial cells in transwell culture demonstrated an initial increase in 55Fe flux with IL-6 that is reduced by 6h coinciding with upregulation of hepcidin. Overall, this research details one pathway by which inflammatory signaling mediated by calcium can regulate iron metabolism, likely contributing to inflammatory disease mechanisms.

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

BackgroundTriptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp).MethodsWe investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice.ResultsWe demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice.ConclusionsWe have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.Graphical

Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

BackgroundLocal brain tissue can suffer from ischaemia/reperfusion (I/R) injury, which lead to vascular endothelial damage. The peptide δ opioid receptor (δOR) agonist [D-ala2, D-leu5]-Enkephalin (DADLE) can reduce apoptosis caused by...

Elevation of miR-150-5p in Brain Microvascular Endothelial Cells as a Result of Prenatal Alcohol Exposure Is Mediated by Transcriptional Mechanisms

Fetal Alcohol Spectrum Disorders (FASD) encompass a variety of conditions impacting development in children exposed to alcohol in utero. Vascular alterations such as defective angiogenesis, blood brain barrier permeability, vessel reactivity, and blood flow have been found in various animal models of prenatal alcohol exposure (PAE). The underlying mechanisms of these vascular defects are not fully understood. Our lab previously showed that miR-150-5p was upregulated in brain microvascular endothelial cells (BMVECs) isolated from embryonic mouse cortices following moderate PAE. We identified downstream vascular targets of miR-150-5p in BMVECs and demonstrated that miR-150-5p inhibition could counter alcohol-mediated effects on BMVECS and the cortical microvasculature in vitro and in vivo, respectively. Here, we investigate upstream mechanisms that contribute to miR-150-5p elevation in BMVECs following PAE. We hypothesized that multiple mechanisms, including pathways at the transcriptional level, contribute to increased miR-150-5p abundance in BMVECs following ethanol (EtOH) exposure. Overall transcriptional activity at the miR-150 promoter was increased in BMVECs with EtOH treatment, and pri-miR-150, the primary transcript for miR-150-5p, was elevated in EtOH-treated BMVECs. Additionally, miR-150 promoter methylation was decreased in PAE cortices compared to saccharine-exposed controls (SAC). Current work aims to confirm the role of DNA methylation in transcriptional regulation of the miR-150 promoter in BMVECs and characterize DNA methyltransferase activity in BMVECs during PAE. We also assessed transcription factor binding at the miR-150 promoter and found that miR-150 promoter activation by several transcription factors was blunted with EtOH treatment. Current work involves the investigation of global transcription factor activation in BMVECs in PAE versus SAC. In summary, our research sheds light on multiple mechanisms of miR-150-5p regulation that may contribute to brain microvascular defects as a result of PAE. American Physiological Society Porter Physiology Development Fellowship University of New Mexico Health School of Medicine Department of Cell Biology and Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3020: The Effects Of High Glucose On The Release Of Exosomes From Adipocytes

Type 2 Diabetes Mellitus is primarily characterized by hyperglycemia accompanied by adipose tissue dysfunction. More importantly, diabetes is a risk factor for stroke and other cerebrovascular diseases, which might be related to poorly explored adipose tissue dysfunction. Even though adipose tissue is comprised of various cells, it is found that predominantly adipocyte dysfunction contributes to diabetes and related complications. Exosomes are extracellular vesicles released from almost all cells with varying functions in different conditions. Here, we aim to determine the function of adipocyte-derived exosomes (Adp-EXs) in high glucose (HG) conditions. We hypothesized that Adp-EXs from the HG group would induce cytotoxicity and oxidative stress in primary human brain microvascular endothelial cells (HBMECs). First, the level of Adp-EXs was measured after HG treatment. Secondly, their effects were studied on HBMECs after co-incubation. Adipocytes were cultured in normal glucose (NG) or HG (25mM) media to isolate Adp-EXs by serial ultracentrifugation method. The size and concentration of Adp-EXs were determined via the Nanoparticle Tracking Analysis system. Adp-EXs at three concentrations (1x10 8 , 5x10 8 and 1x10 9 particles/mL) were co-incubated with HBMECs for 24 to 48 hours to evaluate optimal cellular communication. PKH26-labelling (2uL/mL) of Adp-EX was performed to track its incorporation into HBMECs. Dihydroethidium staining and flow cytometry quantified reactive oxygen species production and cytotoxicity was determined by lactate dehydrogenase assay. For all experiments, n=5-10 and mean SEM=8.600±6.194. Results showed: 1) HG increased Adp-EX levels without size differences between NG and HG groups. 2) Adp-EXs integrated with HBMECs in a time and dose-dependent manner, more prominently in the HG group. 3) Adp-EXs from the HG group increased Dihydroethidium-positive HBMECs. 4) Adp-EXs from the HG group increased fluorescence for lactate dehydrogenase. In conclusion, the data suggest that HG could promote Adp-EX release, and trigger oxidative stress and cytotoxicity in HBMECs.

Role of L-PGDS in Light Alcohol Consumption-Induced Cerebral Angiogenesis in Male Mice

Ischemic stroke is one of the leading causes of death and permanent disability. Promoting cerebral angiogenesis before or after ischemic stroke reduces ischemic brain damage and improves functional recovery. Alcohol is one of the most commonly consumed chemical substances. Light alcohol consumption (LAC) reduces the incidence and improves the prognosis of ischemic stroke. Recently, we found that LAC promotes cerebral angiogenesis under basal conditions and following ischemic stroke. In addition, LAC upregulates lipocalin-type prostaglandin D2 synthase (L-PGDS), the principal PGH2 isomerase mainly expressed in the central nervous and cardiovascular systems. Therefore, we determined the role of L-PGDS in LAC-induced cerebral angiogenesis. In in vitro studies, low-concentration ethanol significantly upregulated L-PGDS and increased angiogenic capability (tube formation, migration, and proliferation) in cultured C57BL/6J mouse brain microvascular endothelial cells (MBMVECs). AT-56, a selective L-PGDS inhibitor, abolished the increased angiogenic capability. In in vivo studies, 8-week gavage feeding with low-dose ethanol significantly upregulated L-PGDS and increased vessel density and vessel branches in the cerebral cortex and subcortical area under physiological conditions and following ischemic stroke in C57BL/6J male mice. AT-56 and endothelial cell-specific L-PGDS conditional knockout did not affect vessel density and vessel branches of the cerebral cortex and subcortical area in water-fed control mice but alleviated the proangiogenic effect in low-dose ethanol-fed mice under basal conditions and following ischemic stroke. Therefore, our findings suggest that L-PGDS may be crucial in LAC-induced cerebral angiogenesis. NIH 2R01AA023610. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A human pluripotent stem cell-derived in vitro model of the blood–brain barrier in cerebral malaria

BackgroundBlood–brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but their physiological relevance remains uncertain. Using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs), we aimed to develop a novel in vitro model of the BBB in CM, exhibiting enhanced barrier properties.MethodshiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 h. Barrier integrity was measured using transendothelial electrical resistance (TEER) and sodium fluorescein permeability assays. Localization and expression of tight junction (TJ) proteins (occludin, zonula occludens-1, claudin-5), cellular adhesion molecules (ICAM-1, VCAM-1), and endothelial surface markers (EPCR) were determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were measured using multiplex proteome profiler arrays.ResultsAfter 6-h of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and increased sodium fluorescein permeability compared to co-culture with uninfected RBCs, indicative of a leaky barrier. We observed disruptions in localization of occludin, zonula occludens-1, and claudin-5 by IF, but no change in protein expression by WB in Pf-iRBC co-cultures. Expression of ICAM-1 and VCAM-1 but not EPCR was elevated in hiPSC-BMECs with Pf-iRBC co-culture compared to uninfected RBC co-culture. In addition, there was an increase in expression of angiogenin, platelet factor-4, and phospho-heat shock protein-27 in the Pf-iRBCs co-culture compared to uninfected RBC co-culture.ConclusionThese findings demonstrate the validity of our hiPSC-BMECs based model of the BBB, that displays enhanced barrier integrity and appropriate TJ protein localization. In the hiPSC-BMEC co-culture with Pf-iRBCs, reduced TEER, increased paracellular permeability, changes in TJ protein localization, increase in expression of adhesion molecules, and markers of angiogenesis and cellular stress all point towards a novel model with enhanced barrier properties, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.

Targeted Nanoparticles to Mitigate Radiation-Induced Blood-Brain Barrier Disruption and Cognitive Impairment

Background: Damage to neurovascular endothelium contributes to cognitive decline through increased blood-brain barrier (BBB) permeability. In our studies of mice and endothelial cells (ECs) subject to irradiation, we detect increases in mitochondrial reactive oxygen species (mtROS), mitochondrial DNA (mtDNA) damage, and cell-free release of mtDNA. Cell-free mtDNA acts as a damage associated molecular pattern (DAMP), and similarly to bacterial DNA, binds Toll-like receptor 9, triggering a pro-inflammatory and pro-oxidative response. However, these irradiation-induced effects are attenuated with TLR9 inhibition or mtROS scavenging. Our preliminary data implicate TLR9’s critical role in propagating a feedforward mechanism of sustained oxidative endothelial damage and dysfunction following radiation therapy. We hypothesize that specific inhibition of TLR9 in neurovascular ECs during radiation will prevent BBB permeability and cognitive decline. Thus, the purpose of the study is to use a novel nanoparticle (NP)-based tool to examine the sequela of cranial irradiation and provide proof-of-concept for its use as a mitigator. Methods: To target TLR9 in irradiated ECs, poly (lactic-co-glycolic acid)-based NP coated with cyclic Arg-Gly-Asp (cRGD)-peptide were designed and loaded with TLR9 antagonist (aTLR9, ODN-2088, 2.23ug/mg particle). cRGD-peptide is a ligand for integrin αvβ3, which is highly expressed in ECs post-irradiation (IR). C57Bl6 mice were administered NP-ODN-2088 (aTLR9-NP) or empty-NP (3mg injection) and subjected to unilateral cranial Xray IR (dose of 12Gy). 30d post-IR, memory and learning ability were assessed with the Novel Object Recognition (NOR) and Y-maze tests. The functionality of the NPs was assessed in vivo via BBB permeability, neuronal density, mtDNA damage, and serum levels of mtDNA and in vitro via mtROS production levels and inflammatory markers measurement in human brain microvascular endothelial cells (HBMECs) 5 days post-IR (IR dose of 4Gy). Results: In cultured HBMECs, treatment with aTLR9-NP prevented mtROS production 120hours post-IR. This correlated with attenuated inflammatory intermediates (p65, TNFa, IL6, and IL8), as compared to treatment with vehicle. In mice, aTLR9-NP treatment preserved integrity of mtDNA isolated from brain tissue and decreased blood levels of circulating cell-free mtDNA. aTLR9-NP treatment showed protective effects on BBB permeability and neuronal dropout, and these findings corresponded with preserved cognition in both, NOR and Y-maze tests. NP treatment caused no changes in alkaline phosphatase, alanine transaminase, aspartate aminotransferase, or bilirubin levels, indicating no liver injury. Conclusion: Inhibition of TLR9 in ECs was observed to prevent radiation-induced BBB permeability, neuronal dropout, and cognitive decline. Our findings support a pathway through which TLR9 links oxidative endothelial injury to neurological pathology. Currently there are no pharmacological options for treating radiation-induced normal tissue injury and targeting TLR9 poses a promising strategy. American Heart Association 2021CDA 853499. NIH R01 EY 031544-02 Veterans Affairs I01 BX000163-12. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PM2.5 Temporally Decreases Human Brain Endothelial Barrier Proteins and Concomitantly Increases Inflammation and Autophagy in a Dose Dependent Manner

Exposure to airborne particulate matter 2.5 (particle size <PM2.5 um) pollution is a significant co-morbidity for stroke and other cardio- and cerebrovascular diseases, causing premature mortality. However, mechanisms underlying PM2.5-mediated endothelial dysfunction and integrity loss are not known. Our goal was to elucidate how PM2.5 alters brain endothelial phenotype causing inflammation and loss of barrier integrity to provide better insight to the potential pathogenesis of PM2.5. We hypothesized that PM2.5 would alter integral endothelial barrier proteins as well as inflammatory mediators in a dose and temporal dependent manner, subsequently exacerbating the impact of acute ischemic-like injury. Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with PM2.5 (300, 75, or 15 μg/m3; 12, 24, or 36h) or vehicle, then exposed to normoxia (21% O2) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O2), for 3h. HBMEC barrier function was assessed via trans-endothelial electrical resistance (TEER). HBMEC levels or expression of barrier markers (claudin-5, occludin, ZO-1), adhesion molecules (ICAM-1 and PECAM-1), inflammatory proteins (iNOS, COX-2, LOX-1, TNF-α, IL-1β), and autophagic protein (Beclin-1) were examined using qRT-PCR, in cell western, and standard immunoblotting. Ischemia-like injury as well as PM2.5 decreased HBMEC barrier function. PM2.5 also induced a concomitant temporal and dose dependent decrease in claudin-5 and increase in iNOS as well as TNF-α levels. Additionally, PM2.5 increased protein levels of beclin-1 and LOX-1 like that of HGD alone; however, combination of PM2.5 and HGD decreased levels of beclin-1. HGD alone or in combination with PM2.5 decreased levels of PECAM-1, but PM2.5 alone did not alter PECAM-1. PM2.5 in the presence or absence of HGD induced differential alteration in claudin-5 isoform protein levels. Intriguingly, PM2.5 decreased claudin-5 mRNA levels like that observed in HBMECs exposed to HGD alone. PM2.5 plus HGD significantly increased mRNA levels of both ICAM-1 and IL-1β. In conclusion, PM2.5 mediated inflammatory and vascular pathogenesis involves a potential complex intracellular molecular framework associated with the brain endothelium which is compounded by ischemic like injury. These findings have revealed detrimental effects of PM2.5 on the brain vascular endothelium which will help to identify targets in the endothelium to restore or preserve cerebrovascular homeostasis. University of Arizona Valley Research Partnership Grant VRP37 P2 (RJG), American Heart Association 19AIREA34480018 (RJG), UA VRP Grant VRP55 P1a (TSW). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unraveling the Impact of Extracellular Vesicle-Depleted Serum on Endothelial Cell Characteristics over Time.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

RBM3 enhances the stability of MEF2C mRNA and modulates blood-brain barrier permeability in AD microenvironment

Blood-brain barrier (BBB) changes are acknowledged as early indicators of Alzheimer's disease (AD). The permeability and integrity of the BBB rely significantly on the essential role played by the tight junction proteins (TJPs) connecting endothelial cells. This study found the reduced RNA binding motif protein 3 (RBM3) expression in brain microvascular endothelial cells (BMECs) incubated with Aβ1–42. This downregulation of RBM3 caused a decrease in the levels of ZO-1 and occludin and increased the permeability of BBB cell model in AD microenvironment. Myocyte enhancer factor 2C (MEF2C) expression was also inhibited in BMECs incubated with Aβ1–42. A decrease in MEF2C expression led to increased permeability of BBB cell model in AD microenvironment and reductions in the levels of ZO-1 and occludin. Further analysis of the underlying mechanism revealed that RBM3 binds to and stabilizes MEF2C mRNA. MEF2C binds to the promoters of ZO-1 and occludin, enhancing their transcriptional activities and modulating BBB permeability. RBM3 increases the stability of MEF2C mRNA and subsequently modulates BBB permeability through the paracellular pathway of TJPs. This may provide new insights for AD research.

Genomic characterization of Cronobacter sakazakii strains in the neonatal meningitis clonal complex 4

AbstractCronobacter sakazakii clonal complex 4 (CC4) is strongly associated with neonatal meningitis. However, C. sakazakii CC4‐specific pathogenicity traits have not been determined. In this study, the comparative genomic analysis of 144 genomes of C. sakazakii CC4 strains and 376 non‐CC4 C. sakazakii strains was undertaken. Twenty‐four CC4 strains were previously undescribed clinical and nonclinical strains that had been collected from various regions in China (2006–2017). The remaining genomes were of multiple sequence types of C. sakazakii strains, which had been isolated worldwide (1973–2021). The pan‐genome of C. sakazakii comprised 32,332 genes, of which 2.58% (832 genes) constituted the core genome. More C. sakazakii CC4 strains have a complete cusABCFR efflux system with a significant difference compared with non‐CC4 strains, and the ibeB‐homologous cusC gene in the cusABCFR efflux system is probably associated with the invasion of human brain microvascular endothelial cells (BMEC). In addition, the isolates with K2:CA2 capsule or type IV pili‐associated genes associated with neonatal meningitis were statistically more present in CC4 strains than non‐CC4 strains (p < .0001). The thorough description of VirB/VirD4 gene cluster for the type IV secretion system and the impB/impF and vasL/vasJ gene clusters for the type VI secretion system in C. sakazakii were provided. Overall, the specific and diverse virulence factors and genes may have led to C. sakazakii CC4 clone increasing its ability to invade human BMEC and leading to neonatal meningitis. Our findings should facilitate the development of novel strategies to prevent, diagnose, and treat C. sakazakii CC4 infections.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Study on the role of CCM3 gene and lead exposure induced neurotoxicity through neurovascular units

This study aimed to determine the toxic effects of vascular CCM3 gene deficiency and lead (Pb) exposure on the nervous system. Lentiviral transfection was performed to generate a stable strain of brain microvascular endothelial cells with low CCM3 expression. MTT assay assessed the survival rate of cells exposed to Pb, determining the dose and duration of Pb exposure in vitro. Proteomic analysis was performed on the differentially expressed proteins in bEnd3 and HT22 cells and flow cytometry was used to detect cell apoptosis. Finally, urine samples from pregnant and postpartum women were subjected to ICP-MS to detect Pb levels and HPLC to detect neurotransmitter metabolites. Based on the proteomic analysis of bEnd3 (CCM3-/-) cells co-cultured with HT22 cells, it was determined that HT22 cells and CCM3 genes interfered with bEnd3 cell differential proteins,22Abbreviations: SNP, FGF2, EGF, AD including apoptosis and ferroptosis pathways. Electron microscopy observation, ICP-MS iron ion loading detection, and WB determination of protein GPX4 expression confirmed that HT22 cells undergo apoptosis, while bEnd3 cells undergo multiple pathways of iron death and apoptosis regulation. Furthermore, a linear regression model showed the interaction between maternal urine Pb levels, the rs9818496 site of the CCM3 SNP in peripheral blood DNA, and the concentration of the neurotransmitter metabolite 5-HIAA in maternal urine (F=4.198, P < 0.05). bEnd3 cells with CCM3 gene deficiency can induce HT22 cell apoptosis through iron death and apoptosis pathways under Pb exposure in a combined cell culture Pb exposure model, and CCM3 gene deficiency in endothelial cells and Pb exposure interacts with neural cell HT22. Epidemiological studies on maternal and newborn infants further confirmed the interaction between urine Pb levels in mothers and the SNP rs9818496 site of the CCM3 gene in peripheral blood DNA.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

CircRNA-PTPN4 mediated regulation of FOXO3 and ZO-1 expression: implications for blood–brain barrier integrity and cognitive function in uremic encephalopathy

Uremic encephalopathy (UE) poses a significant challenge in neurology, leading to the need to investigate the involvement of non-coding RNA (ncRNA) in its development. This study employed ncRNA-seq and RNA-seq approaches to identify fundamental ncRNAs, specifically circRNA and miRNA, in the pathogenesis of UE using a mouse model. In vitro and in vivo experiments were conducted to explore the circRNA-PTPN4/miR-301a-3p/FOXO3 axis and its effects on blood–brain barrier (BBB) function and cognitive abilities. The research revealed that circRNA-PTPN4 binds to and inhibits miR-301a-3p, leading to an increase in FOXO3 expression. This upregulation results in alterations in the transcriptional regulation of ZO-1, affecting the permeability of human brain microvascular endothelial cells (HBMECs). The axis also influences the growth, proliferation, and migration of HBMECs. Mice with UE exhibited cognitive deficits, which were reversed by overexpression of circRNA-PTPN4, whereas silencing FOXO3 exacerbated these deficits. Furthermore, the uremic mice showed neuronal loss, inflammation, and dysfunction in the BBB, with the expression of circRNA-PTPN4 demonstrating therapeutic effects. In conclusion, circRNA-PTPN4 plays a role in promoting FOXO3 expression by sequestering miR-301a-3p, ultimately leading to the upregulation of ZO-1 expression and restoration of BBB function in mice with UE. This process contributes to the restoration of cognitive abilities.Graphical 1. The circRNA-PTPN4/miR-301a-3p/FOXO3 axis is identified as a key regulator of blood–brain barrier integrity and cognitive function in uremic encephalopathy.2. circRNA-PTPN4 sequestration of miR-301a-3p enhances FOXO3 expression, leading to upregulation of ZO-1 and improved endothelial permeability.3. Overexpression of circRNA-PTPN4 in uremic mice restores cognitive abilities and reduces neuronal loss and inflammatory infiltration.

Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4-NOTCH-CLDN5 pathway and is vulnerable to neonatal hyperoxia.

The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long-term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH-NICD-RBPJ-CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood-brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. KEY POINTS: The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4 regulates endothelial integrity through NOTCH-NICD-RBPJ-CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.