Cerebral Endothelial Cells Research Articles

Identification of plasminogen-binding sites in Streptococcus suis enolase that contribute to bacterial translocation across the blood-brain barrier.

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Plasma-derived exosomes contributes to endothelial-to-mesenchymal transition in Moyamoya disease

BackgroundMoyamoya disease (MMD) is a cerebrovascular disease with a high disability rate; however, its pathogenesis remains unknown. Endothelial-mesenchymal transition (EndMT) is the pathological basis of many vascular diseases; however, the key role of EndMT in MMD has not yet been reported. MethodWe collected vascular tissues from three control samples and six patients with MMD to detect the expression of EndMT-related genes. To elucidate the mechanism of EndMT in MMD, we performed in vitro cell experiments. Plasma-derived exosomes (PDEs) can transmit information between cells and tissues and are of considerable importance in several disease studies. PDEs were used to stimulate EndMT phenotype in cerebrovascular endothelial cells. ResultsMultiplex fluorescent immunohistochemistry staining confirmed that CD31, VE-cadherin and E-cadherin down-regulated, whereas α-SMA and vimentin were significantly up-regulated in moyamoya vascular endothelial cells than in control samples. PDEs from MMD patients significantly promoted cell proliferation and migration, resulting in slender cells. PDEs induce EndMT-related phenotype changes in cerebral vascular endothelial cells, including decreased endothelial cell marker expression and increased mesenchymal cell marker expression. We demonstrated that EndMT phenotypic alterations are mediated, in part, by microRNA(miRNAs). ConclusionThis study was the first to propose that EndMT may exist in the vessels of patients with MMD. PDEs induce the EndMT phenotype to promote the development of MMD. This study aimed to provide a new theoretical basis for elucidating the pathogenesis of MMD.

Molecular mechanisms of naringenin modulation of mitochondrial permeability transition acting on F1FO-ATPase and counteracting saline load-induced injury in SHRSP cerebral endothelial cells

Naringenin (NRG) was characterized for its ability to counteract mitochondrial dysfunction which is linked to cardiovascular diseases. The F1FO-ATPase can act as a molecular target of NRG. The interaction of NRG with this enzyme can avoid the energy transmission mechanism of ATP hydrolysis, especially in the presence of Ca2+ cation used as cofactor. Indeed, NRG was a selective inhibitor of the hydrophilic F1 domain displaying a binding site overlapped with quercetin in the inside surface of an annulus made by the three α and the three β subunits arranged alternatively in a hexamer. The kinetic constant of inhibition suggested that NRG preferred the enzyme activated by Ca2+ rather than the F1FO-ATPase activated by the natural cofactor Mg2+. From the inhibition type mechanism of NRG stemmed the possibility to speculate that NRG can prevent the activation of F1FO-ATPase by Ca2+. The event correlated to the protective role in the mitochondrial permeability transition pore opening by NRG as well as to the reduction of ROS production probably linked to the NRG chemical structure with antioxidant action. Moreover, in primary cerebral endothelial cells (ECs) obtained from stroke prone spontaneously hypertensive rats NRG had a protective effect on salt-induced injury by restoring cell viability and endothelial cell tube formation while also rescuing complex I activity.

Homocysteine potentiates amyloid -induced death receptor 4- and 5-mediated cerebral endothelial cell apoptosis, blood brain barrier dysfunction and angiogenic impairment.

Cerebrovascular dysfunction has been implicated as a major contributor to Alzheimer's Disease (AD) pathology, with cerebral endothelial cell (cEC) stress promoting ischemia, cerebral-blood flow impairments and blood-brain barrier (BBB) permeability. Recent evidence suggests that cardiovascular (CV)/cerebrovascular risk factors, including hyperhomocysteinemia (Hhcy), exacerbate AD pathology and risk. Yet, the underlying molecular mechanisms for this interaction remain unclear. Our lab has demonstrated that amyloid beta 40 (Aβ40) species, and particularly Aβ40-E22Q (AβQ22; vasculotropic Dutch mutant), promote death receptor 4 and 5 (DR4/DR5)-mediated apoptosis in human cECs, barrier permeability, and angiogenic impairment. Previous studies show that Hhcy also induces EC dysfunction, but it remains unknown whether Aβ and homocysteine function through common molecular mechanisms. We tested the hypotheses that Hhcy exacerbates Aβ-induced cEC DR4/5-mediated apoptosis, barrier dysfunction, and angiogenesis defects. This study was the first to demonstrate that Hhcy specifically potentiates AβQ22-mediated activation of the DR4/5-mediated extrinsic apoptotic pathway in cECs, including DR4/5 expression, caspase 8/9/3 activation, cytochrome-c release and DNA fragmentation. Additionally, we revealed that Hhcy intensifies the deregulation of the same cEC junction proteins mediated by Aβ, precipitating BBB permeability. Furthermore, Hhcy and AβQ22, impairing VEGF-A/VEGFR2 signaling and VEGFR2 endosomal trafficking, additively decrease cEC angiogenic capabilities. Overall, these results show that the presence of the CV risk factor Hhcy exacerbates Aβ-induced cEC apoptosis, barrier dysfunction, and angiogenic impairment. This study reveals specific mechanisms through which amyloidosis and Hhcy jointly operate to produce brain EC dysfunction and death, highlighting new potential molecular targets against vascular pathology in comorbid AD/CAA and Hhcy conditions.

Abstract WP296: Human Vascular Smooth Muscle Cells Derived Small Extracellular Vesicles Reduce the Neurovascular Damage After Stroke in Diabetic Mice

Introduction: Stroke patients with type-2 diabetes exhibit exacerbated cerebrovascular dysfunction and increased neurological deficits compared to non-diabetic stroke patients. Vascular smooth muscle cells (VSMCs) play an important role in mediating neurovascular function, however, how VSMCs communicate with cerebral endothelial cells (CECs) has not been studied. In this study, we tested the hypothesis that small extracellular vesicles (sEVs) derived from healthy human VSMCs (VSMC-sEVs) reduce diabetes- and ischemia-neurovascular damage. Methods and Results: VSMC-sEVs were characterized. In vitro CEC permeability assay showed that treatment of hypoxia-challenged human brain microvascular endothelial cells (BMECs) or primary CECs isolated from type-2 diabetic mice (n=6/group) with VSMC-sEVs significantly reduced and augmented cell permeability and trans-endothelial electrical resistance, respectively, compared to non VSMC-sEVs control. Inhibition of sEV endocytosis in BMECs with clathrin (CPZ) or caveolin (Nystatin) inhibitors abrogated the effect of VSMC-sEVs on CEC proliferation and migration. We then examined the effect of VSMC-sEVs on diabetic ischemia in which diabetic mice, with diabetes induced by a high fat diet and streptozotocin, were subjected to permanent MCAo. Middle-age diabetic male mice (10-12m old) were treated with intranasal administration of VSMC-sEVs (1x10 9 particles per injection) or saline (n=12/group) at 24h after cerebral ischemia. We found that VSMC-sEVs treatment significantly increased regional cerebral blood flow and FITC-dextran perfused functional microvessels (17.55% vs 13.32% control) as well as decreased brain hemorrhage and BBB leakage (1.51% vs 1.76% of albumin, p<0.05) in the ischemic border zone 28 days after MCAo compared with the saline treatment mice. Moreover, compared to the saline treatment, VSMC-sEVs significantly improved neurological function after stroke as measured by modified neurological severity score, and foot-fault and adhesive removal tests, respectively. Conclusion: This study suggests that VSMC-sEVs have a therapeutic effect to improve neurological outcome after diabetic stroke, likely by reducing diabetes and ischemia impaired neurovascular function.

Abstract WMP110: Endogenous Angiogenic Cerebral Endothelial Cell Derived Small Extracellular Vesicles Actively Communicate to Neurons and Astrocytes After Stroke

While ex-vivo studies have shown that small extracellular vesicles (sEVs) isolated from cerebral endothelial cells (CEC-sEVs) can be internalized by neurons leading to enhancement of neurite remodeling, whether such communication are present in ischemic brain is not known. Using CEC-sEV reporter mice, we investigated whether endogenous CEC-sEV mediate communication between CECs and neural cells after stroke. A CEC-specific/sEV reporter mouse line was generated by crossing Slco1c1- CreERT2 mice with CD63-GFP loxP/loxP mice. The adult reporter mice were subjected to middle cerebral artery occlusion or sham operation after tamoxifen injection (n=4/group). 3D confocal microscopic analysis revealed that GFP signals in sham operated reporter mice were primarily localized within cerebral vessels, while extravascular GFP signals were also detected throughout the entire brain, suggesting that endogenous sEVs are specific to CECs and CEC-sEVs are released into brain parenchyma. Compared to the sham mice, ischemia induced robust angiogenesis in the peri-infarct areas assessed by increasing vascular density at day 7 and 42 post stroke, where the GFP signals within endothelium increased by 56% and 28%, respectively. The GFP signals within parenchyma (outside of vessels) of the peri-infarct areas were augmented by 21% and 24% at day 7 and 42 post stroke, respectively, suggesting that stroke increases CEC-sEVs secretion at peri-infarct angiogenic sites. Immunofluorescent staining showed that the increased parenchymal GFP signals in peri-infarct areas were localized within GFAP + astrocytes by 36% and 21% of total parenchymal GFP signals at day 7 and 42 post stroke, respectively. Moreover, the GFP signals within the peri-infarct MAP2 + neurons were significantly augmented by 22% at day 42 but not at day 7 after stroke compared to the sham group. Stroke did not alter GFP signals within IBA1 + microglia in the reporter mice. In conclusion, our in vivo data for the first time demonstrate that stroke increases the release of CEC-sEVs at peri-infarct angiogenic sites, where they actively communicate with astrocytes and neurons during stroke recovery. These data suggest that endogenous CEC-sEVs may regulate neurovascular function contributing to stroke recovery.

Cellular iron depletion enhances behavioral rhythm by limiting brain Per1 expression in mice.

Disturbances in the circadian rhythm are positively correlated with the processes of aging and related neurodegenerative diseases, which are also associated with brain iron accumulation. However, the role of brain iron in regulating the biological rhythm is poorly understood. In this study, we investigated the impact of brain iron levels on the spontaneous locomotor activity of mice with altered brain iron levels and further explored the potential mechanisms governing these effects invitro. Our results revealed that conditional knockout of ferroportin 1 (Fpn1) in cerebral microvascular endothelial cells led to brain iron deficiency, subsequently resulting in enhanced locomotor activity and increased expression of clock genes, including the circadian locomotor output cycles kaput protein (Clock) and brain and muscle ARNT-like 1 (Bmal1). Concomitantly, the levels of period circadian regulator 1 (PER1), which functions as a transcriptional repressor in regulating biological rhythm, were decreased. Conversely, the elevated brain iron levels in APP/PS1 mice inhibited autonomous rhythmic activity. Additionally, our findings demonstrate a significant decrease in serum melatonin levels in Fpn1cdh5 -CKO mice compared with the Fpn1flox/flox group. In contrast, APP/PS1 mice with brain iron deposition exhibited higher serum melatonin levels than the WT group. Furthermore, in the human glioma cell line, U251, we observed reduced PER1 expression upon iron limitation by deferoxamine (DFO; iron chelator) or endogenous overexpression of FPN1. When U251 cells were made iron-replete by supplementation with ferric ammonium citrate (FAC) or increased iron import through transferrin receptor 1 (TfR1) overexpression, PER1 protein levels were increased. Additionally, we obtained similar results to U251 cells in mouse cerebellar astrocytes (MA-c), where we collected cells at different time points to investigate the rhythmic expression of core clock genes and the impact of DFO or FAC treatment on PER1 protein levels. These findings collectively suggest that altered iron levels influence the circadian rhythm by regulating PER1 expression and thereby modulating the molecular circadian clock. In conclusion, our study identifies the regulation of brain iron levels as a potential new target for treating age-related disruptions in the circadian rhythm.

Abstract WMP111: Intra-Arterial Administration of Cerebral Endothelial Cell Derived Small Extracellular Vesicles Reduces Neurovascular Damage and Improves Neurological Outcome After Acute Ischemia in the Aged Rat

Stroke is a leading cause of death and disability worldwide, mainly affecting the elderly. Endovascular thrombectomy (EVT) is a standard of care therapy for stroke patients with large-vessel occlusion, however, the majority of these patients do not achieve full functional improvement. The present study tested the hypothesis that intra-arterial (IA) administration of cerebral endothelial cell derived small extracellular vesicles (CEC-sEVs) following transient middle cerebral artery occlusion (tMCAO) improves stroke outcome in the aged rat. CEC-sEVs (1x10 11 particles/injection) isolated from healthy adult rats were employed. Male aged rats (18 months, n=10/group) subjected to 3h tMCAO were randomly divided into the experimental groups. To examine whether IA injection of CEC-sEVs improves stroke outcome, CEC-sEVs were administered via the internal carotid artery (ICA) immediately following reperfusion by withdrawal of the occlusion filament. To examine whether intravenous administration (IV) of CEC-sEVs during tMCAO and then followed by IA CEC-sEVs upon reperfusion further enhances stroke outcome, CEC-sEVs were given at 30 min of tMCAO via a tail vein and upon reperfusion via the ICA. The ischemic rats treated with IA saline were used as a control. Neurological outcome and infarct volume were assessed in a blinded manner. IA CEC-sEVs upon reperfusion significantly reduced infarct volume (24±5% vs 31±6% in saline, p<0.05) which was associated with robust improvement of neurological function from weeks 2 to 4 after tMCAO compared to saline treated rats. Moreover, rats treated with CEC-sEVs IV at 30min followed by IA treatment upon reperfusion further reduced infarct volume (16±6%) compared to rats treated with IA saline or CEC-sEVs. These rats also exhibited robust and early improvement of neurological function starting at 1d after tMCAO. In conclusion, IA administration of CEC-sEVs upon reperfusion significantly reduces ischemic damage, while an additional early (30min post stroke) dose of IV CEC-sEVs further enhances and accelerates the therapeutic effect of IA CEC-sEVs in the aged rats after tMCAO. Our data suggest that CEC-sEVs could potentially may be a therapy to amplify the efficacy of EVT even for elderly patients with acute ischemic stroke.

Temperature Dependence of Membrane Permeability Parameters for Five Cell Types Using Nonideal Thermodynamic Assumptions to Mathematically Model Cryopreservation Protocols.

Cryopreservation is the process of preserving biological matter at subzero temperatures for long-term storage. During cryopreservation, cells are susceptible to various injuries that can be mitigated by controlling the cooling and warming profiles and cryoprotective agent (CPA) addition and removal procedures. Mathematical modeling of the changing cell volume at different temperatures can greatly reduce the experiments needed to optimize cryopreservation protocols. Such mathematical modeling requires as inputs the cell membrane permeabilities to water and CPA and the osmotically inactive fraction of the cell. Since the intra- and extracellular solutions are generally thermodynamically nonideal, our group has been incorporating the osmotic virial equation to model the solution thermodynamics that underlie the cell volume change equations, adding the second and third osmotic virial coefficients of the grouped intracellular solute to the cell osmotic parameters that must be measured. In our previous work, we reported methods to obtain cell osmotic parameters at room temperature by fitting experimental cell volume kinetic data with equations that incorporated nonideal solution thermodynamics assumptions. Since the relevant cell volume excursions occur at different temperatures, the temperature dependence of the osmotic parameters plays an important role. In this work, we present a new two-part fitting method to obtain five cell-type-specific parameters (water permeability, dimethyl sulfoxide permeability, osmotically inactive fraction, and the second and third osmotic virial coefficients of the intracellular solution) from experimental measurements of equilibrium cell volume and cell volume as a function of time at room temperature and 0 °C for five cell types, namely, human umbilical vein endothelial cells (HUVECs), H9c2 rat myoblasts, porcine corneal endothelial cells (PCECs), the Jurkat T-lymphocyte cell line, and human cerebral microvascular endothelial cells (hCMECs/D3 cell line). The fitting method in this work is based on both equilibrium and kinetic cell volume data, enabling us to solve some technical challenges and expand our previously reported measurement technique to 0 °C. Finally, we use the measured parameters to model the cell volume changes for a HUVEC cryopreservation protocol to demonstrate the impact of the nonideal thermodynamic assumptions on predicting the changing cell volume during freezing and thawing.

Heparan Sulfate Modulation Affects Breast Cancer Cell Adhesion and Transmigration across In Vitro Blood-Brain Barrier.

The disruption of endothelial heparan sulfate (HS) is an early event in tumor cell metastasis across vascular barriers, and the reinforcement of endothelial HS reduces tumor cell adhesion to endothelium. Our recent study showed that while vascular endothelial growth factor (VEGF) greatly reduces HS at an in vitro blood-brain barrier (BBB) formed by human cerebral microvascular endothelial cells (hCMECs), it significantly enhances HS on a breast cancer cell, MDA-MB-231 (MB231). Here, we tested that this differential effect of VEGF on the HS favors MB231 adhesion and transmigration. We also tested if agents that enhance endothelial HS may affect the HS of MB231 and reduce its adhesion and transmigration. To test these hypotheses, we generated an in vitro BBB by culturing hCMECs on either a glass-bottom dish or a Transwell filter. We first quantified the HS of the BBB and MB231 after treatment with VEGF and endothelial HS-enhancing agents and then quantified the adhesion and transmigration of MB231 across the BBB after pretreatment with these agents. Our results demonstrated that the reduced/enhanced BBB HS and enhanced/reduced MB231 HS increase/decrease MB231 adhesion to and transmigration across the BBB. Our findings suggest a therapeutic intervention by targeting the HS-mediated breast cancer brain metastasis.

Deferoxamine reduces endothelial ferroptosis and protects cerebrovascular function after experimental traumatic brain injury

Cerebrovascular dysfunction resulting from traumatic brain injury (TBI) significantly contributes to poor patient outcomes. Recent studies revealed the involvement of iron metabolism in neuronal survival, yet its effect on vasculature remains unclear. This study aims to explore the impact of endothelial ferroptosis on cerebrovascular function in TBI. A Controlled Cortical Impact (CCI) model was established in mice, resulting in a significant increase in iron-related proteins such as TfR1, FPN1, and FTH, as well as oxidative stress biomarker 4HNE. This was accompanied by a decline in expression of the ferroptosis inhibitor GPX4. Moreover, Perls' staining and nonhemin iron content assay showed iron overload in brain microvascular endothelial cells (BMECs) and the ipsilateral cortex. Immunofluorescence staining revealed more FTH-positive cerebral endothelial cells, consistent with impaired perfusion vessel density and cerebral blood flow. As a specific iron chelator, deferoxamine (DFO) treatment inhibited such ferroptotic proteins expression and the accumulation of lipid‐reactive oxygen species following CCI, enhancing glutathione peroxidase (GPx) activity. DFO treatment significantly reduced iron deposition in BMECs and brain tissue, and increased density of the cerebral capillaries as well. Consequently, DFO treatment led to improvements in cerebral blood flow (as measured by laser speckle imaging) and behavioral performance (as measured by the neurological severity scores, rotarod test, and Morris water maze test). Taken together, our results indicated that TBI induces remarkable iron disorder and endothelial ferroptosis, and DFO treatment may help maintain iron homeostasis and protect vascular function. This may provide a novel therapeutic strategy to prevent cerebrovascular dysfunction following TBI.

Erythrocyte membrane-coated nanocarriers modified by TGN for Alzheimer's disease

Alzheimer's disease (AD) is an aging-related neurodegenerative disease, and the main pathological feature was β-amyloid protein (Aβ) deposition. Recently, bioactive materials-based drug delivery system has been widely investigated for the treatment of AD. In this study, we developed a red blood cells (RBC) membrane-coated polycaprolactone (PCL) nanoparticles (NPs) loading with a therapeutic agent for AD, curcumin (Cur). A functional peptide TGNYKALHPHN (TGN) was conjugated to the surface of membrane for blood-brain barrier (BBB) transport (TGN-RBC-NPs-Cur). TGN peptide can be recognized by receptors on the BBB and has great potential for brain transport. To confirm the targeted delivery of Cur to the brain, a cell co-culturing immortalized human cerebral microvascular endothelial cells and human brain astrocytes glioblastoma (hCMEC/D3 and U-118MG) in vitro model was established. As a result, the BBB transporting ratio of TGN-RBC-NPs-FITC was 29.64% at 12 h which was approximately eight-fold than RBC-NPs-FITC. The improvement of drug accumulation in the AD lesion was confirmed by the NPs modified with the BBB-penetrating peptide in the fluorescence imaging and quantitative analysis with UPLC-MS/MS in vivo. The neuroprotective effects were evaluated with new object recognition behavioral test, in vitro AD cell model, dendritic spine stain, GFAP and IBA1 immunofluorescence stain. The spatial learning and memory abilities of the AD model mice treated with TGN-RBC-NPs-Cur were obviously enhanced compared with the AD control mice and were also better than Cur at the same dosage. These results were consistent with the values of protection index of rat adrenal pheochromocytoma cells (PC12 cells) treated by Aβ25–35. TGN-RBC-NPs-Cur increased the dendritic segments densities and restrained activation of microglia and astrocytes of AD mice, as well as reversed cognitive function of AD mice. All of the results demonstrated TGN-RBC-NPs-Cur a promising therapeutic strategy for delaying the progression of AD by designing biomimetic nanosystems to deliver drugs into the brain.

Knockdown of FOXO4 protects against OGD/R‑induced cerebral microvascular endothelial cell injury and regulates the AMPK/Nrf2/HO‑1 pathway through transcriptional activation of CTRP6.

Cerebral ischemia is a type of cerebrovascular disease with high disability and mortality rates. The expression of forkhead box protein O4 (FOXO4) in the brain is increased following traumatic brain injury. To the best of our knowledge, however, the role of FOXO4 as well as its mechanism in cerebral ischemia has not been reported so far. For the establishment of an in vitro cellular injury model, human brain microvascular endothelial HCMEC/D3 cells were induced by oxygen-glucose deprivation/reoxygenation (OGD/R). mRNA and protein expressions of FOXO4 and C1q/tumor necrosis factor-related protein 6 (CTRP6) in OGD/R-induced HCMEC/D3 cells were detected by reverse transcription-quantitative (RT-q)PCR and western blotting. The transfection efficacy of small interfering (si)- and overexpression (Ov)-FOXO4 and si-CTRP6 was assessed using RT-qPCR and western blotting. Cell Counting Kit-8 and TUNEL assay were used to assess viability and apoptosis of HCMEC/D3 cells induced by OGD/R, respectively. A FITC-Dextran assay kit was applied to determine endothelial permeability and immunofluorescence assay was used for the measurement of the tight junction protein zonula occludens-1. The levels of oxidative stress markers and inflammatory cytokines were assessed with corresponding assay kits. The binding sites of transcription factor, FOXO4 and CTRP6 promoter were predicted using HDOCK SERVER. Luciferase reporter assay was used to detect the activity of the CTRP6 promoter while chromatin immunoprecipitation assay was used to evaluate the binding ability of the FOXO4 and CTRP6 promoter. Western blotting was used for the detection of apoptosis- and AMPK/Nrf2/heme oxygenase-1 (HO-1) pathway-associated proteins, along with tight junction proteins. The expression of FOXO4 was increased in OGD/R-induced HCMEC/D3 cells. After interfering with FOXO4 in cells, the viability of the OGD/R-induced HCMEC/D3 cells was increased while apoptosis was decreased. Furthermore, FOXO4 interference improved cellular barrier dysfunction but inhibited oxidative stress and the inflammatory response in HCMEC/D3 cells induced by OGD/R. FOXO4 knockdown regulated CTRP6 transcription in HCMEC/D3 cells. Knockdown of FOXO4 regulated expression of CTRP6 and protected OGD/R-induced HCMEC/D3 cell injury via the AMPK/Nrf2/HO-1 pathway. The present study indicated that FOXO4 knockdown activated CTRP6 to protect against cerebral microvascular endothelial cell injury induced by OGD/R via the AMPK/Nrf2/HO-1 pathway.

Impaired cerebral microvascular endothelial cells integrity due to elevated dopamine in myasthenic model

Myasthenia gravis is an autoimmune disease characterized by pathogenic antibodies that target structures of the neuromuscular junction. However, some patients also experience autonomic dysfunction, anxiety, depression, and other neurological symptoms, suggesting the complex nature of the neurological manifestations. With the aim of explaining the symptoms related to the central nervous system, we utilized a rat model to investigate the impact of dopamine signaling in the central nervous and peripheral circulation. We adopted several screening methods, including western blot, quantitative PCR, mass spectrum technique, immunohistochemistry, immunofluorescence staining, and flow cytometry. In this study, we observed increased and activated dopamine signaling in both the central nervous system and peripheral circulation of myasthenia gravis rats. Furthermore, changes in the expression of two key molecules, Claudin5 and CD31, in endothelial cells of the blood–brain barrier were also examined in these rats. We also confirmed that dopamine incubation reduced the expression of ZO1, Claudin5, and CD31 in endothelial cells by inhibiting the Wnt/β-catenin signaling pathway. Overall, this study provides novel evidence suggesting that pathologically elevated dopamine in both the central nervous and peripheral circulation of myasthenia gravis rats impair brain–blood barrier integrity by inhibiting junction protein expression in brain microvascular endothelial cells through the Wnt/β-catenin pathway.Graphical

Dynamic A-to-I RNA editing during acute neuroinflammation in sepsis-associated encephalopathy.

The activation of cerebral endothelial cells (CECs) has recently been reported to be the earliest acute neuroinflammation event in the CNS during sepsis-associated encephalopathy (SAE). Importantly, adenosine-to-inosine (A-to-I) RNA editing mediated by ADARs has been associated with SAE, yet its role in acute neuroinflammation in SAE remains unclear. Our current study systematically analyzed A-to-I RNA editing in cerebral vessels, cerebral endothelial cells (CECs), and microglia sampled during acute neuroinflammation after treatment in a lipopolysaccharide (LPS)-induced SAE mouse model. Our results showed dynamic A-to-I RNA editing activity changes in cerebral vessels during acute neuroinflammation. Differential A-to-I RNA editing (DRE) associated with acute neuroinflammation were identified in these tissue or cells, especially missense editing events such as S367G in antizyme inhibitor 1 (Azin1) and editing events in lincRNAs such as maternally expressed gene 3 (Meg3), AW112010, and macrophage M2 polarization regulator (Mm2pr). Importantly, geranylgeranyl diphosphate synthase 1 (Ggps1) and another three genes were differentially edited across cerebral vessels, CECs, and microglia. Notably, Spearman correlation analysis also revealed dramatic time-dependent DRE during acute neuroinflammation, especially in GTP cyclohydrolase1 (Gch1) and non-coding RNA activated by DNA damage (Norad), both with the editing level positively correlated with both post-LPS treatment time and edited gene expression in cerebral vessels and CECs. The findings in our current study demonstrate substantial A-to-I RNA editing changes during acute neuroinflammation in SAE, underlining its potential role in the disease.

Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P<0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P<0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P<0.05, P<0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P<0.05, P<0.01, P<0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

Identification of a Novel Mesenchymal Stem Cell–Related Signature for Predicting the Prognosis and Therapeutic Responses of Bladder Cancer

Background: Mesenchymal stem cells (MSCs) have been identified to have a unique migratory pattern toward tumor sites across diverse cancer types, playing a crucial role in cancer progression, treatment resistance, and immunosuppression. This study aims to formulate a prognostic model focused on MSC‐associated markers to efficiently predict the clinical outcomes and responses to therapy in individuals with bladder cancer (BC).Methods: Clinical and transcriptome profiling data were extracted from The Cancer Genome Atlas Urothelial Bladder Carcinoma (TCGA‐BLCA) and GSE31684 databases. Systematic quantification of MSC prevalences and stromal indices was undertaken, culminating in the discernment of genes correlated with stromal MSCs following a thorough application of weighted gene coexpression network analysis techniques. Subsequently, an exhaustive risk signature pertinent to MSC was formulated by amalgamating methods from univariate and Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression models. Drugs targeting genes associated with MSCs were screened using molecular docking.Results: The prognostic model for MSC incorporated five critical genes: ZNF165, matrix remodeling‐associated 7 (MXRA7), CEMIP, ADP‐ribosylation factor‐like 4C (ARL4C), and cerebral endothelial cell adhesion molecule (CERCAM). In the case of BC patients, stratification was performed into discrete risk categories, utilizing the median MSC risk score as a criterion. It was striking that those classified within the high‐MSC‐risk bracket demonstrated correlations with unfavorable prognostic implications. Enhanced responsiveness to immunotherapy in low‐MSC‐risk patients was delineated compared to their high‐MSC‐risk counterparts. A heightened receptivity was noted toward particular chemotherapy drugs, encompassing gemcitabine, vincristine, paclitaxel, gefitinib, and sorafenib, within this high‐risk group. Conversely, a superior reaction to cisplatin was distinctly evident among those marked by low MSC scores. The results of molecular docking demonstrated that kaempferol exhibited favorable docking with ZNF165, quercetin exhibited favorable docking with MXRA7, mairin exhibited favorable docking with CEMIP, and limonin diosphenol exhibited favorable docking with ARL4C.Conclusions: The five‐gene MSC prognostic model demonstrates substantial efficacy in prognosticating clinical outcomes and gauging responsiveness to chemotherapy and immunotherapy regimens. The genes ZNF165, MXRA7, CEMIP, ARL4C, and CERCAM are underscored as promising candidates warranting further exploration for anti‐MSC therapeutic strategies, thereby offering novel insights for personalized treatment approaches in BC.

Shexiang Tongxin dropping pills protect against ischemic stroke-induced cerebral microvascular dysfunction via suppressing TXNIP/NLRP3 signaling pathway

Ethnopharmacological relevancePatients with ischemic stroke (IS) often continue to exhibit cerebral microcirculatory dysfunction even after receiving thrombolytic therapy. Enhancing the function of cerebral microvascular endothelia represents a pivotal advancement in the therapeutic strategy for ischemic microcirculatory disturbances. A traditional Chinese medicinal formulation named Shexiang Tongxin Dropping Pills (STDP), has been clinically employed to ameliorate microcirculatory abnormalities. Existing literature attests to the beneficial role of STDP on endothelial cells (ECs). Nevertheless, specific impacts and underlying mechanisms of STDP in rectifying IS-induced cerebral microvascular dysfunction warrant further exploration. Aim of the studyThis investigation seeks to delineate the effects of STDP on cerebral microvascular endothelial damage induced by ischemic stroke and to elucidate the underlying mechanism involved. Materials and methodsMiddle cerebral artery occlusion and reperfusion (MCAO/R) technique was employed to established ischemic stroke model in mice. The therapeutic efficacy of STDP on cerebral microvascular function was assessed through laser speckle contrast imaging, behavioral assays, and histological evaluations. Biochemical markers in the brain tissue, including GSH, SOD, MDA, and ROS, were quantified using specific assay kits. In vitro study, oxygen-glucose deprivation and reperfusion (OGD/R) was performed in bEnd.3 cells. The cytoprotective potential of STDP was then evaluated by measuring cell viability, LDH activity, endothelial permeability, and oxidative stress parameters. Important targets in critical pathway were verified by immunoblotting and immunofluorescence both in mice brain slices and bEnd.3 cells. ResultsSTDP decrease brain infarct size, repaired microvascular cerebral blood flow and attenuated neurological deficiency in MCAO/R mice. Moreover, STDP abolished MCAO/R-induced oxidative stress which was reflected by rescuing GSH content, restoration of SOD activity and T-AOC, reduction of MDA and ROS. Ex vivo, STDP increased cerebral microvascular endothelial cells viability, abolished oxidative stress and decreased their permeability after ODG/R. Mechanistically, STDP significantly suppressed endothelial ROS-TXNIP mediated the activation of NLRP3 inflammasome in vivo and in vitro. ConclusionSTDP improves ischemic stroke-induced cerebral microcirculatory deficits by regulating cerebral microvascular endothelial ROS/TXNIP/NLRP3 signaling pathway.

P-Glycoprotein Exacerbates Brain Injury Following Experimental Cerebral Ischemia by Promoting Proinflammatory Microglia Activation.

Microglia are activated following cerebral ischemic insult. P-glycoprotein (P-gp) is an efflux transporter on microvascular endothelial cells and upregulated after cerebral ischemia. This study evaluated the effects and possible mechanisms of P-gp on microglial polarization/activation in mice after ischemic stroke. P-gp-specific siRNA and adeno-associated virus (p-AAV) were used to silence and overexpress P-gp, respectively. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were performed in mice and cerebral microvascular endothelial cells (bEnd.3) in vitro, respectively. OGD/R-injured bEnd.3 cells were cocultured with mouse microglial cells (BV2) in Transwell. Influences on acute ischemic stroke outcome, the expression of inflammatory cytokines, and chemokines and chemokines receptors, microglial polarization, glucocorticoid receptor (GR) nuclear translocation, and GR-mediated mRNA decay (GMD) activation were evaluated via reverse transcription real-time polymerase chain reaction, western blot, or immunofluorescence. Silencing P-gp markedly alleviated experimental ischemia injury as indicated by reduced cerebral infarct size, improved neurological deficits, and reduced the expression of interleukin-6 (IL-6) and IL-12 expression. Silencing P-gp also mitigated proinflammatory microglial polarization and the expression of C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 expression, whereas promoted anti-inflammatory microglia polarization. Additionally, P-gp silencing promoted GR nuclear translocation and the expression of GMD relative proteins in endothelial cells. Conversely, overexpressing P-gp via p-AAV transfection offset all these effects. Furthermore, silencing endothelial GR counteracted all effects mediated by silencing or overexpressing P-gp. Elevated P-gp expression aggravated inflammatory response and brain damage after ischemic stroke by augmenting proinflammatory microglial polarization in association with increased endothelial CCL2 release due to GMD inhibition by P-gp.

Self-Assembled nanoparticles of natural bioactive molecules enhance the delivery and efficacy of paclitaxel in glioblastoma.

Glioblastoma (GBM) is the most common primary malignant tumor in the central nervous system. Paclitaxel (PTX) is a well-established and highly effective anti-cancer drug for peripheral solid tumors. However, the application of PTX in GBM is hindered by several limitations, including poor water solubility, restricted entry across the blood-brain barrier (BBB), and enhanced excretion by efflux transporters. P-glycoprotein (P-gp) is a crucial efflux transporter that is abundantly present in cerebral vascular endothelial cells and GBM cells. It plays a significant role in the exocytosis of PTX within tumor tissues. Recently, we have developed a novel technique for creating self-assembled nanoparticles utilizing a range of natural bioactive molecules. These nanoparticles can encapsulate insoluble drugs and effectively cross the BBB. In additional, we revealed that certain nanoparticles have the potential to act as P-gp inhibitors, thereby reducing the excretion of PTX. In this study, we conducted a screening of bioactive molecular nanoparticles to identify those that effectively inhibit the function of P-gp transporters. Among the candidates, we identified ursolic acid nanoparticles (UA NPs) as the P-gp inhibitors. Furthermore, we prepared co-assembled UA NPs embedded with paclitaxel, referred to as UA-PTX NPs. Our results demonstrate that UA-PTX NPs can enhance the blood concentration of PTX, facilitate its entry into the BBB, and inhibit the function of P-gp, resulting in a decrease in the excretion of PTX. This discovery effectively addressed the above three issues associated with the use of PTX in glioma treatment. UA-PTX NPs demonstrate strong anti-tumor effects and show great potential for treating GBM.