Cultured Brain Microvascular Endothelial Cells Research Articles

Subarachnoid hemorrhage-associated brain injury and neurobehavioral deficits are reversed with synthetic adropin treatment through sustained Ser1179 phosphorylation of endothelial nitric oxide synthase

BackgroundSubarachnoid hemorrhage (SAH) is a life-threatening vascular condition without satisfactory treatment options. The secreted peptide adropin is highly expressed in the human brain and has neuroprotective effects in brain injury models, including actions involving the cerebrovasculature. Here, we report an endothelial nitric oxide synthase (eNOS)-dependent effect of synthetic adropin treatment that reverses the deleterious effects of SAH.MethodsWe tested the molecular, cellular, and physiological responses of cultured brain microvascular endothelial cells and two mouse models of SAH to treatment using synthetic adropin peptide or vehicle.ResultsSAH decreases adropin expression in cultured brain microvascular endothelial cells and in murine brain tissue. In two validated mouse SAH models, synthetic adropin reduced cerebral edema, preserved tight junction protein expression, and abolished microthrombosis at 1 day post-SAH. Adropin treatment also prevented delayed cerebral vasospasm, decreased neuronal apoptosis, and reduced sensorimotor deficits at seven days post-SAH. Delaying initial treatment of adropin until 24 h post-SAH preserved the beneficial effect of adropin in preventing vasospasm and sensorimotor deficits. Mechanistically, adropin treatment increased eNOS phosphorylation (Ser1179) at 1 & 7 days post-SAH. Treating eNOS−/− mice with adropin failed to prevent vasospasm or behavioral deficits, indicating a requirement of eNOS signaling.ConclusionsAdropin is an effective treatment for SAH, reducing cerebrovascular injury in both the acute (1 day) and delayed (7 days) phases. These findings establish the potential of adropin or adropin mimetics to improve outcomes following subarachnoid hemorrhage.

Catalpol Alleviates Ischemic Stroke Through Promoting Angiogenesis and Facilitating Proliferation and Differentiation of Neural Stem Cells via the VEGF-A/KDR Pathway.

Stroke is one of the leading causes of disability and death globally with a lack of effective therapeutic strategies. Catalpol is a bioactive compound derived from the traditional Chinese medicine Rehmannia glutinosa and it has been shown to be protective against various neurological diseases. The potential roles of catalpol against ischemic stroke are still not completely clear. In this study, we examined the effect and mechanism of catalpol against ischemic stroke using in vivo rat distal middle cerebral artery occlusion (dMCAO) and in vitro oxygen-glucose deprivation (OGD) models. We demonstrated that catalpol indeed attenuated the neurological deficits caused by dMCAO and improved neurological function. Catalpol remarkably promoted angiogenesis, promoted proliferation and differentiation of neural stem cells (NSCs) in the subventricular zone (SVZ), and prevented neuronal loss and astrocyte activation in the ischemic cortex or hippocampal dentate gyrus (DG) in vivo. The vascular endothelial growth factor receptor 2 (KDR, VEGFR-2) inhibitor SU5416 and VEGF-A shRNA were used to investigate the underlying mechanisms. The results showed that SU5416 administration or VEGF-A-shRNA transfection both attenuated the effects of catalpol. We also found that catalpol promoted the proliferation of cultured brain microvascular endothelial cells (BMECs) and the proliferation and differentiation of NSCs subjected to OGD insult in vitro. Interestingly, the impact of catalpol on cultured cells was also inhibited by SU5416. Moreover, catalpol was shown to protect NSCs against OGD indirectly by promoting BMEC proliferation in the co-cultured system. Taken together, catalpol showed therapeutic potential in cerebral ischemia by promoting angiogenesis and NSC proliferation and differentiation. The protective effects of catalpol were mediated through VEGF-A/KDR pathway activation.

Methods to Study Drug Uptake at the Blood-Brain Barrier Following Experimental Ischemic Stroke: In Vitro and In Vivo Approaches.

Drug permeability across the blood-brain barrier (BBB) is an important concept in the development of therapeutic strategies to treat neurological diseases such as ischemic stroke. These mechanisms can be evaluated in detailusing cultured brain microvascular endothelial cells or intact animals subjected to experimental stroke. Here, we describe state-of-the-art approaches to study BBB transport of therapeutics using our in vitro and in vivo approaches. These methodologies allow for precise determination of transporter kinetic properties for currently marketed therapeutics or fornew chemical entities that areunder development as stroke drugs.

Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice.

The neurovascular unit (NVU) is a multicellular structure comprising of neurons, glial cells, and non-neural cells, and it is supported by a specialized extracellular matrix, the basal lamina. Astrocytes, brain microvascular endothelial cells (BMECs), pericytes, and smooth muscle cells constitute the blood–brain barrier (BBB). BMECs have a mesodermal origin and invade the nervous system early in neural tube development, forming the BBB anatomical core. BMECs are connected by adherent junction complexes composed of integral membrane and cytoplasmic proteins. In vivo and in vitro studies have shown that, given the proximity and relationship with neural cells, BMECs acquire a unique gene expression profile, proteome, and specific mechanical and physical properties compared to endothelial cells from the general vasculature. BMECs are fundamental in maintaining brain homeostasis by regulating transcellular and paracellular transport of fluids, molecules, and cells. Therefore, it is essential to gain in-depth knowledge of the dynamic cellular structure of the cells in the NVU and their interactions with health and disease. Here we describe a significantly improved and simplified protocol using C57BL/6 newborn mice at postnatal day 1 (PND1) to isolate, purify, and culture BMECs monolayers in two different substrates (glass coverslips and transwell culture inserts). In vitro characterization and validation of the BMEC primary culture monolayers seeded on glass or insert included light microscopy, immunolabeling, and gene expression profile. Transendothelial electrical resistance (TEER) measurement and diffusion test were used as functional assays for adherent junction complexes and integrity and permeability of BMECs monolayers. The protocol presented here for the isolation and culture of BMECs is more straightforward than previously published protocols and yields a high number of purified cells. Finally, we tested BMECs function using the oxygen–glucose deprivation (OGD) model of hypoxia. This protocol may be suitable as a bioscaffold for secondary cell seeding allowing the study and better understanding of the NVU.

Transfer of neuron-derived α-synuclein to astrocytes induces neuroinflammation and blood–brain barrier damage after methamphetamine exposure: Involving the regulation of nuclear receptor-associated protein 1

The α-synuclein (α-syn) is involved in methamphetamine (METH)-induced neurotoxicity. Neurons can transfer excessive α-syn to neighboring neurons and glial cells. The effects of α-syn aggregation in astrocytes after METH exposure on the blood–brain barrier (BBB) remains unclear. Our previous study demonstrated that nuclear receptor-related protein 1 (Nurr1), a member of the nuclear receptor family widely expressed in the brain, was involved in the process of METH-induced α-syn accumulated in astrocytes to activate neuroinflammation. The role Nurr1 plays in astrocyte-mediated neuroinflammation, which results in BBB injury induced by METH, remains uncertain. This study found that METH up-regulated α-syn expression in neurons extended to astrocytes, thereby eliciting astrocyte activation, increasing and decreasing IL-1β, IL-6, TNF-α, and GDNF levels by down-regulating Nurr1 expression, and ultimately damaging the BBB. Specifically, the permeability of BBB to Evans blue and sodium fluorescein (NaF) increased; IgG deposits in the brain parenchyma increased; the Claudin5, Occludin, and PDGFRβ levels decreased. Several ultrastructural pathological changes occurred in the BBB, such as abnormal cerebral microvascular diameter, astrocyte end-foot swelling, decreased pericyte coverage, and loss of tight junctions. However, knockout or inhibition of α-syn or astrocyte-specific overexpression of Nurr1 partially alleviated these symptoms and BBB injury. Moreover, the in vitro experiments confirmed that METH increased α-syn level in the primary cultured neurons, which could be further transferred to primary cultured astrocytes, resulting in decreased Nurr1 levels. The decreased Nurr1 levels mediated the increase of IL-1β, IL-6, and TNF-α, and the decrease of GDNF, thereby changing the permeability to NaF, transendothelial electrical resistance, and Claudin5 and Occludin levels of primary cultured brain microvascular endothelial cells. Based on our findings, we proposed a new mechanism to elucidate METH-induced BBB injury and presented α-syn and Nurr1 as promising drug intervention targets to reduce BBB injury and resulting neurotoxicity in METH abusers.

Insulin-Like Growth Factor-1 Differentially Modulates Glutamate-Induced Toxicity and Stress in Cells of the Neurogliovascular Unit.

The age-related reduction in circulating levels of insulin-like growth factor-1 (IGF-1) is associated with increased risk of stroke and neurodegenerative diseases in advanced age. Numerous reports highlight behavioral and physiological deficits in blood-brain barrier function and neurovascular communication when IGF-1 levels are low. Administration of exogenous IGF-1 reduces the extent of tissue damage and sensorimotor deficits in animal models of ischemic stroke, highlighting the critical role of IGF-1 as a regulator of neurovascular health. The beneficial effects of IGF-1 in the nervous system are often attributed to direct actions on neurons; however, glial cells and the cerebrovasculature are also modulated by IGF-1, and systemic reductions in circulating IGF-1 likely influence the viability and function of the entire neuro-glio-vascular unit. We recently observed that reduced IGF-1 led to impaired glutamate handling in astrocytes. Considering glutamate excitotoxicity is one of the main drivers of neurodegeneration following ischemic stroke, the age-related loss of IGF-1 may also compromise neural function indirectly by altering the function of supporting glia and vasculature. In this study, we assess and compare the effects of IGF-1 signaling on glutamate-induced toxicity and reactive oxygen species (ROS)-produced oxidative stress in primary neuron, astrocyte, and brain microvascular endothelial cell cultures. Our findings verify that neurons are highly susceptible to excitotoxicity, in comparison to astrocytes or endothelial cells, and that a prolonged reduction in IGFR activation increases the extent of toxicity. Moreover, prolonged IGFR inhibition increased the susceptibility of astrocytes to glutamate-induced toxicity and lessened their ability to protect neurons from excitotoxicity. Thus, IGF-1 promotes neuronal survival by acting directly on neurons and indirectly on astrocytes. Despite increased resistance to excitotoxic death, both astrocytes and cerebrovascular endothelial cells exhibit acute increases in glutamate-induced ROS production and mitochondrial dysfunction when IGFR is inhibited at the time of glutamate stimulation. Together these data highlight that each cell type within the neuro-glio-vascular unit differentially responds to stress when IGF-1 signaling was impaired. Therefore, the reductions in circulating IGF-1 observed in advanced age are likely detrimental to the health and function of the entire neuro-glio-vascular unit.

Combined Analysis of Surface Protein Profile and microRNA Expression Profile of Exosomes Derived from Brain Microvascular Endothelial Cells in Early Cerebral Ischemia.

Endothelial cell damage is an important pathological basis for the deterioration of acute ischemia stroke. Our previous studies have been exploring the mechanism of blood–brain barrier (BBB) endothelial cell injury in the early stage of cerebral ischemia. Exosomes act as an important intercellular player in neurovascular communication. However, the characteristic of exosomes derived from BBB endothelial cells in early ischemic stroke is poorly understood. We exposed cultured brain microvascular endothelial cells (bEnd.3) to 3 h oxygen glucose deprivation (OGD) to mimic early cerebral ischemia in vitro and compared miRome and surface protein contents of exosomes derived from bEnd.3 cells by miRNA sequencing and the proximity barcoding assay (PBA). A total of 346 differentially miRNA (159 upregulated and 187 downregulated) were identified via miRNA-Seq in bEnd.3 cells after exposure to OGD for 3 h. Moreover, Gene Ontology (GO) and KEGG pathway analyses showed that cell proliferation- and angiogenesis-associated miRNAs were significantly affected. The abnormal changes in top eight miRNAs were further verified by a quantitative polymerase chain reaction (qPCR). PBA experiments showed that the numbers of exosomes carrying the following proteins increased significantly under ischemia, including bFGF, CD146, EPHA2, ABCB5, and ITGB2. These proteins were related to angiogenesis, cell proliferation, and cell inflammation. The network analysis combining PBA data with miRNA-Seq data showed that 79 miRNAs were related to 24 membrane proteins and predicted that there were surface proteins associated with a variety of miRNA molecules, such as ITGA9, XIAP, ADAM1, ITGA2, ITGA3, PDPN, and ITGB1. Meanwhile, there were miRNAs related to various surface proteins including miR-410-3p, miR-378b, and miR-1960. Taken together, our data demonstrated for the first time the changes of exosomal miRNAs and surface protein profiles derived from ischemic microvascular endothelial cells, which may provide new therapeutic targets for BBB protection in ischemic stroke.

ErbB3 is a critical regulator of cytoskeletal dynamics in brain microvascular endothelial cells: Implications for vascular remodeling and blood brain barrier modulation

Neuregulin (NRG)1 - ErbB receptor signaling has been shown to play an important role in the biological function of peripheral microvascular endothelial cells. However, little is known about how NRG1/ErbB signaling impacts brain endothelial function and blood-brain barrier (BBB) properties. NRG1/ErbB pathways are affected by brain injury; when brain trauma was induced in mice in a controlled cortical impact model, endothelial ErbB3 gene expression was reduced to a greater extent than that of other NRG1 receptors. This finding suggests that ErbB3-mediated processes may be significantly compromised after injury, and that an understanding of ErbB3 function would be important in the of study of endothelial biology in the healthy and injured brain. Towards this goal, cultured brain microvascular endothelial cells were transfected with siRNA to ErbB3, resulting in alterations in F-actin organization and microtubule assembly, cell morphology, migration and angiogenic processes. Importantly, a significant increase in barrier permeability was observed when ErbB3 was downregulated, suggesting ErbB3 involvement in BBB regulation. Overall, these results indicate that neuregulin-1/ErbB3 signaling is intricately connected with the cytoskeletal processes of the brain endothelium and contributes to morphological and angiogenic changes as well as to BBB integrity.

Occludin degradation makes brain microvascular endothelial cells more vulnerable to reperfusion injury in vitro.

Intracerebral hemorrhage is the most dangerous complication in tPA thrombolytic therapy for ischemic stroke, which occurs as a consequence of endothelial cell death at the blood-brain barrier (BBB) during thrombolytic reperfusion. We have previously shown that cerebral ischemia-induced rapid occludin degradation and BBB disruption. Here we demonstrated an important role of occludin degradation in facilitating the evolution of ischemic endothelial cells toward death. Cultured brain microvascular endothelial cells (bEnd.3 cells) were exposed to oxygen-glucose deprivation (OGD) or incubated with occludin siRNA or occludin AAV to achieve an occludin deficiency or over-expression status before exposing to reoxygenation (R) or TNF-α treatment. Cell death was assessed by measuring lactate dehydrogenase release, TUNEL staining, and flow cytometry analysis. Inhibition of OGD-induced occludin degradation with SB-3CT or over-expression of occludin with occludin AAV both significantly attenuated OGD/R-induced apoptosis and pyroptosis in bEnd.3 cells. Consistently, knockdown of occludin with siRNA potentiated TNF-α-induced apoptosis, supporting an important role of occludin integrity in endothelial cell survival. Similar results were observed for pyroptosis, in which occludin knockdown with siRNA led to a significant augmentation of cytokines secretion, inflammasome activation, and pyroptosis occurrence in TNF-α-treated bEnd.3 cells. Lastly, up-regulation of c-Yes, PI3K/AKT, and ERK concurrently occurred with occludin degradation after OGD/R or TNF-α treatment, and the level of these proteins were further increased when inhibition of occludin degradation or over-expression of occludin. These data indicate that occludin degradation inflicted during ischemia makes BBB endothelial cells more vulnerable to reperfusion-associated stress stimuli.

Hydrogen improves cell viability partly through inhibition of autophagy and activation of PI3K/Akt/GSK3β signal pathway in a microvascular endothelial cell model of traumatic brain injury

ABSTRACTObjective：Traumatic brain injury (TBI) is one of the most serious public health problems in the world. Hydrogen (H2), a flammable, colorless, and odorless gas, has been observed to have preventive and therapeutic effects on brain trauma and other neurological disorders, but its exact mechanism has not been fully clarified.Methods: To further study the mechanism underlying the role of hydrogen gas in alleviating BBB damage after TBI, we performed the scratch injury model on cultured brain microvascular endothelial cells (bEnd.3), which formed the microvascular endothelial barrier – an integral part of the highly specialized BBB.Results: In the case of TBI, hydrogen was able to improve the decline of cell viability induced by TBI. More importantly, inhibition of PI3 K/Akt/GSK3β signal pathway or activation of autophagy reduced the protective effect of hydrogen on cell viability, indicating that such protective effect was regulated by PI3 K/Akt/GSK3β signal pathway and was related to the inhibition of autophagy.Conclusion: So we concluded that hydrogen improved the cell viability in a microvascular endothelial cell model of TBI partly through inhibition of autophagy, and inhibitory effect of hydrogen on autophagy was exerted by activating PI3 K/Akt/GSK3β signal pathway. These findings enriched our knowledge about the mechanism of hydrogen therapy against TBI.

Endothelium-Targeted Deletion of microRNA-15a/16-1 Promotes Poststroke Angiogenesis and Improves Long-Term Neurological Recovery.

Angiogenesis promotes neurological recovery after stroke and is associated with longer survival of stroke patients. Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/16-1 cluster, among others. However, the function of the miR-15a/16-1 cluster in endothelium on postischemic cerebral angiogenesis is not known. To investigate the functional significance and molecular mechanism of endothelial miR-15a/16-1 cluster on angiogenesis in the ischemic brain. Endothelial cell-selective miR-15a/16-1 conditional knockout (EC-miR-15a/16-1 cKO) mice and wild-type littermate controls were subjected to 1 hour middle cerebral artery occlusion followed by 28-day reperfusion. Deletion of miR-15a/16-1 cluster in endothelium attenuates post-stroke brain infarction and atrophy and improves the long-term sensorimotor and cognitive recovery against ischemic stroke. Endothelium-targeted deletion of the miR-15a/16-1 cluster also enhances post-stroke angiogenesis by promoting vascular remodeling and stimulating the generation of newly formed functional vessels, and increases the ipsilateral cerebral blood flow. Endothelial cell-selective deletion of the miR-15a/16-1 cluster up-regulated the protein expression of pro-angiogenic factors VEGFA (vascular endothelial growth factor), FGF2 (fibroblast growth factor 2), and their receptors VEGFR2 (vascular endothelial growth factor receptor 2) and FGFR1 (fibroblast growth factor receptor 1) after ischemic stroke. Consistently, lentiviral knockdown of the miR-15a/16-1 cluster in primary mouse or human brain microvascular endothelial cell cultures enhanced in vitro angiogenesis and up-regulated pro-angiogenic proteins expression after oxygen-glucose deprivation, whereas lentiviral overexpression of the miR-15a/16-1 cluster suppressed in vitro angiogenesis and down-regulated pro-angiogenic proteins expression. Mechanistically, miR-15a/16-1 translationally represses pro-angiogenic factors VEGFA, FGF2, and their receptors VEGFR2 and FGFR1, respectively, by directly binding to the complementary sequences within 3'-untranslated regions of those messenger RNAs. Endothelial miR-15a/16-1 cluster is a negative regulator for postischemic cerebral angiogenesis and long-term neurological recovery. Inhibition of miR-15a/16-1 function in cerebrovascular endothelium may be a legitimate therapeutic approach for stroke recovery.

Transcriptome clarifies mechanisms of lesion genesis versus progression in models of Ccm3 cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are dilated capillaries causing epilepsy and stroke. Inheritance of a heterozygous mutation in CCM3/PDCD10 is responsible for the most aggressive familial form of the disease. Here we studied the differences and commonalities between the transcriptomes of microdissected lesional neurovascular units (NVUs) from acute and chronic in vivo Ccm3/Pdcd10ECKO mice, and cultured brain microvascular endothelial cells (BMECs) Ccm3/Pdcd10ECKO.We identified 2409 differentially expressed genes (DEGs) in acute and 2962 in chronic in vivo NVUs compared to microdissected brain capillaries, as well as 121 in in vitro BMECs with and without Ccm3/Pdcd10 loss (fold change ≥ |2.0|; p < 0.05, false discovery rate corrected). A functional clustered dendrogram generated using the Euclidean distance showed that the DEGs identified only in acute in vivo NVUs were clustered in cellular proliferation gene ontology functions. The DEGs only identified in chronic in vivo NVUs were clustered in inflammation and immune response, permeability, and adhesion functions. In addition, 1225 DEGs were only identified in the in vivo NVUs but not in vitro BMECs, and these clustered within neuronal and glial functions. One miRNA mmu-miR-3472a was differentially expressed (FC = − 5.98; p = 0.07, FDR corrected) in the serum of Ccm3/Pdcd10+/− when compared to wild type mice, and this was functionally related as a putative target to Cand2 (cullin associated and neddylation dissociated 2), a DEG in acute and chronic lesional NVUs and in vitro BMECs. Our results suggest that the acute model is characterized by cell proliferation, while the chronic model showed inflammatory, adhesion and permeability processes. In addition, we highlight the importance of extra-endothelial structures in CCM disease, and potential role of circulating miRNAs as biomarkers of disease, interacting with DEGs. The extensive DEGs library of each model will serve as a validation tool for potential mechanistic, biomarker, and therapeutic targets.

Longitudinal examination of mitochondrial dynamics in vivo in cerebral vascular endothelial cells

Mitochondria are important regulators of vascular function and provide resiliency against stress and disease but in vivo mitochondrial dynamics in the cerebral circulation are unexplored. To remedy this deficit we used multi‐photon microscopy, permanent mitochondrial labeling using mitochondrial Dendra2 fluorescent protein (mitoDendra2 FP) specific to the endothelium, and intravascular Rhodamine dextran to visualize the microcirculation in anesthetized mice. We determined optimal imaging conditions, validated the specificity of mitochondrial labeling in endothelium, developed approaches to data analysis, and explored usefulness of this approach in preliminary studies.Cranial windows were implanted in adult mice and the brain microcirculation and mitoDendra2 FP characteristics were examined in the same animal starting at 5 mo of age and at 2, 4, and 12 wks (Groups I–IV). Stacks of 100 consecutive images were taken at 1 μm increments. Immunohistochemistry and cultured brain microvascular cells confirmed the location of the mitochondrial signal in endothelium. Endothelial expression of mitoDendra2 FP was confirmed in vitro on brain slices using endothelium specific VWF and CD31 antibodies. In addition, we observed an overlapping mitoDendra2 FP and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Mitochondrial and vessel area (Ma; Va; respectively; expressed in μm2), their ratio (Ma/Va), and the mean vessel diameter [Vd; expressed in μm] were determined using ImageJ. Mitochondrial and vessel area, as well as the vessel diameters were similar among groups [Ma I: 3,157 ± 488.9*; Ma II: 2,556 ± 512.9; Ma III: 2,174 ± 368.8; Ma IV: 3,178 ± 627.9; n = 24–27/session; * p &lt;0.05 Ma I vs. II–IV; Va I: 16,138 ± 1,503; Va II: 16,627 ± 2,439; Va III: 14,634 ± 1,673; Va IV: 22,056 ± 2,940; n = 24–27/session; Ma/Va I: 0.182 ± 0.013#; Ma/Va II: 0.138 ± 0.009; Ma/Va III: 0.144 ± 0.011; Ma/Va IV: 0.138 ± 0.015; n= 24–27/session; #p &lt;0.05 Ma/Va I vs. IV; Vd I: 5.00 ± 0.05; Vd II: 4.95 ± 0.07; Vd III: 4.84 ± 0.08; Vd IV: 5.00 ± 0.12; n = 8–9/session). In preliminary studies, we also found that exposure of the cerebral vasculature to 30–60 min of ischemia followed by reperfusion led to changes in mitochondrial dynamics including changes in appearance suggesting fragmentation. In addition, the mitoDendra2 FP signal was particularly pronounced in the vasculature of a juvenile mouse, possibly indicating the need for more mitochondrial‐derived ATP to promote blood vessel growth as the brain matures.Our results indicate that mito‐Dendra2 FP signal and vascular parameters were stable and suitable for longitudinal examination of the in vivo cerebral circulation in mice and that these methods are useful in examining the mechanisms of vascular development and remodeling and the responses of the cerebral microcirculation to disease processes.Support or Funding InformationAHA 17SDG33410366, U54 GM104940 and HL‐093554.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke.

Angiogenesis is a crucial defense response to hypoxia that regulates the process of raising the promise of long-term neurologic recovery during the management of stroke. A high expression of antiangiogenic factors leads to the loss of neovascularization capacity in pathologic conditions. We have previously documented an impairment of the cerebral vessel perfusion and neovascularization in the cortex neighboring the stroke-induced lesion, which was accompanied by an activation of semaphorin 3E (Sema3E)/PlexinD1 after ischemic stroke. In this study, we employed micro-optical sectioning tomography to fully investigate the details of the vascular pattern, including the capillaries. We found that after transient middle cerebral artery occlusion, inhibiting PlexinD1 signaling led to an organized recovery of the vascular network in the ischemic area. We then further explored the possible mechanisms. In vivo, Sema3E substantially decreased dynamic delta-like 4 (DLL4) expression. In cultured brain microvascular endothelial cells, Sema3E down-regulated DLL4 expression via inhibiting Ras-related C3 botulinum toxin substrate 1-induced JNK phosphorylation. At the microcosmic level, Sema3E/PlexinD1 signaling promoted F-actin disassembly and focal adhesion reduction by activating the small guanosine triphosphatase Ras homolog family member J by releasing RhoGEF Tuba from direct binding to PlexinD1, thus mediating endothelial cell motility and filopodia retraction. Our study reveals that Sema3E/PlexinD1 signaling, which suppressed endothelial DLL4 expression, cell motility, and filopodia formation, is expected to be a novel druggable target for angiogenesis during poststroke progression.-Zhou, Y.-F., Chen, A.-Q., Wu, J.-H., Mao, L., Xia, Y.-P., Jin, H.-J., He, Q.-W., Miao, Q. R., Yue, Z.-Y., Liu, X.-L., Huang, M., Li, Y.-N., Hu, B. Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke.

MiRNA-27b Regulates Angiogenesis by Targeting AMPK in Mouse Ischemic Stroke Model

Stroke is a leading cause of mortality and serious disability worldwide with limited treatment options. Angiogenesis has been reported to be involved in post-stroke recovery. Although the molecular mechanisms that regulate angiogenesis remain ambiguous, microRNAs have emerged as effective regulators of angiogenesis, involved in neurological function outcome. The present study aims to investigate the regulatory effects of miRNA-27b on post-stroke angiogenesis. In primary cultured brain microvascular endothelial cells (BMECs), the inhibition of miRNA-27b induced the activation of adenosine monophosphate-activated protein kinase (AMPK), which increased tube formation and migration. This action was attenuated when AMPKα2 was knocked down. Mice were subjected to middle cerebral artery occlusion (MCAo) surgery and administrated with Lentivirus miR-27b inhibitor. Enhanced angiogenesis in ischemic boundary zone (IBZ) was observed, and the neurological outcome during the entire study period was improved. The number of phosphate-AMPKα2+ cells that co-expressed endothelial cell marker CD31 was significantly increased. Taken together, the present study demonstrated that downregulated miRNA-27b promoted recovery after ischemic stroke via AMPK stimulus.

In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation.

Focussed radiosurgery may provide a means of inducing molecular changes on the luminal surface of diseased endothelium to allow targeted delivery of novel therapeutic compounds. We investigated the potential of ionizing radiation to induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (EC) in vitro and in vivo, to assess their suitability as vascular targets in irradiated arteriovenous malformations (AVMs). Cultured brain microvascular EC were irradiated by linear accelerator at single doses of 0, 5, 15 or 25 Gy and expression of ICAM-1 and VCAM-1 measured by qRT-PCR, Western, ELISA and immunocytochemistry. In vivo, near-infrared (NIR) fluorescence optical imaging using Xenolight 750-conjugated ICAM-1 or VCAM-1 antibodies examined luminal biodistribution over 84 days in a rat AVM model after Gamma Knife surgery at a single 15 Gy dose. ICAM-1 and VCAM-1 were minimally expressed on untreated EC in vitro. Doses of 15 and 25 Gy stimulated expression equally; 5 Gy was not different from the unirradiated. In vivo, normal vessels did not bind or retain the fluorescent probes, however binding was significant in AVM vessels. No additive increases in probe binding were found in response to radiosurgery at a dose of 15 Gy. In summary, radiation induces adhesion molecule expression in vitro but elevated baseline levels in AVM vessels precludes further induction in vivo. These molecules may be suitable targets in irradiated vessels without hemodynamic derangement, but not AVMs. These findings demonstrate the importance of using flow-modulated, pre-clinical animal models for validating candidate proteins for vascular targeting in irradiated AVMs.

Lipopolysaccharide‐Induced Activation of Bone Marrow‐Derived Macrophages Disrupts Brain Microvascular Endothelial Cell Function in a Mouse Model of Psychological Resilience

Psychological resilience, as measured by the ability of an individual to overcome the development of psychological disorders following exposure to stressful or traumatic events, varies significantly from individual to individual. Inflammation, and the breakdown of the blood‐brain barrier (BBB) are thought to be key factors in the development and progression of psychological disorders such as depression, schizophrenia, and post‐traumatic stress disorder. The activation of macrophages is associated with the disruption of barrier function of the BBB, and thus may play an important role in determining resilience to stress. It is unknown whether the macrophage phenotype and/or BBB phenotype of resilient individuals is significantly different from that of non‐resilient individuals, or whether these phenotypic differences and their interaction contribute to the degree of resilience in individuals. In this study, we tested the hypothesis that resilient individuals possess macrophage and blood brain barrier phenotypes that are more resistant to the activating effects of lipopolysaccharide (LPS), than non‐resilient individuals. We used Electric Cell‐substrate Impedance Signaling (ECIS) technology (Applied BioPhysics, Troy, NY) to measure transendothelial electrical resistance (TEER) of cultured brain microvascular endothelial cells of the resilient C57BL6 mouse (BMECC57BL6), and the non‐resilient BalbC mouse (BMECBalbC). BMECs were plated at a density of 4.8 × 104 cells/cm2, and allowed to establish a monolayer for 48 hours. BMECs were then treated with LPS derived from E. coli (0, 10, or 100 ng/ml). After 24 hours, bone marrow‐derived macrophages of C57BL6 mice (BMM C57BL6) or BalbC mice (BMM BalbC) (4.0 × 104 cells/cm2) were added to the BMEC monolayer. TEER was continuously recorded for an additional 72 hours. Barrier function (Rb), membrane capacitance (Cm), and constraint on current flow beneath the cells (α) were derived via ECIS mathematical modeling. At baseline, BMECC57BL6 had significantly lower Rb (1.90±0.26 versus 2.81±0.26 Ωcm2, p = 0.03, n = 7) and α (7.01±0.14 versus 8.01±0.16 Ω0.5cm, p = 0.0006, n =7), and significantly higher Cm (1.42±0.03 versus 1.22±0.02 μF/cm2, p = 0.0003, n = 7) when compared to BMECBalbC. LPS treatment alone or BMM alone did not have significantly differential effects on any of these parameters between BMECC57BL6 and BMECBalbC; however, in the presence of 100 ng/ml LPS, only α value of BMECBalbC significantly declined over the 6 hours after addition of BMM C57BL6 (from 7.50±0.211 to 6.71±0.221 Ω0.5cm; one‐way ANOVA, p = 0.02, n = 4), or BMM BalbC (from 7.54±0.17 to 6.16±0.17 Ω0.5cm; one‐way ANOVA, p &gt; 0.0001, n = 4). These findings indicate that variability in psychological resilience among individuals may be associated with functional differences in blood brain barrier and macrophages.Support or Funding InformationThe views expressed in this presentation are those of the authors and do not reflect the official policy or position of the Department of the Army, the Department of Defense, or the U.S. government.

Involvement of Nox2 and Nox4 NADPH oxidases in early brain injury after subarachnoid hemorrhage

Oxidative stress is responsible for a poor prognosis of subarachnoid hemorrhage (SAH) patients. Nox2 has been shown to participate in SAH-induced early brain injury (EBI). Nox4 is another major subtype of Nox family widely expressed in central nervous system (CNS). Here, we investigated the role of Nox4 and whether there was a synergistic effect of Nox2 and Nox4 in SAH-induced EBI. Clinical brain biopsies of four patients with traumatic brain injury (TBI) and perihematomal brain tissue from six subjects with SAH were examined. Gp91ds-tat (a specific inhibitor of Nox2), GKT137831 (a specific inhibitor of Nox4), and apocynin (a non-specific Nox inhibitor) were used to test the role of Nox2 and Nox4. The protein levels of Nox2 and Nox4 were elevated in rat neurons and astrocytes at 12 h after SAH, and in cultured brain microvascular endothelial cells at 24 h after exposure to OxyHb. Similarly, there were higher Nox2 and Nox4 protein levels in perihematomal neurons and astrocytes in SAH patients than that in brain tissue from subjects with TBI. In SAH rat model, gp91ds-tat and GKT137831 could reduce SAH-induced neuronal death and degeneration, whereas apocynin did not induce a more intense neuroprotection. Consistently, in in vitro SAH model, siRNA-mediated silencing of Nox2 and Nox4 suppressed the OxyHb-induced neuronal apoptosis, whereas Nox2 and Nox4 co-knockdown also did not show a remarkable overlay effect. In conclusion, Nox4 should contribute to the pathological processes in SAH-induced EBI, and there was not an overlay effect of Nox2 inhibition and Nox4 inhibition on preventing SAH-induced EBI.

Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

MiR-21-5p alleviates leakage of injured brain microvascular endothelial barrier in vitro through suppressing inflammation and apoptosis

Our recent researches have identified increased expression of miR-21-5p in rats brain following traumatic brain injury (TBI), which protected against blood-brain barrier (BBB) damage. To further study the mechanism underlying the role of miR-21-5p on alleviating BBB damage after TBI, we performed the scratch injury model on cultured brain microvascular endothelial cells (BMVECs), which formed the microvascular endothelial barrier – an integral part of the highly specialized BBB. The expression level of miR-21-5p in BMVECs was observed to be increased after scratch injury, and could be further up-regulated by transfecting miR-21-5p mimics. We found that up-regulation of miR-21-5p level in BMVECs can alleviate endothelial barrier damage and loss of tight junction proteins. To further investigate the mechanism of this protective effect, we evaluated the impact of miR-21-5p on inflammation and apoptosis in injured BMVECs. On one hand, miR-21-5p suppressed inflammation by regulating the expression of inflammatory cytokines and NF-kB signaling. On the other hand, miR-21-5p inhibited cellular apoptosis by regulating the expression of apoptosis factors and Akt signaling. In addition, we also detected the activity of Ang-1/Tie-2 axis (associated with BBB stabilization) in BMVECs after scratch injury, and found that miR-21-5p can promote its activation. Taken together, miR-21-5p alleviates leakage of injured brain microvascular endothelial barrier through suppressing inflammation and apoptosis, while impacting the activities of NF-kB, Akt and Ang-1/Tie-2 signaling. Thus, miR-21-5p could be a potential therapeutic target for interventions of BBB damage after TBI.