Rat Brain Microvascular Endothelial Cells Research Articles

DPP-4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high-glucose conditions, potentially through the SIRT1/HIF-1/VEGF pathway.

Vascular disease in diabetes, for example, stroke, presents a significant public health burden. Recently, the dipeptidyl peptidase 4 (DPP-4) inhibitor linagliptin has been found to counteract stroke among diabetic patients, showing great promise in drug repurposing and indication expansion. However, the molecular basis of this protection mechanism remains unknown. The expression and localization of DPP-4 in rat brain microvascular endothelial cells (rBMVECs) were assessed with immunofluorescent staining and Western blotting. The effects of DPP-4 inhibitors on cell proliferation and migration of rBMVECs were determined using MTT and transwell assays, separately. The influence of DPP-4 inhibition on the expression of molecular markers (eg, VEGF, eNOS, HIF-1α. SIRT1) was examined at both mRNA and protein levels with qRT-PCR and Western blotting, individually. DPP-4 inhibitors (40nmol/L linagliptin, 30μmol/L berberine) offer protection from hypoxia/high glucose induced impairments in the proliferation and migration of rBMVECs. Treatment with DPP-4 inhibitors counteracted the attenuating effects of hypoxic/high-glucose conditions on the expression of VEGF, eNOS, HIF-1α, and SIRT1, which can be completely eliminated by the inhibition of SIRT1 with 1mmol/L nicotinamide. The protection of rBMVECs from hypoxia/high-glucose induced impairment by DPP-4 inhibitors may be mediated by the SIRT1/HIF-1α/VEGF pathway.

Novel Hemoglobin-Based Oxygen Carrier Bound With Albumin Shows Neuroprotection With Possible Antioxidant Effects

Background and Purpose- A hemoglobin-albumin cluster, 1 core of hemoglobin covalently bound with 3 shell albumins, designated as HemoAct was developed as a hemoglobin-based oxygen carrier. We aim to investigate neuroprotection by HemoAct in transient cerebral ischemia and elucidate its underlying mechanisms. Methods- Male rats were subjected to 2-hour transient middle cerebral artery occlusion and were then administered HemoAct transarterially at the onset of reperfusion. Neurological and pathological findings were examined after 24 hours of reperfusion to identify neuroprotection by HemoAct. Intermittent measurements of cortical blood flow and oxygen content were performed, and a histopathologic analysis was conducted on rats during the early phase of reperfusion to assess the therapeutic mechanism of HemoAct. In addition, the antioxidant effects of HemoAct were examined in hypoxia/reoxygenation-treated rat brain microvascular endothelial cells. Results- Neurological deterioration, infarct and edema development, and the activation of MMP-9 (matrix metalloprotease-9) and lipid peroxidation after 24 hours of reperfusion were significantly ameliorated by the HemoAct treatment. Reductions in blood flow and tissue partial oxygen pressure in the cortical penumbra after 6 hours of reperfusion were significantly ameliorated by the HemoAct treatment. The histopathologic analysis of the cortical penumbra revealed that HemoAct in HemoAct-treated rats showed superior microvascular perfusion with the mitigation of microvascular narrowing changes than autologous erythrocytes in nontreated rats. Although HemoAct extravasated into the ischemic core with serum protein, it did not induce an increase in serum extravasation or reactive oxygen species production in the ischemic core. In vitro experiments with rat brain microvascular endothelial cells revealed that HemoAct significantly suppressed cellular reactive oxygen species production in hypoxia/reoxygenation-treated cells, similar to albumin. Conclusions- HemoAct exerted robust neuroprotection in transient cerebral ischemia. Superior microvascular perfusion with an oxygen delivery capability and possible antioxidant effects appear to be the underlying neuroprotective mechanisms.

Vitexin reduces epilepsy after hypoxic ischemia in the neonatal brain via inhibition of NKCC1

BackgroundNeonatal hypoxic-ischemic brain damage, characterized by tissue loss and neurologic dysfunction, is a leading cause of mortality and a devastating disease of the central nervous system. We have previously shown that vitexin has been attributed various medicinal properties and has been demonstrated to have neuroprotective roles in neonatal brain injury models. In the present study, we continued to reinforce and validate the basic understanding of vitexin (45 mg/kg) as a potential treatment for epilepsy and explored its possible underlying mechanisms.MethodsP7 Sprague-Dawley (SD) rats that underwent right common carotid artery ligation and rat brain microvascular endothelial cells (RBMECs) were used for the assessment of Na+-K+-Cl− co-transporter1 (NKCC1) expression, BBB permeability, cytokine expression, and neutrophil infiltration by western blot, q-PCR, flow cytometry (FCM), and immunofluorescence respectively. Furthermore, brain electrical activity in freely moving rats was recorded by electroencephalography (EEG).ResultsOur data showed that NKCC1 expression was attenuated in vitexin-treated rats compared to the expression in the HI group in vivo. Oxygen glucose deprivation/reoxygenation (OGD) was performed on RBMECs to explore the role of NKCC1 and F-actin in cytoskeleton formation with confocal microscopy, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide, and FCM. Concomitantly, treatment with vitexin effectively alleviated OGD-induced NKCC1 expression, which downregulated F-actin expression in RBMECs. In addition, vitexin significantly ameliorated BBB leakage and rescued the expression of tight junction-related protein ZO-1. Furthermore, inflammatory cytokine and neutrophil infiltration were concurrently and progressively downregulated with decreasing BBB permeability in rats. Vitexin also significantly suppressed brain electrical activity in neonatal rats.ConclusionsTaken together, these results confirmed that vitexin effectively alleviates epilepsy susceptibility through inhibition of inflammation along with improved BBB integrity. Our study provides a strong rationale for the further development of vitexin as a promising therapeutic candidate treatment for epilepsy in the immature brain.

Hydroxysafflor yellow a protects brain microvascular endothelial cells against oxygen glucose deprivation/reoxygenation injury: Involvement of inhibiting autophagy via class I PI3K/Akt/mTOR signaling pathway

The present study aimed to test whether Hydroxysafflor yellow A (HSYA) protects the brain microvascular endothelial cells (BMECs) injury induced by oxygen glucose deprivation/reoxygenation (OGD/R) via the PI3K/Akt/mTOR autophagy signaling pathway. Primary rat BMECs were cultured and identified by the expression of factor VIII-related antigen before being exposed to OGD/R to imitate ischemia/reperfusion (I/R) damage in vitro. The protective effect of HSYA was evaluated by assessing (1) cellular morphologic and ultrastructural changes; (2) cell viability and cytotoxicity; (3) transendothelial electrical resistance (TEER) of monolayer BMECs; (4) cell apoptosis; (5) fluorescence intensity of LC3B; (6) LC3 mRNA expression; (7) protein expressions of LC3, Beclin-1, Zonula occludens-1 (ZO-1), phospho-Akt (p-Akt), Akt, phospho-mTOR (p-mTOR) and mTOR. It was found that HSYA (20, 40, and 80 μM) and 3-MA effectively reversed the cellular morphological and ultrastructural changes, increased cell survival, normalized the permeability of BMECs, and suppressed apoptosis induced by OGD/R (2 h OGD followed by 24 h reoxygenation). Concurrently, HSYA and 3-MA also inhibited OGD/R-induced autophagy evidenced by the decreased number of autophagosomes and down-regulated levels of LC3 and Beclin-1 proteins and mRNAs. HSYA (80 μM), in combination with 3-MA showed a synergistic effect. Mechanistic studies revealed that HSYA (80 μM) markedly increased the levels of p-Akt and p-mTOR proteins. Blockade of PI3K activity by ZSTK474 abolished its anti-autophagic and pro-survival effect and lowered both Akt and mTOR phosphorylation levels. Taken together, these results suggest that HSYA protects BMECs against OGD/R-induced injury by inhibiting autophagy via the Class I PI3K/Akt/mTOR signaling pathway.

Brain Transport Profiles of Ginsenoside Rb1 by Glucose Transporter 1: In Vitro and in Vivo.

Ginsenoside Rb1 (Rb1) has been demonstrated its protection for central nervous system and is apparently highly distributed to the brain. The objective of this study was to characterize Rb1 transport at the blood–brain barrier (BBB) using primary cultured rat brain microvascular endothelial cells (rBMEC), an in vitro BBB model. The initial uptake velocity of Rb1 in rBMEC was temperature- and concentration-dependent, and was significantly reduced by phloretin, an inhibitor of GLUT1 transporter, but was independent of metabolic inhibitor. Furthermore, the transport of Rb1 into rBMEC was significantly diminished in the presence of natural substrate α-D-glucose, suggesting a facilitated transport of Rb1 via GLUT1 transporter. The impact of GLUT1 on the distribution of Rb1 between brain and plasma was studied experimentally in rats. Administration of phloretin (5 mg/kg, i.v.) to normal rats for consecutive 1 week before Rb1 (10 mg/kg, i.v.) at 0.5, 2, and 6 h did not alter Rb1 concentrations in plasma, but resulted in significant decreased brain concentrations of Rb1 compared to in the phloretin-untreated normal rats (489.6 ± 58.3 versus 105.1 ± 15.1 ng/g, 193.8 ± 11.1 versus 84.8 ± 4.1 ng/g, and 114.2 ± 24.0 versus 39.9 ± 4.9 ng/g, respectively). The expression of GLUT1 in the phloretin-treated group by western blotting analysis in vitro and in vivo experiments was significantly decreased, indicating that the decreased transport of Rb1 in brain was well related to the down-regulated function and level of GLUT1. Therefore, our in vitro and in vivo results indicate that the transport of Rb1 at the BBB is at least partly mediated by GLUT1 transporter.

Effects of Platelet-Activating Factor on Brain Microvascular Endothelial Cells

Platelet-activating factor (PAF) is a potent phospholipid mediator that exerts various pathophysiological effects by interacting with a G protein-coupled receptor. PAF has been reported to increase the permeability of the blood–brain barrier (BBB) via incompletely characterized mechanisms. We investigated the effect of PAF on rat brain microvascular endothelial cells (RBMVEC), a critical component of the BBB. PAF produced a dose-dependent increase in cytosolic Ca2+ concentration; the effect was prevented by the PAF receptor antagonist, WEB2086. The effect of PAF on cytosolic Ca2+ was abolished in Ca2+-free saline or in the presence of L-type voltage-gated Ca2+ channel inhibitor, nifedipine, indicating that Ca2+ influx is critical for PAF-induced increase in cytosolic Ca2+. PAF produced RBMVEC depolarization; the effect was inhibited by WEB2086. In cells loaded with [(4-amino-5-methylamino-2′,7′-difluoro-fluorescein)diacetate] (DAF-FM), a nitric oxide (NO)-sensitive fluorescent dye, PAF increased the NO level; the effect was prevented by WEB2086, nifedipine or by l-NAME, an inhibitor of NO synthase. Immunocytochemistry studies indicate that PAF reduced the immunostaining of ZO-1, a tight junction-associated protein, increased F-actin fibers, and produced intercellular gaps. PAF produced a decrease in RBMVEC monolayer electrical resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS), indicative of a disruption of endothelial barrier function. In vivo studies indicate that PAF increased the BBB permeability, assessed with sodium fluorescein and Evans Blue methods, via PAF receptor-dependent mechanisms, consequent to Ca2+ influx and increased NO levels. Our studies reveal that PAF alters the BBB permeability by multiple mechanisms, which may be relevant for central nervous system (CNS) inflammatory disorders.

A195 GLUTAMINE SYNTHETASE IN ENDOTHELIAL CELLS OF THE BLOOD-BRAIN BARRIER: NEW TARGET FOR THE TREATMENT OF HEPATIC ENCEPHALOPATHY?

The liver plays a major role in regulating ammonia levels in the blood. Therefore, in liver disease the loss of hepatic function leads to hyperammonemia and increased brain ammonia and consequently hepatic encephalopathy (HE). Ammonia-lowering strategies remain the mainstay therapeutic strategy. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. Therefore, the aim is to evaluate GS expression in endothelial cells of the BBB. Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS expression by western blot using brain lysates as positive control and by immunohistochemistry (co-localized with caveolin-1, a marker for ECs). In addition, to understand the effect of ammonia on GS, ECs were exposed to 1mM of ammonia chloride for 48h. Finally, ECs were subjected to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) or sham-operated controls. We have characterized this BDL model and found both systemic oxidative stress and inflammation, in addition to hyperammonemia. ECs expressed GS mRNA, protein and activity. However, expression of GS was lower compared to brain lysate control samples (p<0.05). GS expression in CMV showed similar results to brain but GS activity was less (p<0.05). Using immunohistochemistry, GS was detected in ECs and in vessels of brain from naïve rats. When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS was decreased when compared to sham-operated controls (p<0.01). These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme compared to that found in the brain, could explain why GS has never been reported in these cells. Interestingly, ammonia stimulates GS in ECs, but its activity is decreased in the presence of other pathogenic factors in plasma from cirrhotic rats such as oxidative stress and inflammation. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may be implicated in the pathogenesis of HE. We anticipate increasing GS in ECs of the BBB could become a new therapeutic target for HE. CIHR

ROS responsive resveratrol delivery from LDLR peptide conjugated PLA-coated mesoporous silica nanoparticles across the blood\u2013brain barrier

BackgroundOxidative stress acts as a trigger in the course of neurodegenerative diseases and neural injuries. An antioxidant-based therapy can be effective to ameliorate the deleterious effects of oxidative stress. Resveratrol (RSV) has been shown to be effective at removing excess reactive oxygen species (ROS) or reactive nitrogen species generation in the central nervous system (CNS), but the delivery of RSV into the brain through systemic administration is inefficient. Here, we have developed a RSV delivery vehicle based on polylactic acid (PLA)-coated mesoporous silica nanoparticles (MSNPs), conjugated with a ligand peptide of low-density lipoprotein receptor (LDLR) to enhance their transcytosis across the blood–brain barrier (BBB).ResultsResveratrol was loaded into MSNPs (average diameter 200 nm, pore size 4 nm) at 16 μg/mg (w/w). As a gatekeeper, the PLA coating prevented the RSV burst release, while ROS was shown to trigger the drug release by accelerating PLA degradation. An in vitro BBB model with a co-culture of rat brain microvascular endothelial cells (RBECs) and microglia cells using Transwell chambers was established to assess the RSV delivery across BBB. The conjugation of LDLR ligand peptides markedly enhanced the migration of MSNPs across the RBECs monolayer. RSV could be released and effectively reduce the activation of the microglia cells stimulated by phorbol-myristate-acetate or lipopolysaccharide.ConclusionsThese ROS responsive LDLR peptides conjugated PLA-coated MSNPs have great potential for oxidative stress therapy in CNS.

The Effect of Therapeutic Mild Hypothermia on Brain Microvascular Endothelial Cells During Ischemia-Reperfusion Injury.

To determine the cerebral protective effects of mild hypothermia (MH) on cerebral microcirculation. We established ischemia-reperfusion (I/R) injury and MH treatment models with rat brain microvascular endothelial cells (RBMECs) in vitro and examined the apoptotic changes. The cultured RBMECs were randomly divided into the control group, I/R group, and MH group, which was further divided into two subgroups: intra-ischemia hypothermia (IIH) and post-ischemia hypothermia (PIH). Cell morphological changes were assessed using fluorescence microscopy. Apoptotic rates were obtained by flow cytometry. Expressions of caspase-3, Bax, and Bcl-2 were analyzed by Western blot. I/R injury in vitro induced apoptosis of RBMECs. The apoptotic rates in the control group, I/R group, and MH group were 0.13, 19.04, and 13.13%, respectively (P<0.01). Compared with the I/R group, the MH group showed a significant decrease in the number of apoptotic cells, mainly in stage I apoptotic cells (P<0.0083). The caspase-3 and Bax expressions were significantly enhanced (P<0.05) in RBMECs after I/R injury, while substantial decreases in Bcl-2 expression were noted (P<0.05). Following MH intervention, the increase in caspase-3 and Bax expression was suppressed (P<0.05), while Bcl-2 expression significantly increased. The apoptotic rates or protein expressions between the two subgroups were not different significantly (P>0.05). These results indicate that MH could inhibit RBMEC apoptosis by preventing pro-apoptotic cells and early apoptotic cells from progressing to intermediate and advanced stages. This may be due to the effect of MH on I/R-induced apoptotic gene expression changes.

Protective effect of Cordyceps sinensis extract on rat brain microvascular endothelial cells injured by oxygen–glucose deprivation

ObjectiveTo investigate the protective effect of Cordyceps sinensis extract (CSE) on injury of primary cultured rat brain microvascular endothelial cells (rBMECs) induced by oxygen–glucose deprivation (OGD). MethodsWe isolated and cultured primary rBMECs in order to establish an in vitro OGD model. Cellular activity was detected using a cell counting kit to determine the appropriate dosage. The rBMECs were divided into control, model, low-, mid-, and high-dose (5, 10, 20 μg·mL−1) CSE groups under OGD for 6 hours. CSE was dissolved in cell culture medium to the appropriate concentration, passed through a 0.22 μm sterile filter, and administered for 12 hours before and during OGD. Cellular morphology was observed under a microscope. Lactate dehydrogenase level in cultural supernatant, superoxide dismutase activity, and the content of nitric oxide and malondialdehyde in cells were tested by colorimetric methods. Levels of tumor necrosis factor-α and interleukin-1 beta in cells were determined by enzyme-linked immunosorbent assay. ResultsAfter 12-hour administration of CSE at the concentration of 5, 10, 20 μg·mL−1 before and during OGD, compared with the model group, the CSE groups obviously alleviated the damage of rBMECs induced by OGD, inhibited the apoptosis and the necrosis of the cells, and improved cellular morphology of rBMECs. Additionally, compared with the model group, CSE also restrained lactate dehydrogenase leakage in hypoxic cells (P < .01), significantly increased superoxide dismutase activity (P < .05), and reduced the levels of nitric oxide, malondialdehyde, tumor necrosis factor-α, and interleukin-1 beta (P < .05). ConclusionC. sinensis extract plays a significant role in protecting injured primary cultured rBMECs induced by OGD. The mechanism may be related with the increase of cellular anti-oxidative capacity and anti-inflammatory effect.

Troxerutin and Cerebroprotein Hydrolysate Injection Protects Neurovascular Units from Oxygen-Glucose Deprivation and Reoxygenation-Induced Injury In Vitro.

Cerebral ischemia/reperfusion (I/R) injury involves complex events of cellular and molecular processes. Previous studies suggest that a neurovascular unit (NVU) acts as an intricate network to maintain the neuronal homeostatic microenvironment. The present study established an NVU model for oxygen-glucose deprivation and reoxygenation (OGD/R) damage, trying to target the major components of the NVU using a coculture of rat neurons, astrocytes, and rat brain microvascular endothelial cells (rBMECs) to investigate the therapeutic effects of troxerutin and cerebroprotein hydrolysate injections (TCHis). The study observed that OGD/R downregulated the expressions of GAP-43, Claudin-5, and AQP-4 obviously detected by Western blotting and immunocytochemical analysis, respectively, while TCHi ameliorated the effect of OGD/R significantly. Meanwhile, TCHi alleviated the abnormalities of ultrastructure of neurons and rBMECs induced by OGD/R. Furthermore, both levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and cell adhesion molecules (VCAM-1 and ICAM-1) detected by ELISA in NVU supernatant were found elevated significantly through OGD/R, but TCHi ameliorated the trend. In addition, TCHi also mitigated the increase of proapoptotic factors (Bax, p53, and caspase-3) induced by OGD/R in NVU model statistically. All these findings demonstrated that TCHis played a protective role, which was reflected in anti-inflammation, antiapoptosis, and blood–brain barrier maintenance. The results of the study concluded that the NVU is an ideal target and TCHi acts as a neuroprotective agent against cerebral I/R injuries.

Biospecific isolation and characterization of angiogenesis-promoting ingredients in Buyang Huanwu decoction using affinity chromatography on rat brain microvascular endothelial cells combined with solid-phase extraction, and HPLC-MS/MS

Buyang Huanwu decoction (BHD) was reported to exert angiogenesis-promoting effects, but its active ingredients remain unknown. In this study, we developed a method to screen potential angiogenesis-promoting compounds in BHD, which involved biospecific isolation using live rat brain microvascular endothelial cells (rBMECs) and characterization using solid-phase extraction (SPE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Six compounds showed binding affinity to rBMECs and were further identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-β-D-glucoside, galloylpaeoniflorin, formononetin-7-O-β-D-glucoside, and (3R)-7,2′-hydroxy-3′,4′-dimethoxy-isoflavan. The results indicated that five of them except 6-hydroxykaempferol-di-O-glucoside showed a protective effect against oxygen glucose deprivation/reperfusion injury in rBMECs and upregulated the secretion of vascular endothelial growth factor and basic fibroblast growth factor, suggesting a mechanism underlying their angiogenic activity. Our findings suggest that biospecific live cell-based isolation combined with SPE and HPLC-MS/MS is an effective method for screening potential bioactive components in traditional Chinese medicines.

MicroRNA-126-3p attenuates blood-brain barrier disruption, cerebral edema and neuronal injury following intracerebral hemorrhage by regulating PIK3R2 and Akt

MiR-126, a microRNA implicated in blood vessel integrity, angiogenesis and vascular inflammation, is markedly decreased in the sera of patients with intracerebral hemorrhage (ICH). The current study aims to evaluate the potential therapeutic effect of miR-126-3p on brain injuries in a rat model of collagenase-induced ICH. Intracerebroventricular administration of a miR-126-3p mimic significantly alleviated behavioral defects 24 h after ICH, as examined by paw placement and corner tests. ICH led to increased blood-brain barrier (BBB) permeability and cerebral edema, both of which were attenuated by miR-126-3p mimic. Treatment with miR-126-3p mimic reduced the numbers of myeloperoxidase (MPO)-positive, OX42-positive, Fluoro Jade B (FJB)-positive and NEUN/TUNEL double-positive cells around the hematoma, implying that miR-126-3p inhibited neutrophil infiltration, microglial activation and neuronal apoptosis following hemorrhage. In addition, miR-126-3p mimic suppressed the upregulation of phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) in the perihematomal area and maintained the activation of Akt. Furthermore, in vitro assays confirmed upregulation of PIK3R2 upon knockdown of miR-126-3p in rat brain microvascular endothelial cells (BMECs), and silencing of miR-126-3p resulted in impaired BMEC barrier permeability and reversed vascular endothelial growth factor (VEGF)- and angiopoietin-1 (Ang-1)-induced activation of Akt and inhibition of BMEC apoptosis. In summary, our results suggest that exogenous miR-126-3p may alleviate BBB disruption, cerebral edema and neuronal injury following ICH by targeting PIK3R2 and the Akt signaling pathway in brain vascular endothelium.

MiR-377 Regulates Inflammation and Angiogenesis in Rats After Cerebral Ischemic Injury.

Ischemic stroke is the leading cause of disabilities worldwide. MicroRNA-377 (miR-377) plays important roles in ischemic injury. The present study focused on the mechanisms of miR-377 in protecting ischemic brain injury in rats. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Primary rat microglial cells and brain microvascular endothelial cells (BMECs) were exposed to oxygen-glucose deprivation (OGD). The concentrations of cytokines (TNF-α, IL-1β, IL-6, IFN-γ, TGF-β, MMP2, COX2, and iNOS) in the culture medium were measured by specific ELISA. Tube formation assay was for the in vitro study of angiogenesis. Luciferase reporter assay was performed to confirm whether VEGF and EGR2 were direct targets of miR-377. The MCAO rats were intracerebroventricular (ICV) injection of miR-377 inhibitor to assess its protective effects in vivo. MiR-377 levels were decreased in the rat brain tissues at 1, 3, and 7 d after MCAO. Both microglia cells and BMECs under OGD showed markedly lower expression levels of miR-377 while higher expression levels of EGR2 and VEGF compared to those under normoxia conditions. Knockdown of miR-377 inhibited microglial activation and the release of pro-inflammatory cytokines after OGD. Suppression of miR-377 promoted the capillary-like tube formation and cell proliferation and migration of BMECs. The anti-inflammation effect of EGR2 and the angiogenesis effect of VEGF were regulated by miR-377 after OGD. Inhibition of miR-377 decreased cerebral infarct volume and suppressed cerebral inflammation but promoted angiogenesis in MCAO rats. Knockdown of miR-377 lessened the ischemic brain injury through promoting angiogenesis and suppressing cerebral inflammation. J. Cell. Biochem. 119: 327-337, 2018. © 2017 Wiley Periodicals, Inc.

Characterization of Biaxial Stretch as an In Vitro Model of Traumatic Brain Injury to the Blood-Brain Barrier.

Traumatic brain injury (TBI) is one of the major causes of disability in the USA. It occurs when external mechanical forces induce brain damage that causes deformation of brain tissue. TBI is also associated with alterations of the blood-brain barrier (BBB). Using primary rat brain microvascular endothelial cells as an in vitro BBB model, the effects of biaxial stretch were characterized at 5, 10, 15, 25, and 50% deformation using a commercially available system. The results were compared to the effects of mild and moderate TBI in vivo, induced by the weight-drop method in mice. In vitro, live/dead cells, lactate dehydrogenase (LDH) release, caspase 3/7 staining, and tight junction (TJ) protein expression were evaluated 24h after a single stretch episode. In vivo, Evans blue extravasation, serum levels of S100β, and TJ protein expression were evaluated. Stretch induced a deformation-dependent increase in LDH release, cell death, and activation of caspase 3/7, suggesting the induction of apoptosis. Interestingly, low magnitudes of deformation increased the expression of TJ proteins, likely in an attempt to compensate for stretch damage. High magnitudes of deformation decreased the expression of TJ proteins, suggesting that the damage was too severe to counteract. In vivo, mild TBI did not affect BBB permeability or the expression of TJ proteins. However, moderate TBI significantly increased BBB permeability and decreased the expression of these proteins, similar to the results obtained with a high magnitude deformation. These data support the use biaxial stretch as valuable tool in the study of TBI in vitro.

Long noncoding RNA MEG3 mediated angiogenesis after cerebral infarction through regulating p53/NOX4 axis

ObjectiveThis study aimed to explore the mechanism of lncRNA MEG3 on angiogenesis after cerebral infarction (CI). MethodsThe rat brain microvascular endothelial cells (RBMVECs) isolated from rat was used to establish CI model, which were treated with oxygen-glucose deprivation/reoxygenation (OGD/R). The genes mRNA and protein expression levels in RBMVECs were determined by the quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, respectively. The flow cytometry was used to measured cell apoptosis and intracellular reactive oxygen species (ROS) generation. The RBMVECs activities was detected by MTT method. The RNA-immunoprecipitation (RIP) assay was used to detect the interaction between MEG3 and p53, and the relationship between p53 and NOX4 was proved by chromatin co-immunoprecipitation (chip) assay. ResultsThe results showed that OGD or OGD/R increased MEG3 and NOX4 expression, and there was positive correlation between MEG3 and NOX4 expression in RBMVECs. Next, knockdown of MEG3 indicated that inhibition of MEG3 was conducive to protect RBMVECs against OGD/R-induced apoptosis, with decreased NOX4 and p53 expression, further enhanced pro-angiogenic factors (HIF-1α and VEGF) expression, and reduced intracellular ROS generation. And then the RIP and CHIP assay demonstrated that MEG3 could interacted with p53 and regulated its expression, and p53 exerted significant binding in the promoters for NOX4, suggesting that MEG3 regulated NOX4 expression via p53. At last, knockdown of NOX4 indicated that inhibition of NOX4 protected RBMVECs against OGD/R-induced apoptosis, with increased cell viability and pro-angiogenic factors expression, and reduced ROS generation. ConclusionLncRNA MEG3 was an important regulator in OGD/R induced-RBMVECs apoptosis and the mechanism of MEG3 on angiogenesis after CI was reduced ROS by p53/NOX4 axis.

Administration of sonic hedgehog protein induces angiogenesis and has therapeutic effects after stroke in rats

The Sonic hedgehog (Shh) signaling pathway is recapitulated in response to ischemic injury. Here, we investigated the clinical implications of Shh protein in the ischemic stroke and explored the underlying mechanism. Intracerebroventricular injection of Shh, Cyclopamine, or anti-vascular endothelial growth factor (VEGF) was performed immediately after permanent middle cerebral artery occlusion (pMCAO) surgery and lasted for 7days (d). Phosphate-buffered saline (PBS) was used as control. Neurological deficits and infarct volume were examined 7d after pMCAO. Microvascular density with fluorescein-iso-thiocyanate (FITC) assay and double staining with CD31 and Ki-67 was measured at 7d. To observe in vitro angiogenesis, rat brain microvascular endothelial cells (RBMECs) were incubated under oxygen glucose deprivation (OGD) for 6h (h) and treated with Shh/anti-VEGF. We found that (1) Shh improved neurological scores and reduced infarct volume, which was blocked by Cyclopamine, (2) Shh improved the microvascular density and promoted angiogenesis and neuron survival in the ischemic boundary zone, (3) Shh enhanced VEGF expression and VEGF antibody could reverse angiogenic and protective effect of Shh in vivo and in vitro. These data demonstrate that the administration of Shh protein could protect brain from ischemic injury, in part by promoting angiogenic repair.

A Novel Functional Variant of nNOS Mediates Anoxic Injury in Brain Microvascular Endothelial Cells via Mitochondrial Superoxide Generation

Deletions of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms in mice have been shown to elicit opposite effects on ischemic brain injury following experimental stroke. Recently, we have demonstrated the expression of nNOS in human, rat, and mouse primary brain microvascular endothelial cells (MECs). The constitutively active endothelial nNOS (enNOS) was found to be functionally distinct from the nNOS expressed in neurons and the eNOS but sensitive to inhibition by pharmacological nNOS inhibitors. Under physiological conditions, enNOS was found to produce superoxide instead of nitric oxide (NO), unlike all known NOS isoforms. The objective of the present study was to examine the pathophysiological role of enNOS in endothelial injury. We hypothesized that enNOS mediates the anoxic injury underlying the blood‐brain barrier (BBB) dysfunction and cell death by instigating mitochondrial oxidative stress. We utilized primary human brain MECs (passage# 7–9) and employed inhibitors of nNOS (N‐ω‐Propyl‐L‐arginine or NPA and ARL‐17477; 1 μmol/L each) and eNOS (L‐N5‐(1‐Iminoethyl)ornithine or NIO, 1 μmol/L). Electron spin resonance spectrometry measurements with mitochondria‐targeted mito‐TEMPO‐H (50 μmol/L) showed that cells treated with enNOS inhibitor (ARL‐17477, 165±16 a.u, n=7; p &lt; 0.05) reduced the mitochondrial superoxide in compared to the untreated MECs (108±10 a.u, n=7). Oxygen consumption rate (OCR; pmol/minute/pg protein) measured in MECs by Seahorse Analyzer showed increase in maximal respiration and decreased non‐mitochondrial OCR with NPA treatment (355 ± 60 and 7 ± 4, respectively; n=11; p &lt; 0.05) compared to untreated cells (189 ± 49 and 19 ± 4, respectively; n=11). Transendothelial electrical resistance (TEER) measurements were made to quantitate the BBB integrity in confluent monolayers of hMECs grown in transwells and subjected to oxygen‐glucose deprivation (OGD; 6 h). Inhibition of enNOS with ARL‐17477 (50 ± 3 vs. 39 ± 2 untreated, n=6–8; p &lt; 0.05) and NPA (46 ± 2 vs. 36 ± 2 untreated, n=7–9; p &lt; 0.05) increased TEER (Ω.cm2) in MECs. In addition, ARL‐17477 and NPA (119 ± 15 and 116 ± 9, n=9 each; p&lt;0.05) sustained recovery of TEER (% change) at 30 hours following OGD compared to the eNOS inhibited (89 ± 5, n=9; p=NS) and untreated cells (75 ± 8, n=9). Western blot studies have shown that treatment with enNOS inhibitors increases the levels of the tight junction protein, Zona Occludens‐1, in MECs compared to untreated cells. In contrast, viability measurements using CCK‐8 assay (% viable cells) observed that MECs treated with each NOS inhibitor (NPA: 42 ± 3; ARL‐17477: 52 ± 3; NIO: 70 ± 5; n = 16, p &lt; 0.05) and exposed to OGD increased cell survival compared to untreated cells (26 ± 2; n = 16). Thus, enNOS mediates anoxic‐injury leading to the impairment of BBB integrity as well as cell death.Support or Funding InformationAHA Predoctoral (VNS: 16PRE31450006) and postdoctoral (IR: 15POST23040005) Fellowship Grant, AHA Scientist Development Grant (PVK: 14SDG20490359), and NIH grants (DWB: HL093554 and PVK: NS094834).

Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells

Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood–brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH.

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843.