Disrupt Blood-brain Barrier Integrity Research Articles

The novel ROCK2 selective inhibitor NRL-1049 preserves the blood-brain barrier after acute injury.

Endothelial blood-brain barrier (BBB) dysfunction is critical in the pathophysiology of brain injury. Rho-associated protein kinase (ROCK) activation disrupts BBB integrity in the injured brain. We aimed to test the efficacy of a novel ROCK2 inhibitor in preserving the BBB after acute brain injury. We characterized the molecular structure and pharmacodynamic and pharmacokinetic properties of a novel selective ROCK2 inhibitor, NRL-1049, and its first metabolite, 1-hydroxy-NRL-1049 (referred to as NRL-2017 hereon) and tested the efficacy of NRL-1049 on the BBB integrity in rodent models of acute brain injury. Our data show that NRL-1049 and NRL-2017 both inhibit ROCK activity and are 44-fold and 17-fold more selective towards ROCK2 than ROCK1, respectively. When tested in a mouse model of cortical cryoinjury, NRL-1049 significantly attenuated the increase in water content. Interestingly, 60% of the mice in the vehicle arm developed seizures within 2 hours after cryoinjury versus none in the NRL-1049 arm. In spontaneously hypertensive rats, NRL-1049 attenuated the dramatic surge in Evans Blue extravasation compared with the vehicle arm after transient middle cerebral artery occlusion. Hemorrhagic transformation was also reduced. We show that NRL-1049, a selective ROCK2 inhibitor, is a promising drug candidate to preserve the BBB after brain injury.

Depletion of Endothelial-Derived 2-AG Reduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-Proteome.

Mounting evidence supports the role of the endocannabinoid system in neurophysiology, including blood-brain barrier (BBB) function. Recent work has demonstrated that activation of endocannabinoid receptors can mitigate insults to the BBB during neurological disorders like traumatic brain injury, cortical spreading depression, and stroke. As alterations to the BBB are associated with worsening clinical outcomes in these conditions, studies herein sought to examine the impact of endocannabinoid depletion on BBB integrity. Barrier integrity was investigated in vitro via bEnd.3 cell monolayers to assess endocannabinoid synthesis, barrier function, calcium influx, junctional protein expression, and proteome-wide changes. Inhibition of 2-AG synthesis using DAGLα inhibition and siRNA inhibition of DAGLα led to loss of barrier integrity via altered expression of VE-cadherin, which could be partially rescued by exogenous application of 2-AG. Moreover, the deleterious effects of DAGLα inhibition on BBB integrity showed both calcium and PKC (protein kinase C)-dependency. These data indicate that disruption of 2-AG homeostasis in brain endothelial cells, in the absence of insult, is sufficient to disrupt BBB integrity thus supporting the role of the endocannabinoid system in neurovascular disorders.

Bradykinin-bradykinin receptor (B1R) signalling is involved in the blood-brain barrier disruption in moyamoya disease.

Moyamoya disease (MMD) is a rare disorder of the cerebrovascular system. It is a steno-occlusive disease that involves angiogenesis and blood-brain barrier (BBB) disruption. Bradykinin (BK), its metabolite des-Arg9-BK, and receptor (B1R) affect angiogenesis and BBB integrity. In this study, we aimed to investigate the changes in BK, B1R and des-Arg9-BK levels in the serum and brain tissues of patients with MMD and explore the underlying mechanism of these markers in MMD. We obtained the serum samples and superficial temporal artery (STA) tissue of patients with MMD from the Department of Neurosurgery of the Jining First People's Hospital. First, we measured BK, des-Arg9-BK and B1R levels in the serum of patients by means of ELISA. Next, we performed immunofluorescence to determine B1R expression in STA tissues. Finally, we determined the underlying mechanism through Western blot, angiogenesis assay, immunofluorescence, transendothelial electrical resistance and transcytosis assays. Our results demonstrated a significant increase in the BK, des-Arg9-BK and B1R levels in the serum of patients with MMD compared to healthy controls. Furthermore, an increase in the B1R expression level was observed in the STA tissues of patients with MMD. BK and des-Arg9-BK could promote the migratory and proliferative abilities of bEnd.3 cells and inhibited the formation of bEnd.3 cell tubes. In vitro BBB model showed that BK and des-Arg9-BK could reduce claudin-5, ZO-1 and occluding expression and BBB disruption. To the best of our knowledge, our results show an increase in BK and B1R levels in the serum and STA tissues of patients with MMD. BK and Des-Arg9-BK could inhibit angiogenesis, promote migratory and proliferative capacities of cells, and disrupt BBB integrity. Therefore, regulating BK, des-Arg9-BK and B1R levels in the serum and the brain could be potential strategies for treating patients with MMD.

CNSC-03. EXPLORING STRUCTURAL DISRUPTION OF TRICELLULAR JUNCTIONS TO ENHANCE BLOOD-BRAIN-BARRIER PERMEABILITY

Abstract The blood-brain barrier (BBB) hinders CNS chemotherapy entry for malignant glioma treatment. It is predominantly composed of brain endothelium linked by bicellular (BJ) and tricellular (TJ) tight junctions. Previous studies demonstrated,angubindin-1 inhibited TJ angulin-1 to increase brain and CSF targeted therapy in rodent lung cancer models. We hypothesize angubindin-1 can transiently decrease BBB junctional integrity to increase chemotherapy permeability and prolong rodent glioma model survival. Rat brain endothelial cells were treated with TJ and BJ inhibitors against angulin-1 (angubindin-1 600µg/mL), claudin-3 (C-CPE 200µg/mL) or claudin-5 (C-CPE-MT 200µg/mL). Endothelial integrity was assessed by immunoblotting and cell-cell electrical impedance. Effects of angubindin-1 on efflux transporter P-glycoprotein (PGP) and migration were studied using rat and human derived glioma cells. Rat glioma models were treated with doxil (3 mg/kg), angubindin-1 (10 mg/kg or 30 mg/kg), or combination therapy for assessments on tumor volume, BBB permeability and survival. We observed decreased angulin-1 expression 5 hours after angubindin-1 treatment, with return to baseline by 24 hours (p<0.05). Angubindin-1, CCPE, and CCPE-mt globally reduced endothelial cell-cell integrity, maximally at 4 hours with a return to baseline by 12 hours; with angubindin-1 demonstrating the largest decreased cell-adhesion (angubindin-1 vs control, p< 0.0001). Angubindin-1 also decreased PGP efflux of rhodamine in both endothelial and glioma cells, along with demonstrating a pro-migratory dose-dependent effect on rat glioma cells. Combined angubindin-1 and doxil increased survival in rat glioma models compared with doxil alone (24 days vs. 18 days, p < 0.0001). Day 14 tumor volume was significantly decreased with angubindin-1 and combination treatment respectively (77.5 % vs 81.6 %, p<0.05). Ongoing studies are exploring, angubindin-1’s effect on large CNS drug permeability, TJ localization post BBB disruption and additional combinational treatments in glioma models. Collectively, these findings pose a unique opportunity to disrupt BBB integrity and improve malignant glioma treatment options.

TMIC-23. EXPLORING IBRUTINIB'S EFFECTS ON BLOOD-BRAIN BARRIER INTEGRITY AND GLIOMA PROGRESSION

Abstract BACKGROUND The blood-brain barrier (BBB), among malignant gliomas limits certain agents from impairing tumor growth. Thus, identifying agents that can transiently increase BBB permeability may prove useful in enhancing glioma treatment response. Previous ibrutinib studies showed it independently disrupted gut epithelial integrity and hampered glioma growth through BMX (bone marrow x-linked tyrosine kinase) inhibition. We propose BMX inhibition via ibrutinib disrupts BBB integrity while impairing glioma progression. METHODS To evaluate ibrutinib’s effect on brain endothelium, we evaluated electrical cell-cell impedance, junctional/cytoskeletal expression, downstream protein expression and functional ABC transporter activity; monitoring changes over time (0 to 8h) and at varied drug concentrations (1-10μM ibrutinib). Using rat glioma cells (S635) in vitro and in models, we examined cytotoxicity, apoptosis, model survival, and drug concentrations with ibrutinib alone or combined with poorly permeable Abcb1 substrate doxorubicin. RESULTS Ibrutinib dose-dependently decreased brain endothelial cell-cell impedance by 60% at 2h, without affecting cell viability. We observed decreased ZO-1 junctions, actin cytoskeletal rearrangement, and downstream pErk and pMek decreased expression optimally 2h (10μM ibrutinib). Dose-dependently ibrutinib inhibited Abcb1 efflux activity, further favoring a more permeable endothelium. Synergy with doxorubicin was seen in rat glioma cells treated with ibrutinib via increased apoptosis and cytotoxicity. However, while combined treatment resulted in decreased systemic doxorubicin concentrations, no differences in brain:plasma or tumor:plasma concentrations were evident. Combination therapy also did not increase rodent glioma model survival nor decrease tumor size. CONCLUSION Our results suggest that while ibrutinib induces brain endothelial permeability by junctional/cytoskeletal disruption and inhibition of Abcb1 efflux, no sustained effect can be seen in rodent glioma models in combination with a cytotoxic agent. Additional studies exploring similar agents that can enhance BBB permeability while slowing glioma growth are warranted, so as to improve upon current bleak malignant glioma treatment options.

Astrocytic responses to high glucose impair barrier formation in cerebral microvessel endothelial cells.

Hyperglycemic conditions are prodromal to blood-brain barrier (BBB) impairment. The BBB comprises cerebral microvessel endothelial cells (CMECs) that are surrounded by astrocytic foot processes. Astrocytes express high levels of gap junction connexin 43 (Cx43), which play an important role in autocrine and paracrine signaling interactions that mediate gliovascular cross talk through secreted products. One of the key factors of the astrocytic "secretome" is vascular endothelial growth factor (VEGF), a potent angiogenic factor that can disrupt BBB integrity. We hypothesize that high-glucose conditions change the astrocytic expression of Cx43 and increase VEGF secretion leading to impairment of CMEC barrier properties in vitro and in vivo. Using coculture of neonatal rat astrocytes and CMEC, we mimic hyperglycemic conditions using high-glucose (HG) feeding media and show a significant decrease in Cx43 expression and the corresponding increase in secreted VEGF. This result was confirmed by the analyses of Cx43 and VEGF protein levels in the brain cortex samples from the type 2 diabetic rat (T2DN). To further characterize inducible changes in BBB, we measured transendothelial cell electrical resistance (TEER) and tight junction protein levels in cocultured conditioned astrocytes with isolated rat CMEC. The coculture monolayer's integrity and permeability were significantly compromised by HG media exposure, which was indicated by decreased TEER without a change in tight junction protein levels in CMEC. Our study provides insight into gliovascular adaptations to increased glucose levels resulting in impaired cellular cross talk between astrocytes and CMEC, which could be one explanation for cerebral BBB disruption in diabetic conditions.

Ribosomal Protein SA-Positive Neutrophil Elicits Stronger Phagocytosis and Neutrophil Extracellular Trap Formation and Subdues Pro-Inflammatory Cytokine Secretion Against Streptococcus suis Serotype 2 Infection.

Streptococcus suis serotype 2 (SS2), an important zoonotic pathogen that causes septicemia, arthritis, and irreversible meningitis in pigs and humans, can be transmitted to humans from pigs. S. suis causes huge economic losses to the swine industry and poses a serious threat to public health. Previously, we found that the brain tissues of mice with SS2-induced meningitis showed disrupted structural integrity and significantly enhanced polymorphonuclear neutrophil (PMN) infiltration. We showed that the brain tissues of SS2-infected mice had increased ribosomal protein SA (RPSA)-positive PMN counts. However, the inflammatory responses of RPSA+ PMNs to SS2 and their effects on the blood-brain barrier (BBB) remain unclear. Therefore, in studying the pathogenesis of SS2-induced meningitis, it is essential that we explore the functions of RPSA+ PMNs and their effects on the BBB. Herein, using flow cytometry and immunofluorescence microscopy analyses, we found that RPSA expression enhances PMN-induced phagocytosis and PMN-induced formation of neutrophil extracellular traps (NETs), which facilitate further elimination of bacteria. PMN surface expression of RPSA also alleviates local inflammation and tissue injuries by inhibiting secretion of the pro-inflammatory cytokines, TNF-α and IL-6. Moreover, the single-cell BBB model showed that RPSA disrupts BBB integrity by downregulating expression of tight junction-associated membrane proteins on PMNs. Taken together, our data suggest that PMN-surface expression of RPSA is a double-edged sword. RPSA+ PMN owns a stronger ability of bacterial cleaning and weakens inflammatory cytokines release which are useful to anti-infection, but does hurt BBB. Partly, RPSA+ PMN may be extremely useful to control the infection as a therapeutic cellular population, following novel insights into the special PMN population.

DDRE-01. ACQUIRED AND INTRINSIC RESISTANCE TO VEMURAFENIB IN BRAFV600E-DRIVEN MELANOMA BRAIN METASTASES

Abstract BRAF V600-mutated melanoma brain metastases (MBMs) are responsive to BRAF inhibitors, but clinical responses are less durable than those of extracranial metastases. We studied the impact of the drug efflux proteins P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) at the blood-brain barrier (BBB) on the efficacy of vemurafenib against BRAFV600E-mutated A375 MBMs. We intracranially implanted A375 tumor cells in wild-type and Abcb1a/b;Abcg2-/- mice. We characterized the tumor BBB, analyzed drug levels in plasma and brain lesions after oral vemurafenib and determined the efficacy against brain metastases and subcutaneous lesions. MRI shows that A375 MBMs disrupt BBB integrity, but vemurafenib accumulation in MBMs was still reduced by P-gp/BCRP. Vemurafenib is also less efficacious against MBMs in wild-type mice compared to Abcb1a/b;Abcg2-/- mice. Vemurafenib efficacy against subcutaneous A375 tumors was similar in both strains. Even in Abcb1a/b;Abcg2-/- mice, A375 MBMs rapidly developed resistance, which was unrelated to pharmacokinetic issues or insufficient inhibition of MAPK/PI3K pathways. Taken together, these studies demonstrate that although the BBB is disrupted in MBMs, P-gp/BCRP still limit the efficacy of vemurafenib. Moreover, the response to vemurafenib is less and of shorter duration also due to rapidly acquired resistance, most likely by resorting to non-canonical growth signaling.

EXTH-35. LASER INTERSTITIAL THERMAL THERAPY INCREASES BLOOD-BRAIN BARRIER AND BLOOD-TUMOR BARRIER PERMEABILITY IN A MOUSE MODEL OF GLIOBLASTOMA

Abstract INTRODUCTION A central challenge in glioblastoma treatment is the presence of the blood-brain barrier (BBB) and blood-tumor barrier (BTB), which prevent access of drugs to the brain and tumor respectively. Recent evidence in patients suggests laser interstitial thermal therapy (LITT), used clinically for tumor ablation, locally disrupts BBB integrity, potentially creating a therapeutic window to deliver otherwise brain-impermeant agents. METHODS A mouse model for LITT, established using a Nd-YAG laser coupled to a 600 mm fiber optic and thermocouple probe, was inserted via burrhole to target the somatosensory cortex. Syngeneic GL261 tumor cells were stereotactically implanted prior to LITT. BBB and BTB permeability were assessed through measurement of fluorescein and doxorubicin after IV injection. Permeability of IV dextran (10 and 70 kDa) and human IgG was monitored by immunohistochemistry (IHC) analysis. Mechanisms of BBB breakdown in vivo were explored utilizing electron microscopy and IHC. RESULTS By fluorescein assay, LITT-induced BBB and BTB permeability began one day post-treatment and was sustained for at least 2 weeks. Additionally, both normal brain and brain tumors demonstrated an increase in Dextran 10 kDa, Dextran 70 kDa, and human IgG extravasation after IV injection in vivo. Mechanistically, we provide evidence that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis. As proof-of-concept that LITT can enhance tumor delivery of systemic drugs, LITT increased IV doxorubicin permeability in brain in vivo. Moreover, LITT plus doxorubicin significantly increased survival in brain tumor-bearing mice compared to doxorubicin or LITT alone. CONCLUSIONS Our data suggest that LITT increases BBB and BTB permeability over a defined time window to large molecular weight agents, including antibodies, through multiple cellular mechanisms. Our preclinical results with LITT plus doxorubicin, which mirror a current clinical trial, indicate LITT can enhance the efficacy of systemically delivered drugs.

Endothelial Desert Hedgehog and ectopic Sonic Hedgehog act antagonistically to regulate blood vessel integrity and angiogenesis

Sonic Hedgehog (Shh), when administered as recombinant protein or via gene therapy was shown to be proangiogenic. However, studies performed in mice deficient for Hedgehog (Hh) signaling pathway elements have revealed that, on the contrary, endogenous Hh signaling is necessary for endothelium integrity and limits angiogenesis, suggesting that Hh ligands may have antagonistic effects. Notably, we recently demonstrated that endothelium integrity depends on endothelial cell (EC)-derived Desert Hedgehog (Dhh). We wish to clarify mechanisms underlying regulation of angiogenesis and vascular barrier integrity by Hh ligands. First we confirmed that Shh recombinant protein induces angiogenesis in the mouse corneal angiogenesis model. Then, we investigated the role of EC-derived Dhh in the same model, and found that, unexpectedly, VEGFA-induced angiogenesis was significantly increased in the absence of EC-derived Dhh suggesting that endogenous Dhh is anti-angiogenic. Besides, we administered Shh-encoding lentiviruses in the brain of WT mice and found that ectopic Shh, on the contrary to EC-derived Dhh disrupts blood brain barrier integrity, demonstrating, once again, that EC-derived Dhh and ectopic Shh have antagonistic effects. We then performed in vitro experiments in cultured ECs and confirmed that both treatment of ECs by Shh recombinant protein and Dhh KD in ECs disrupt intercellular junction integrity and promote EC migration. Notably, Smoothened inhibition recapitulates the phenotype of Dhh deficiency both in vivo and in vitro suggesting that Dhh autocrinally activates Hh signaling in ECs while ectopic Shh inhibits it. Finally, we performed immunoprecipitation assays and found that Dhh binding to Patched-1 is decreased in the presence of Shh. Altogether these results suggest for the first time that Shh, when administered ectopically may counteract Dhh-induced blood vessel integrity and quiescence.

Blast exposure elicits blood-brain barrier disruption and repair mediated by tight junction integrity and nitric oxide dependent processes

Mild blast-induced traumatic brain injury (TBI) is associated with blood-brain barrier (BBB) disruption. However, the mechanisms whereby blast disrupts BBB integrity are not well understood. To address this issue BBB permeability to peripherally injected 14C-sucrose and 99mTc-albumin was quantified in ten brain regions at time points ranging from 0.25 to 72 hours. In mice, repetitive (2X) blast provoked BBB permeability to 14C-sucrose that persisted in specific brain regions from 0.25 to 72 hours. However, 99mTc-albumin revealed biphasic BBB disruption (open-closed-open) over the same interval, which was most pronounced in frontal cortex and hippocampus. This indicates that blast initiates interacting BBB disruption and reparative processes in specific brain regions. Further investigation of delayed (72 hour) BBB disruption revealed that claudin-5 (CLD5) expression was disrupted specifically in the hippocampus, but not in dorsal striatum, a brain region that showed no blast-induced BBB permeability to sucrose or albumin. In addition, we found that delayed BBB permeability and disrupted CLD5 expression were blocked by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). These data argue that latent nitric oxide-dependent signaling pathways initiate processes that result in delayed BBB disruption, which are manifested in a brain-region specific manner.

Brain microvascular endothelial-astrocyte cell responses following Japanese encephalitis virus infection in an in vitro human blood-brain barrier model

Japanese encephalitis virus (JEV) remains a leading cause of encephalitis, globally, which continues to grow in importance despite the availability of vaccines. Viral entry into the brain can occur via the blood-brain barrier (BBB), and inflammation at the BBB is a common final pathway in many brain infections. However, the role of the BBB during JEV infection and the contribution of the endothelial and astrocytic cell inflammation in facilitating virus entry into the brain are incompletely understood. We established a BBB model using human brain endothelial cells (HBECs) and human astrocytes. HBECs are polarised, and therefore the model was inoculated by JEV from the apical side to simulate the in vivo situation. The effects of JEV on the BBB permeability and release of inflammatory mediators from both apical and basolateral sides, representing the blood and the brain side respectively were investigated. JEV infected HBECs with limited active virus production, before crossing the BBB and infecting astrocytes. Control of JEV production by HBECs was associated with a significant increase in permeability, and with elevation of many host mediators, including cytokines, chemokines, cellular adhesion molecules, and matrix metalloproteases. When compared to the controls, significantly higher amounts of mediators were released from the apical side as opposed to the basolateral side. The increased release of mediators over time also correlated with increased BBB permeability. Treatment with dexamethasone led to a significant reduction in the release of interleukin 6 (IL6), C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10) from the apical side with a reduction in BBB disruption and no change in JEV production. The results are consistent with the hypothesis that JEV infection of the BBB triggers the production of a range of host mediators from both endothelial cells and astrocytes, which control JEV production but disrupt BBB integrity thus allowing virus entry into the brain. Dexamethasone treatment controlled the host response and limited BBB disruption in the model without increasing JEV production, supporting a re-investigation of its use therapeutically.

TNFα disrupts blood brain barrier integrity to maintain prolonged depressive-like behavior in mice

Recovery from major depressive disorder is difficult, particularly in patients who are refractory to antidepressant treatments. To examine factors that regulate recovery, we developed a prolonged learned helplessness depression model in mice. After the induction of learned helplessness, mice were separated into groups that recovered or did not recover within 4 weeks. Comparisons were made between groups in hippocampal proteins, inflammatory cytokines, and blood brain barrier (BBB) permeability. Compared with mice that recovered and control mice, non-recovered mice displaying prolonged learned helplessness had greater hippocampal activation of glycogen synthase kinase-3 (GSK3), higher levels of tumor necrosis factor-α (TNFα), interleukin-17A, and interleukin-23, increased permeability of the blood brain barrier (BBB), and lower levels of the BBB tight junction proteins occludin, ZO1, and claudin-5. Treatment with the GSK3 inhibitor TDZD-8 reduced inflammatory cytokine levels, increased tight junction protein levels, and reversed impaired recovery from learned helplessness, demonstrating that prolonged learned helplessness is reversible and is maintained by abnormally active GSK3. In non-recovered mice with prolonged learned helpless, stimulation of sphingosine 1-phosphate receptors by Fingolimod or administration of the TNFα inhibitor etanercept repaired the BBB and reversed impaired recovery from prolonged learned helplessness. Thus, disrupted BBB integrity mediated in part by TNFα contributes to blocking recovery from prolonged learned helplessness depression-like behavior. Overall, this report describes a new model of prolonged depression-like behavior and demonstrates that stress-induced GSK3 activation contributes to disruption of BBB integrity mediated by inflammation, particularly TNFα, which contributes to impaired recovery from prolonged learned helplessness.

Sema4D/PlexinB1 inhibition ameliorates blood-brain barrier damage and improves outcome after stroke in rats.

The inflammatory process in stroke is the major contributor to blood-brain barrier (BBB) breakdown. Previous studies indicated that semaphorin 4D (Sema4D), an axon guidance molecule, initiated inflammatory microglial activation and disrupted endothelial function in the CNS. However, whether Sema4D disrupts BBB integrity after stroke remains unclear. To study the effect of Sema4D on BBB disruption in stroke, rats were subjected to transient middle cerebral artery occlusion and targeted injection of lentivirus-mediated clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene disruption of PlexinB1. We found that Sema4D synchronously increased with BBB permeability and accumulated in the perivascular area after stroke. Suppressing Sema4D/PlexinB1 signaling in the periinfarct cortex significantly decreased BBB permeability as detected by MRI and fibrin deposition, and thereby improved stroke outcome. In vitro, we confirmed that Sema4D disrupted BBB integrity and endothelial tight junctions. Moreover, we found that Sema4D induced pericytes to acquire a CD11b-positive phenotype and express proinflammatory cytokines. In addition, Sema4D inhibited AUF1-induced proinflammatory mRNA decay effect. Taken together, our data provides evidence that Sema4D disrupts BBB integrity and promotes an inflammatory response by binding to PlexinB1 in pericytes after transient middle cerebral artery occlusion. Our study indicates that Sema4D may be a novel therapeutic target for treatment in the acute phase of stroke.-Zhou, Y.-F., Li, Y.-N., Jin, H.-J., Wu, J.-H., He, Q.-W., Wang, X.-X., Lei, H., Hu, B. Sema4D/PlexinB1 inhibition ameliorates blood-brain barrier damage and improves outcome after stroke in rats.

Abstract 146: Deficiency of Cystathionine beta Synthase Alters Blood Brain Barrier Integrity and Exacerbates Cerebral Ischemia Reperfusion Injury in Mice

Hyperhomocysteinemia is a risk factor for stroke; however the mechanisms by which elevated homocysteine leads to stroke are poorly defined. In a murine model of cystathionine beta synthase (CBS) deficiency, we investigated whether mild to severe elevation in plasma total homocysteine (tHcy) increases susceptibility to cerebral infarction and if this phenotype is associated with loss of blood brain barrier (BBB) integrity. We studied male Cbs -/- mice conditionally expressing a zinc-inducible mutated human CBS (I278T) transgene along with Cbs+/- and Cbs+/+ littermates at 10-14 weeks of age. The human transgene was allowed to express only until weaning to overcome the early mortality of Cbs-/- mice. Experimental stroke was induced with middle cerebral artery occlusion for one hour followed by 24 hours of reperfusion. TTC stained coronal sections were used to quantify the infarcted area. BBB integrity was assessed prior to injury using a standard Evan’s blue (EB) infusion method. Leakage of EB was quantified in brain homogenates and normalized to brain weight. Mild to severe increases in plasma tHcy were observed in Cbs +/- and Cbs -/- mice (6.1±0.3 and 309±18 μM, respectively) compared with Cbs+/+ littermates (3.1±0.6 μM, P<0.01). Both Cbs +/- and Cbs -/- mice exhibited significant increases in infarct size following ischemia-reperfusion injury as compared to Cbs+/+ mice (12.2±3% in Cbs+/+ mice vs. 35.1±7.7% in Cbs+/- and 27.7±7.8% in Cbs-/- mice, P<0.05). A significant increase in EB extravasation was observed in Cbs+/- and Cbs -/- mice (3.8±0.8 and 4.8±0.2 μg/g, respectively) compared to Cbs+/+ mice (1.7±0.4 μg/g, P<0.05). The similarity in extent of cerebral infarction observed in Cbs+/- and Cbs-/- mice suggests a threshold effect and that a mild increase in tHcy is enough to disrupt BBB integrity and produce a severe stroke phenotype in experimental murine models.

High-Throughput Screening forIdentification of Blood-Brain Barrier Integrity Enhancers: A Drug Repurposing Opportunity to Rectify Vascular Amyloid Toxicity.

The blood-brain barrier (BBB) is a dynamic interface that maintains brain homeostasis and protects it from free entry of chemicals, toxins, and drugs. The barrier function of the BBB is maintained mainly by capillary endothelial cells that physically separate brain from blood. Several neurological diseases, such as Alzheimer's disease (AD), are known to disrupt BBB integrity. In this study, a high-throughput screening (HTS) was developed to identify drugs that rectify/protect BBB integrity from vascular amyloid toxicity associated with AD progression. Assessing Lucifer Yellow permeation across in-vitro BBB model composed from mouse brain endothelial cells (bEnd3) grown on 96-well plate inserts was used to screen 1280 compounds of Sigma LOPAC®1280 library for modulators of bEnd3 monolayer integrity. HTS identified 62 compounds as disruptors, and 50 compounds as enhancers of the endothelial barrier integrity. From these 50 enhancers, 7 FDA approved drugs were identified with EC50 values ranging from 0.76-4.56 μM. Of these 7 drugs, 5 were able to protect bEnd3-based BBB model integrity against amyloid toxicity. Furthermore, to test the translational potential to humans, the 7 drugs were tested for their ability to rectify the disruptive effect of Aβ in the human endothelial cell line hCMEC/D3. Only 3 (etodolac, granisetron, and beclomethasone) out of the 5 effective drugs in the bEnd3-based BBB model demonstrated a promising effect to protect the hCMEC/D3-based BBB model integrity. These drugs are compelling candidates for repurposing as therapeutic agents that could rectify dysfunctional BBB associated with AD.

Depressive symptoms in chronic hepatitis C are associated with plasma apolipoprotein E deficiency

Neuro-psychiatric and cognitive disorders are frequent in patients with chronic hepatitis C (CHC) virus (HCV) infection which adversely impact quality of life, antiviral treatment adherence and outcome. HCV has neurotrophic properties and affects lipid metabolism, essential for cognitive function. We evaluated the relationship of lipid profiles with depression and anxiety symptoms and the effects of 12-weeks of therapy with fluvastatin and omega-3 ethyl esters (n-3 PUFA) in a randomised pilot study of CHC prior non-responders. Participants (n = 60) had fasting lipid profiles and assessment of depression and anxiety symptoms using the Hospital Anxiety and Depression Scale (HADS) questionnaire at each study visit. At screening 26/60 (43 %) had HADS-A score ≥8 and 13/60 (22 %) had HADS-D scores ≥8. Depressed patients had significantly lower apolipoprotein-E concentrations (30 mg/l vs 39 mg/l, P = 0.029) than those without depression and a tendency toward lower total cholesterol (3.8 vs 4.4 mmol/l, P = 0.053). 3 patients discontinued lipid-modifying treatment because of worsening depression. However, there was a small but significant improvement in anxiety symptoms after 12-weeks of high-dose (2-4 g daily) n-3 PUFA. In conclusion, depression in CHC is associated with plasma apoE deficiency. We postulate that apoE deficiency disrupts blood brain barrier integrity to promote HCV infection of the CNS. High-dose n-PUFAs may alleviate anxiety in some CHC patients but the use of lipid lowering therapy must be balanced against risks of worsening depression.

Abstract 49: Deletion of Mirna 17-92 in Cerebral Endothelial Cells Induces Disruption of BBB

Background: The MicroRNA 17-92 (miR17-92) cluster regulates endothelial homeostasis. However, the effect of the miR17-92 cluster in cerebral endothelial cells on blood brain barrier (BBB) has not been investigated. We examined BBB integrity in a transgenic mouse line in which miR17-92 cluster is ablated in endothelial cells. Methods and Results: Adult Tie2-Cre:miR17-92 flox/flox (Tie2/miR17-92) mice and Tie2-Cre:Tomato (Tie2/red) reporter mice were used. Cerebral vessels in Tie2/red reporter mice exhibited red fluorescence, indicating specific expression of Tie2 in cerebral endothelial cells. RT-PCR analysis revealed ~70% reduction of miRNA levels of individual members of the miR17-92 cluster in endothelial cells harvested from Tie2/miR17-92 mouse brain, confirming deletion of the miR17-92 cluster. Time-lapse microscopic analysis of cultured endothelial cells showed a substantial reduction of capillary formation by miR17-92 cluster knockout endothelial cells compared to the cells harvested from Tie2/red reporter mice. In vivo, analysis of 3D cerebral microvessels showed that ablation of the miR17-92 markedly (P<0.05) increased capillary diameter in the cortex (7.1±0.4μm vs 5.1±0.3μm in reporter, n=4 mice/group), corpus callosum (CC, 6.6±0.4 vs 4.7±0.3), and striatum (6.7±0.4 vs 5.0±0.3). Triple immunofluorescent staining showed that the absence of this cluster substantially (p<0.05) reduced pericytes identified by platelet-derived growth factor-β positive cells (32±6 cells vs 80±6 cells in cortex, 34±7 vs 77±6 in CC, and 36±6 vs 79±5 in striatum) and decreased astrocyte end-foot processes identified by aquaporin 4 positive cells (42±6 cells vs 72±6 cells in cortex, 40±10 vs 70±6 in CC, and 36±6 vs 65±8 in striatum). These data suggest that cerebral endothelil cells lacking miR17-92 causes disruption of vascular coverage by pericytes and astrocyte end-foot processes. Deletion of the miR17-92 cluster also increased extravasation of Evans blue dye into the parenchyma and reduced the number of doublecortin positive neuroblasts in the subventricular zone. Conclusion: Our data indicate that ablation of the miR17-92 cluster in cerebral endothelial cells disrupts BBB integrity, which could exacerbate ischemic damage.

Disruption of the blood-brain barrier in parkinson's disease: curse or route to a cure?

The vertebrate blood-brain barrier (BBB) is critical for ensuring the maintenance of brain homeostasis, whilst protecting the brain against toxic insults. Various pathological events disrupt BBB integrity, holding several important clinical implications. In instances where the normal mechanisms controlling passage of substances into the brain are compromised, these could sensitize or even worsen endogenous pathological conditions. Recognition has grown recently that patients diagnosed with Parkinson's disease (PD) present with concurrent medical problems, including cerebrovascular lesions. However, cerebrovascular disturbances may also result from PD-related disease processes; the pathological mechanisms which could entail interaction between environment-derived and genetic factors. The current review addresses the accumulation of studies aimed at better understanding the series of processes affecting the neurovascular unit in human Parkinsonism, due in part to the BBB presenting as a formidable opponent in the effective delivery of therapeutics that have shown promise as therapeutic strategies for treating aspects of PD when tested in vitro.

A novel bioactivity of andrographolide from Andrographis paniculata on cerebral ischemia/reperfusion-induced brain injury through induction of cerebral endothelial cell apoptosis

Context: Andrographolide, extracted from the leaves of Andrographis paniculata (Burm. f.) Nees (Acanthaceae), is a labdane diterpene lactone. It is widely reported to possess anti-inflammatory and antitumorigenic activities. Cerebral endothelial cells (CECs) play a crucial role in supporting the integrity and the function of the blood–brain barrier (BBB). However, no data are available concerning the effects of andrographolide in CECs. The aim of this study was to examine the detailed mechanisms of andrographolide on CECs.Objective: This study investigated a novel bioactivity of andrographolide on cerebral ischemia/reperfusion-induced brain injury.Materials and methods: CECs were treated with andrographolide (20–100 µΜ) for the indicated times (0–24 h). After the reactions, cell survival rate and cytotoxicity were tested by the MTT assay and the lactate dehydrogenase (LDH) test, respectively. Western blotting was used to detect caspase-3 expression. In addition, analysis of cell cycle and apoptosis using PI staining and annexin V-FITC/PI labeling, respectively, was performed by flow cytometry. We also investigated the effect of andrographolide on middle cerebral artery occlusion (MCAO)/reperfusion-induced brain injury in a rat model.Results: In the present study, we found that andrographolide (50–100 µΜ) markedly inhibited CEC growth according to an MTT assay and caused CEC damage according to a LDH test. Our data also revealed that andrographolide (50 µM) induced CEC apoptosis and caspase-3 activation as respectively detected by PI/annexin-V double staining and western blotting. Moreover, andrographolide arrested the CEC cell cycle at the G0/G1 phase by PI staining. In addition, andrographolide (5 mg/kg) caused deterioration of MCAO/reperfusion-induced brain injury in a rat model.Conclusions: These data suggest that andrographolide may disrupt BBB integrity, thereby deteriorating MCAO/reperfusion-induced brain injury, which are, in part, associated with its capacity to arrest cell-cycle and induce CEC apoptosis.