Dong-Xin Liu

Inactivation of EphA2 promotes tight junction formation and impairs angiogenesis in brain endothelial cells.

Eph receptor tyrosine kinases and ephrin ligands participate in the regulation of a wide variety of biological processes, such as axon guidance, synaptic plasticity, angiogenesis, and tumorigenesis. The role of Eph receptors and ephrin ligands in brain endothelial cells remains unknown. Here, we examined the expression profile of EphA receptors and ephrin-A ligands in human brain microvascular endothelial cell line (HBMEC). Our results showed that multiple EphA receptors and ephrin-A ligands are expressed in HBMEC. We found that the phosphorylation of EphA2, but not other EphA receptors, was significantly increased in HBMEC treated with recombinant ephrin-A1/Fc. Meanwhile, elevated EphA2 phosphorylation was accompanied by disassembly of tight junctions in HBMEC. Furthermore, EphA2 RNAi in HBMEC could promote tight junction formation and prevent the ephrin-A1-induced tight junction disruption. Also, when a kinase-inactive mutant of EphA2 (EphA2-K646M) was expressed in HBMEC, the tight junction was enhanced and the ephrin-A1-induced tight junction disruption was blocked. In addition, EphA2 RNAi and expression of EphA2-K646M in HBMEC inhibited in vitro cell migration and angiogenesis of HBMEC. These data indicated an important role of EphA2 in regulating both tight junction formation and angiogenesis in brain endothelial cells.

Ephrin-A3 and Ephrin-A4 Contribute to Microglia-Induced Angiogenesis in Brain Endothelial Cells

The association of microglia with brain vasculature during development and the reduced brain vascular complexity in microglia‐deficient mice suggest the role of microglia in cerebrovascular angiogenesis. However, the underlying molecular mechanism remains unclear. Here, using an in vitro angiogenesis model, we found the culture supernatant of BV2 microglial cells significantly enhanced capillary‐like tube formation and migration of brain microvascular endothelial cells (BMECs). The expression of angiogenic factors, ephrin‐A3 and ephrin‐A4, were specifically upregulated in BMECs exposed to BV2‐derived culture supernatant. Knockdown of ephrin‐A3 and ephrin‐A4 in BMECs by siRNA significantly attenuated the enhanced angiogenesis and migration of BMECs induced by BV2 supernatant. Our further results indicated that the ability of BV2 supernatant to promote endothelial angiogenesis was caused by the soluble tumor necrosis factor α (TNF‐α) released from BV2 microglial cells. Moreover, the upregulations of ephrin‐A3 and ephrin‐A4 in BMECs in response to BV2 supernatant were effectively abolished by neutralization antibody against TNF‐α and TNF receptor 1, respectively. The present study provides evidence that microglia upregulates endothelial ephrin‐A3 and ephrin‐A4 to facilitate in vitro angiogenesis of brain endothelial cells, which is mediated by microglia‐released TNF‐α. Anat Rec, 297:1908–1918, 2014.

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF‐induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC‐dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF‐induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF‐induced redistribution of ZO‐1 and the recruitment of detergent‐soluble occludin and claudin‐5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time‐ and dose‐dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF‐induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant‐negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF‐mediated changes in paracellular permeability. Anat Rec, 297:964–972, 2014.

Decreased expression of cathepsin D in monocytes is related to the defective degradation of amyloid-β in Alzheimer's disease.

Alzheimers disease (AD) is a progressive neurodegenerative dementia characterized by pathological senile plaques composed of amyloid-β (Aβ) in the cerebral cortex and hippocampus. Bone marrow-derived monocytes of patients with AD migrate across the blood-brain barrier into the brain, but are defective at clearing Aβ in the neuritic plaques. However, the underlying mechanisms remain unclear. Here, in patients with AD, we found that cathepsin D, a major lysosomal aspartic protease, was underexpressed in monocytes, resulting in the defective degradation of Aβ by monocytes/macrophages. Further, downregulation of cathepsin D in THP-1 cells significantly reduced the clearance of amyloid plaques in the brain sections of AβPP/PS1 mice. The clearance ability was recovered by the overexpression of cathepsin D in AD monocytes. These results suggest that decreased expression of cathepsin D in the peripheral monocytes is a potential signature of AD, and that this decreased expression is involved in Aβ degradation and AD pathogenesis.

cPLA2α-mediated actin rearrangements downstream of the Akt signaling is required for Cronobacter sakazakii invasion into brain endothelial cells.

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that causes sepsis and meningitis in neonate. The molecular mechanism involved in the pathogenesis of C. sakazakii meningitis remains unclear. In this study, we found that C. sakazakii invasion was significantly decreased in human brain microvascular endothelial cells (HBMEC) treated with cytosolic phospholipases A(2)α (cPLA(2)α) inhibitor. Increased phosphorylation of cPLA(2)α was observed in HBMEC infected with C. sakazakii, which was prevented by treatment with cPLA(2)α inhibitor. cPLA(2)α knockdown in HBMEC significantly attenuated C. sakazakii invasion into HBMEC. Immunofluorescence demonstrated that the rearrangements of actin filaments in HBMEC induced by C. sakazakii were effectively blocked by either treatment with cPLA(2)α inhibitor or transfection with cPLA(2)α siRNA. Interestingly, we found that C. sakazakii infection promoted the aggregation of phosphorylated cPLA(2)α, which was associated with depolymerized actin filaments in HBMEC. Furthermore, our data revealed that cPLA(2)α acts downstream of Akt signaling pathway in HBMEC stimulated with C. sakazakii. Taken together, our results illustrated that cPLA(2)α-mediated actin filament rearrangements downstream of Akt activation is required for C. sakazakii invasion into brain endothelial cells.

Cystatin C Shifts APP Processing from Amyloid-β Production towards Non-Amyloidgenic Pathway in Brain Endothelial Cells

Amyloid-β (Aβ), the major component of neuritic plaques in Alzheimer’s disease (AD), is derived from sequential proteolytic cleavage of amyloid protein precursor (APP) by secretases. In this study, we found that cystatin C (CysC), a natural cysteine protease inhibitor, is able to reduce Aβ40 secretion in human brain microvascular endothelial cells (HBMEC). The CysC-induced Aβ40 reduction was caused by degradation of β-secretase BACE1 through the ubiquitin/proteasome pathway. In contrast, we found that CysC promoted secretion of soluble APPα indicating the activated non-amyloidogenic processing of APP in HBMEC. Further results revealed that α-secretase ADAM10, which was transcriptionally upregulated in response to CysC, was required for the CysC-induced sAPPα secretion. Knockdown of SIRT1 abolished CysC-triggered ADAM10 upregulation and sAPPα production. Taken together, our results demonstrated that exogenously applied CysC can direct amyloidogenic APP processing to non-amyloidgenic pathway in brain endothelial cells, mediated by proteasomal degradation of BACE1 and SIRT1-mediated ADAM10 upregulation. Our study unveils previously unrecognized protective role of CysC in APP processing.

ZO-1 expression is suppressed by GM-CSF via miR-96/ERG in brain microvascular endothelial cells:

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases in some disorders such as vascular dementia, Alzheimer’s disease, and multiple sclerosis. We previously reported that in Alzheimer’s disease patients, a high level of GM-CSF in the brain parenchyma downregulated expression of ZO-1, a blood–brain barrier tight junction protein, and facilitated the infiltration of peripheral monocytes across the blood–brain barrier. However, the molecular mechanism underlying regulation of ZO-1 expression by GM-CSF is unclear. Herein, we found that the erythroblast transformation-specific (ETS) transcription factor ERG cooperated with the proto-oncogene protein c-MYC in regulation of ZO-1 transcription in brain microvascular endothelial cells (BMECs). The ERG expression was suppressed by miR-96 which was increased by GM-CSF through the phosphoinositide-3 kinase (PI3K)/Akt pathway. Inhibition of miR-96 prevented ZO-1 down-regulation induced by GM-CSF both in vitro and in vivo. Our results revealed the mechanism of ZO-1 expression reduced by GM-CSF, and provided a potential target, miR-96, which could block ZO-1 down-regulation caused by GM-CSF in BMECs.

Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

The formation of brain vasculature is an essential step during central nervous system development. The molecular mechanism underlying brain angiogenesis remains incompletely understood. The role of Atg7, an autophagy-related protein, in brain angiogenesis was investigated in this study. We found that the microvessel density in mice brains with endothelial-specific knockout of Atg7 (Atg7 EKO) was significantly decreased compared to wild-type control. Consistently, in vitro angiogenesis assays showed that Atg7 knockdown impaired angiogenesis in brain microvascular endothelial cells. Further results indicated that knockdown of Atg7 reduced interleukin-6 (IL-6) expression in brain microvascular endothelial cells, which is mediated by NF-κB-dependent transcriptional control. Interestingly, exogenous IL-6 restored the impaired angiogenesis and reduced cell motility caused by Atg7 knockdown. These results demonstrated that Atg7 has proangiogenic activity in brain angiogenesis which is mediated by IL-6 production in a NF-κB-dependent manner.

Caspr1 is a host receptor for meningitis-causing Escherichia coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but the pathogenesis of E. coli meningitis remains elusive. E. coli penetration of the blood–brain barrier (BBB) is the critical step for development of meningitis. Here, we identify Caspr1, a single-pass transmembrane protein, as a host receptor for E. coli virulence factor IbeA to facilitate BBB penetration. Genetic ablation of endothelial Caspr1 and blocking IbeA–Caspr1 interaction effectively prevent E. coli penetration into the brain during meningitis in rodents. IbeA interacts with extracellular domain of Caspr1 to activate focal adhesion kinase signaling causing E. coli internalization into the brain endothelial cells of BBB. E. coli can invade hippocampal neurons causing apoptosis dependent on IbeA–Caspr1 interaction. Our results indicate that E. coli exploits Caspr1 as a host receptor for penetration of BBB resulting in meningitis, and that Caspr1 might be a useful target for prevention or therapy of E. coli meningitis.Penetration of the blood–brain barrier (BBB) is crucial for development of E. coli-caused meningitis. Here, the authors show that a host membrane protein, Caspr1, acts as a receptor for a bacterial virulence factor to facilitate BBB penetration and entry of E. coli into brain neurons.

Escherichia coli hijack Caspr1 receptor to invade cerebral vascular and neuronal hosts

Escherichia coli (E. coli) penetration of the blood-brain barrier (BBB) is the key step essential for the development of meningitis. In a recent paper (Nat Commun 9:2296), we identify Caspr1 as a host receptor for E. coli virulence factor IbeA to pave the way the penetration of bacteria through the BBB. Bacterial IbeA interacts with endothelial Caspr1 to trigger intracellular focal adhesion kinase activation, leading to E. coli internalization into the brain endothelial cells. Importantly, endothelial knockout of Caspr1 in mice significantly reduced E. coli crossing through the BBB. Based on the results that extracellular aa 203-355 of Caspr1 bind with IbeA, we tested the blocking effect of recombinant Caspr1(203-355) peptides in neonatal rat model of meningitis. The results showed that Caspr1(203-355) peptides effectively attenuated E. coli penetration into the brain during meningitis, indicating that Caspr1(203-355) peptides could be used to neutralize the virulent IbeA to prevent meningitis. We further found that E. coli can directly invade into hippocampal neurons causing apoptosis which required the interaction between bacterial IbeA and neuronal Caspr1. These findings demonstrate that E. coli hijack Caspr1 as a host receptor for penetration of BBB and invasion of hippocampal neurons, resulting in progression of meningitis.