Human Cerebral Microvascular Endothelial Cells Research Articles

Major involvement of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells.

The purpose of this study was to clarify the expression of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood-brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody-free method established by Kubo etal. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock-down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [(3) H]biotin and [(3) H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6-mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood-brain barrier. In humans, it was unclear (not concluded) about what transport system at the blood-brain barrier (BBB) is responsible for the brain uptakes of two vitamins, biotin and pantothenic acid, which are necessary for brain proper function. This study clarified for the first time that the solute carrier 5A6/Na(+) -dependent multivitamin transporter SLC5A6/SMVT is responsible for the supplies of biotin and pantothenic acid into brain across the BBB in humans. DHA, docosahexaenoic acid; NSAID, non-steroidal anti-inflammatory drug; PGE2, prostaglandin E2.

Aryl hydrocarbon receptor regulates CYP1B1 but not ABCB1 and ABCG2 in hCMEC/D3 human cerebral microvascular endothelial cells after TCDD exposure

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor activated by a variety of widespread persistent environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It can transactivate the expression of several target genes. Recently AhR transcripts were detected in isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line, an in vitro model of the human cerebral endothelium. To date AhR implication in the co-regulation of ABCB1, ABCG2 and CYP1B1 at human cerebral endothelium has not been addressed. Here we investigated whether AhR could co-regulate ABCB1, ABCG2 and CYP1B1 expressions in the hCMEC/D3 cell line. Exposure to TCDD induced a concentration-dependent increase in CYP1B1 expression. We demonstrated AhR involvement in the TCDD-mediated increase in CYP1B1 expression by using small interfering RNA against AhR. Western blotting analysis also revealed an increase in CYP1B1 protein expression following TCDD exposure in hCMEC/D3. Regarding ABCB1 and ABCG2, exposure to TCDD had no effect on their protein expressions and functional activities. In conclusion our data indicated a differential modulation of CYP1B1 and ABCB1/ABCG2 expressions in hCMEC/D3 cells following TCDD exposure.

Altered Nrf2 signaling mediates hypoglycemia-induced blood-brain barrier endothelial dysfunction in vitro.

Hypoglycemia impairs blood-brain barrier (BBB) endothelial function; a major hallmark in the pathogenesis of various CNS disorders. Previously, we have demonstrated that prolonged hypoglycemic exposure down-regulated BBB endothelial NF-E2 related factor-2 (Nrf2) expression; a redox-sensitive transcriptional factor that regulates endothelial function. Here, we sought to determine the functional role of Nrf2 in preserving BBB integrity and molecular mechanisms underlying hypoglycemia-induced Nrf2 down-regulation in vitro using human cerebral microvascular endothelial cell line (hCMEC/D3). Cell monolayers were exposed to normal or hypoglycemic (5.5 or 2.2mM D-glucose) media for 3-24h. Pharmacological or gene manipulation (by silencing RNA) approaches were used to investigate specific molecular pathways implicated in hypoglycemia-induced Nrf2 degradation. BBB integrity was assessed by paracellular permeability to labeled dextrans of increasing molecular sizes (4-70kDa). Silencing Nrf2 expression in hCMEC/D3 cells abrogated the expression of claudin-5 and VE-cadherin, while ZO-1 was up-regulated. These effects were paralleled by a decrease in electrical resistance of hCMEC/D3 monolayers and potential increase in permeability to all labeled dextrans. Hypoglycemic exposure (3-24h) led to progressive and sustained down-regulation of Nrf2 (without affecting mRNA) and its target, NQO-1, with a concomitant increase in the cytosolic pool of E3 ubiquitin ligase, Siah2 (but not Keap1). Pretreatment with protease inhibitor MG132, or selective knock-down of Siah2 (but not Keap1) significantly attenuated hypoglycemia-induced Nrf2 destabilization. While hypoglycemic exposure triggered a significant increase in BBB permeability to dextrans, silencing Siah2 gene abrogated the effects of hypoglycemia and restored BBB integrity. In summary, our data indicate a potential role for Nrf2 signaling in regulating tight junction integrity and maintaining BBB function. Nrf2 suppression by increased Siah2-driven proteasomal degradation mediates hypoglycemia-evoked endothelial dysfunction and loss of BBB integrity. Overall, this study suggests that sustained activation of endothelial Nrf2 signaling could have therapeutic potential to prevent hypoglycemia-induced cerebrovascular dysfunction.

Brain endothelial miR-146a negatively modulates T-cell adhesion through repressing multiple targets to inhibit NF-κB activation.

Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation.

Endophilin-1 regulates blood-brain barrier permeability via EGFR-JNK signaling pathway

Endophilin-1 (Endo1), a multifunctional protein, is essential for synaptic vesicle endocytosis. However, the role and mechanism of endophilin-1 in blood–brain barrier (BBB) function are still unclear. This study was performed to determine whether endophilin-1 regulated BBB permeability via the EGFR-JNK signaling pathway. In the present study, we found that endophilin-1 over-expression in human cerebral microvascular endothelial cell (hCMEC/D3) increased BBB permeability and meanwhile reduced the expression levels of epidermal growth factor receptor (EGFR), phosphorylated c-Jun N-terminal kinase (p-JNK). While endophilin-1 knockdown led to the contrary results. After JNK inhibitor SP600125 was administered to the endophilin-1 silenced hCMEC/D3 cells, the transendothelial electrical resistance (TEER) value was decreased and the permeability coefficient values to 4kDa and 40kDa FITC–dextran were increased. Results observed by Transmission electron microscopy (TEM) showed that tight junctions (TJs) were opened. Moreover, immunofluorescence and Western blot assays revealed the discontinuous distribution of TJ-associated proteins ZO-1, occludin on cell–cell boundaries and a significant decrease in protein expressing levels. Therefore, these results indicated that endophilin-1 positively regulated BBB permeability via the EGFR-JNK signaling pathway in hCMEC/D3 cells, which would provide an experimental basis for further research on endophilin-1 mediated the opening of BBB.

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood-brain barrier.

Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.

LPS Induces Occludin Dysregulation in Cerebral Microvascular Endothelial Cells via MAPK Signaling and Augmenting MMP-2 Levels.

Disrupted blood-brain barrier (BBB) integrity contributes to cerebral edema during central nervous system infection. The current study explored the mechanism of lipopolysaccharide- (LPS-) induced dysregulation of tight junction (TJ) proteins. Human cerebral microvascular endothelial cells (hCMEC/D3) were exposed to LPS, SB203580 (p38MAPK inhibitor), or SP600125 (JNK inhibitor), and cell vitality was determined by MTT assay. The proteins expressions of p38MAPK, JNK, and TJs (occludin and zonula occludens- (ZO-) 1) were determined by western blot. The mRNA levels of TJ components and MMP-2 were measured with quantitative real-time polymerase chain reaction (qRT-PCR), and MMP-2 protein levels were determined by enzyme-linked immunosorbent assay (ELISA). LPS, SB203580, and SP600125 under respective concentrations of 10, 7.69, or 0.22 µg/mL had no effects on cell vitality. Treatment with LPS decreased mRNA and protein levels of occludin and ZO-1 and enhanced p38MAPK and JNK phosphorylation and MMP-2 expression. These effects were attenuated by pretreatment with SB203580 or SP600125, but not in ZO-1 expression. Both doxycycline hyclate (a total MMP inhibitor) and SB-3CT (a specific MMP-2 inhibitor) partially attenuated the LPS-induced downregulation of occludin. These data suggest that MMP-2 overexpression and p38MAPK/JNK pathways are involved in the LPS-mediated alterations of occludin in hCMEC/D3; however, ZO-1 levels are not influenced by p38MAPK/JNK.

Hypoxia interferes with aryl hydrocarbon receptor pathway in hCMEC/D3 human cerebral microvascular endothelial cells.

The expression of aryl hydrocarbon receptor (AhR) transcription factor was detected at transcript level in freshly isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line. Recent studies have demonstrated that AhR pathway is able to crosstalk with other pathways such as hypoxia signaling pathway. Therefore, we used the hCMEC/D3 cell line to investigate the potential crosstalk between AhR and hypoxia signaling pathways. First, we performed two different hypoxia-like procedures in hCMEC/D3 cells; namely, exposition of cells to 150 μM deferoxamine or to glucose and oxygen deprivation for 6 h. These two procedures led to hypoxia-inducible factor (HIF)-1α and HIF-2α proteins accumulation together with a significant induction of the two well-known hypoxia-inducible genes VEGF and GLUT-1. Both HIF-1α and -2α functionally mediated hypoxia response in the hCMEC/D3 cells. Then, we observed that a 6 h exposure to 25 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin, a strong AhR ligand, up-regulated CYP1A1 and CYP1B1 expression, and that this effect was AhR dependent. Regarding AhR and hypoxia crosstalk, our experiments revealed that an asymmetric interference between these two pathways effectively occurred in hCMEC/D3 cells: hypoxia pathway interfered with AhR signaling but not the other way around. We studied the putative crosstalk of AhR and hypoxia pathways in hCMEC/D3 human cerebral microvascular endothelial cells. While hypoxia decreased the expression of the two AhR target genes CYP1A1 and CYP1B1, AhR activation results in no change in hypoxia target gene expression. This is the first sign of AhR and hypoxia pathway crosstalk in an in vitro model of the human cerebral endothelium.

In vitro modeling of the neurovascular environment by coculturing adult human brain endothelial cells with human neural stem cells.

Brain and vascular cells form a functionally integrated signalling network that is known as the neurovascular unit (NVU). The signalling (autocrine, paracrine and juxtacrine) between different elements of this unit, especially in humans, is difficult to disentangle in vivo. Developing representative in vitro models is therefore essential to better understand the cellular interactions that govern the neurovascular environment. We here describe a novel approach to assay these cellular interactions by combining a human adult cerebral microvascular endothelial cell line (hCMEC/D3) with a fetal ganglionic eminence-derived neural stem cell (hNSC) line. These cell lines provide abundant homogeneous populations of cells to produce a consistently reproducible in vitro model of endothelial morphogenesis and the ensuing NVU. Vasculature-like structures (VLS) interspersed with patches of differentiating neural cells only occurred when hNSCs were seeded onto a differentiated endothelium. These VLS emerged within 3 days of coculture and by day 6 were stabilizing. After 7 days of coculture, neuronal differentiation of hNSCs was increased 3-fold, but had no significant effect on astrocyte or oligodendrocyte differentiation. ZO1, a marker of adherens and tight junctions, was highly expressed in both undifferentiated and differentiated endothelial cells, but the adherens junction markers CD31 and VE-cadherin were significantly reduced in coculture by approximately 20%. A basement membrane, consisting of laminin, vitronectin, and collagen I and IV, separated the VLS from neural patches. This simple assay can assist in elucidating the cellular and molecular signaling involved in the formation of VLS, as well as the enhancement of neuronal differentiation through endothelial signaling.

Complete nucleotide sequence of pRS218, a large virulence plasmid, that augments pathogenic potential of meningitis-associated Escherichia coli strain RS218.

BackgroundEscherichia coli is the most predominant Gram-negative bacterial pathogen associated with neonatal meningitis. Previous studies indicated that the prototypic neonatal meningitis E. coli (NMEC) strain RS218 (O18:K1:H7) harbors one large plasmid. Objectives of the present study were to analyze the complete nucleotide sequence of this large plasmid (pRS218) and its contribution to NMEC pathogenesis using in vitro and in vivo models of neonatal meningitis.ResultsThe plasmid is 114,231 bp in size, belongs to the incompatibility group FIB/IIA (IncFIB/IIA), and contains a genetic load region that encodes several virulence and fitness traits such as enterotoxicity, iron acquisition and copper tolerance. The nucleotide sequence of pRS218 showed a 41- 46% similarity to other neonatal meningitis-causing E. coli (NMEC) plasmids and remarkable nucleotide sequence similarity (up to 100%) to large virulence plasmids of E. coli associated with acute cystitis. Some genes located on pRS218 were overly represented by NMEC strains compared to fecal E. coli isolated from healthy individuals. The plasmid-cured strain was significantly attenuated relative to the RS218 wild-type strain as determined in vitro by invasion potential to human cerebral microvascular endothelial cells and in vivo by mortalities, histopathological lesions in the brain tissue, and bacterial recovery from the cerebrospinal fluid of infected rat pups.ConclusionsThe pRS218 is an IncFIB/IIA plasmid which shares a remarkable nucleotide sequence similarity to large plasmids of E. coli associated with cystitis. Both in vitro and in vivo experiments indicated that pRS218 plays an important role in NMEC pathogenesis.

Microvascular lesions by estrogen-induced ID3: its implications in cerebral and cardiorenal vascular disease.

Severe symptoms of cerebral and cardiorenal vascular diseases can be triggered when cerebral, coronary, or glomerular arterioles grow inappropriately as a result of abnormal cell proliferation. The risk factor(s) and molecular mechanisms responsible for microvascular lesion formation are largely unknown. Although controversial, both animal and epidemiological studies have shown that estrogen increases the risk of stroke which may be due to microvascular lesions. Since microvascular diseases are characterized by excessive vessel growth, it is plausible that estrogen-induced neovascularization contributes to the growth of microvascular lesions. We present evidence for how ID3 overexpression in endothelial cells contributes to the development of an estrogen-induced neovascular phenotype with an additional focus on Pyk2 kinase. Our data showed that ID3 overexpression increased neovascularization, cell migration, and spheroid growth of human cerebral microvascular endothelial cells, hCMEC/D3. ID3-overexpressing cells showed significant estrogen-induced G2/M phase transition. Estrogen treatment increased both ID3 phosphorylation; total protein that was inhibited by tamoxifen, and Pyk2-mediated estrogen-induced ID3 mRNA expression. These findings suggest that Pyk2 signals ID3 expression and ID3 is necessary for estrogen-induced neovascularization in hCMEC/D3 cells. A better understanding of how microvascular lesions depend on ID3 may open new avenues for prevention and treatment of neurological diseases.

Traumatic injury elicits JNK-mediated human astrocyte retraction in vitro

Brain injury causes dysfunction of the blood–brain barrier (BBB). The BBB is comprised of perivascular astrocytes whose end-feet ensheath brain microvascular endothelial cells. We investigated trauma-induced morphological changes of human astrocytes (HA) and human cerebral microvascular endothelial cells (hCMEC/D3) in vitro, including the potential role of mitogen-activated protein kinase (MAPK) signal-transduction pathways. HA or hCMEC/D3 were grown on flexible culture membranes and subjected to single traumatic injury normalized to 20%, 30% or 55% membrane deformation. Cells were assayed for morphological changes (i.e. retraction) and MAPK phosphorylation and/or expression (c-Jun NH2-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and p38). HA retraction was rapidly elicited with a single traumatic injury (55% membrane deformation; p<0.01). Morphological recovery of HA was observed within 2h (p<0.05). Traumatic injuries increased phospho-JNK1/2 (p<0.05) in HA, indicating MAPK activation. Pre-treatment of HA with structurally distinct JNK inhibitors (25μM), either SP600125 or SU3327, reduced JNK phosphorylation (p<0.05) and trauma-induced HA retraction (P<0.05). In contrast to HA, traumatic injury failed to induce either morphological changes or MAPK activation in hCMEC/D3. In summary, traumatic injury induces JNK-mediated HA retraction in vitro, while sparing morphological changes in cerebral microvascular endothelial cells. Astrocyte retraction from microvascular endothelial cells in vivo may occur after brain trauma, resulting in cellular uncoupling and BBB dysfunction. JNK may represent a potential therapeutic target for traumatic brain injuries.

A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding.

We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3), to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R) was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR) was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation.

Concussive injury elicits JNK‐mediated human astrocyte retraction (651.10)

Background: The pathology of traumatic brain injury (TBI) is poorly understood, but likely involves blood‐brain barrier (BBB) dysfunction. The BBB is lined with microvascular endothelial cells, ensheathed by astrocyte endfeet. We investigated human astrocytes and endothelial cell responses to concussive injury in vitro. Methods: Concussive injury in vitro was elicited by using a Cell Injury Controller II, a device which induces regulated and repeatable pulse of gas pressure to deform a flexible membrane. Human astrocytes (HA) or human immortalized cerebral microvascular endothelial cells (hCMEC/D3) grown on flexible culture membranes were subjected to a single (20, 35, or 55% deformation) or repeated (20% deformation; 6‐consecutive injuries spanned every 2 minutes) concussive injuries. Cells were assayed for morphological changes (retraction), detachment/cell death, and MAPK activation (phosphorylation of JNK1/2, ERK1/2, and p38).Results: Significant HA retraction was elicited 30 min following either a single (55% deformation) or repeated concussive injury. HA retraction was not associated with cell death or detachment. HA recovered morphologically after 2 hrs post‐injury. Concussive injury increased phospho‐JNK1/2, but not phospho‐ERK1/2 or phospho‐p38. Pre‐treatment of HA with JNK inhibitors (SP600125 or SU3327) reduced injury‐induced HA retraction. In contrast, concussive injury failed to induce either morphological changes or MAPK activation in hCMEC/D3 Conclusion: Concussive injury induces JNK‐mediated astrocyte retraction in vitro, while sparing morphological changes in endothelial cells. In vivo, astrocyte retraction from endothelial cells may result in BBB dysfunction. JNK inhibition may be a potential therapeutic target for TBI.Grant Funding Source: Supported by the Children's Health Foundation (London ON) and HSFO Grant NA6914

CXCL1/CXCL8 (GROα/IL-8) in human diabetic ketoacidosis plasma facilitates leukocyte recruitment to cerebrovascular endothelium in vitro

Diabetic ketoacidosis (DKA) in children is associated with intracranial vascular complications, possibly due to leukocyte-endothelial interactions. Our aim was to determine whether DKA-induced inflammation promoted leukocyte adhesion to activated human cerebrovascular endothelium. Plasma was obtained from children with type 1 diabetes either in acute DKA or in an insulin-controlled state (CON). Plasma concentrations of 21 inflammatory analytes were compared between groups. DKA was associated with altered circulating levels of ↑CXCL1 (GROα), ↑CXCL8 (IL-8), ↑IL-6, ↑IFNα2, and ↓CXCL10 (IP-10) compared with CON. These plasma analyte measurements were then used to create physiologically relevant cytokine mixtures (CM). Human cerebral microvascular endothelial cells (hCMEC/D3) were stimulated with either plasma (DKA-P or CON-P) or CM (DKA-CM or CON-CM) and assessed for polymorphonuclear leukocyte (PMN) adhesion. Stimulation of hCMEC/D3 with DKA-P or DKA-CM increased PMN adhesion to hCMEC/D3 under "flow" conditions. PMN adhesion to hCMEC/D3 was suppressed with neutralizing antibodies to CXCL1/CXCL8 or their hCMEC/D3 receptors CXCR1/CXCR2. DKA-P, but not DKA-CM, initiated oxidative stress in hCMEC/D3. Expression of ICAM-1, VCAM-1, and E-selectin were unaltered on hCMEC/D3 by either DKA-P or DKA-CM. In summary, DKA elicits inflammation in children associated with changes in circulating cytokines/chemokines. Increased CXCL1/CXCL8 instigated PMN adhesion to hCMEC/D3, possibly contributing to DKA-associated intracranial vascular complications.

Immature mesenchymal stem cell-like pericytes as mediators of immunosuppression in human malignant glioma

Malignant gliomas are primary brain tumors characterized by profound local immunosuppression. While the remarkable plasticity of perivascular cells – resembling mesenchymal stem cells (MSC) – in malignant gliomas and their contribution to angiogenesis is increasingly recognized, their role as potential mediators of immunosuppression is unknown. Here we demonstrate that FACS-sorted malignant glioma-derived pericytes (HMGP) were characterized by the expression of CD90, CD248, and platelet-derived growth factor receptor-β (PDGFR-β). HMGP shared this expression profile with human brain vascular pericytes (HBVP) and human MSC (HMSC) but not human cerebral microvascular endothelial cells (HCMEC). CD90+PDGFR-β+perivascular cells distinct from CD31+ endothelial cells accumulated in human gliomas with increasing degree of malignancy and negatively correlated with the presence of blood vessel-associated leukocytes and CD8+ T cells. Cultured CD90+PDGFR-β+HBVP were equally capable of suppressing allogeneic or mitogen-activated T cell responses as human MSC. HMGP, HBVP and HMSC expressed prostaglandin E synthase (PGES), inducible nitric oxide synthase (iNOS), human leukocyte antigen-G (HLA-G), hepatocyte growth factor (HGF) and transforming growth factor-β (TGF-β). These factors but not indoleamine 2,3-dioxygenase-mediated conversion of tryptophan to kynurenine functionally contributed to immunosuppression of immature pericytes. Our data provide evidence that human cerebral CD90+ perivascular cells possess T cell inhibitory capability comparable to human MSC and suggest that these cells, besides their critical role in tumor vascularization, also promote local immunosuppression in malignant gliomas and possibly other brain diseases.

Cytokine-induced changes in the gene expression profile of a human cerebral microvascular endothelial cell-line, hCMEC/D3

BackgroundThe human cerebral microvascular endothelial cell line, hCMEC/D3, has been used extensively to model the blood–brain barrier (BBB) in vitro. Recently, we reported that cytokine-treatment induced loss of brain endothelial barrier properties. In this study, we further determined the gene expression pattern of hCMEC/D3 cells in response to activation with TNFα and IFNγ.FindingsUsing a microarray approach, we observed that expression of genes involved in the control of barrier permeability, including inter-brain endothelial junctions (e.g. claudin-5, MARVELD-2), integrin-focal adhesions complexes (e.g. integrin β1, ELMO-1) and transporter systems (e.g. ABCB1, SLC2A1), are altered by pro-inflammatory cytokines.ConclusionsOur study shows that previously-described cytokine-induced changes in the pattern of gene expression of endothelium are reproduced in hCMEC/D3 cells, suggesting that this model is suitable to study inflammation at the BBB, while at the same time it has provided insights into novel key molecular processes that are altered in brain endothelium during neuroinflammation, such as modulation of cell-to-matrix contacts.

Extracellular RNA Liberates Tumor Necrosis Factor-α to Promote Tumor Cell Trafficking and Progression

Extracellular RNA (eRNA) released from injured cells promotes tissue permeability, thrombosis, and inflammation in vitro and in vivo, and RNase1 pretreatment can reduce all these effects. In this study, we investigated the role of the eRNA/RNase1 system in tumor progression and metastasis. Under quiescent and stimulatory conditions, tumor cells released much higher levels of endogenous extracellular RNA (eRNA) than nontumor cells. In glioblastomas, eRNA was detected at higher levels in tumors than nontumor tissue. eRNA induced tumor cells to adhere to and migrate through human cerebral microvascular endothelial cells (HCMEC/D3), in a manner requiring activation of VEGF signaling. In addition, eRNA liberated TNF-α from macrophages in a manner requiring activation of the TNF-α-converting enzyme TACE. Accordingly, supernatants derived from eRNA-treated macrophages enhanced tumor cell adhesion to HCMEC/D3. TNF-α release evoked by eRNA relied upon signaling activation of mitogen-activated protein kinases and the NF-κB pathway. In subcutaneous xenograft models of human cancer, administration of RNase1 but not DNase decreased tumor volume and weight. Taken together, these results suggest that eRNA released from tumor cells has the capacity to promote tumor cell invasion through endothelial barriers by both direct and indirect mechanisms, including through a mechanism involving TNF-α release from tumor-infiltrating monocytes/macrophages. Our findings establish a crucial role for eRNA in driving tumor progression, and they suggest applications for extracellular RNase1 as an antiinvasive regimen for cancer treatment.

TWEAK/Fn14 pathway modulates properties of a human microvascular endothelial cell model of blood brain barrier

BackgroundThe TNF ligand family member TWEAK exists as membrane and soluble forms and is involved in the regulation of various human inflammatory pathologies, through binding to its main receptor, Fn14. We have shown that the soluble form of TWEAK has a pro-neuroinflammatory effect in an animal model of multiple sclerosis and we further demonstrated that blocking TWEAK activity during the recruitment phase of immune cells across the blood brain barrier (BBB) was protective in this model. It is now well established that endothelial cells in the periphery and astrocytes in the central nervous system (CNS) are targets of TWEAK. Moreover, it has been shown by others that, when injected into mice brains, TWEAK disrupts the architecture of the BBB and induces expression of matrix metalloproteinase-9 (MMP-9) in the brain. Nevertheless, the mechanisms involved in such conditions are complex and remain to be explored, especially because there is a lack of data concerning the TWEAK/Fn14 pathway in microvascular cerebral endothelial cells.MethodsIn this study, we used human cerebral microvascular endothelial cell (HCMEC) cultures as an in vitro model of the BBB to study the effects of soluble TWEAK on the properties and the integrity of the BBB model.ResultsWe showed that soluble TWEAK induces an inflammatory profile on HCMECs, especially by promoting secretion of cytokines, by modulating production and activation of MMP-9, and by expression of cell adhesion molecules. We also demonstrated that these effects of TWEAK are associated with increased permeability of the HCMEC monolayer in the in vitro BBB model.ConclusionsTaken together, the data suggest a role for soluble TWEAK in BBB inflammation and in the promotion of BBB interactions with immune cells. These results support the contention that the TWEAK/Fn14 pathway could contribute at least to the endothelial steps of neuroinflammation.

Endothelial Gene Expression and Molecular Changes in Response to Radiosurgery inIn VitroandIn VivoModels of Cerebral Arteriovenous Malformations

Radiosurgery for cerebral arteriovenous malformations (AVMs) is limited to 2-year latency. There is no early marker to monitor whether the lesion is responsive to radiosurgery. In this study, we examined endothelial gene expression and molecular changes in response to radiosurgery. Gene expression of E- and P-selectin, ICAM-1, PECAM-1, VCAM-1, tissue factor, and vWF in human cerebral microvascular endothelial cells was quantified by RT-qPCR at different radiation doses and time points. Soluble E- and P-selectin, ICAM-1, VCAM-1, and tissue factor in an animal model of AVMs were quantified by ELISA at different time after radiosurgery. We found that gene expression of E- and P-selectin, ICAM-1, PECAM-1, and VCAM-1 was upregulated by radiation in a dose-dependent manner (P < .05). Gene expression of E- and P-selectin and ICAM-1 was more sensitive to irradiation than that of PECAM-1 and VCAM-1. Radiosurgery induced gene expression of P-selectin, ICAM-1, PECAM-1, and VCAM-1 was linearly correlated with time (P < .05). Radiosurgery induced elevation of soluble E- and P-selectin, ICAM-1, VCAM-1, and tissue factor in a rat model of AVMs (P < .05). Thus, a combination of these molecules measured at different time points may serve as an early predictor of responsiveness of AVMs to radiosurgery.